Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

PubMed

Taupin, Philippe

2011-09-01

The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

Klotho protein lowered in senile patients with brady sinus arrhythmia.

PubMed

Wang, Ying; Yang, Wei; Zheng, Ernv; Zhang, Wei; Su, Xianming

2015-01-01

To explore the correlationship between brady sinus arrhythmia and the levels of serum klotho protein in aged. 104 patients over 75 years old with brady sinus arrhythmia (experiment group) were enrolled, including 34 cases of sinus arrest, 43 cases of sinus bradycardia and 25 cases of atrioventricular block. 109 patients over 75 years old without brady sinus arrhymia were chosen as control group. All subjects were monitored by Holter. The levels of serum klotho protein were detected and compared among three groups. The correlation between the frequency of sinus arrest and the levels of serum klotho protein was analyzed simultaneously. The levels of serum klotho protein in experiment group were lower than that in control group (P<0.01); the sinus arrest frequency was negatively correlated with the levels of serum klotho protien. The levels of serum klotho protein in patients with sinus arrest were lower than that with sinus bradycardia and atrioventricularblock (P<0.05). But there was no significant difference between sinus bradycardia group and atrioventricular block group. The levels of serum klotho protein may reflect the function of sinoatrial node and could be used as an index to estimate the function of sinoatrial node.

Klotho protein lowered in senile patients with brady sinus arrhythmia

PubMed Central

Wang, Ying; Yang, Wei; Zheng, Ernv; Zhang, Wei; Su, Xianming

2015-01-01

Objective: To explore the correlationship between brady sinus arrhythmia and the levels of serum klotho protein in aged. Methods: 104 patients over 75 years old with brady sinus arrhythmia (experiment group) were enrolled, including 34 cases of sinus arrest, 43 cases of sinus bradycardia and 25 cases of atrioventricular block. 109 patients over 75 years old without brady sinus arrhymia were chosen as control group. All subjects were monitored by Holter. The levels of serum klotho protein were detected and compared among three groups. The correlation between the frequency of sinus arrest and the levels of serum klotho protein was analyzed simultaneously. Results: The levels of serum klotho protein in experiment group were lower than that in control group (P<0.01); the sinus arrest frequency was negatively correlated with the levels of serum klotho protien. The levels of serum klotho protein in patients with sinus arrest were lower than that with sinus bradycardia and atrioventricularblock (P<0.05). But there was no significant difference between sinus bradycardia group and atrioventricular block group. Conclusion: The levels of serum klotho protein may reflect the function of sinoatrial node and could be used as an index to estimate the function of sinoatrial node. PMID:26550342

A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

PubMed Central

Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

2013-01-01

The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Comparison of high-sensitivity C-reactive protein and fetuin-A levels before and after treatment for subjects with subclinical hyperthyroidism.

PubMed

Bilgir, Oktay; Bilgir, Ferda; Topcuoglu, Tuba; Calan, Mehmet; Calan, Ozlem

2014-03-01

This study was designed to show the effect of propylthiouracil treatment on sCD40L, high-sensitivity C-reactive protein, and fetuin-A levels on subjects with subclinical hyperthyroidism. After checking sCD40L, high-sensitivity C-reactive protein, and fetuin-A levels of 35 patients with subclinical hyperthyroidism, each was given 50 mg tablets of propylthiouracil three times daily. After 3 months, sCD40L, high-sensitivity C-reactive protein, and fetuin-A levels were then compared to the levels before treatment. Although high-sensitivity C-reactive protein and sCD40L levels were normal in the subclinical hyperthyroidism patients compared to the healthy controls, fetuin-A levels were statistically significantly higher (*p = 0.022). After treatment, fetuin-A levels of subclinical hyperthyroidism patients decreased statistically significantly compared to the levels before treatment (**p = 0.026). sCD40L and high-sensitivity C-reactive protein levels did not have a statistically significant difference compared to the control group and post-propylthiouracil treatment. In subclinical hyperthyroidism patients, high fetuin-A levels before propylthiouracil treatment and decreases in these levels after treatment in cases with subclinical hyperthyroidism indicated the possibility of preventing long-term cardiac complications with propylthiouracil treatment.

Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis

PubMed Central

Zhou, Hao; Shen, Fengxian; Li, Juan; Xie, Zhenwei

2017-01-01

Objective To explore the expression level of Nrf2 in adenomyosis and study the mechanism of abnormal expression of Nrf2 in the pathogenesis of adenomyosis. Methods Western blot, immunohistochemistry(IHC) and real time PCR were used to measure Nrf2 expression levels in tissue and cell samples. Knockdown and overexpression of Nrf2 were used to investigate the variation of migration ability of endometrial glandular cells as well as the regulatory mechanism. Results Nrf2 protein levels were significantly higher in the eutopic and ectopic endometrial glands when compared with control cases using IHC and western blot methods. (p< 0.05). However, there was no statistical difference in Nrf2 mRNA expression levels between the adenomyosis and control groups. Using an agonist and Nrf2 siRNA, we regulated the Nrf2 protein levels of primary cultured endometrial glandular cells. With increased expression of Nrf2, cell scratch assay showed that the agonist-treated group migrated significantly faster than the control group, with MMP9 protein level markedly elevated. In contrast, Nrf2 siRNA-treated group migrated slower than the control group, with decreased expression of MMP9 protein. All of the scratching healing spaces and protein levels between the treated and control groups were statistically significant (p< 0.05). Conclusions Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis. Specified reduction of Nrf2 expression could prove to be a new therapeutic target in the clinical treatment of adenomyosis. PMID:28817677

Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis.

PubMed

Chen, Ning; Du, Baoying; Zhou, Hao; Shen, Fengxian; Li, Juan; Xie, Zhenwei

2017-01-01

To explore the expression level of Nrf2 in adenomyosis and study the mechanism of abnormal expression of Nrf2 in the pathogenesis of adenomyosis. Western blot, immunohistochemistry(IHC) and real time PCR were used to measure Nrf2 expression levels in tissue and cell samples. Knockdown and overexpression of Nrf2 were used to investigate the variation of migration ability of endometrial glandular cells as well as the regulatory mechanism. Nrf2 protein levels were significantly higher in the eutopic and ectopic endometrial glands when compared with control cases using IHC and western blot methods. (p< 0.05). However, there was no statistical difference in Nrf2 mRNA expression levels between the adenomyosis and control groups. Using an agonist and Nrf2 siRNA, we regulated the Nrf2 protein levels of primary cultured endometrial glandular cells. With increased expression of Nrf2, cell scratch assay showed that the agonist-treated group migrated significantly faster than the control group, with MMP9 protein level markedly elevated. In contrast, Nrf2 siRNA-treated group migrated slower than the control group, with decreased expression of MMP9 protein. All of the scratching healing spaces and protein levels between the treated and control groups were statistically significant (p< 0.05). Abnormal expression of Nrf2 may play an important role in the pathogenesis and development of adenomyosis. Specified reduction of Nrf2 expression could prove to be a new therapeutic target in the clinical treatment of adenomyosis.

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Mitochondrial Protein Profile in Mice with Low or Excessive Selenium Diets

PubMed Central

Hu, Lianmei; Wang, Congcong; Zhang, Qin; Yan, Hao; Li, Ying; Pan, Jiaqiang; Tang, Zhaoxin

2016-01-01

Dietary selenium putatively prevents oxidative damage, whereas excessive selenium may lead to animal disorder. In this study, we investigated the effects of low and excessive levels of dietary selenium on oxidative stress and mitochondrial proteins in mouse liver. Six to eight week old mice were fed a diet with low, excessive, or moderate (control) levels of selenium (sodium selenite). The selenium concentration and oxidative stress-related parameters in hepatic mitochondria were evaluated. Two-dimensional electrophoresis and mass spectrometry were applied to identify the differentially-expressed proteins associated with dietary selenium. The selenium content of the livers in mice with the low selenium diet was significantly lower than that of the control, while that of mice fed excessive levels was significantly higher. In both groups oxidative stress in hepatic mitochondria was found; accompanied by lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) levels and higher malondialdehyde (MDA) content, compared with the control group. Furthermore, ten proteins in the hepatic mitochondria of the selenium-low or -excessive groups with more than two-fold differences in abundance compared with the control group were identified. The differentially-expressed proteins in hepatic mitochondria may be associated with dietary (low or excessive) selenium-induced oxidative stress. PMID:27428959

In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks

PubMed Central

Orabona, Emanuele; De Stefano, Luca; Ferry, Mike; Hasty, Jeff; di Bernardo, Mario; di Bernardo, Diego

2014-01-01

We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available. PMID:24831205

Estimation of Serum Triglycerides, Serum Cholesterol, Total Protein, IgG Levels in Chronic Periodontitis Affected Elderly Patients: A Cross-Sectional Study

PubMed Central

Saravanan, A. V.; Ravishankar, P. L.; Kumar, Pradeep; Rajapandian, K.; Kalaivani, V.; Rajula, M. Prem Blaisie

2017-01-01

Aim: The present study was conducted to evaluate the serum triglycerides, serum cholesterol, total protein, and IgG levels in elderly patients who were affected by periodontal disease. Materials and Methods: This study was conducted at the Rajah Muthiah Dental College and Hospital in the periodontics division. The study was conducted for a period of 3 months. This study is a prospective analytical study. Sixty individuals who were systemically healthy in the age group of 50 and above were included in this study. Control and experimental groups of 30 participants each were included. Plaque index, gingival index, probing pocket depth, and clinical attachment loss were recorded. Biochemical parameters such as serum cholesterol, serum triglycerides, total protein, and IgG levels were also evaluated and correlated with the periodontal parameters. Data was analyzed using SPSS version 16.0 (IBM Corp., Armonk, NY). The relationship between periodontal status and the biochemical parameters such as serum cholesterol, serum triglycerides, total protein, and IgG levels were evaluated by Student's t-test. Results: There was no significant difference in the plaque and gingival scores between the experimental and control group. It was observed that serum cholesterol level and total protein level was lower in participants suffering from chronic periodontitis. Triglycerides level was significantly elevated in the experimental group. IgG, a level which is not significant, concluded that there is no difference in control and experimental group. Conclusion: It was concluded from the results obtained from the study that there is an association between serum triglycerides, serum cholesterol, total protein, and periodontal disease. However, further longitudinal and well-controlled studies are required to evaluate the relationship between these biochemical parameters and periodontal disease. PMID:28462181

Estimation of Serum Triglycerides, Serum Cholesterol, Total Protein, IgG Levels in Chronic Periodontitis Affected Elderly Patients: A Cross-Sectional Study.

PubMed

Saravanan, A V; Ravishankar, P L; Kumar, Pradeep; Rajapandian, K; Kalaivani, V; Rajula, M Prem Blaisie

2017-01-01

The present study was conducted to evaluate the serum triglycerides, serum cholesterol, total protein, and IgG levels in elderly patients who were affected by periodontal disease. This study was conducted at the Rajah Muthiah Dental College and Hospital in the periodontics division. The study was conducted for a period of 3 months. This study is a prospective analytical study. Sixty individuals who were systemically healthy in the age group of 50 and above were included in this study. Control and experimental groups of 30 participants each were included. Plaque index, gingival index, probing pocket depth, and clinical attachment loss were recorded. Biochemical parameters such as serum cholesterol, serum triglycerides, total protein, and IgG levels were also evaluated and correlated with the periodontal parameters. Data was analyzed using SPSS version 16.0 (IBM Corp., Armonk, NY). The relationship between periodontal status and the biochemical parameters such as serum cholesterol, serum triglycerides, total protein, and IgG levels were evaluated by Student's t-test. There was no significant difference in the plaque and gingival scores between the experimental and control group. It was observed that serum cholesterol level and total protein level was lower in participants suffering from chronic periodontitis. Triglycerides level was significantly elevated in the experimental group. IgG, a level which is not significant, concluded that there is no difference in control and experimental group. It was concluded from the results obtained from the study that there is an association between serum triglycerides, serum cholesterol, total protein, and periodontal disease. However, further longitudinal and well-controlled studies are required to evaluate the relationship between these biochemical parameters and periodontal disease.

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

PubMed

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

The ever-evolving role of mTOR in translation.

PubMed

Fonseca, Bruno D; Smith, Ewan M; Yelle, Nicolas; Alain, Tommy; Bushell, Martin; Pause, Arnim

2014-12-01

Control of translation allows for the production of stoichiometric levels of each protein in the cell. Attaining such a level of fine-tuned regulation of protein production requires the coordinated temporal and spatial control of numerous cellular signalling cascades impinging on the various components of the translational machinery. Foremost among these is the mTOR signalling pathway. The mTOR pathway regulates both the initiation and elongation steps of protein synthesis through the phosphorylation of numerous translation factors, while simultaneously ensuring adequate folding of nascent polypeptides through co-translational degradation of misfolded proteins. Perhaps most remarkably, mTOR is also a key regulator of the synthesis of ribosomal proteins and translation factors themselves. Two seminal studies have recently shown in translatome analysis that the mTOR pathway preferentially regulates the translation of mRNAs encoding ribosomal proteins and translation factors. Therefore, the role of the mTOR pathway in the control of protein synthesis extends far beyond immediate translational control. By controlling ribosome production (and ultimately ribosome availability), mTOR is a master long-term controller of protein synthesis. Herein, we review the literature spanning the early discoveries of mTOR on translation to the latest advances in our understanding of how the mTOR pathway controls the synthesis of ribosomal proteins. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Serum Heat Shock Protein 70 Level as a Biomarker of Exceptional Longevity

PubMed Central

Terry, Dellara F.; Wyszynski, Diego F.; Nolan, Vikki G.; Atzmon, Gil; Schoenhofen, Emily A.; Pennington, JaeMi Y.; Andersen, Stacy L.; Wilcox, Marsha A.; Farrer, Lindsay A.; Barzilai, Nir; Baldwin, Clinton T.; Asea, Alexzander

2006-01-01

Heat shock proteins are highly conserved proteins that, when produced intracellularly, protect stress exposed cells. In contrast, extracellular Hsp70 has been shown to have both protective and deleterious effects. In this study, we assessed heat shock protein 70 (Hsp70) for its potential role in human longevity. Because of the importance of HSP to disease processes, cellular protection, and inflammation, we hypothesized that: (1) Hsp70 levels in centenarians and centenarian offspring are different from controls and (2) alleles in genes associated with Hsp70 explain these differences. In this cross-sectional study, we assessed serum Hsp70 levels from participants enrolled in either the New England Centenarian Study (NECS) or the Longevity Genes Project (LGP): 87 centenarians (from LGP), 93 centenarian offspring (from NECS), and 126 controls (43 from NECS, 83 from LGP). We also examined genotypic and allelic frequencies of polymorphisms in HSP70-A1A and HSP70-A1B in 347 centenarians (266 from the NECS, 81 from the LGP), 260 NECS centenarian offspring, and 238 controls (NECS: 53 spousal controls and 106 septuagenarian offspring controls; LGP: 79 spousal controls). The adjusted mean serum Hsp70 levels (ng/mL) for the NECS centenarian offspring, LGP centenarians, LGP spousal controls, and NECS controls were 1.05, 1.13, 3.05, 6.93, respectively, suggesting that a low serum Hsp70 level is associated with longevity; however, no genetic associations were found with two SNPs within two hsp70 genes. PMID:17027907

Alterations of the levels of primary antioxidant enzymes in different grades of human astrocytoma tissues.

PubMed

Yen, Hsiu-Chuan; Lin, Chih-Lung; Chen, Bing-Shian; Chen, Chih-Wei; Wei, Kuo-Chen; Yang, Mei-Lin; Hsu, Jee-Ching; Hsu, Yung-Hsing

2018-06-03

Malignant astrocytoma is the most commonly occurring brain tumor in humans. Oxidative stress is implicated in the development of cancers. Superoxide dismutase 2 (SOD2) was found to exert tumor suppressive effect in basic research, but increased SOD2 protein level was associated with higher aggressiveness of human astrocytomas. However, studies reporting alterations of antioxidant enzymes in human astrocytomas often employed less accurate methods or included different types of tumors. Here we analyzed the mRNA levels, activities, and protein levels of primary antioxidant enzymes in control brain tissues and various grades of astrocytomas obtained from 40 patients. SOD1 expression, SOD1 activity, and SOD1 protein level were lower in Grade IV astrocytomas. SOD2 expression was lower in low-grade (Grades I and II) and Grade III astrocytomas than in controls, but SOD2 expression and SOD2 protein level were higher in Grade IV astrocytomas than in Grade III astrocytomas. Although there was no change in SOD2 activity and a lower activity of citrate synthase (CS), the MnSOD:CS ratio increased in Grade IV astrocytomas compared with controls and low-grade astrocytomas. Furthermore, SOD1 activity, CS activity, SOD1 expression, GPX4 expression, and GPX4 protein level were inversely correlated with the malignancy, whereas catalase activity, catalase protein, SOD2 protein level, and the SOD2:CS ratio were positively correlated with the degree of malignancy. Lower SOD2:CS ratio was associated with poor outcomes for Grade IV astrocytomas. This is the first study to quantify changes of various primary antioxidant enzymes in different grades of astrocytomas at different levels concurrently in human astrocytomas.

Does soy protein affect circulating levels of unbound IGF-1?

PubMed

Messina, Mark; Magee, Pamela

2018-03-01

Despite the enormous amount of research that has been conducted on the role of soyfoods in the prevention and treatment of chronic disease, the mechanisms by which soy exerts its physiological effects are not fully understood. The clinical data show that neither soyfoods nor soy protein nor isoflavones affect circulating levels of reproductive hormones in men or women. However, some research suggests that soy protein, but not isoflavones, affects insulin-like growth factor I (IGF-1). Since IGF-1 may have wide-ranging physiological effects, we sought to determine the effect of soy protein on IGF-1 and its major binding protein insulin-like growth factor-binding protein (IGFBP-3). Six clinical studies were identified that compared soy protein with a control protein, albeit only two studies measured IGFBP-3 in addition to IGF-1. Although the data are difficult to interpret because of the different experimental designs employed, there is some evidence that large amounts of soy protein (>25 g/day) modestly increase IGF-1 levels above levels observed with the control protein. The clinical data suggest that a decision to incorporate soy into the diet should not be based on its possible effects on IGF-1.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

PubMed

Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

PubMed Central

Rodríguez, Andrea E.; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

ABSTRACT Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells. PMID:26654586

Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

NASA Astrophysics Data System (ADS)

Li, Erchao; Arena, Leticia; Lizama, Gabriel; Gaxiola, Gabriela; Cuzon, Gerard; Rosas, Carlos; Chen, Liqiao; van Wormhoudt, Alain

2011-03-01

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture. The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp ( L. vannamei) were investigated. This involved an examination of growth performance, glutamate dehydrogenase (GDH) and Na+-K+ ATPase mRNA expression,, and GDH activity in muscles and gills. Three experimental diets were formulated, containing 25%, 40%, and 50% dietary protein, and fed to the shrimp at a salinity of 25. After 20 days, no significant difference was observed in weight gain, though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels. Subsequently, shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5, respectively, and sampled at weeks 1 and 2. Shrimp fed with 40% protein at 25 in salinity (optimal conditions) were used as a control. Regardless of the salinities, shrimp fed with 50% dietary protein had significantly higher growth performance than other diets; no significant differences were found in comparison with the control. Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks. Ambient salinity change also stimulated the hepatosomatic index, which increased in the first week and then recovered to a relatively normal level, as in the control, after 2 weeks. These findings indicate that in white shrimp, the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism. Increased dietary protein level could improve the osmoregulation capacity of L. vannamei with more energy resources allocated to GDH activity and expression.

Protein Z G79A polymorphism in Turkish pediatriccerebral infarct patients.

PubMed

Öztürk, Ayşenur; Eğin, Yonca; Deda, Gülhis; Teber, Serap; Akar, Nejat

2008-09-05

Protein Z (PZ) plays an enhancer role in coagulation as an anticoagulant. This is the first study in which G79A polymorphism investigated in Turkish paediatric stroke patients. Ninety-one paediatric stroke patients with cerebral ischemia and 70 control subjects were analyzed for PZ G79A and also FVL, PT mutations. PZ 79 'A' allele in homozygous state was found in five patients (5,5%), while it was found only in one control subject (1,4%) and it was seemed as a risk factor for peadiatric ischemia [OR=3,94 (0,44-35,1)]. When patients and controls who had FVL and PT carriers were excluded, AA genotype carried a risk [OR=3,88 (0,41-36,5)]. Also plasma protein Z levels measured in 21 stroke patients and 52 controls. Plasma protein Z levels were not different between stroke patients (500,95 ngmL-1±158,35) and controls (447,34 ngmL-1±165,97). But the plasma levels of protein Z was decreased in patients with AA genotype. Our data showed that carrying 79 AA genotype could be a genetic risk factor for cerebral infarct in peadiatric patients.

Expression of fas protein on CD4+T cells irradiated by low level He-Ne

NASA Astrophysics Data System (ADS)

Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

2005-07-01

Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2（60 minutes）and 45.84J/cm2（120minutes）, the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

Alteration in G Proteins and Prolactin Levels in Pituitary After Ethanol and Estrogen Treatment

PubMed Central

Chaturvedi, Kirti; Sarkar, Dipak K.

2010-01-01

Background Chronic administration of ethanol increases plasma prolactin levels and enhances estradiol’s mitogenic action on the lactotropes of the pituitary gland. The present study was conducted to determine the changes in the pituitary levels of G proteins during the tumor development following alcohol and ethanol treatments. Methods Using ovariectomized Fischer-344 female rats, we have determined ethanol and estradiol actions at 2 and 4 weeks on pituitary weight and pituitary cell contents of prolactin, Gs. Gq11, Gi1, Gi2, and Gi3 proteins. Western blots were employed to measure protein contents. Results Ethanol increased basal and estradiol-enhanced wet weight and the prolactin content in the pituitary in a time-dependent manner. Chronic exposure of estradiol increased the levels of Gs protein in the pituitary. Unlike estradiol, ethanol exposure did not show significant effect on the basal level of Gs protein, but moderately increased the estradiol-induced levels of this protein. Estradiol exposure enhanced Gq11 protein levels in the pituitary after 2 and 4 weeks, while ethanol treatment failed to alter these protein levels in the pituitary in control-treated or estradioltreated ovariectomized rats. In the case of Gi1, estradiol but not ethanol increased the level of this protein at 4 weeks of treatment. However, estradiol and ethanol alone reduced the levels of both Gi2 and Gi3 proteins at 2 and 4 weeks of treatment. Ethanol also significantly reduced the estradiol-induced Gi2 levels at 4 weeks and Gi3 level at 2 and 4 weeks. Conclusions These results confirm ethanol’s and estradiol’s growth-promoting and prolactin stimulating actions on lactotropes of the pituitary and further provide evidence that ethanol and estradiol may control lactotropic cell functions by altering expression of specific group of G proteins in the pituitary. PMID:18336630

Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

PubMed

Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

2016-09-01

Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

PubMed Central

Amândio, Ana Rita; Gaspar, Pedro; Whited, Jessica L.; Janody, Florence

2014-01-01

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth. PMID:24788460

Increased expression of sex determining region Y-box 11 (SOX11) in cutaneous malignant melanoma.

PubMed

Jian, Jiao; Guoying, Wang; Jing, Zhao

2013-08-01

To observe sex determining region Y-box 11 (SOX11) gene expression in cutaneous malignant melanoma and its effect on tumour cell proliferation. Clinicopathological data and tissue samples from patients with cutaneous malignant melanoma, together with tissue samples from healthy volunteers (controls), were retrospectively reviewed. Protein levels of SOX11 and the antigen identified by monoclonal antibody Ki-67 (Ki-67) in skin lesions were analysed using immunohistochemistry. The correlation between protein levels and clinipathological parameters was investigated. Out of 40 patient samples, 25 (62.5%) were positive for SOX11 protein in malignant melanoma tissue. This was significantly higher than in 40 control tissue samples, in which no SOX11 protein was detected. Presence of SOX11 protein was positively related to the proliferation index of cutaneous malignant melanoma tumour cells. Presence of SOX11 protein in cutaneous malignant melanoma was related to tumour type, tumour location, lymph node metastasis and 5-year survival rate. Human cutaneous malignant melanoma tissues expressed high levels of SOX11 compared with healthy controls, suggesting that SOX11 may be a new prognostic marker for malignant melanoma.

Level of supplemental protein does not influence the ruminally undegradable protein value.

PubMed

Legleiter, L R; Mueller, A M; Kerley, M S

2005-04-01

Two experiments were conducted to determine whether elevating the percentage of ruminally undegradable protein (RUP) in the diet would influence the RUP value of the protein feedstuff. A single-effluent, continuous-culture study was designed to test the effect of RUP inclusion rate in the diet on ruminal degradability of the protein. Treatments consisted (DM basis) of a control diet with no supplemental protein, control + 2.5% bloodmeal (BM-L), control + 5% bloodmeal (BM-H), control + 4.45% soybean meal (SBM-L), and control + 8.89% soybean meal (SBM-H). Proteolytic activity and total VFA concentration were not affected (P = 0.73 and P = 0.13) by treatment. Within protein source, dietary RUP value was not affected (P = 0.94) by level of inclusion. When corrected for control diet RUP flow, the RUP value of the blood meal (BM) protein was higher (P = 0.01) than soybean meal (SBM); however, level of supplementation did not affect (P = 0.07) the RUP value of BM or SBM. In Exp. 2, 32 British x Continental crossbred steers (276 +/- 26.3 kg) were fed for 72 d to examine the effects of balancing the AA:energy ratio, using BM as a RUP source, on ADG, G:F, and lean tissue deposition. Diets were formulated to provide increasing levels of arginine, while ruminally degradable protein and energy were held constant. Four dietary treatments provided 0.5, 1, 1.5, and 2x the required amount of arginine, whereas the control diet had no BM included. Daily DMI averaged 7.6 kg/steer and did not differ (P = 0.71) among treatments. Steers gained an average of 1.9 kg/d and average G:F was 0.260, with no differences (P = 0.60 and P = 0.97, respectively) among treatments. There was no difference (P = 0.48) in the change in 12th-rib fat depth during the study; however, change in LM area was affected quadratically as the level of BM increased in the diet, with the greatest increase in LM area occurring in steers fed the 1x and 1.5x required arginine treatments. Balancing the AA:energy ratio did not affect G:F, DMI, or ADG; however, it increased deposition of lean in the LM quadratically. Level of dietary inclusion of BM as an RUP source does not affect its RUP value or efficacy of providing postruminal AA in growing steers.

Senile dementia of Lewy body type and Alzheimer type are biochemically distinct in terms of paired helical filaments and hyperphosphorylated tau protein.

PubMed

Harrington, C R; Perry, R H; Perry, E K; Hurt, J; McKeith, I G; Roth, M; Wischik, C M

1994-01-01

We have used biochemical assays to examine cingulate and occipital cortices from age-matched cases of Alzheimer's disease (AD; n = 12), senile dementia of the Lewy body type (SDLT; n = 13), Parkinson's disease (PD; 5 non-demented cases and 7 cognitively impaired cases) and controls (n = 11) for paired helical filaments (PHFs), phosphorylated and normal tau protein and beta/A4-protein. Whereas cingulate cortex is characterised by relatively high densities of cortical Lewy bodies in the SDLT cases and lower numbers in PD, these inclusion bodies were absent in the cingulate cortex from AD and control cases. Protease-resistant PHFs and hyperphosphorylated tau protein were found in AD and, at low levels, in a minority of SDLT cases. Qualitatively, both of these preparations were indistinguishable in SDLT from those found in AD but levels of both parameters in SDLT were less than 5% of those in AD. SDLT, PD and control groups did not differ from each other in terms of the quantity of protease-resistant PHFs or the level of hyperphosphorylated tau. Furthermore, PHF accumulation did not distinguish between PD cases with or without dementia. The levels of normal tau protein did not differ between the four groups. beta/A4 protein levels did not distinguish between PD and control groups, between AD and SDLT groups, or between SDLT and control groups for either cingulate or occipital cortices. Thus extensive accumulation of PHFs in either neurofibrillary tangles or dystrophic neurites is not a feature of either SDLT or PD. Our findings provide molecular support for the neuropathological and clinical separation of SDLT as a form of dementia that is distinct from AD.

The roles of connective tissue growth factor in the development of anastomotic esophageal strictures.

PubMed

Zhao, Haibin; Zhao, Lingna; Zhou, Zhihua; Wu, Yaoyi

2015-08-12

The aim of this study was to investigate the roles of connective tissue growth factor (CTGF) in the development of anastomotic strictures after surgical repair of the esophagus. Tissues collected from the patients were divided into three groups based on the results of endoscopy and clinical grading. Patients without dysphagia after esophagectomy were used as the control population. The protein levels of CTGF, TGF-β1, Smad2, and Smad4 were determined by immunohistochemistry (IHC) and western blot analyses, while the mRNA levels of the two growth factors were evaluated by real-time polymerase chain reaction. Compared with the control group, significantly increased (p < 0.01) levels of CTGF and TGF-β1 protein were observed in the anastomotic stenosis (AS) group, and levels of the two proteins detected by the IHC and western blot analyses were also significantly increased with the increasing severity of stenosis (p < 0.05). The mRNA levels of CTGF and TGF-β1 in the tissues collected from the patients with stenosis were significantly up-regulated (p < 0.05) as compared with those from the control group. In addition, the levels of Smad2 and Smad4 protein were also significantly increased (p < 0.05) with the increasing severity of stenosis, and the protein levels were positively correlated with the levels of CTGF (r = 0.59, p < 0.05) and TGF-β1 (r = 0.63, p < 0.05). Inhibition of CTGF protein or mRNA expression may be a distinctive and effective therapy for the treatment of postoperative anastomotic strictures.

Neural restrictive silencer factor and choline acetyltransferase expression in cerebral tissue of Alzheimer’s Disease patients: A pilot study

PubMed Central

González-Castañeda, Rocío E.; Sánchez-González, Víctor J.; Flores-Soto, Mario; Vázquez-Camacho, Gonzalo; Macías-Islas, Miguel A.; Ortiz, Genaro G.

2013-01-01

Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer’s Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels. PMID:23569405

Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate.

PubMed

Park, Gun Wook; Hwang, Heeyoun; Kim, Kwang Hoe; Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Ji Yeong; Ji, Eun Sun; Park, Sung-Kyu Robin; Yates, John R; Kwon, Kyung-Hoon; Park, Young Mok; Lee, Hyoung-Joo; Paik, Young-Ki; Kim, Jin Young; Yoo, Jong Shin

2016-11-04

In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

Dietary protein intake, energy deficit, and nitrogen balance in normal-weight adults: a randomized controlled

USDA-ARS?s Scientific Manuscript database

Consuming protein at levels higher than the recommended dietary allowance (RDA) may be metabolically advantageous for overweight and obese individuals attempting weight loss. However, the dose-response characteristics that define the optimal level of dietary protein necessary to sustain measures of...

The impact of carbohydrate and protein level and sources on swine manure foaming properties

USDA-ARS?s Scientific Manuscript database

This study explored the impact of swine diet on the composition, methane production potential, and foaming properties of manure. Samples of swine manure were collected from controlled feeding trials with diets varying in protein and carbohydrate levels and sources. Protein sources consisted of corn ...

Controlled levels of protein modification through a chromatography-mediated bioconjugation

DOE PAGES

Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; ...

2015-02-27

Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety usingmore » an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.« less

Protein S is protective in pulmonary fibrosis.

PubMed

Urawa, M; Kobayashi, T; D'Alessandro-Gabazza, C N; Fujimoto, H; Toda, M; Roeen, Z; Hinneh, J A; Yasuma, T; Takei, Y; Taguchi, O; Gabazza, E C

2016-08-01

Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis. © 2016 International Society on Thrombosis and Haemostasis.

Increased neuroinflammatory and arachidonic acid cascade markers, and reduced synaptic proteins, in the postmortem frontal cortex from schizophrenia patients

PubMed Central

Rao, Jagadeesh Sridhara; Kim, Hyung-Wook; Harry, Gaylia Jean; Rapoport, Stanley Isaac; Reese, Edmund Arthur

2013-01-01

Schizophrenia (SZ) is a progressive, neuropsychiatric disorder associated with cognitive impairment. A number of brain alterations have been linked to cognitive impairment, including neuroinflammation, excitotoxicity, increased arachidonic acid (AA) signaling and reduced synaptic protein. On this basis, we tested the hypothesis that SZ pathology is associated with these pathological brain changes. To do this, we examined postmortem frontal cortex from 10 SZ patients and 10 controls and measured protein and mRNA levels of cytokines, and astroglial, microglial, neuroinflammatory excitotoxic, AA cascade, apoptotic and synaptic markers. Mean protein and mRNA levels of interleukin-1β, tumor necrosis factor-α, glial acidic fibrillary protein (GFAP), a microglial marker CD11b, and nuclear factor kappa B subunits were significantly increased in SZ compared with control brain. Protein and mRNA levels of cytosolic and secretory phospholipase A2 and cyclooxygenase were significantly elevated in postmortem brains from SZ patients. N-methyl-D-aspartate receptor subunits 1 and 2B, inducible nitric oxide synthase and c-FOS were not significantly different. In addition, reduced protein and mRNA levels of brain-derived neurotrophic factor, synaptophysin and drebrin were found in SZ compared with control frontal cortex. Increased neuroinflammation and AA cascade enzyme markers with synaptic protein loss could promote disease progression and cognitive defects in SZ patients. Drugs that downregulate these changes might be considered for new therapies in SZ. PMID:23566496

Dysregulated glutamate and dopamine transporters in postmortem frontal cortex from bipolar and schizophrenic patients

PubMed Central

Rao, Jagadeesh Sridhara; Kellom, Matthew; Reese, Edmund Arthur; Rapoport, Stanley Isaac; Kim, Hyung-Wook

2012-01-01

Background Dysregulated glutamate, serotonin and dopamine neurotransmission has been reported in bipolar disorder (BD) and schizophrenia (SZ), but the underlying mechanisms of dysregulation are not clear. We hypothesized that they involve alterations in excitatory amino acid transporters (EAATs), the serotonin reuptake transporter (SERT), and the dopamine reuptake transporter (DAT). Methods To test this hypothesis, we determined protein and mRNA levels of EAAT subtypes 1–4, of the SERT and of the DAT in postmortem frontal cortex from BD (n=10) and SZ (n=10) patients and from healthy control (n=10) subjects. Results Compared to control levels, protein and mRNA levels of EAAT1 were increased significantly in cortex from both BD and SZ patients. EAAT2 protein and mRNA levels were decreased significantly in BD but not in SZ cortices. EAAT3 and EAAT 4 protein and mRNA levels were significantly higher in SZ but not in BD compared with control. DAT protein and mRNA levels were decreased significantly in both BD and SZ cortex. There was no significant change in SERT expression in either BD or SZ. Conclusions The altered EAATs and DAT expression could result in altered glutamatergic and hyperdopaminergic function in BD and SZ. Differently altered EAATs involved in glutamatergic transmission could be therapeutic targets for treating BD and SZ. PMID:21925739

High Protein Intake Does Not Prevent Low Plasma Levels of Conditionally Essential Amino Acids in Very Preterm Infants Receiving Parenteral Nutrition.

PubMed

Morgan, Colin; Burgess, Laura

2017-03-01

We have shown that increasing protein intake using a standardized, concentrated, added macronutrients parenteral (SCAMP) nutrition regimen improves head growth in very preterm infants (VPIs) compared with a control parenteral nutrition (PN) regimen. VPIs are at risk of conditionally essential amino acid (CEAA) deficiencies because of current neonatal PN amino acid (AA) formulations. We hypothesized that the SCAMP regimen would prevent low plasma levels of CEAAs. To compare the plasma AA profiles at approximately day 9 of life in VPIs receiving SCAMP vs a control PN regimen. VPIs (<29 weeks' gestation) were randomized to receive SCAMP (30% more PN AA) or a control regimen. Data were collected to measure parenteral and enteral protein, energy, and individual AA intake and the first plasma AA profile. Plasma profiles of the 20 individual protogenic AA levels were measured using ion exchange chromatography. Plasma AA profiles were obtained at median (interquartile range [IQR]) age of 9 (8-10) days in both SCAMP (n = 59) and control (n = 67) groups after randomizing 150 VPIs. Median (IQR) plasma levels of individual essential AAs were higher than the reference population mean (RPM) in both groups, especially for threonine. SCAMP infants had higher plasma levels of essential AAs than did the controls. Median (IQR) plasma levels of glutamine, arginine, and cysteine (CEAAs) were lower than the RPM in both groups. Plasma AA levels in PN-dependent VPIs indicate there is an imbalance in essential and CEAA provision in neonatal PN AA formulations that is not improved by increasing protein intake.

Altered binding of /sup 125/I-labeled calmodulin to a 46. 5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tallant, E.A.; Wallace, R.W.

1987-02-01

The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca/sup 2 +//calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblastsmore » or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of /sup 125/I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.« less

Levels of metacaspase1 and chaperones related to protein quality control in alcoholic and nonalcoholic steatohepatitis.

PubMed

Mendoza, Alejandro S; Dorce, Jacques; Peng, Yue; French, Barbara A; Tillman, Brittany; Li, Jun; French, Samuel W

2015-02-01

Efficient management of misfolded or aggregated proteins in ASH and NASH is crucial for continued hepatic viability. Cellular protein quality control systems play an important role in the pathogenesis and progression of ASH and NASH. In a recent study, elevated Mca1 expression counteracted aggregation and accumulation of misfolded proteins and extended the life span of the yeast Saccharomyces cerevisiae (Hill et al, 2014). Mca1 may also associate with Ssa1 and Hsp104 in disaggregation and fragmentation of aggregated proteins and their subsequent degradation through the ER-associated degradation (ERAD) pathway. If degradation is not available, protection of the cellular environment from a misfolded protein is accomplished by its sequestration into two distinct inclusion bodies (Kaganovich et al., 2008) called the JUNQ (JUxta Nuclear Quality control compartment) and the IPOD (Insoluble Protein Deposit). Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 all play important roles in protein quality control systems. This study aims to measure the expression of Mca1 and related chaperones involved in protein quality control in alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) compared with normal control liver biopsies. Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 expressions were measured in three to six formalin-fixed paraffin embedded ASH and NASH liver biopsies and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. Mca1, Hsp104, Ydj1 and p62 were significantly upregulated compared to control (p<0.05) in ASH specimens. Hsp40 and VCP/p97 were also uptrending in ASH. In NASH, the only significant difference was the increased expression of Hsp104 compared to control (p<0.05). Ssa1 levels were uptrending in both ASH and NASH specimens. The upregulation of Mca1, Hsp104, Ydj1 and p62 in ASH may be elicited as a response to the chronic exposure of the hepatocytes to the toxicity of alcohol. Recruitment of Mca1, Hsp104, Ydj1 and p62 may indicate that autophagy, the ERAD, JUNQ, and IPOD systems are active in ASH. Whereas in NASH, elevated Hsp104 and uptrending Ssa1 levels may indicate that autophagy and IPOD may be the only active protein quality control systems involved. Published by Elsevier Inc.

Consumption of protein-enriched milk has minor effects on inflammation in older adults-A 12-week double-blind randomized controlled trial.

PubMed

Gjevestad, Gyrd O; Ottestad, Inger; Biong, Anne Sofie; Iversen, Per Ole; Retterstøl, Kjetil; Raastad, Truls; Skålhegg, Bjørn S; Ulven, Stine M; Holven, Kirsten B

2017-03-01

Aging is associated with increased levels of circulating inflammatory markers and reduced muscle mass and strength. We investigated whether intake of protein-enriched milk for 12 weeks would influence markers of inflammation among adults ≥70years of age with reduced physical strength. In a double-blind randomized controlled intervention study, subjects were randomly allocated into two groups, receiving a protein-enriched milk (2×20g protein/d, n=14, mean (±SD) age 76.9±4.9 yrs) or an isocaloric carbohydrate drink (n=17, age 77.7±4.8 yrs) for 12 weeks. We measured serum and mRNA expression levels of inflammatory markers in PBMCs. Significant differences in the mRNA expression of nuclear receptor subfamily, group H, member 3 (NR1H3, encoding the LXRα transcription factor) and interferon gamma (INFG) were observed between groups. The mRNA level of TNFRSF1A was significantly reduced, while the mRNA level of dipeptidyl-peptidase 4 (DPP4) was significantly increased, in the control group. The serum level of TNFα increased significantly in the control group, while sTNFRSF1A increased significantly in both groups, but with no significant differences between groups. Consumption of a low-fat, protein-enriched milk for 12 weeks had minor effects on inflammatory related markers in older adults compared to an isocaloric carbohydrate drink. Copyright © 2017 Elsevier B.V. All rights reserved.

GCN5L1 modulates cross-talk between mitochondria and cell signaling to regulate FoxO1 stability and gluconeogenesis.

PubMed

Wang, Lingdi; Scott, Iain; Zhu, Lu; Wu, Kaiyuan; Han, Kim; Chen, Yong; Gucek, Marjan; Sack, Michael N

2017-09-12

The mitochondrial enriched GCN5-like 1 (GCN5L1) protein has been shown to modulate mitochondrial protein acetylation, mitochondrial content and mitochondrial retrograde signaling. Here we show that hepatic GCN5L1 ablation reduces fasting glucose levels and blunts hepatic gluconeogenesis without affecting systemic glucose tolerance. PEPCK and G6Pase transcript levels are downregulated in hepatocytes from GCN5L1 liver specific knockout mice and their upstream regulator, FoxO1 protein levels are decreased via proteasome-dependent degradation and via reactive oxygen species mediated ERK-1/2 phosphorylation. ERK inhibition restores FoxO1, gluconeogenic enzyme expression and glucose production. Reconstitution of mitochondrial-targeted GCN5L1 blunts mitochondrial ROS, ERK activation and increases FoxO1, gluconeogenic enzyme expression and hepatocyte glucose production. We suggest that mitochondrial GCN5L1 modulates post-translational control of FoxO1, regulates gluconeogenesis and controls metabolic pathways via mitochondrial ROS mediated ERK activation. Exploring mechanisms underpinning GCN5L1 mediated ROS signaling may expand our understanding of the role of mitochondria in gluconeogenesis control.Hepatic gluconeogenesis is tightly regulated at transcriptional level and is essential for survival during prolonged fasting. Here Wang et al. show that the mitochondrial enriched GCN5-like 1 protein controls hepatic glucose production by regulating FoxO1 protein levels via proteasome-dependent degradation and, in turn, gluconeogenic gene expression.

Effects of dietary corn gluten meal on growth performance and protein metabolism in relation to IGF-I and TOR gene expression of juvenile cobia ( Rachycentron canadum)

NASA Astrophysics Data System (ADS)

Luo, Yiwen; Ai, Qinghui; Mai, Kangsen; Zhang, Wenbing; Xu, Wei; Zhang, Yanjiao; Liufu, Zhiguo

2013-09-01

A growth experiment was conducted on cobia ( Rachycentron canadum, initial weight 108.2 g ± 3.0 g) to investigate the effects of dietary corn gluten meal (CGM) levels on the fish growth, whole body composition and protein metabolism in relation to specific gene expression. Five isonitrogenous (crude protein 45%) and isoenergetic (gross energy 20 kJ g-1) practical diets were formulated by replacing 0% (the control), 17.5%, 35.0%, 52.5%, and 70.0% of fish meal (FM) protein with CGM protein. No significant differences were observed in the survival, feed intake (FI), specific growth rate (SGR), feed efficiency (FE) and protein productive value (PPV) among fish fed diets with 0%, 17.5%, 35.0%, and 52.5% of CGM protein. However, these indices were significantly lower in fish fed the diet with 70.0% of CGM protein than those in fish fed the control diet ( P < 0.05). The whole-body crude protein and lipid contents were significantly lower while the whole-body moisture content was significantly higher in fish fed the diet with 70.0% of CGM protein compared with the control group ( P < 0.05). When 70.0% of FM protein was replaced by CGM, plasma total protein and cholesterol contents were significantly lower than those in the control group ( P < 0.05). Fish fed the diet with 70.0% of CGM protein had significantly lower hepatic insulin-like growth factor I (IGF-I) expression levels than those in the control group ( P < 0.05). However, no significant differences were observed in hepatic target of rapamycin (TOR), dorsal muscle IGF-I and TOR expression levels among dietary treatments. Results of the present study indicated that 52.5% of FM protein could be replaced by CGM in the diets without significant influences on the growth, feed utilization and protein metabolism of juvenile cobia. The present results might be useful for developing cost effective and sustainable cobia dietary formulations.

Selenium Deficiency Induces Autophagy in Immune Organs of Chickens.

PubMed

Khoso, Pervez Ahmed; Pan, Tingru; Wan, Na; Yang, Zijiang; Liu, Ci; Li, Shu

2017-05-01

The aim of the present study was to investigate the effects of selenium (Se) deficiency on autophagy-related genes and on ultrastructural changes in the spleen, bursa of Fabricius, and thymus of chickens. The Se deficiency group was fed a basal diet containing Se at 0.033 mg/kg and the control group was fed the same basal diet containing Se at 0.15 mg/kg. The messenger RNA (mRNA) levels of the autophagy genes microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin 1, dynein, autophagy associated gene 5 (ATG5), and target of rapamycin complex 1 (TORC1) were assessed using real-time qPCR. The protein levels of LC3-II, Beclin 1, and dynein were investigated using western blot analysis. Furthermore, the ultrastructure was observed using an electron microscope. The results indicated that spleen mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of LC3-II, Beclin 1, and dynein were increased in the Se deficiency group compared with the control group. In the bursa of Fabricius, the mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of Beclin 1 and dynein were increased; furthermore, the protein level of LC3-II was decreased in the Se deficiency group compared to the control group. In the thymus, the mRNA levels of LC3-I, Beclin 1, and ATG5 increased; the levels of LC3-II, dynein, and TORC1 were decreased; the protein level of Beclin 1 increased; and the levels of LC3-II and dynein decreased in the Se deficiency group compared to those in the control group. Further cellular morphological changes, such as autophagy vacuoles, autolysosomes, and lysosomal degradation, were observed in the spleen, bursa of Fabricius, and thymus of the Se-deficiency group. In summary, Se deficiency caused changes in autophagy-related genes, which increased the autophagic process and also caused structural damages to the immune organs of chickens.

Association of ubiquitin carboxy-terminal hydrolase-L1 in cerebrospinal fluid with clinical severity in a cohort of patients with Guillain-Barré syndrome.

PubMed

Nagamine, Satoshi; Fujiwara, Yuuki; Shimizu, Toshio; Kawata, Akihiro; Wada, Keiji; Isozaki, Eiji; Kabuta, Tomohiro

2015-06-01

Guillain-Barré syndrome (GBS) is an acute immune-mediated polyneuropathy. Although its pathogenic mechanism has been revealed and various therapeutic trials have been performed, a proportion of patients experience the severe sequelae associated with GBS. In this paper, we investigated whether the amount of the neuron-specific protein, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), in the cerebrospinal fluid of patients with GBS was correlated with the clinical course of the disease. UCH-L1 protein levels were greater in patients with GBS than in controls. The patients with GBS whose UCH-L1 protein levels were higher than those of the controls presented with more severe symptoms at peak. UCH-L1 protein levels tended to become elevated as the total protein levels were increased; however, elevated UCH-L1 without an increase in total protein might be correlated with severe disease course (bedridden or ventilator supported). These results suggest that UCH-L1 could be a biomarker associated with the severity of the disease at the acute phase of GBS.

Associations between expression levels of nucleotide excision repair proteins in lymphoblastoid cells and risk of squamous cell carcinoma of the head and neck.

PubMed

Han, Peng; Liu, Hongliang; Shi, Qiong; Liu, Zhensheng; Troy, Jesse D; Lee, Walter T; Zevallos, Jose P; Li, Guojun; Sturgis, Erich M; Wei, Qingyi

2018-06-01

Squamous cell carcinoma of head and neck (SCCHN) is one of the most common malignancies worldwide, and nucleotide excision repair (NER) is involved in SCCHN susceptibility. In this analysis of 349 newly diagnosed SCCHN patients and 295 cancer-free controls, we investigated whether expression levels of eight core NER proteins were associated with risk of SCCHN. We quantified NER protein expression levels in cultured peripheral lymphocytes using a reverse-phase protein microarray. Compared with the controls, SCCHN patients had statistically significantly lower expression levels of ERCC3 and XPA (P = 0.001 and 0.001, respectively). After dividing the subjects by controls' median values of expression levels, we found a dose-dependent association between an increased risk of SCCHN and low expression levels of ERCC3 (adjusted OR, 1.75, and 95% CI: 1.26-2.42; P trend  = 0.008) and XPA (adjusted OR, 1.88; 95% CI, 1.35-2.60; P trend  = 0.001). We also identified a significant multiplicative interaction between smoking status and ERCC3 expression levels (P = 0.014). Finally, after integrating demographic and clinical variables, we found that the addition of ERCC3 and XPA expression levels to the model significantly improved the sensitivity of the expanded model on SCCHN risk. In conclusion, reduced protein expression levels of ERCC3 and XPA were associated with an increased risk of SCCHN. However, these results need to be confirmed in additional large studies. © 2018 Wiley Periodicals, Inc.

TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

PubMed

Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

2012-02-01

TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

Systems Mechanobiology: Tension-Inhibited Protein Turnover Is Sufficient to Physically Control Gene Circuits

PubMed Central

Dingal, P.C. Dave P.; Discher, Dennis E.

2014-01-01

Mechanotransduction pathways convert forces that stress and strain structures within cells into gene expression levels that impact development, homeostasis, and disease. The levels of some key structural proteins in the nucleus, cytoskeleton, or extracellular matrix have been recently reported to scale with tissue- and cell-level forces or mechanical properties such as stiffness, and so the mathematics of mechanotransduction becomes important to understand. Here, we show that if a given structural protein positively regulates its own gene expression, then stresses need only inhibit degradation of that protein to achieve stable, mechanosensitive gene expression. This basic use-it-or-lose-it module is illustrated by application to meshworks of nuclear lamin A, minifilaments of myosin II, and extracellular matrix collagen fibers—all of which possess filamentous coiled-coil/supercoiled structures. Past experiments not only suggest that tension suppresses protein degradation mediated and/or initiated by various enzymes but also that transcript levels vary with protein levels because key transcription factors are regulated by these structural proteins. Coupling between modules occurs within single cells and between cells in tissue, as illustrated during embryonic heart development where cardiac fibroblasts make collagen that cardiomyocytes contract. With few additional assumptions, the basic module has sufficient physics to control key structural genes in both development and disease. PMID:25468352

Combined effect of tumor necrosis factor (TNF)-alpha and heat shock protein (HSP)-70 in reducing apoptotic injury in hypoxia: a cell culture study.

PubMed

Goel, Gunjan; Guo, Miao; Ding, Jamie; Dornbos, David; Ali, Ahmer; Shenaq, Mohammed; Guthikonda, Murali; Ding, Yuchuan

2010-10-15

Studies have demonstrated neuroprotective effects of either TNF-alpha or HSP-70 in ischemia/reperfusion injury following exercise. However, the protective mechanisms involving combined effect of the two proteins, particularly in neuronal apoptosis, remain unclear. This study aims to elucidate the beneficial role of TNF-alpha and HSP-70 in the regulation of apoptotic proteins and ERK signaling in hypoxic injury. Cortical neurons from 20 Sprague-Dawley rat embryos were isolated and cultured in five groups with or without pretreatment with recombinant TNF-alpha, HSP-70 protein or both prior to hypoxic conditions: (1) control; (2) control/hypoxia; (3) TNF-alpha/hypoxia; (4) HSP-70/hypoxia and (5) TNF-alpha/HSP-70/hypoxia. Western blotting was used to detect pro- and anti-apoptotic proteins, including Bax, AIF, Bcl-xL, Bcl-2, and pERK1/2 protein. TNF-alpha and HSP-70 significantly (p<0.05) reduced the levels of pro-apoptotic proteins, Bax and AIF. Also, pretreatment of hypoxic brain tissue with TNF-alpha and HSP-70 significantly (p<0.05) enhanced the levels of anti-apoptotic protein, Bcl-xL. TNF-alpha and HSP-70 together increased Bcl-2 levels by 70%. Hypoxia caused a significant (p<0.05) increase in ERK1/2 phosphorylation levels by 224%. The most effective inhibition of ERK levels was obtained by the combined administration of TNF-alpha and HSP-70. This study suggested that TNF-alpha and HSP-70 together enhance the decrease in pro-apoptotic protein levels and the increase in anti-apoptotic protein levels in the event of neuronal hypoxia through ERK1/2 signal transduction. 2010. Published by Elsevier Ireland Ltd.

Influence of carbohydrate and fat intake on diet-induced thermogenesis and brown fat activity in rats fed low protein diets.

PubMed

Rothwell, N J; Stock, M J

1987-10-01

Voluntary intake of protein, fat and carbohydrate (CHO) was modified by feeding young rats either a control purified diet [% metabolizable energy (ME): protein 21, fat 7, CHO 72], a control diet plus sucrose solution (20%) to drink (final intakes 17, 6 and 77% ME as protein, fat and CHO, respectively) or a low protein diet substituted with either CHO (8, 7 and 85% ME as protein, fat and CHO, respectively) or fat (8, 20 and 72% ME as protein, fat and CHO, respectively). Total ME intakes corrected for body size were similar for all rats, but body weight, energy gain and net energetic efficiency were lower in both low protein-fed groups than in the control group. The acute thermogenic response (% rise in oxygen consumption) to a standard balanced-nutrient meal was higher (12%) in sucrose-supplemented and in low protein groups (15-16%) than in control rats (8%). Brown adipose tissue protein content and thermogenic capacity (assessed from purine nucleotide binding to isolated mitochondria) were greater than control values in sucrose-fed and protein-deficient animals, and the greatest levels of activity were seen in low protein-fed rats with a high fat intake. The results demonstrate that the changes in energy balance, thermogenesis and brown adipose tissue activity that result from protein deficiency cannot be ascribed to changes in the level of energy intake or to a specific increase in the amount or proportion of either CHO or fat. They suggest that the protein-to-energy ratio must be the primary influence on thermogenesis and brown fat activity in these animals.

Studies of protein oxidation as a product quality attribute on a scale-down model for cell culture process development.

PubMed

Lee, Nacole D; Kondragunta, Bhargavi; Uplekar, Shaunak; Vallejos, Jose; Moreira, Antonio; Rao, Govind

2015-01-01

Of importance to the biological properties of proteins produced in cell culture systems are the complex post-translational modifications that are affected by variations in process conditions. Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by such process variations. Dissolved oxygen is a parameter of increasing interest since studies have shown that despite the necessity of oxygen for respiration, there may also be some detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to proteins as a result of oxidation of the cell and cellular components. Variation, or changes to cell culture products, can affect function, clearance rate, immunogenicity, and specific activity, which translates into clinical implications. The effect of increasing dissolved oxygen on protein oxidation in immunoglobulin G3-producing mouse hybridoma cells was studied using 50 mL high-throughput mini-bioreactors that employ non-invasive optical sensor technology for monitoring and closed feedback control of pH and dissolved oxygen. Relative protein carbonyl concentration of proteins produced under varying levels of dissolved oxygen was measured by enzyme-linked immunosorbent assay and used as an indicator of oxidative damage. A trend of increasing protein carbonyl content in response to increasing dissolved oxygen levels under controlled conditions was observed. Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by variations in dissolved oxygen levels in cell culture systems. Studies have shown that despite the necessity of oxygen for respiration, there may be detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to proteins as a result of oxidation of the cell and cellular components, affecting function, clearance rate, immunogenicity, and specific activity, which translates into clinical implications. The effect of increasing dissolved oxygen on protein oxidation in immunoglobulin G3-producing mouse hybridoma cells was studied using 50 mL high-throughput mini-bioreactors that employ non-invasive optical sensor technology for monitoring and closed feedback control of pH and dissolved oxygen. Protein carbonyl concentration of proteins produced under varying levels of dissolved oxygen was measured by enzyme-linked immunosorbent assay and used as an indicator of oxidative damage. A trend of increasing protein carbonyl content in response to increasing dissolved oxygen levels under controlled conditions was observed. © PDA, Inc. 2015.

[Effects of postnatal lambda-cyhalothrin exposure on synaptic proteins in ICR mouse brain].

PubMed

Bao, Xun-Di; Wang, Qu-Nan; Li, Fang-Fang; Chai, Xiao-Yu; Gao, Ye

2011-04-01

To evaluate the influence on the synaptic protein expression in different brain regions of ICR mice after lambda-cyhalothrin (LCT) exposure during postnatal period. Two male and 4 female healthy ICR mice were put in one cage. It was set as pregnancy if vaginal plug was founded. Offspring were divided into 5 groups randomly, and exposed to LCT (0.01% DMSO solution) at the doses of 0.1, 1.0 and 10.0 mg/kg by intragastric rout every other day from postnatal days (PND) 5 to PND13, control animals were treated with normal saline or DMSO by the same route. The brains were removed from pups on PND 14, the synaptic protein expression levels in cortex, hippocampus and striatum were measured by western blot. GFAP levels of cortex and hippocampus in the LCT exposure group increased with doses, as compared with control group (P < 0.05), while Tuj protein expression did not change significantly in the various brain regions of ICR mice. GAP-43 protein expression levels in the LCT exposed mouse hippocampus and in female ICR mouse cortex increased with doses, as compared with control group (P < 0.05). Presynaptic protein (Synapsin I) expression levels did not change obviously in various brain regions. However, postsynaptic density protein 95 (PSD95) expression levels of the hippocampus and striatum in male offspring of 10.0 mg/kg LCT group, of cortex of female LCT groups, and of female offspring in all exposure groups, of striatum, in 1.0 or 10.0 mg/kg LCT exposure groups significantly decreased (P < 0.05). Early postnatal exposure to LCT affects synaptic protein expression. These effects may ultimately affect the construction of synaptic connections.

Dual transcriptional-translational cascade permits cellular level tuneable expression control

PubMed Central

Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

2016-01-01

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

PubMed

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration

PubMed Central

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds. PMID:21994173

High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector

PubMed Central

Werner, Stefan; Breus, Oksana; Symonenko, Yuri; Marillonnet, Sylvestre; Gleba, Yuri

2011-01-01

We describe here a unique ethanol-inducible process for expression of recombinant proteins in transgenic plants. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the uninduced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. Transgenic Nicotiana benthamiana plants incorporating this double-inducible system demonstrate negligible background expression, high (over 0.5 × 104-fold) induction multiples, and high absolute levels of protein expression upon induction (up to 4.3 mg/g fresh biomass). The process can be easily scaled up, supports expression of practically important recombinant proteins, and thus can be directly used for industrial manufacturing. PMID:21825158

High infestation levels of Schizotetranychus oryzae severely affects rice metabolism.

PubMed

Blasi, Édina A R; Buffon, Giseli; Rativa, Angie G S; Lopes, Mara C B; Berger, Markus; Santi, Lucélia; Lavallée-Adam, Mathieu; Yates, John R; Schwambach, Joséli; Beys-da-Silva, Walter O; Sperotto, Raul A

2017-12-01

High levels of Schizotetranychus oryzae phytophagous mite infestation on rice leaves can severely affect productivity. Physiological characterization showed that S. oryzae promotes a decrease in chlorophyll concentration and the establishment of a senescence process in rice leaves. Late-infested leaves also present high levels of superoxide radical and hydrogen peroxide accumulation, along with high levels of membrane integrity loss, which is indicative of cell death. To better understand the rice molecular responses to high levels of mite infestation, we employed the Multidimensional Protein Identification Technology (MudPIT) approach to identify differentially expressed proteins. We identified 83 and 88 proteins uniquely present in control and late-infested leaves, respectively, along with 11 and one proteins more abundant in control and late-infested leaves, respectively. S. oryzae infestation induces a decreased abundance of proteins related to translation, protease inhibition, and photosynthesis. On the other hand, infestation caused increased abundance of proteins involved in protein modification and degradation. Our results also suggest that S. oryzae infestation interferes with intracellular transport, DNA structure maintenance, and amino acid and lipid metabolism in rice leaves. Proteomic data were positively correlated with enzymatic assays and RT-qPCR analysis. Our findings describe the protein expression patterns of late-infested rice leaves and suggest several targets which could be tested in future biotechnological approaches aiming to avoid the population increase of phytophagous mite in rice plants. Copyright © 2017 Elsevier GmbH. All rights reserved.

Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation

PubMed Central

Ciccosanti, Fabiola

2016-01-01

Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51–like kinase 1 (ULK1) is a conserved serine–threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold. PMID:27932573

Antioxidants and biomarkers of oxidative damage in the saliva of patients with Down's syndrome.

PubMed

de Sousa, Michelle Cardoso; Vieira, Rafael Brizola; Dos Santos, Danielle Sá; Carvalho, Claudio Antonio Talge; Camargo, Samira Esteves Afonso; Mancini, Maria Nadir Gasparoto; de Oliveira, Luciane Dias

2015-04-01

The aim of this study was to investigate enzymatic and non-enzymatic antioxidant systems and levels of biomarker levels of oxidative damage in the saliva of patients with Down's syndrome (DS). Saliva samples were collected from 30 patients with DS and control group (age: 14-24 years). Subsequently, the concentrations of superoxide dismutase, concentration of malondialdehyde, carbonylated proteins, uric acid, vitamin C and total protein, peroxidase activity and total antioxidant capacity were analyzed. Patients with DS presented significantly higher concentrations of superoxide dismutase, higher levels of malondialdehyde and salivary total protein content than controls (p<0.05). Conversely, no difference in carbonylated proteins or antioxidants (uric acid, vitamin C, peroxidase, and total antioxidant capacity) was observed between DS patients and controls (p>0.05). Patients with DS are more vulnerable to oxidative stress in saliva as indicated by the significant increase in malondialdehyde and superoxide dismutase concentrations found in this study. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inflammation associated anemia and ferritin as disease markers in SLE

PubMed Central

2012-01-01

Introduction In a recent screening to detect biomarkers in systemic lupus erythematosus (SLE), expression of the iron storage protein, ferritin, was increased. Given that proteins that regulate the storage, transfer and release of iron play an important role in inflammation, this study aims to determine the serum and urine levels of ferritin and of the iron transfer protein, transferrin, in lupus patients and to correlate these levels with disease activity, inflammatory cytokine levels and markers of anemia. Methods A protein array was utilized to measure ferritin expression in the urine and serum of SLE patients and healthy controls. To confirm these results as well as the role of the iron transfer pathway in SLE, ELISAs were performed to measure ferritin and transferrin levels in inactive or active SLE patients and healthy controls. The relationship between ferritin/transferrin levels and inflammatory markers and anemia was next analyzed. Results Protein array results showed elevated ferritin levels in the serum and urine of lupus patients as compared to controls, which were further validated by ELISA. Increased ferritin levels correlated with measures of disease activity and anemia as well as inflammatory cytokine titers. Though active SLE patients had elevated urine transferrin, serum transferrin was reduced. Conclusion Urine ferritin and transferrin levels are elevated significantly in SLE patients and correlate with disease activity, bolstering previous reports. Most importantly, these changes correlated with the inflammatory state of the patients and anemia of chronic disease. Taken together, altered iron handling, inflammation and anemia of chronic disease constitute an ominous triad in SLE. PMID:22871034

Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

PubMed Central

Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

2012-01-01

Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study. PMID:22272336

[Study on proteomics of familial systemic lupus erythematosus patients in one family from Sichuan, China].

PubMed

Wu, Yong-kang; Huang, Zhuo-chun; Shi, Yun-ying; Cai, Bei; Wang, Lan-lan; Ying, Bin-wu; Hu, Chao-jun; Li, Yong-zhe

2009-05-01

To investigate the proteomic characteristics of systemic lupus erythematosus (SLE) in a SLE family from Sichuan, China which consisting of 7 members with 3 SLE cases, and to find the proteins correlated with the heredity of SLE. A total of 153 serum samples were collected from 7 members including 3 SLE sisters in this SLE family, 63 individual SLE patients, as well as 83 healthy controls. The diagnosis of SLE is based on the American College of Rheumatology criteria (1997). All serum samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with magnetic beads technology. Serum protein profiles were obtained by MALDI-TOF-MS combined with magnetic beads in order to identify predictive biomarkers of risk of suffering SLE. The resulting spectra were analyzed with Biomarker Wizard software 3.1.0. Four discriminative mass/charge (m/z) proteins serving as pathogenic biomarkers were identified on arrays for family SLE cases versus individual SLE and healthy controls. The protein level of peak intensities at m/z of 9342.23 was significantly greater in SLE family group compared with that in individual SLE patients and healthy controls (P<0.05), those of individual SLE patients were significantly greater compared with healthy controls (P<0.05); the proteins level of peak intensities at m/z of 4094.03, 5905.35 and 7973.53 in SLE family group were significantly lower compared with that in individual SLE patients and healthy controls (P<0.05), those of individual SLE patients were significantly lower compared with healthy controls (P<0.05). The proteins of m/z of 9342.23, 4094.03, 5905.35 and 7973.53 maybe play a great role in assemble pathogenesis of SLE and predict the risk of suffering SLE. The higher protein level of m/z of 9342.23 and the lower protein level of m/z of 4094.03, 5905.35 and 7973.53, the higher risk of sufferring with SLE.

[The effect of UV-irradiation on telomerase activity and other stress-related proteins in human lens epithelial cells].

PubMed

Wu, Zhi-hong; Zhang, Jin-song

2005-05-01

To investigate the changes and the role of telomerase activity and other stress-related proteins in the process of UV-induced DNA damage and repair in human lens epithelial cells. Human lens epithelial cells were irradiated at UV-doses 0.0 (control group) and 0.5, 1.5, 2.5, 3.5, 5.0, 7.5, 10.0 mJ/cm(2) (treated 1-7 group). Telomerase activity was determined by Telomerase Repeat Amplification Protocol-Enzyme Linked Immunosorbent Assay (TRAP-ELISA), p53, growth arrest and DNA damage inducible (GADD45), proliferating cell nuclear antigen (PCNA) and p16 protein levels were analyzed by Western blotting. Telomerase activity in control group and treated 1-7 group showed increased tendency, the differences of telomerase activity in 8 groups were significantly (P < 0.01). The expression of p53, GADD45, PCNA, p16 proteins showed increased tendency in experimental group, comparing with the control group, there were significant difference (P < 0.01). During UV-induced DNA damage and repair in human lens epithelial cells, telomerase activity was upregulated and the expression of stress-related proteins levels was increased. Upregulated telomerase activity may play both a protective and a proliferative role in human lens epithelial cells. Increased stress-related proteins level is critic in UV-induced DNA damage and repair in human lens epithelial. Increased telomerase activity is associated with increased levels of the stress-related proteins.

May the thyroid gland and thyroperoxidase participate in nitrosylation of serum proteins and sporadic Parkinson's disease?

PubMed

Fernández, Emilio; García-Moreno, José-Manuel; Martín de Pablos, Angel; Chacón, José

2014-11-20

The research group has detected nitrosative stress and a singular version of nitrosylated serum α-synuclein in serum of Parkinson's disease (PD) patients. Dysfunction of the thyroid gland has been proposed to be linked to this disease. The aim of the study was to know if the thyroid gland is involved in idiopathic PD and nitrosative stress. We studied 50 patients (early and advanced disease patients), 35 controls, and 6 subjects with thyroidectomy. Clinical characteristics, serum thyroperoxidase levels, and 3-nitrotyrosine proteins were analyzed. Enzyme-linked immunosorbent assay and immunoblotting methods were employed. The findings indicated that the prevalence of two thyroid dysfunctions (hyper- or hypothyroidism) was not found to be different in patients relative to controls. However, the levels of the enzyme thyroperoxidase were found to be elevated in early disease patients (p<0.006), not in advanced disease subjects, and these levels were negatively correlated with serum 3-nitrotyrosine proteins (p<0.05), the indicators of nitrosative stress. The thyroidectomized subjects showed very low levels of serum 3-nitrotyrosine proteins (78% reduction vs. controls) and, among these proteins, the nitrosylated serum α-synuclein was nearly absent. These observations lead to the hypothesis that the thyroid gland and thyroperoxidase participate in nitrosylation of serum proteins and they could influence Parkinsonian nitrosative stress as well as nitrosylation of serum α-synuclein, a potentially pathogenic factor.

Protein-expression profiles in mouse blood-plasma following acute whole-body exposure to (137)Cs gamma rays.

PubMed

Rithidech, Kanokporn Noy; Honikel, Louise; Rieger, Robert; Xie, Weiping; Fischer, Thomas; Simon, Sanford R

2009-05-01

To compare the pattern of protein-expression profiles in blood-plasma after exposure of CBA/CaJ mice to 0 or 3 Gy of (137)Cs gamma rays. Two-dimensional electrophoresis gel coupled with mass spectrometry was used to analyze blood-samples collected at days 2 and 7 post-irradiation. At each sacrifice-time, alterations in expression-level of protein spots between control- and exposed-groups were analyzed statistically by the PDQuest software using Student's t-test (at the significance level of p < 0.05). Mass spectrometry was used to identify the identity of protein-spots with significantly altered expression-level. At day 2, 18 proteins were significantly up-regulated in exposed-mice. These included: alpha-2-Heremans-Schmid (HS)-glycoprotein, apolipoprotein (Apo)-AII-precursor, Apo-E, beta-2-glycoprotein-I, clusterin, fibrinogen-alpha-chain, fibrinogen-gamma-polypeptide, fetuin-B, haptoglobin, high-molecular-weight (HMW)-kininogen (Kng), low-MW-Kng, Kng1-precursor, liver-carboxylesterase-I-precursor, major-urinary-protein-6-precursor, mannose-binding-protein-C-precursor, mannose-binding-lectin-C, and prothrombin-precursor. Gelsolin was detected in control-mice only. At day 7, high expression-levels of 14 proteins were detected in control-mice (i.e., alpha-1-antitrypsin-precursor, carboxylesterase-N, cholesterol-7-alpha-hydroxylase, contraspin, coagulation-factor-II, coagulation-factor-XIII, gelsolin, immunoglobulin-G-heavy-chain, neurexin, prothrombin-precursor, protein-phosphatase, putative-calcium-influx-channel, vitamin-D-binding-protein, and 1110018G07Rik); while 15 proteins were highly expressed in exposed-mice. These included: alpha-1-acid-glycoprotein, alpha-2-HS-glycoprotein, alpha-1-protease-inhibitor-2, ApoA-IV, ApoC-I, ApoH, beta-1-globin, clusterin, complement-component-3, fibrinogen-beta-chain, HMW-Kng, major-histocompatibility-complex-class-Ia-H2-K, serine-(cysteine)-proteinase-inhibitor, retinoblastoma-associated-protein-140, and vascular-cell-adhesion-molecule-1. Although different proteins (mostly involved in inflammatory responses) were detected in exposed-mice, alterations in expression-levels of clusterin, gelsolin, kininogen, and alpha-2-HS-glycoproteins were found at both times. Despite the need for validation, the results suggested that alterations in expression-levels of specific proteins may be indicative of radiation-exposure. The results also provided the important step in an eventual establishment of blood-based biomarkers of radiation-exposure in vivo.

Glutamine supplementation stimulates protein-synthetic and inhibits protein-degradative signaling pathways in skeletal muscle of diabetic rats.

PubMed

Lambertucci, Adriana C; Lambertucci, Rafael H; Hirabara, Sandro M; Curi, Rui; Moriscot, Anselmo S; Alba-Loureiro, Tatiana C; Guimarães-Ferreira, Lucas; Levada-Pires, Adriana C; Vasconcelos, Diogo A A; Sellitti, Donald F; Pithon-Curi, Tania C

2012-01-01

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats

PubMed Central

Lambertucci, Adriana C.; Lambertucci, Rafael H.; Hirabara, Sandro M.; Curi, Rui; Moriscot, Anselmo S.; Alba-Loureiro, Tatiana C.; Guimarães-Ferreira, Lucas; Levada-Pires, Adriana C.; Vasconcelos, Diogo A. A.; Sellitti, Donald F.; Pithon-Curi, Tania C.

2012-01-01

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes. PMID:23239980

Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

PubMed

Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

2015-11-06

The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

The effects of dietary protein levels on the population growth, performance, and physiology of honey bee workers during early spring.

PubMed

Zheng, Benle; Wu, Zaifu; Xu, Baohua

2014-01-01

This study was conducted to investigate the effects of dietary protein levels on honey bee colonies, specifically the population growth, physiology, and longevity of honey bee workers during early spring. Diets containing four different levels of crude protein (25.0, 29.5, 34.0, or 38.5%) and pure pollen (control) were evaluated. Twenty-five colonies of honey bees with sister queens were used in the study. We compared the effects of the different bee diets by measuring population growth, emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland development, and survival. After 48 d, the cumulative number of workers produced by the colonies ranged from 22,420 to 29,519, providing a significant fit to a quadratic equation that predicts the maximum population growth when the diet contains 31.7% crude protein. Significantly greater emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland acini, and survival were observed in the colonies that were fed diets containing 34.0% crude protein compared with the other crude protein levels. Although higher emergent worker weight and survival were observed in the colonies that were fed the control diet, there were no significant differences between the control colonies and the colonies that were fed 34.0% crude protein. Based on these results, we concluded that a dietary crude protein content of 29.5-34.0% is recommended to maximize the reproduction rate of honey bee colonies in early spring. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Transgenic Biofortification of the Starchy Staple Cassava (Manihot esculenta) Generates a Novel Sink for Protein

PubMed Central

Abhary, Mohammad; Siritunga, Dimuth; Stevens, Gene; Taylor, Nigel J.; Fauquet, Claude M.

2011-01-01

Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz). (Montagnac JA, Davis CR, Tanumihardjo SA. (2009) Compr Rev Food Sci Food Saf 8:181–194). Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications. PMID:21283593

Transgenic biofortification of the starchy staple cassava (Manihot esculenta) generates a novel sink for protein.

PubMed

Abhary, Mohammad; Siritunga, Dimuth; Stevens, Gene; Taylor, Nigel J; Fauquet, Claude M

2011-01-25

Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz). (Montagnac JA, Davis CR, Tanumihardjo SA. (2009) Compr Rev Food Sci Food Saf 8:181-194). Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications.

Alterations in Protein Expression in Tree Shrew Sclera during Development of Lens-Induced Myopia and Recovery

PubMed Central

Norton, Thomas T.

2012-01-01

Purpose. During the development of, and recovery from, negative lens-induced myopia there is regulated remodeling of the scleral extracellular matrix (ECM) that controls the extensibility of the sclera. Difference gel electrophoresis (DIGE) was used to identify and categorize proteins whose levels are altered in this process. Methods. Two groups of five tree shrews started monocular lens wear 24 days after eye opening (days of visual experience [VE]). The lens-induced myopia (LIM) group wore a −5 D lens for 4 days. The recovery (REC) group wore a −5 D lens for 11 days and then recovered for 4 days. Two normal groups (28 and 39 days of VE; n = 5 each) were also examined, age-matched to each of the treatment groups. Refractive and A-scan measures confirmed the effect of the treatments. Scleral proteins were isolated and resolved by DIGE. Proteins that differed in abundance were identified by mass spectrometry. Ingenuity pathway analysis was used to investigate potential biological pathway interactions. Results. During normal development (28–39 days of VE), eight proteins decreased and one protein increased in relative abundance. LIM-treated eyes were myopic and longer than control eyes; LIM-control eyes were slightly myopic compared with 28N eyes, indicating a yoking effect. In both the LIM-treated and the LIM-control eyes, there was a general downregulation from normal of proteins involved in transcription, cell adhesion, and protein synthesis. Additional proteins involved in cell adhesion, actin cytoskeleton, transcriptional regulation, and ECM structural proteins differed in the LIM-treated eyes versus normal but did not differ in the control eyes versus normal. REC-treated eyes were recovering from the induced myopia. REC-control eye refractions were not significantly different from the 39N eyes, and few proteins differed from age-matched normal eyes. The balance of protein expression in the REC-treated eyes, compared with normal eyes and REC-control eyes, shifted toward upregulation or a return to normal levels of proteins involved in cell adhesion, cell division, cytoskeleton, and ECM structural proteins, including upregulation of several cytoskeleton-related proteins not affected during myopia development. Conclusions. The DIGE procedure revealed new proteins whose abundance is altered during myopia development and recovery. Many of these are involved in cell-matrix adhesions, cytoskeleton, and transcriptional regulation and extend our understanding of the remodeling that controls the extensibility of the sclera. Reductions in these proteins during minus lens wear may produce the increased scleral viscoelasticity that results in faster axial elongation. Recovery is not a mirror image of lens-induced myopia—many protein levels, decreased during LIM, returned to normal, or slightly above normal, and additional cytoskeleton proteins were upregulated. However, no single protein or pathway appeared to be responsible for the scleral changes during myopia development or recovery. PMID:22039233

Differential hippocampal protein expression between normal aged rats and aged rats with postoperative cognitive dysfunction: A proteomic analysis.

PubMed

Li, Yang; Wang, Saiying; Ran, Ke; Hu, Zhonghua; Liu, Zhaoqian; Duan, Kaiming

2015-08-01

The aim of the present study was to investigate the differences in the expression of hippocampal proteins between normal control aged rats and aged rats with postoperative cognitive dysfunction (POCD). A total of 24 aged rats were randomly divided into a surgery group (n=12) and a control group (n=12). The rats in the surgery group were treated with 2 h isoflurane anesthesia and splenectomy, while the rats in the control group received 40% oxygen for 2 h without surgery. The cognitive functions of the two groups were examined using a Y-maze test. The protein expression profiles of the hippocampus of six aged rats (three rats with POCD and three from the normal control group) were assessed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. A total of three differential proteins were further confirmed between the POCD rats and normal rats using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The expression levels of 21 proteins in the rats with POCD were significantly different compared with the normal control rats. These proteins were functionally clustered to synaptic plasticity (three proteins), oxidative stress (four proteins), energy production (six proteins), neuroinflammation (three proteins) and glutamate metabolism (two proteins). In addition, three proteins (fatty acid binding protein 7, brain, glutamate dehydrogenase 1 and glutamine synthetase), associated with astrocytic function, were significantly different in the rats with POCD compared with those in the normal control (P<0.05). Similar changes in the mRNA expression levels of the three proteins in the hippocampi of POCD rats were also detected using RT-qPCR. Neuroinflammation, glutamate toxicity and oxidative stress were possibly involved in the pathological mechanism underlying POCD in aged rats. In addition, astrocytes may also be important in POCD in aged rats.

Serum heart type fatty acid binding protein levels are not changed in hyperthyroidism.

PubMed

Ozbek, Mustafa; Gungunes, Askin; Sahin, Mustafa; Ginis, Zeynep; Ucan, Bekir; Sayki, Muyesser; Tutal, Esra; Cakal, Erman; Kuşkonmaz, Serife M; Öztürk, Mehmet A; Delibasi, Tuncay

2016-09-01

Heart type fatty acid binding protein (H-FABP) is a small protein and released into the circulation when myocardial damage has occurred. Previous studies have demonstrated that H-FABP is closely associated with cardiac and some endocrinologic disorders including prediabetes, metabolic syndrome, and acromegaly. Hyperthyroism is a well-known disorder associated with cardiovascular diseases. We aimed to investigate the effect of hyperthyrodism on H-FABP levels. Forty six patients with hyperthyroidism with no known history of coronary artery disease and 40 healthy controls are involved in the study. Serum H-FABP levels are measured using sandwich enzyme-linked immunosorbent assay. There was no significant difference between serum H-FABP levels of patients with hyperthyroidism and controls (871±66 pg/mL, and 816±66 pg/mL, respectively P=0.56). There was no significant correlation between H-FABP, free triiodothyronine (fT3), free thyroxine (fT4), and thyroid stimulating hormone (TSH) levels in patients and controls. Serum H-FABP levels are not altered in patients with hyperthyroidism.

Matrix metalloproteinases and tissue damage in HIV-tuberculosis immune reconstitution inflammatory syndrome

PubMed Central

Tadokera, Rebecca; Meintjes, Graeme A; Wilkinson, Katalin A; Skolimowska, Keira H; Walker, Naomi; Friedland, Jon S; Maartens, Gary; Elkington, Paul T G; Wilkinson, Robert J

2014-01-01

The HIV-TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) can complicate combined treatments for HIV-1 and TB. Little is known about tissue damage in TB-IRIS. Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix and consequently may play a role in such immunopathology. Here we investigated the involvement of MMPs in TB-IRIS. We determined MMP transcript abundance and secreted protein in Mycobacterium tuberculosis stimulated PBMCs from 22 TB-IRIS patients and 22 non-IRIS controls. We also measured MMP protein levels in corresponding serum and the effect of prednisone — which reduces the duration of symptoms in IRIS patients — or placebo treatment on MMP transcript and circulating MMP protein levels. PBMCs from TB-IRIS had increased MMP-1,-3,-7, and-10 transcript levels when compared with those of controls at either 6 or 24 h. Similarly, MMP-1,-3,-7, and-10 protein secretion in stimulated cultures was higher in TB-IRIS than in controls. Serum MMP-7 concentration was elevated in TB-IRIS and 2 weeks of corticosteroid therapy decreased this level, although not significantly. TB-IRIS is associated with a distinct pattern of MMP gene and protein activation. Modulation of dysregulated MMP activity may represent a novel therapeutic approach to alleviate TB-IRIS in HIV-TB patients undergoing treatment. PMID:24136296

Protein levels of ADAM10, BACE1, and PSEN1 in platelets and leukocytes of Alzheimer's disease patients.

PubMed

Bram, Jessyka Maria de França; Talib, Leda Leme; Joaquim, Helena Passarelli Giroud; Sarno, Tamires Alves; Gattaz, Wagner Farid; Forlenza, Orestes Vicente

2018-05-29

The clinical diagnosis of Alzheimer's disease (AD) is a probabilistic formulation that may lack accuracy particularly at early stages of the dementing process. Abnormalities in amyloid-beta precursor protein (APP) metabolism and in the level of APP secretases have been demonstrated in platelets, and to a lesser extent in leukocytes, of AD patients, with conflicting results. The aim of the present study was to compare the protein level of the APP secretases A-disintegrin and metalloprotease 10 (ADAM10), Beta-site APP-cleaving enzyme 1 (BACE1), and presenilin-1 (PSEN1) in platelets and leukocytes from 20 non-medicated older adults with AD and 20 healthy elders, and to determine the potential use of these biomarkers to discriminate cases of AD from controls. The protein levels of all APP secretases were significantly higher in platelets compared to leukocytes. We found statistically a significant decrease in ADAM10 (52.5%, p < 0.0001) and PSEN1 (32%, p = 0.02) in platelets from AD patients compared to controls, but not in leukocytes. Combining all three secretases to generate receiver-operating characteristic (ROC) curves, we found a good discriminatory effect (AD vs. controls) when using platelets (the area under the curve-AUC-0.90, sensitivity 88.9%, specificity 66.7%, p = 0.003), but not in leukocytes (AUC 0.65, sensitivity 77.8%, specificity 50.0%, p = 0.2). Our findings indicate that platelets represent a better biological matrix than leukocytes to address the peripheral level of APP secretases. In addition, combining the protein level of ADAM10, BACE1, and PSEN1 in platelets, yielded a good accuracy to discriminate AD from controls.

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a.

PubMed

Carter, Javier A; Jiménez, Juan C; Zaldívar, Mercedes; Alvarez, Sergio A; Marolda, Cristina L; Valvano, Miguel A; Contreras, Inés

2009-10-01

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded Wzz(pHS-2) proteins, respectively. In this study, genes wzzB, wzz(pHS-2) and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz(pHS-2) revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and Wzz(pHS-2) proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.

The Time Required for Dormancy Release in Arabidopsis Is Determined by DELAY OF GERMINATION1 Protein Levels in Freshly Harvested Seeds[OA

PubMed Central

Nakabayashi, Kazumi; Bartsch, Melanie; Xiang, Yong; Miatton, Emma; Pellengahr, Silke; Yano, Ryoichi; Seo, Mitsunori; Soppe, Wim J.J.

2012-01-01

Seed dormancy controls the start of a plant’s life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA. PMID:22829147

An Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

PubMed Central

McClintock, Carlee S; Hettich, Robert L.

2012-01-01

Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent – hydroxyl radicals – for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins. PMID:23210708

High protein consumption in trained women: bad to the bone?

PubMed

Antonio, Jose; Ellerbroek, Anya; Evans, Cassandra; Silver, Tobin; Peacock, Corey A

2018-01-01

It has been posited that the consumption of extra protein (> 0.8 g/kg/d) may be deleterious to bone mineral content. However, there is no direct evidence to show that consuming a high-protein diet results in a demineralization of the skeleton. Thus, the primary endpoint of this randomized controlled trial was to determine if a high-protein diet affected various parameters of whole body and lumbar bone mineral content in exercise-trained women. Twenty-four women volunteered for this 6-month investigation ( n  = 12 control, n = 12 high-protein). The control group was instructed to consume their habitual diet; however, the high-protein group was instructed to consume ≥2.2 g of protein per kilogram body weight daily (g/kg/d). Body composition was assessed via dual-energy x-ray absorptiometry (DXA). Subjects were instructed to keep a food diary via the mobile app MyFitnessPal ® . Exercise or activity level was not controlled. Subjects were asked to maintain their current levels of exercise. During the 6-month treatment period, there was a significant difference in protein intake between the control and high-protein groups (mean±SD; control: 1.5±0.3, high-protein: 2.8±1.1 g/kg/d); however, there were no differences in the consumption total calories, carbohydrate or fat. Whole body bone mineral density did not change in the control (pre: 1.22±0.08, post: 1.22±0.09 g/cm 2 ) or high-protein group (pre: 1.25±0.11, post: 1.24±0.10 g/cm 2 ). Similarly, lumbar bone mineral density did not change in the control (pre: 1.08±0.16, post: 1.05±0.13 g/cm 2 ) or high-protein group (pre: 1.07±0.11, post: 1.08±0.12 g/cm 2 ). In addition, there were no changes in whole body or lumbar T-Scores in either group. Furthermore, there were no changes in fat mass or lean body mass. Despite an 87% higher protein intake (high-protein versus control), 6 months of a high-protein diet had no effect on whole body bone mineral density, lumbar bone mineral density, T-scores, lean body mass or fat mass.

Anti-Angiogenic Action of Neutral Endopeptidase

DTIC Science & Technology

2007-11-01

message levels of NEP in hypoxia treated PC cells. Messenger RNA levels of NEP decreased between 50-75% relative to normoxic controls with high...GAGCATC-3 (sense) and 5-ATATGAATTCTCAGCTCT- TAGCAGACATGGAAGAAAG-3 ( antisense ) for glutathione S-transferase (GST) fusion proteins and 5-ATGGCAGCCGG...GAGCATC-3 (sense) and 5-CCCCAAGCTTTTAGCTCT- TAGCAGACAT-3 ( antisense ) for maltose-binding protein fusion proteins, as previously described (13

THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS.

PubMed

A, Güllü; S, Dede

2016-01-01

TQ has been used as treatment and preventive agent for many diseases over the years. The goal of this study was to investigate the effects of TQ supplement on fractions of serum proteins. Fourteen male Wistar-Albino rats (200-250 g weight) were used as material for two groups; (control (C) and thymoquinone (TQ) respectively. Each group contained seven rats. The control group had only corn oil, while the TQ group was dissolved in corn oil. 30 mg/kg/day were given by oral gavage for four weeks. The serum protein fractions were identified using cellulose acetate technique. The total protein level and albumin, α-1, α-2 fractions and A/G ratio have showed no difference between groups (p>0.05). β-globulin fractions of TQ group were higher than control's (p<0.05). In addition, it was observed that the γ-globulin levels of TQ group were lower than that of the control group's (p<0.05). From the results, it was observed that the changes of these fractions may have originated from elevation or decline synthesis, or activities of containing proteins.

Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells.

PubMed

Lin, Chi-Hung; Jarvis, Donald L

2013-05-10

Genetically transformed lepidopteran insect cell lines have biotechnological applications as constitutive recombinant protein production platforms and improved hosts for baculovirus-mediated recombinant protein production. Insect cell transformation is often accomplished with a DNA construct(s) encoding a foreign protein(s) under the transcriptional control of a baculovirus immediate early promoter, such as the ie1 promoter. However, the potential utility of increasingly stronger promoters from later baculovirus gene classes, such as delayed early (39K), late (p6.9), and very late (polh), has not been systematically assessed. Hence, we produced DNA constructs encoding secreted alkaline phosphatase (SEAP) under the transcriptional control of each of the four temporally distinct classes of baculovirus promoters, used them to transform insect cells, and compared the levels of SEAP RNA and protein production obtained before and after baculovirus infection. The ie1 construct was the only one that supported SEAP protein production by transformed insect cells prior to baculovirus infection, confirming that only immediate early promoters can be used to isolate transformed insect cells for constitutive recombinant protein production. However, baculovirus infection activated transgene expression by all four classes of baculovirus promoters. After infection, cells transformed with the very late (polh) and late (p6.9) promoter constructs produced the highest levels of SEAP RNA, but only low levels of SEAP protein. Conversely, cells transformed with the immediate early (ie1) and delayed early (39K) promoter constructs produced lower levels of RNA, but equal or higher levels of SEAP protein. Unexpectedly, the 39K promoter construct provided tightly regulated, baculovirus-inducible protein production at higher levels than the later promoter constructs. Thus, this study demonstrated the utility of the 39K promoter for insect cell engineering, particularly when one requires higher levels of effector protein production than obtained with ie1 and/or when constitutive transgene expression adversely impacts host cell fitness and/or genetic stability. Copyright © 2013 Elsevier B.V. All rights reserved.

Early alterations in soleus GLUT-4, glucose transport, and glycogen in voluntary running rats

NASA Technical Reports Server (NTRS)

Henriksen, Erik J.; Halseth, Amy E.

1994-01-01

Voluntary wheel running (WR) by juvenile female rats was used as a noninterventional model of soleus muscle functional overload to study the regulation of insulin-stimulated glucose transport activity by the glucose transporter (GLUT-4 isoform) protein level and glycogen concentration. Soleus total protein content was significantly greater (+18%;P greater than 0.05) than in age-matched controls after 1 wk of WR, and this hypertrophic response continued in weeks 2-4 (+24-32%). GLUT-4 protein was 39% greater than in controls in 1-wk WR soleus, and this adaptation was accompanied by a similar increase in in vitro insulin-stimulated glucose transport activity(+29%). After 2 and 4 wk of WR, however, insulin-stimulated glucose transport activity had returned to control levels, despite a continued elevation (+25-28%) of GLUT-4 protein. At these two time points, glycogen concentration was significantly enhanced in WR soleus (+21-42%), which coincided with significant reductions in glycogen synthase activity ratios (-23 to-41%). These results indicate that, in this model of soleus muscle functional overload, the GLUT-4 protein level may initially regulate insulin-stimulated glucose transport activity in the absence of changes in other modifying factors. However,this regulation of glucose transport activity by GLUT-4 protein may be subsequently overridden by elevated glycogen concentration.

Expression of APP pathway mRNAs and proteins in Alzheimer's disease.

PubMed

Matsui, Toshifumi; Ingelsson, Martin; Fukumoto, Hiroaki; Ramasamy, Karunya; Kowa, Hisatomo; Frosch, Matthew P; Irizarry, Michael C; Hyman, Bradley T

2007-08-03

In both trisomy 21 and rare cases of triplication of amyloid precursor protein (APP) Alzheimer's disease (AD) pathological changes are believed to be secondary to increased expression of APP. We hypothesized that sporadic AD may also be associated with changes in transcription of APP or its metabolic partners. To address this issue, temporal neocortex of 27 AD and 21 non-demented control brains was examined to assess mRNA levels of APP isoforms (total APP, APP containing the Kunitz protease inhibitor domain [APP-KPI] and APP770) and APP metabolic enzymatic partners (the APP cleaving enzymes beta-secretase [BACE] and presenilin-1 [PS-1], and putative clearance molecules, low-density lipoprotein receptor protein [LRP] and apolipoprotein E [apoE]). Furthermore, we evaluated how changes in APP at the mRNA level affect the amount of Tris buffer extractable APP protein and Abeta40 and 42 peptides in AD and control brains. As assessed by quantitative PCR, APP-KPI (p=0.007), APP770 (p=0.004), PS-1 (p=0.004), LRP (p=0.003), apoE (p=0.0002) and GFAP (p<0.0001) mRNA levels all increased in AD, and there was a shift from APP695 (a neuronal isoform) towards KPI containing isoforms that are present in glia as well. APP-KPI mRNA levels correlated with soluble APPalpha-KPI protein (sAPPalpha-KPI) levels measured by ELISA (tau=0.33, p=0.015 by Kendall's rank correlation); in turn, soluble APPalpha-KPI protein levels positively correlated with Tris-extractable, soluble Abeta40 (p=0.046) and 42 levels (p=0.007). The ratio of soluble APPalpha-KPI protein levels to total APP protein increased in AD, and also correlated with GFAP protein levels in AD. These results suggest that altered transcription of APP in AD is proportionately associated with Abeta peptide, may occur in the context of gliosis, and may contribute to Abeta deposition in sporadic AD.

Infrapatellar fat pad features in osteoarthritis: a histopathological and molecular study.

PubMed

Favero, Marta; El-Hadi, Hamza; Belluzzi, Elisa; Granzotto, Marnie; Porzionato, Andrea; Sarasin, Gloria; Rambaldo, Anna; Iacobellis, Claudio; Cigolotti, Augusto; Fontanella, Chiara Giulia; Natali, Arturo; Ramonda, Roberta; Ruggieri, Pietro; De Caro, Raffaele; Vettor, Roberto; Rossato, Marco; Macchi, Veronica

2017-10-01

The infrapatellar fat pad (IFP) is considered a local producer of adipocytokines, suggesting a potential role in OA. The objective of this study was to evaluate the histopathological and molecular characteristics of OA IFPs compared with controls. The histopathological characteristics of IFPs were evaluated in patients undergoing total knee replacements and in control patients (without OA), considering the following parameters: presence of inflammatory cells, vascularization, adipose lobules dimension and thickness of the interlobular septa. Immunohistochemistry was performed to evaluate VEGF, monocyte chemotactic protein 1 (MCP-1) and IL-6 proteins. Quantitative real time PCR was performed to evaluate the expression levels of adipocytokines in the OA IFPs. OA IFPs showed an increase in inflammatory infiltration, vascularization and thickness of the interlobular septa compared with controls. VEGF, MCP-1 and IL-6 proteins were higher in OA IFPs compared with in controls. Inflammatory infiltration, hyperplasia, vascularization and fibrosis were increased in OA IFP synovial membranes compared with in those of controls. VEGF protein levels were associated with an increased number of vessels in the OA IFPs, while MCP-1 and IL-6 protein levels were associated with higher grades of inflammatory infiltration. Leptin levels were positively correlated with adiponectin and MCP-1expression, while adiponectin positively correlated with peroxisome proliferative activated receptor gamma, MCP-1 and IFP vascularity. MCP-1 showed a positive correlation with peroxisome proliferative activated receptor gamma. IFP lobules dimensions were positively correlated with IL-6 expression and negatively with thickness of interlobular septa. VEGF mRNA levels were positively correlated with increased synovial vascularity. OA IFPs and synovial membranes are more inflamed, vascularized and fibrous compared with those of control patients (without OA). © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Expression and clinical significance of connective tissue growth factor in thyroid carcinomas.

PubMed

Wang, Guimin; Zhang, Wei; Meng, Wei; Liu, Jia; Wang, Peisong; Lin, Shan; Xu, Liyan; Li, Enmin; Chen, Guang

2013-08-01

To examine expression of the connective tissue growth factor (CTGF) gene in human thyroid cancer and establish whether a correlation exists between the presence of CTGF protein and clinicopathological parameters of the disease. CTGF protein expression was investigated retrospectively by immunohistochemical analysis of CTGF protein levels in thyroid tumour tissue. Associations between immunohistochemical score and several clinicopathological parameters were examined. In total, 131 thyroid tissue specimens were included. High levels of CTGF protein were observed in papillary thyroid carcinoma tissue; benign thyroid tumour tissue scored negatively for CTGF protein. In papillary thyroid carcinoma, there was a significant relationship between high CTGF protein levels and Union for International Cancer Control disease stage III-IV, and presence of lymph node metastasis. In papillary thyroid carcinomas, CTGF protein levels were not significantly associated with sex or age. These findings suggest that the CTGF protein level is increased in papillary thyroid carcinoma cells compared with benign thyroid tumours. CTGF expression might play a role in the development of malignant tumours in the thyroid.

Macrophage inflammatory protein-1alpha: a link between innate immunity and familial Mediterranean fever?

PubMed

Dizdar, Omer; Kalyoncu, Umut; Karadag, Omer; Akdogan, Ali; Kiraz, Sedat; Ertenli, Ihsan; Barista, Ibrahim; Calguneri, Meral

2007-01-01

The aim of this study is to investigate the relationship between chemokines and the inflammation in Familial Mediterranean Fever (FMF). Forty-nine patients with FMF (41 in remission and 8 in acute attack period) and 20 healthy controls were included in the study. Serum levels of macrophage inflammatory protein-1alpha (MIP-1alpha) were assessed in the patients and the controls, along with other parameters of disease activity, i.e., fibrinogen, C-reactive protein and erythrocyte sedimentation rate. Serum MIP-1alpha levels of the patients with FMF in acute attack period were significantly higher than the patients in remission and healthy controls (p=0.02 and p=0.038, respectively). MIP-1alpha levels were weakly correlated with CRP (r=0.32, p=0.032) levels. MIP-1alpha may have a role in the pathogenesis of FMF attacks. MIP-1alpha and other chemokines may constitute a link between the innate immune system and FMF.

Investigation into the Effects of Boron on Liver Tissue Protein Carbonyl, MDA, and Glutathione Levels in Endotoxemia.

PubMed

Balabanlı, Barbaros; Balaban, Tuba

2015-10-01

Endotoxin has been known to cause the formation and damage of free radical. The importance of boron for human life is increasing each passing day, and its consuming fields are continuing to expand due to the advances in science and technology. Therefore, in our study, we intended to investigate into the effects of boron on liver tissue oxidative events. Eighteen male Wistar albino rats were randomly separated into three equal groups in the experiments; control group, boron + endotoxin group, and endotoxin group. Dissolved in distilled water, boric acid (100 mg/kg) was administered to boron + endotoxin group via gavage procedure for 28 days. Only distilled water was administered to control and endotoxin groups via gavage procedure for 28 days. Then 4 mg/kg endotoxin (LPS; Escherichia coli 0111:B4) was intraperitoneally (ip) administered to boron + endotoxin and endotoxin groups on the 28th day. Sterile saline was injected into control group on the 28th day (ip). Malondialdehyde (MDA), which is the end product of lipid peroxidation in liver tissues, protein carbonyl compounds (PC), which are protein oxidization markers, and glutathione (GSH) levels were measured spectrophotometrically. The results were compared with Mann-Whitney U test. When boron + endotoxin group is compared with endotoxin group, PC levels of endotoxin group showed a significant increase. When GSH levels are compared, GSH level in boron + endotoxin group decreased according to endotoxin group. Variations among all groups in MDA levels were found to be statistically insignificant. We are of the opinion that endotoxin affects the proteins by forming free radicals, and boron may also cause the structural and/or functional changes in proteins in order to protect proteins from oxidization.

Downregulation of plasma SELENBP1 protein in patients with recent-onset schizophrenia.

PubMed

Chau, Edith J; Mostaid, Md Shaki; Cropley, Vanessa; McGorry, Patrick; Pantelis, Christos; Bousman, Chad A; Everall, Ian P

2018-07-13

Upregulation of selenium binding protein 1 (SELENBP1) mRNA expression has been reported in schizophrenia, primarily in the dorsolateral prefrontal cortex. However, peripheral blood studies are limited and results are inconsistent. In this study, we examined SELENBP1 mRNA expression in whole blood and protein expression in plasma from patients with recent-onset schizophrenia (n = 30), treatment-resistant schizophrenia (n = 71) and healthy controls (n = 57). We also examined the effects of SELENBP1 genetic variation on gene and protein expression. We found lower SELENBP1 plasma protein levels in patients with recent-onset schizophrenia (p = 0.042) but not in treatment-resistant schizophrenia (p = 0.81). Measurement of peripheral mRNA levels showed no difference between treatment-resistant schizophrenia and healthy controls (p = 0.234) but clozapine plasma levels (p = 0.036) and duration of illness (p = 0.028) were positively correlated with mRNA levels. Genetic variation was not associated with mRNA or protein expression. Our data represent the first peripheral proteomic study of SELENBP1 in schizophrenia and suggest that plasma SELENBP1 protein is downregulated in patients with recent-onset schizophrenia. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of Dietary Protein Level on the Expression of Proteins in the Gastrointestinal Tract of Young Pigs.

PubMed

Ma, Xianyong; Tian, Zhimei; Deng, Dun; Cui, Yiyan; Qiu, Yueqin

2018-05-02

The objective of this research is to investigate the effect of protein level on proteins expression in the gastrointestinal tract of young pigs. Eighteen piglets (Duroc × Landrace × Yorkshire) were weaned at 28 days of age and randomly assigned to three diets with 20%, 17%, and 14% CP level, and four essential amino acids, Lys, Met, Thr, and Trp, in three diets met the requirements of weaned piglets. The experimental period lasted 45 days. Compared with the control (20% CP level), the average daily feed intake, the average daily gain, and gain feed ratio of the 17% CP group did not decrease ( P > 0.05), but those of 14% CP group decreased ( P < 0.05). The proteomics profiles result of three tissues (gastric antrum, duodenum, and jejunum) showed that, compared with the control, the immune system, protein digestion and absorption, lipid or carbon digestion and absorption, etc. were up-regulated in 17% CP group, while most of them were down-regulated in 14% CP group. Amino acids metabolism of gastric, pancreatic secretion of duodenum or steroid hormone biosynthesis of jejunum was down-regulated in the 17% CP group, but the lipid metabolism was up-regulated in the 14% CP group. Six proteins were selected for identification by Western-blot, and their changes had the same trend as the proteomics results. The protein level decreased from 20% to 17%, the growth performance was not affected, while the nutrient digestion and absorption or the immune function were improved, which implied that 17% protein level maybe benefit for nutrients absorption of pigs.

Alpha-mangostin attenuates diabetic nephropathy in association with suppression of acid sphingomyelianse and endoplasmic reticulum stress.

PubMed

Liu, Tingting; Duan, Wang; Nizigiyimana, Paul; Gao, Lin; Liao, Zhouning; Xu, Boya; Liu, Lerong; Lei, Minxiang

2018-02-05

Diabetic nephropathy is a common complication of diabetes, but there are currently few treatment options. The aim of this study was to gain insight into the effect of alpha-mangostin on diabetic nephropathy and possible related mechanisms. Goto-Kakizaki rats were used as a diabetic model and received alpha-mangostin or desipramine treatment with normal saline as a control. Ten age-matched Sprague Dawley rats were used as normal controls and treated with normal saline. At week 12, blood glucose, albuminuria, apoptosis and renal pathologic changes were assessed. Protein levels for acid sphingomyelinase, glucose-regulated protein 78, phosphorylated PKR-like ER-resident kinase, activated transcription factor 4, CCAAT/enhancer-binding protein, homologous protein), and cleaved-caspase12 were measured. The level of acid sphingomyelinase was significantly increased, and ER stress was activated in diabetic rat kidneys when compared to the control animals. When acid sphingomyelinase was inhibited by alpha-mangostin, the expression of ER stress-related proteins was down-regulated in association with decreased levels of diabetic kidney injury. Alpha-mangostin, an acid sphingomyelinase inhibitor plays a protective role in diabetic neuropathy by relieving ER stress induced-renal cell apoptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification and characterization of a gibberellin-regulated protein, which is ASR5, in the basal region of rice leaf sheaths.

PubMed

Takasaki, Hironori; Mahmood, Tariq; Matsuoka, Makoto; Matsumoto, Hiroshi; Komatsu, Setsuko

2008-04-01

Gibberellins (GAs) regulate growth and development in higher plants. To identify GA-regulated proteins during rice leaf sheath elongation, a proteomic approach was used. Proteins from the basal region of leaf sheath in rice seedling treated with GA(3) were analyzed by fluorescence two-dimensional difference gel electrophoresis. The levels of abscisic acid-stress-ripening-inducible 5 protein (ASR5), elongation factor-1 beta, translationally controlled tumor protein, fructose-bisphosphate aldolase and a novel protein increased; whereas the level of RuBisCO subunit binding-protein decreased by GA(3) treatment. ASR5 out of these six proteins was significantly regulated by GA(3) at the protein level but not at the mRNA level in the basal region of leaf sheaths. Since this protein is regulated not only by abscisic acid but also by GA(3), these results indicate that ASR5 might be involved in plant growth in addition to stress in the basal regions of leaf sheaths.

Serum retinol binding protein 4 in patients with familial partial lipodystrophy.

PubMed

Godoy-Matos, Amélio F; Moreira, Rodrigo O; MacDowell, Renata; Bendet, Izidro; Mory, Patrícia B; Moises, Regina S

2009-07-01

To determine Retinol Binding Protein 4 (RBP4) levels in patients with Familial Partial Lipodystrophy (FPLD). Ten patients with FPLD and a control group (9 patients) were selected to participate in the study. RBP4-log levels were lower in patients with FPLD in comparison to control group (1.52 +/- 0.32 vs 1.84+/-0.25, p=0.029). A statistical trend was observed between Waist-to-Hip Ratio and RBP4-log (r=-0.44, p=0.054). RBP4 levels are decreased in FPLD.

Environmental Mycobiome Modifiers of Inflammation and Fibrosis in Systemic Sclerosis

DTIC Science & Technology

2016-09-01

TUBB), and ribosomal proteins), while others are considered specific to SSc despite trace level detection in controls. For ex- ample, multiple SSc...Strong re- activity was seen against all five proteins in SSc with only trace levels detected in controls (Fig. 3a), indicating widespread immune...sequences in SSc RNA-seq data was used to detect microbial sequences in human tissues in an unbiased, quantitative manner. Our studies suggest that

Serum levels of leptin and adiponectin and clinical parameters in women with fibromyalgia and overweight/obesity.

PubMed

Paiva, Eduardo S; Andretta, Aline; Batista, Emmanuelle Dias; Lobo, Márcia Maria Marques Teles; Miranda, Renata Costa de; Nisihara, Renato; Schieferdecker, Maria Eliana Madalozzo; Boguszewski, César L

2017-01-01

The objectives of this study were to evaluate the serum levels of adipokines in women with fibromyalgia with and without overweight/obesity, and to correlate the adipokines levels with clinical parameters associated with fibromyalgia and adipose tissue mass (body fat). The study included 100 women divided into four groups: (a) fibromyalgia and overweight/obesity; (b) fibromyalgia and normal weight; (c) controls and overweight/obesity; and (d) controls and normal weight. Patients and controls were evaluated for clinical, anthropometric, and fibromyalgia-related parameters. Assessments included serum levels of leptin, adiponectin, monocyte chemoattractant protein-1 (MCP-1), and C-reactive protein (CRP). Levels of adipokines were further adjusted for fat mass. Fibromyalgia patients with overweight/obesity or normal weight had no differences in clinical parameters. Unadjusted leptin levels were lower in fibromyalgia patients than controls, a finding that was more remarkable in fibromyalgia patients with overweight/obesity. Leptin levels had no correlation with clinical parameters of fibromyalgia or inflammation markers (MCP-1 and CRP), and adiponectin levels showed no difference between groups. No correlation was observed between adjusted leptin levels and clinical parameters of fibromyalgia. Patients with fibromyalgia and overweight/obesity presented lower levels of leptin than controls with overweight/obesity.

Effect of soya protein on blood pressure: a meta-analysis of randomised controlled trials.

PubMed

Dong, Jia-Yi; Tong, Xing; Wu, Zhi-Wei; Xun, Peng-Cheng; He, Ka; Qin, Li-Qiang

2011-08-01

Observational studies have indicated that soya food consumption is inversely associated with blood pressure (BP). Evidence from randomised controlled trials (RCT) on the BP-lowering effects of soya protein intake is inconclusive. We aimed to evaluate the effectiveness of soya protein intake in lowering BP. The PubMed database was searched for published RCT in the English language through to April 2010, which compared a soya protein diet with a control diet. We conducted a random-effects meta-analysis to examine the effects of soya protein on BP. Subgroup and meta-regression analyses were performed to explore possible explanations for heterogeneity among trials. Meta-analyses of twenty-seven RCT showed a mean decrease of 2·21 mmHg (95 % CI - 4·10, - 0·33; P = 0·021) for systolic BP (SBP) and 1·44 mmHg (95 % CI - 2·56, - 0·31; P = 0·012) for diastolic BP (DBP), comparing the participants in the soya protein group with those in the control group. Soya protein consumption significantly reduced SBP and DBP in both hypertensive and normotensive subjects, and the reductions were markedly greater in hypertensive subjects. Significant and greater BP reductions were also observed in trials using carbohydrate, but not milk products, as the control diet. Meta-regression analyses further revealed a significantly inverse association between pre-treatment BP and the level of BP reductions. In conclusion, soya protein intake, compared with a control diet, significantly reduces both SBP and DBP, but the BP reductions are related to pre-treatment BP levels of subjects and the type of control diet used as comparison.

Do infants with cow's milk protein allergy have inadequate levels of vitamin D?

PubMed

Silva, Cristiane M; Silva, Silvia A da; Antunes, Margarida M de C; Silva, Gisélia Alves Pontes da; Sarinho, Emanuel Sávio Cavalcanti; Brandt, Katia G

To verify whether infants with cow's milk protein allergy have inadequate vitamin D levels. This cross-sectional study included 120 children aged 2 years or younger, one group with cow's milk protein allergy and a control group. The children were recruited at the pediatric gastroenterology, allergology, and pediatric outpatient clinics of a university hospital in the Northeast of Brazil. A questionnaire was administered to the caregiver and blood samples were collected for vitamin D quantification. Vitamin D levels <30ng/mL were considered inadequate. Vitamin D level was expressed as mean and standard deviation, and the frequency of the degrees of sufficiency and other variables, as proportions. Infants with cow's milk protein allergy had lower mean vitamin D levels (30.93 vs.35.29ng/mL; p=0.041) and higher deficiency frequency (20.3% vs.8.2; p=0.049) than the healthy controls. Exclusively or predominantly breastfed infants with cow's milk protein allergy had higher frequency of inadequate vitamin D levels (p=0.002). Regardless of sun exposure time, the groups had similar frequencies of inadequate vitamin D levels (p=0.972). Lower vitamin D levels were found in infants with CMPA, especially those who were exclusively or predominantly breastfed, making these infants a possible risk group for vitamin D deficiency. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Effects of glycemic control on saliva flow rates and protein composition in non-insulin-dependent diabetes mellitus.

PubMed

Dodds, M W; Dodds, A P

1997-04-01

The objective of this study was to determine whether improvements in the level of diabetic control in a group of subjects with poorly controlled non-insulin-dependent diabetes mellitus influence salivary output and composition. Repeated whole unstimulated and stimulated parotid saliva samples were collected from diabetic patients attending an outpatient diabetes education program and a matched nondiabetic control group. Saliva was analyzed for flow rates, parotid protein concentration and composition, and amylase activity. Subjective responses to questions about salivary hypofunction were tested. There were no significant differences in whole unstimulated and stimulated parotid flow rates or stimulated parotid protein concentration and composition between diabetics and the control group. Amylase activity was higher in diabetics and decreased with improved glycemic control. Subjects reporting taste alterations had higher mean blood glucose levels than subjects with normal taste sensation. Poorly controlled non-insulin-dependent diabetes mellitus has no influence on saliva output, although amylase activity may be elevated, and there may be taste alterations.

FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

PubMed

Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J

2017-04-11

The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Protein Mediated Oxidative Stress in Patients with Diabetes and its Associated Neuropathy: Correlation with Protein Carbonylation and Disease Activity Markers

PubMed Central

Almogbel, Ebtehal

2017-01-01

Introduction Free radicals have been implicated as Diabetes Mellitus (DM) contributors in type 2 DM and its associated Diabetes Mellitus Neuropathy (DMN). However, the potential for protein mediated oxidative stress to contribute disease pathogenesis remains largely unexplored. Aim To investigate the status and contribution of protein mediated oxidative stress in patients with DM or DMN and to explore whether oxidative protein modification has a role in DM progression to DM associated neuropathy. Materials and Methods Sera from 42 DM and 37 DMN patients with varying levels of disease activities biomarkers (HbA1C, patients’ age or disease duration) and 21 age- and sex-matched healthy controls were evaluated for serum levels of protein mediated oxidative stress. Results Serum analysis showed significantly higher levels of protein carbonyl contents in both DM and DMN patients compared with healthy controls. Importantly, not only was there an increased number of subjects positive for protein carbonylation, but also the levels of protein carbonyl contents were significantly higher among DM and DMN patients, whose HbA1C were ≥8.8 as compared with patients with lower HbA1C (HbA1C<8.8). Similar pattern of protein carbonyls formation was also observed with patients’ ages or with patient’s disease durations, suggesting a possible relationship between protein oxidation and disease progression. Furthermore, sera from DMN patients had higher levels of protein carbonylation compared with non-neuropathic DM patients’ sera, suggesting an involvement of protein oxidation in the progression of diabetes to diabetes neuropathy. Conclusion These findings support an association between protein oxidation and DM or DMN progression. The stronger response observed in patients with higher HbA1C or patients’ ages or disease durations suggests, that protein mediated oxidative stress may be useful in evaluating the progression of DM and its associated DMN and in elucidating the mechanisms of these disorders pathogenesis. PMID:28384853

Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice.

PubMed

Pan, Minglin; Han, Ying; Basu, Aninda; Dai, Anzhi; Si, Rui; Willson, Conor; Balistrieri, Angela; Scott, Brian T; Makino, Ayako

2018-03-07

Coronary microvascular rarefaction due to endothelial cell (EC) dysfunction is one of the causes of increased morbidity and mortality in diabetes. Coronary ECs in diabetes are more apoptotic due partly to mitochondrial calcium overload. This study was designed to investigate the role of hexokinase 2 (HK2, an endogenous inhibitor of voltage-dependent anion channel) in coronary endothelial dysfunction in type 2 diabetes. We used mouse coronary ECs (MCECs) isolated from type 2 diabetic mice and human coronary ECs (HCECs) from type 2 diabetic patients to examine protein levels and mitochondrial functions. ECs were more apoptotic and capillary density was lower in the left ventricle of diabetic mice than the control. MCECs from diabetic mice exhibited significant increase in mitochondrial Ca 2+ concentration ([Ca 2+ ] mito ) compared to the control. Among several regulatory proteins for [Ca 2+ ] mito , HK1 and HK2 were significantly lower in MCECs from diabetic mice than control MCECs. We also found that the level of HK2 ubiquitination was higher in MCECs from diabetic mice than in control MCECs. In line with the data from MCECs, HCECs from diabetic patients showed lower HK2 protein levels than HCECs from non-diabetic patients. High-glucose treatment, but not high-fat treatment, significantly decreased HK2 protein levels in the MCEC. HK2 overexpression in MCECs of diabetic mice not only lowered the level of [Ca 2+ ] mito , but also reduced mitochondrial ROS production toward the level seen in control MCECs. These data suggest that HK2 is a potential therapeutic target for coronary microvascular disease in diabetes by restoring mitochondrial function in coronary ECs.

[The relationship between the abnormal behavior and serum C-reactive protein in children with obstructive sleep apnea-hypopnea syndrome].

PubMed

Wang, Yan; Li, Yanzhong; Wang, Xin

2009-12-01

To explore the pathogenesis of abnormal behavior in children with obstructive sleep apnea-hypopnea syndrome (OSAHS). The behavioral problems and C-reactive protein were measured in 40 children with OSAHS and 30 children with habitual snoring who underwent overnight Polysomnography, 40 cases of healthy children for the control group. The ratio of abnormal behavior in OSAHS and habitual snoring children was significantly higher than that of the healthy control group, while no significant difference between the two groups. The content of C-reactive protein in OSAHS children (4.24 mg/L) was significantly higher than habitual snoring (2.76 mg/L) and healthy control group (1.27 mg/L); in habitual snoring children C-reactive protein was higher than in healthy control group. The content of serum C-reactive protein in OSAHS children accompanied by abnormal behavior (4.63 mg/L) was significantly higher than that without abnormal behavior (3.23 mg/L). The content of serum C-reactive protein content in habitual snoring children accompanied by abnormal behavior (3.63 mg/L) was significantly higher than that without abnormal behavior (1.76 mg/L). OSAHS and habitual snoring children have more behavior problems. C-reactive protein levels are higher in children with OSAHS and habitual snoring, and the levels of C-reactive protein are related to the abnormal behavior in these children.

Prevention of Pulmonary Fibrosis via Trichostatin A (TSA) in Bleomycin Induced Rats.

PubMed

Ye, Qing; Li, Yanqin; Jiang, Handong; Xiong, Jianfei; Xu, Jiabo; Qin, Hui; Liu, Bin

2014-10-20

To investigate the effects of non selective histone deacetylase inhibitors Trichostatin A (TSA)on bleomycin-induced pulmonary fibrosis. To investigate the effects of non selective histone deacetylase inhibitors Trichostatin A ( TSA ) on HDAC2, p-SMAD2, HDAC2 mRNA, SMAD2mRNA in pulmonary fibrosis rats and investigate impossible mechanism. 46 SPF level male SD rats were randomly divided into four groups: ten for normal control group, fourteen for model control group I, twelve for model control group II and ten for treatment group. Rat pulmonary fibrosis was induced by bleomycin(5mg/kg) via single intratracheal perfusion in the two model control groups and treatment group. Normal control mice were instilled with a corresponding volume of 0.9% saline intratracheally. Treatment group was treated by the dilution of TSA 2mg/kg DMSO 60ul and0.9% saline 1.2ml intraperitoneal injection from the next day ,once a day for three days. Model control group II was treated by the dilution of DMSO 60ul and0.9% saline 1.2ml intraperitoneal injection from the next day once a day for three days. Model control group I and normal control group were treated by 0.9% saline 1.2ml intraperitoneal injection from the next day once a day for three days. All the animals were sacrificed on the 21 day after modeling. The pathological changes were observed by hematoxylin and eosin(HE)stain and masson trichrome stain. The expression of HDAC2 mRNA,SMAD2 mRNA were measured by real-time PCR. The protein level of HDAC2 and p-SMAD2 in serum was measured by Western blot. The pulmonary fibrosis in treatment group were significantly alleviated compared to the two model control groups (P<0.05). Real-time PCR showed that the treatment group had lower expression of lung tissue HDAC2 mRNA than the two model control groups and normal control group (P<0.05). The expression of lung tissue SMAD2 mRNA increased in the two model control groups and treatment group (P<0.05),but there were no significant differences among the three groups(P>0.05). Western blot indicated that the protein level of HDAC2 and p-SMAD2 in serum increased in the two model control groups compared with normal control group(P<0.05).But treatment group had lower protein level of HDAC2 (P<0.05) and no significant difference in the protein level of p-SMAD2 compared to the two model control groups (P>0.05). Non selective histone deacetylase inhibitors of Trichostatin A (TSA) can reduce the bleomycin induced pulmonary fibrosis in rats. TSA attenuates pulmonary fibrosis and it can inhibit HDAC2 expression at the gene and protein level. Bleomycin induced fibrosis has the relationship with p-SMAD2 in gene and protein levels, but TSA inhibit bleomycin-induced lung fibrosis effect with no relation with SMAD2 phosphorylation pathways.

Proximate composition of CELSS crops grown in NASA's Biomass Production Chamber.

PubMed

Wheeler, R M; Mackowiak, C L; Sager, J C; Knott, W M; Berry, W L

1996-01-01

Edible biomass from four crops of wheat (Triticum aestivum L.), four crops of lettuce (Lactuca sativa L.), four crops of potato (Solanum tuberosum L.), and three crops of soybean (Glycine max (L.) Merr.) grown in NASA's CELSS Biomass Production Chamber were analyzed for proximate composition. All plants were grown using recirculating nutrient (hydroponic) film culture with pH and electrical conductivity automatically controlled. Temperature and humidity were controlled to near optimal levels for each species and atmospheric carbon dioxide partial pressures were maintained near 100 Pa during the light cycles. Soybean seed contained the highest percentage of protein and fat, potato tubers and wheat seed contained the highest levels of carbohydrate, and lettuce leaves contained the highest level of ash. Analyses showed values close to data published for field-grown plants with several exceptions: In comparison with field-grown plants, wheat seed had higher protein levels; soybean seed had higher ash and crude fiber levels; and potato tubers and lettuce leaves had higher protein and ash levels. The higher ash and protein levels may have been a result of the continuous supply of nutrients (e.g., potassium and nitrogen) to the plants by the recirculating hydroponic culture.

Proximate composition of CELSS crops grown in NASA's Biomass Production Chamber

NASA Technical Reports Server (NTRS)

Wheeler, R. M.; Mackowiak, C. L.; Sager, J. C.; Knott, W. M.; Berry, W. L.

1996-01-01

Edible biomass from four crops of wheat (Triticum aestivum L.), four crops of lettuce (Lactuca sativa L.), four crops of potato (Solanum tuberosum L.), and three crops of soybean (Glycine max (L.) Merr.) grown in NASA's CELSS Biomass Production Chamber were analyzed for proximate composition. All plants were grown using recirculating nutrient (hydroponic) film culture with pH and electrical conductivity automatically controlled. Temperature and humidity were controlled to near optimal levels for each species and atmospheric carbon dioxide partial pressures were maintained near 100 Pa during the light cycles. Soybean seed contained the highest percentage of protein and fat, potato tubers and wheat seed contained the highest levels of carbohydrate, and lettuce leaves contained the highest level of ash. Analyses showed values close to data published for field-grown plants with several exceptions: In comparison with field-grown plants, wheat seed had higher protein levels; soybean seed had higher ash and crude fiber levels; and potato tubers and lettuce leaves had higher protein and ash levels. The higher ash and protein levels may have been a result of the continuous supply of nutrients (e.g., potassium and nitrogen) to the plants by the recirculating hydroponic culture.

Proximate composition of CELSS crops grown in NASA's Biomass Production Chamber

NASA Astrophysics Data System (ADS)

Wheeler, R. M.; Mackowiak, C. L.; Sager, J. C.; Knott, W. M.; Berry, W. L.

Edible biomass from four crops of wheat (Triticum aestivum L.), four crops of lettuce (Lactuca sativa L.), four crops of potato (Solanum tuberosum L.), and three crops of soybean (Glycine max (L.) Merr.) grown in NASA's CELSS Biomass Production Chamber were analyzed for proximate composition. All plants were grown using recirculating nutrient (hydroponic) film culture with pH and electrical conductivity automatically controlled. Temperature and humidity were controlled to near optimal levels for each species and atmospheric carbon dioxide partial pressures were maintained near 100 Pa during the light cycles. Soybean seed contained the highest percentage of protein and fat, potato tubers and wheat seed contained the highest levels of carbohydrate, and lettuce leaves contained the highest level of ash. Analyses showed values close to data published for field-grown plants with several exceptions: In comparison with field-grown plants, wheat seed had higher protein levels; soybean seed had higher ash and crude fiber levels; and potato tubers and lettuce leaves had higher protein and ash levels. The higher ash and protein levels may have been a result of the continuous supply of nutrients (e.g., potassium and nitrogen) to the plants by the recirculating hydroponic culture.

O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.

PubMed

Makino, Ayako; Dai, Anzhi; Han, Ying; Youssef, Katia D; Wang, Weihua; Donthamsetty, Reshma; Scott, Brian T; Wang, Hong; Dillmann, Wolfgang H

2015-11-01

Cardiovascular disease is the primary cause of morbidity and mortality in diabetes, and endothelial dysfunction is commonly seen in these patients. Increased O-linked N-acetylglucosamine (O-GlcNAc) protein modification is one of the central pathogenic features of diabetes. Modification of proteins by O-GlcNAc (O-GlcNAcylation) is regulated by two key enzymes: β-N-acetylglucosaminidase [O-GlcNAcase (OGA)], which catalyzes the reduction of protein O-GlcNAcylation, and O-GlcNAc transferase (OGT), which induces O-GlcNAcylation. However, it is not known whether reducing O-GlcNAcylation can improve endothelial dysfunction in diabetes. To examine the effect of endothelium-specific OGA overexpression on protein O-GlcNAcylation and coronary endothelial function in diabetic mice, we generated tetracycline-inducible, endothelium-specific OGA transgenic mice, and induced OGA by doxycycline administration in streptozotocin-induced type 1 diabetic mice. OGA protein expression was significantly decreased in mouse coronary endothelial cells (MCECs) isolated from diabetic mice compared with control MCECs, whereas OGT protein level was markedly increased. The level of protein O-GlcNAcylation was increased in diabetic compared with control mice, and OGA overexpression significantly decreased the level of protein O-GlcNAcylation in MCECs from diabetic mice. Capillary density in the left ventricle and endothelium-dependent relaxation in coronary arteries were significantly decreased in diabetes, while OGA overexpression increased capillary density to the control level and restored endothelium-dependent relaxation without changing endothelium-independent relaxation. We found that connexin 40 could be the potential target of O-GlcNAcylation that regulates the endothelial functions in diabetes. These data suggest that OGA overexpression in endothelial cells improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes. Copyright © 2015 the American Physiological Society.

May the Thyroid Gland and Thyroperoxidase Participate in Nitrosylation of Serum Proteins and Sporadic Parkinson's Disease?

PubMed Central

García-Moreno, José-Manuel; Martín de Pablos, Angel; Chacón, José

2014-01-01

Abstract The research group has detected nitrosative stress and a singular version of nitrosylated serum α-synuclein in serum of Parkinson's disease (PD) patients. Dysfunction of the thyroid gland has been proposed to be linked to this disease. The aim of the study was to know if the thyroid gland is involved in idiopathic PD and nitrosative stress. We studied 50 patients (early and advanced disease patients), 35 controls, and 6 subjects with thyroidectomy. Clinical characteristics, serum thyroperoxidase levels, and 3-nitrotyrosine proteins were analyzed. Enzyme-linked immunosorbent assay and immunoblotting methods were employed. The findings indicated that the prevalence of two thyroid dysfunctions (hyper- or hypothyroidism) was not found to be different in patients relative to controls. However, the levels of the enzyme thyroperoxidase were found to be elevated in early disease patients (p<0.006), not in advanced disease subjects, and these levels were negatively correlated with serum 3-nitrotyrosine proteins (p<0.05), the indicators of nitrosative stress. The thyroidectomized subjects showed very low levels of serum 3-nitrotyrosine proteins (78% reduction vs. controls) and, among these proteins, the nitrosylated serum α-synuclein was nearly absent. These observations lead to the hypothesis that the thyroid gland and thyroperoxidase participate in nitrosylation of serum proteins and they could influence Parkinsonian nitrosative stress as well as nitrosylation of serum α-synuclein, a potentially pathogenic factor. Antioxid. Redox Signal. 21, 2143–2148. PMID:25125346

Gel-Free/Label-Free Proteomic Analysis of Endoplasmic Reticulum Proteins in Soybean Root Tips under Flooding and Drought Stresses.

PubMed

Wang, Xin; Komatsu, Setsuko

2016-07-01

Soybean is a widely cultivated crop; however, it is sensitive to flooding and drought stresses. The adverse environmental cues cause the endoplasmic reticulum (ER) stress due to accumulation of unfolded or misfolded proteins. To investigate the mechanisms in response to flooding and drought stresses, ER proteomics was performed in soybean root tips. The enzyme activity of NADH cytochrome c reductase was two-fold higher in the ER than other fractions, indicating that the ER was isolated with high purity. Protein abundance of ribosomal proteins was decreased under both stresses compared to control condition; however, the percentage of increased ribosomes was two-fold higher in flooding compared to drought. The ER proteins related to protein glycosylation and signaling were in response to both stresses. Compared to control condition, calnexin was decreased under both stresses; however, protein disulfide isomerase-like proteins and heat shock proteins were markedly decreased under flooding and drought conditions, respectively. Furthermore, fewer glycoproteins and higher levels of cytosolic calcium were identified under both stresses compared to control condition. These results suggest that reduced accumulation of glycoproteins in response to both stresses might be due to dysfunction of protein folding through calnexin/calreticulin cycle. Additionally, the increased cytosolic calcium levels induced by flooding and drought stresses might disturb the ER environment for proper protein folding in soybean root tips.

Decreased GRK3 but not GRK2 expression in frontal cortex from bipolar disorder patients

PubMed Central

Rao, Jagadeesh S; Rapoport, Stanley I; Kim, Hyung-Wook

2009-01-01

Overactivation of G-protein mediated functions and altered G-protein regulation have been reported in bipolar disorder (BD) brain. Further, drugs effective in treating BD are reported to upregulate expression of G-protein receptor kinase (GRK) 3 in rat frontal cortex. We therefore hypothesized that some G-protein subunits and GRK levels would be reduced in the brains of BD patients. We determined protein and mRNA levels of G-protein β and γ subunits, GRK2, and GRK3 in postmortem frontal cortex from 10 BD patients and 10 age-matched controls by using immunoblots and real-time RT-PCR. There were the statistically significant decreases in protein and mRNA levels of G-protein subunits β and γ and of GRK3 in the BD brains but not a significant difference in the GRK2 level. Decreased expression of G-protein subunits and of GRK3 may alter neurotransmission, leading to disturbed cognition and behavior in BD. PMID:19400979

Correction of protein metabolic disorders by composite extract of Musa paradisiaca and Coccinia indica in streptozotocin-induced diabetic albino rat: an approach through the pancreas.

PubMed

Mallick, Chhanda; De, Debasis; Ghosh, Debidas

2009-04-01

The study focused on the ability of the extracts of Musa paradisiaca and Coccinia indica on protein metabolic disorders in streptozotocin (STZ)-induced diabetes. Wistar strain rats were divided into 6 groups as control, control + composite extract treated, STZ-induced diabetes, diabetic + composite extract treated, composite extract-pretreated diabetes, and composite extract-pretreated diabetes + composite extract treated. Protein metabolic status was assessed by serum levels of urea, uric acid, albumin, and creatinine along with urine urea and albumin levels. Diabetic therapeutic ability was assessed by blood glucose, glycated hemoglobin, and serum insulin levels. Histology of the pancreas, liver, and kidney was evaluated. Indices of protein metabolic disorders were deviated from control in STZ-induced diabetes, which were protected significantly after the treatment of composite extract of M. paradisiaca and C. indica. This protection was more prominent when the extract-pretreated animals were subjected to diabetes induction by STZ. The composite extract has a protective therapeutic effect against diabetes through beta-cell regeneration capacity.

Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

PubMed Central

Wei, Xiaofan; Wang, Xiang; Zhan, Jun; Chen, Yuhan; Fang, Weigang; Zhang, Lingqiang

2017-01-01

Integrin activation is an indispensable step for various integrin-mediated biological functions. Kindlin-2 is known to coactivate integrins with Talin; however, molecules that restrict integrin activation are elusive. Here, we demonstrate that the E3 ubiquitin ligase Smurf1 controls the amount of Kindlin-2 protein in cells and hinders integrin activation. Smurf1 interacts with and promotes Kindlin-2 ubiquitination and degradation. Smurf1 selectively mediates degradation of Kindlin-2 but not Talin, leading to inhibition of αIIbβ3 integrin activation in Chinese hamster ovary cells and β1 integrin activation in fibroblasts. Enhanced activation of β1 integrin was found in Smurf1-knockout mouse embryonic fibroblasts, which correlates with an increase in Kindlin-2 protein levels. Similarly, a reciprocal relationship between Smurf1 and Kindlin-2 protein levels is found in tissues from colon cancer patients, suggesting that Smurf1 mediates Kindlin-2 degradation in vivo. Collectively, we demonstrate that Smurf1 acts as a brake for integrin activation by controlling Kindlin-2 protein levels, a new mechanism that permits precise modulation of integrin-mediated cellular functions. PMID:28408404

Neurofilament protein levels: quantitative analysis in essential tremor cerebellar cortex.

PubMed

Louis, Elan D; Ma, Karen; Babij, Rachel; Cortés, Etty; Liem, Ronald K; Vonsattel, Jean-Paul G; Faust, Phyllis L

2012-06-14

Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and α-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or α-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

PubMed

Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

2007-11-01

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

Coagulation parameters and platelet function analysis in patients with acromegaly.

PubMed

Colak, A; Yılmaz, H; Temel, Y; Demirpence, M; Simsek, N; Karademirci, İ; Bozkurt, U; Yasar, E

2016-01-01

Acromegaly is associated with increased cardiovascular morbidity and mortality. The data about the evaluation of coagulation and fibrinolysis in acromegalic patients are very limited and to our knowledge, platelet function analysis has never been investigated. So, we aimed to investigate the levels of protein C, protein S, fibrinogen, antithrombin 3 and platelet function analysis in patients with acromegaly. Thirty-nine patients with active acromegaly and 35 healthy subjects were included in the study. Plasma glucose and lipid profile, fibrinogen levels, GH and IGF-1 levels and protein C, protein S and antithrombin III activities were measured in all study subjects. Also, platelet function analysis was evaluated with collagen/ADP and collagen-epinephrine-closure times. Demographic characteristics of the patient and the control were similar. As expected, fasting blood glucose levels and serum GH and IGF-1 levels were significantly higher in the patient group compared with the control group (pglc: 0.002, pGH: 0.006, pIGF-1: 0.001, respectively). But lipid parameters were similar between the two groups. While serum fibrinogen and antithrombin III levels were found to be significantly higher in acromegaly group (p fibrinogen: 0.005 and pantithrombin III: 0.001), protein S and protein C activity values were significantly lower in the patient group (p protein S: 0.001, p protein C: 0.001). Also significantly enhanced platelet function (measured by collagen/ADP- and collagen/epinephrine-closure times) was demonstrated in acromegaly (p col-ADP: 0.002, p col-epinephrine: 0.002). The results did not change, when we excluded six patients with type 2 diabetes in the acromegaly group. There was a negative correlation between serum GH levels and protein S (r: -0.25, p: 0.04)) and protein C (r: -0.26, p: 0.04) values. Likewise, there was a negative correlation between IGF-1 levels and protein C values (r: -0.39, p: 0.002), protein S values (r: -0.39, p: 0.001), collagen/ADP-closure times (r: -0.28, p: 0.02) and collagen/epinephrine-closure times (r:-0.26, p: 0.04). Also, we observed a positive correlation between IGF-1 levels and fibrinogen levels (r: 0.31, p: 0.01). Acromegaly was found to be associated with increased tendency to coagulation and enhanced platelet activity. This hypercoagulable state might increase the risk for cardiovascular and cerebrovascular events in acromegaly.

Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

PubMed

Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

2006-05-01

Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

A dual small-molecule rheostat for precise control of protein concentration in Mammalian cells.

PubMed

Lin, Yu Hsuan; Pratt, Matthew R

2014-04-14

One of the most successful strategies for controlling protein concentrations in living cells relies on protein destabilization domains (DD). Under normal conditions, a DD will be rapidly degraded by the proteasome. However, the same DD can be stabilized or "shielded" in a stoichiometric complex with a small molecule, enabling dose-dependent control of its concentration. This process has been exploited by several labs to post-translationally control the expression levels of proteins in vitro as well as in vivo, although the previous technologies resulted in permanent fusion of the protein of interest to the DD, which can affect biological activity and complicate results. We previously reported a complementary strategy, termed traceless shielding (TShld), in which the protein of interest is released in its native form. Here, we describe an optimized protein concentration control system, TTShld, which retains the traceless features of TShld but utilizes two tiers of small molecule control to set protein concentrations in living cells. These experiments provide the first protein concentration control system that results in both a wide range of protein concentrations and proteins free from engineered fusion constructs. The TTShld system has a greatly improved dynamic range compared to our previously reported system, and the traceless feature is attractive for elucidation of the consequences of protein concentration in cell biology. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Caffeine Increases Apolipoprotein A-1 and Paraoxonase-1 but not Paraoxonase-3 Protein Levels in Human-Derived Liver (HepG2) Cells.

PubMed

Sayılan Özgün, Gülben; Özgün, Eray; Tabakçıoğlu, Kıymet; Süer Gökmen, Selma; Eskiocak, Sevgi; Çakır, Erol

2017-12-01

Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 are antioxidant and anti-atherosclerotic structural high-density lipoprotein proteins that are mainly synthesized by the liver. No study has ever been performed to specifically examine the effects of caffeine on paraoxonase enzymes and on liver apolipoprotein A-1 protein levels. To investigate the dose-dependent effects of caffeine on liver apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels. In vitro experimental study. HepG2 cells were incubated with 0 (control), 10, 50 and 200 μM of caffeine for 24 hours. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels were measured by western blotting. We observed a significant increase on apolipoprotein A-1 and paraoxonase-1 protein levels in the cells incubated with 50 µM of caffeine and a significant increase on paraoxonase-1 protein level in the cells incubated with 200 µM of caffeine. Our study showed that caffeine does not change paraoxonase-3 protein level, but the higher doses used in our study do cause an increase in both apolipoprotein A-1 and paraoxonase-1 protein levels in liver cells.

Serum levels of albumin, triglycerides, total protein and glucose in rats are altered after oral treatment with low doses of 13-cis-retinoic acid or all-trans-retinoic acid.

PubMed

Cisneros, F J; Gough, B J; Patton, R E; Ferguson, S A

2005-01-01

Currently used to treat severe acne, 13-cis-retinoic acid (13-cis-RA) is under investigation for its anticancer effects as is the isomer, all-trans-retinoic acid (all-trans-RA). Here, the effects of oral 13-cis-RA or all-trans-RA treatment on serum chemistry, leptin and adiponectin levels were evaluated. Adult Sprague-Dawley rats were gavaged once daily for 7 consecutive days with 13-cis-RA (7.5 or 15 mg kg(-1)), all-trans-RA (10 or 15 mg kg(-1)) (n=24/sex/dose), or soy oil (n=16/sex) and blood was sampled 30-480 min after the last gavage. The body weight was unaffected; however, the liver/body weight ratios were increased by both doses of all-trans-RA. Sex differences were noted for levels of cholesterol, creatine, triglycerides, albumin, alanine aminotransferase and total protein. Both doses of all-trans-RA reduced albumin levels to approximately 90% of the control and total protein levels to approximately 93% of the control while substantially elevating triglyceride levels to approximately 66%-99% above the control. Additionally, triglyceride levels of the 15 mg kg(-1) 13-cis RA group were approximately 62% higher than the controls and total protein levels were approximately 5% less. Glucose levels were affected by sex and RA treatment in that males treated with 15 mg kg(-1) of 13-cis-RA or 10 mg kg(-1) all-trans-RA had lower (13%-19%) levels than the same-sex controls; however, females were not similarly affected. Neither 13-cis-RA nor all-trans-RA treatment had significant effects on the levels of blood urea nitrogen, aspartate amino transferase, leptin or adiponectin. On a mg kg(-1) basis, all-trans-RA was more potent than 13-cis-RA. These results replicate previous findings of RA-induced increased triglyceride levels. Additionally, several new findings indicate there may be sex-specific effects of RA treatment. Finally, neither treatment appeared to alter the typical diurnal cycles of these endpoints. Copyright (c) 2005 John Wiley & Sons, Ltd.

A biomolecular proportional integral controller based on feedback regulations of protein level and activity.

PubMed

Mairet, Francis

2018-02-01

Homeostasis is the capacity of living organisms to keep internal conditions regulated at a constant level, despite environmental fluctuations. Integral feedback control is known to play a key role in this behaviour. Here, I show that a feedback system involving transcriptional and post-translational regulations of the same executor protein acts as a proportional integral (PI) controller, leading to enhanced transient performances in comparison with a classical integral loop. Such a biomolecular controller-which I call a level and activity-PI controller (LA-PI)-is involved in the regulation of ammonium uptake by Escherichia coli through the transporter AmtB. The P II molecules, which reflect the nitrogen status of the cell, inhibit both the production of AmtB and its activity (via the NtrB-NtrC system and the formation of a complex with GlnK, respectively). Other examples of LA-PI controller include copper and zinc transporters, and the redox regulation in photosynthesis. This scheme has thus emerged through evolution in many biological systems, surely because of the benefits it offers in terms of performances (rapid and perfect adaptation) and economy (protein production according to needs).

GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation.

PubMed

Cai, Shijie; Alp, Nicholas J; McDonald, Denise; Smith, Ian; Kay, Jonathan; Canevari, Laura; Heales, Simon; Channon, Keith M

2002-09-01

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS-GFP fusion protein were significantly increased following GTPCH gene transfer. These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

High Concentrations of Angiopoietin-Like Protein 4 Detected in Serum from Patients with Rheumatoid Arthritis Can Be Explained by Non-Specific Antibody Reactivity.

PubMed

Makoveichuk, Elena; Ruge, Toralph; Nilsson, Solveig; Södergren, Anna; Olivecrona, Gunilla

2017-01-01

Angiopoietin-like protein 4 (ANGPTL4) is suggested to be a master regulator of plasma triglyceride metabolism. Our aim was to study whether the previously reported high levels of ANGPTL4 detected in serum from patients with rheumatoid arthritis (RA) by ELISA was due to any specific molecular form of this protein (oligomers, monomers or fragments). ANGPTL4 levels were first determined in serum from 68 RA patients and 43 age and sex matched control subjects and the mean values differed by a factor of 5.0. Then, ANGPTL4 was analyzed after size exclusion chromatography (SEC) of serum samples. With serum from one of the RA patients with high levels of ANGPTL4, the dominant reactivity was found in fractions corresponding to high-molecular weight proteins. In addition, a minor peak of reactivity eluting late from the column was found both in the patient and in controls. By the use of HeteroBlock®, and by careful selection of antibodies, we documented non-specific reactions for ANGPTL4 in 39% of samples from the RA patients, most likely due to cross-reactivity of the antibodies with rheumatoid factor (RF). The corresponding figure for control subjects was 6.3%. After corrections for non-specific reactions, the mean level of ANGPTL4 in serum from RA patients was still significantly higher than in control individuals (mean levels were 101±62 and 67±39 ng/ml respectively, P = 0.02). We re-analyzed samples from our previously published studies on ANGPL4 levels in patients on hemodialysis and patients with diabetes type 2. These samples did not show false positive reactions. The levels of ANGPTL4 were comparable to those detected previously.

Evaluation of the relationship between passive smoking and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in young children.

PubMed

Avşar, Aysun; Darka, Ozge; Bodrumlu, Ebru Hazar; Bek, Yüksel

2009-05-01

To evaluate the relationship between passive smoking as determined by salivary cotinine levels and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in children. Saliva was collected from 90 passive smoker (PS) subjects (the study group) and 90 healthy age-matched children (the control group). The study group was divided into three subgroups according the number of cigarettes smoked. Socio-economic status, dental and dietary habits were recorded by questionnaire. Stimulated salivary calcium (Ca), phosphate (P), sodium (Na), potassium (P), total protein, amylase activity, sialic acid level, secretory IgA concentration and cotinine level were analysed. All data were analysed using SPSS, version 13.0. Socio-economic status, dental and dietary habits were similar between the two groups. The salivary electrolytes concentrations did not reveal significant difference between the two groups (p>0.05). The mean cotinine levels of PS children were 1.58+/-4.3 ng/mL. The salivary concentrations of protein were similar between the two groups (p>0.05). The salivary secretory IgA concentration was significantly lower in the PS group than controls. The sialic acid level and amylase activity in PS group were found significantly higher compared with the controls (p<0.05). No difference was observed for all these parameters with sex (p>0.05). When saliva samples were analysed for output, the sialic acid level and amylase activity increased significantly in PS subjects (p<0.05). Further, the output of secretory IgA concentration was found significantly lower compared with the controls (p<0.05). In conclusion, we show that passive smoking was associated with a decrease in secretory IgA concentration, whereas with increase in amylase activity and sialic acid level of stimulated whole saliva in young children.

Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

PubMed

Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

2016-08-01

Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

PubMed

Oliveira, Marco A S; Gerhardt, Edileusa C M; Huergo, Luciano F; Souza, Emanuel M; Pedrosa, Fábio O; Chubatsu, Leda S

2015-12-01

Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state. © 2015 FEBS.

Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae.

PubMed

Pedrosa, F O; Benelli, E M; Yates, M G; Wassem, R; Monteiro, R A; Klassen, G; Steffens, M B; Souza, E M; Chubatsu, L S; Rigo, L U

2001-10-04

Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.

Synthesis of the low molecular weight heat shock proteins in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mansfield, M.A.; Key, J.L.

1987-08-01

Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation to these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticummore » asetivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.« less

Elevated interictal serum HSP-70 levels as an indicator of neurodegeneration for chronic migraine.

PubMed

Yon, Mehmet Ilker; Titiz, Ayse Pinar; Bilen, Sule; Ulusoy, Ersin Kasim; Gulunay, Aydin; Karakoc, Mehmet; Yon, Merve Ecem Erdogan; Ak, Fikri

2016-06-01

To investigate whether there is a relationship between chronic migraine and heat shock protein-70. The case-control progressive study was conducted at Ankara Numune Teaching and Research Hospital, Ankara, Turkey, from January to June 2013, and comprised patients over 18 years of age who were diagnosed with chronic migraine and did not have any other known neurological illness. Age and gender-matched volunteers with no history of headache or neurological illness were included as controls. In order to exclude other central nervous system diseases, computed tomography and/or magnetic resonance imaging was carried out. Blood samples to evaluate serum heat shock protein-70 levels were obtained from the patients during headache-free periods and from the controls following 8 hours of fasting. The samples were interpreted using the enzyme-linked immunosorbent assay reader. There were 40 controls and an equal number of cases in the study. Mean heat shock protein-70 levels were higher in the cases 2.37±1.91ng/dl compared to thecontrols1.81±1.30 ng/dl, but the difference was not statistically significant (p=0.12). Serum heat shock protein-70 levels were also compared in terms of the duration of migraine disease, frequency of migraine attacks, Visual Analogue Scale score, migraine attack duration and the presence of aura, but no statistically significant difference was found (p=0.13, p=0.17, p=0.90, p=0.68, p=0.95 respectively). Heat shock protein-70 was not a reliable chronic migraine biomarker.

Induction of the chemotactic S100 protein, CP-10, in monocyte/macrophages by lipopolysaccharide.

PubMed

Hu, S P; Harrison, C; Xu, K; Cornish, C J; Geczy, C L

1996-05-01

The murine S100 protein CP-10 is a potent chemotactic factor for murine and human myeloid cells in vivo and in vitro. This is the first report describing regulations of the CP-10 gene by a proinflammatory stimulus, lipopolysaccharide (LPS), in cells of the monocyte/macrophage lineage. Murine monocyte/macrophage-like WEHI 265 and RAW 264.7 cells preexposed to 5 to 50 ng/mL LPS expressed significant levels of CP-10 mRNA 4 hours, and maximal at 20 hours, after a secondary LPS challenge. This was accompanied by increasing levels of cell-associated and released CP-10 protein. In contrast, a single dose of LPS upregulated CP-10 mRNA in elicited peritoneal macrophages, whereas mRNA and protein levels decreased following LPS challenge. The state of macrophage differentiation may control responsiveness as LPS had no effect on CP-10 basal levels in bone marrow derived macrophages. LPS-induced CP-10 expression was controlled at the transcriptional level and nuclear run-on and protein synthesis inhibition assays indicated that LPS priming and challenge of RAW cells occurred via distinct pathways. MRP14, another S100 protein generally coordinately expressed with human MRP8, was not induced by LPS under the same conditions. We propose that CP-10 may play a key role in recruitment of leukocytes into tissues in response to gram-negative bacterial infection.

Effects of high protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial

USDA-ARS?s Scientific Manuscript database

Context: The benefits of high protein diets for sparing lean body mass and sustaining skeletal muscle protein metabolism during short-term weight loss in normal-weight adults are not well described. Objective: Determine the effects of varying levels of dietary protein intake on body compos...

Cy5 total protein normalization in Western blot analysis.

PubMed

Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

2015-10-01

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

Heritability and genetic basis of protein level variation in an outbred population

PubMed Central

Liu, Yi-Chun; Tekkedil, Manu M.; Steinmetz, Lars M.; Caudy, Amy A.; Fraser, Andrew G.

2014-01-01

The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population’s average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance. PMID:24823668

Upregulation of NLRP3 Inflammasome in the Tears and Ocular Surface of Dry Eye Patients

PubMed Central

Wu, Jihong; Chen, Ling; Wang, Yan

2015-01-01

Purpose To evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye. Methods We recruited 54 patients with Sjögren’s syndrome dry eye (SSDE), 50 patients with non-Sjögren’s syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting. Results NLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p<0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p<0.05) and NSSDE (relative 1.32-fold upregulation, p<0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold (p<0.001) in SSDE and 2.13-fold (p<0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE (p=0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p=0.001) and NSSDE (relative 2.05-fold upregulation, p=0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE (p<0.001) and NSSDE (p<0.05) groups. Conclusions In the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye. PMID:25962072

The effect of cocoa supplementation on hepatic steatosis, reactive oxygen species and LFABP in a rat model of NASH

PubMed Central

2011-01-01

Background Non alcoholic steatohepatitis is hypothesised to develop via a mechanism involving fat accumulation and oxidative stress. The current study aimed to investigate if an increase in oxidative stress was associated with changes in the expression of liver fatty acid binding protein in a rat model of non alcoholic steatohepatitis and whether cocoa supplementation attenuated those changes. Methods Female Sprague Dawley rats were fed a high fat control diet, a high fat methionine choline deficient diet, or one of four 12.5% cocoa supplementation regimes in combination with the high fat methionine choline deficient diet. Results Liver fatty acid binding protein mRNA and protein levels were reduced in the liver of animals with fatty liver disease when compared to controls. Increased hepatic fat content was accompanied by higher levels of oxidative stress in animals with fatty liver disease when compared to controls. An inverse association was found between the levels of hepatic liver fatty acid binding protein and the level of hepatic oxidative stress in fatty liver disease. Elevated NADPH oxidase protein levels were detected in the liver of animals with increased severity in inflammation and fibrosis. Cocoa supplementation was associated with partial attenuation of these pathological changes, although the severity of liver disease induced by the methionine choline deficient diet prevented complete reversal of any disease associated changes. Red blood cell glutathione was increased by cocoa supplementation, whereas liver glutathione was reduced by cocoa compared to methionine choline deficient diet fed animals. Conclusion These findings suggest a potential role for liver fatty acid binding protein and NADPH oxidase in the development of non alcoholic steatohepatitis. Furthermore, cocoa supplementation may have be of therapeutic benefit in less sever forms of NASH. PMID:22081873

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart

PubMed Central

Ashmore, Tom; Fernandez, Bernadette O; Branco-Price, Cristina; West, James A; Cowburn, Andrew S; Heather, Lisa C; Griffin, Julian L; Johnson, Randall S; Feelisch, Martin; Murray, Andrew J

2014-01-01

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l−1 NaCl (as control) or 0.7 mmol l−1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics. PMID:25172947

Effect of Ginkgo biloba extract on apoptosis of brain tissues in rats with acute cerebral infarction and related gene expression.

PubMed

Wu, C; Zhao, X; Zhang, X; Liu, S; Zhao, H; Chen, Y

2015-06-11

We investigated the effect of Ginkgo biloba extract on apoptosis of brain tissues in rats with acute cerebral infarction and apoptosis-related gene expression. Rat models of acute cerebral infarction were constructed using the suture method, and randomly divided into the control group, model, and treatment groups. In the treatment group, 4 mg/kg G. biloba extract was intravenously injected into the rat tail vein. Phosphate-buffered saline solution was injected in the model group. Seventy-two hours after treatment, rats were euthanized, and brain tissues were removed to analyze the changes in caspase-3, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) mRNA and protein levels, and variation in brain tissue cells' apoptosis indices was measured. Compared with the control group, the model and treatment groups showed significantly upregulated caspase-3, Bcl-2, and Bax mRNA and protein levels in brain tissues, but remarkably downregulated Bcl-2 mRNA and protein levels (P < 0.05). After treatment, in treatment group brain tissues, caspase-3 and Bax mRNA and protein levels were significantly lower than those in the model group, while Bcl-2 mRNA and protein levels were higher than that in the model group (P < 0.05). The model and treatment groups showed increased cell apoptosis indices of brain tissues compared to the control group; after treatment, the apoptosis index in the treatment group was significantly downregulated compared with that in the model group (P < 0.05). In conclusion, G. biloba extract significantly reduced apoptosis in rat brain tissue cells with acute cerebral infarction and thus protected brain tissues.

Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes.

PubMed

Gu, Feng; Chauhan, Ved; Chauhan, Abha

2013-12-01

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM. Copyright © 2013 Elsevier Inc. All rights reserved.

[Effect of starvation on blood protein levels in the population of Dobrinja (1992-1995)].

PubMed

Hasković, E

2000-01-01

In nutritional protein deficiency, numerous studies verified utilization of amino acids generated from tissue degradation in intensive protein synthesis. Unlike liver, muscle protein synthesis is extremely dependent on external supplies of essential amino acids. Prolonged nutritional protein deficiency results in decrease of body weight as well as total protein concentration, in particular in early days of starvation. In prolonged starvation during the war, significant decrease of body weight was registered in 70 subjects while their total protein concentration remained within the expected range and did not significantly differ the values recorded in the control group. Concentration of serum albumines in the control group was lower than the concentration recorded in the tested group, while the serum globulins concentration was higher in the control group. Although the difference in body weight between the tested and the control group was statistically significant, no significant difference in the concentration of total proteins, albumines and globulines was recorded.

Measurement of amino acid levels in the vitreous humor of rats after chronic intraocular pressure elevation or optic nerve transection.

PubMed

Levkovitch-Verbin, Hana; Martin, Keith R G; Quigley, Harry A; Baumrind, Lisa A; Pease, Mary Ellen; Valenta, Danielle

2002-10-01

To investigate whether the levels of free amino acids and protein in the vitreous of rat eyes are altered with chronic intraocular pressure (IOP) elevation or after optic nerve transection. The concentrations of 20 amino acids in the vitreous humor were measured by high-performance liquid chromatography in both eyes of 41 rats with unilateral IOP elevation induced by translimbal photocoagulation. Eyes were studied 1 day and 1, 2, 4, and 9 weeks after initial IOP elevation. The same amino acids were measured in 41 rats 1 day and 2, 4, and 9 weeks after unilateral transection of the orbital optic nerve. The intravitreal protein level was assayed in additional 22 rats with IOP elevation and 12 rats after nerve transection. Two masked observers evaluated the amount of optic nerve damage with a semiquantitative, light-microscopic technique. In rats with experimental glaucoma, amino acid concentrations were unchanged 1 day after treatment. At 1 week, 4 of 20 amino acids (aspartate, proline, alanine, and lysine) were higher than in control eyes ( < or = 0.01), but this difference was nonsignificant after Bonferroni correction for multiple simultaneous amino acid comparisons (none achieved < 0.0025). No amino acid was significantly different from control in the nerve transection groups (all > 0.05). Vitreous protein level was significantly higher in glaucomatous eyes than their paired controls at 1 day ( < 0.0001) and 1 week ( < 0.002). One day and 1 week after optic nerve transection, vitreal proteins were significantly elevated compared with control eyes from untreated animals ( < 0.0020 and < 0.0022, respectively), though not compared with their fellow eyes ( = 0.25 and 0.10). Chronic experimental glaucoma and transection of the optic nerve increase the amount of protein in the rat vitreous above control levels. In the vitreous of rats with experimental glaucoma, a number of free amino acids were transiently elevated to a modest degree, but no significant difference in vitreous glutamate concentration was detected ( > 0.01).

Metabotropic glutamate receptor 5 upregulation in children with autism is associated with underexpression of both Fragile X mental retardation protein and GABAA receptor beta 3 in adults with autism

PubMed Central

Fatemi, S. Hossein; Folsom, Timothy D.; Kneeland, Rachel E.; Liesch, Stephanie B.

2011-01-01

Recent work has demonstrated the impact of dysfunction of the GABAergic signaling system in brain and the resultant behavioral pathologies in subjects with autism. In animal models, altered expression of Fragile X mental retardation protein (FMRP) has been linked to downregulation of GABA receptors. Interestingly, the autistic phenotype is also observed in individuals with Fragile X syndrome. This study was undertaken to test previous theories relating abnormalities in levels of FMRP to GABAA receptor underexpression. We observed a significant reduction in levels of FMRP in the vermis of adults with autism. Additionally, we found that levels of metabotropic glutamate receptor 5 (mGluR5) protein were significantly increased in vermis of children with autism vs. age and postmortem interval (PMI) matched controls. There was also a significant decrease in level of GABAA receptor beta 3 (GABRβ3) protein in vermis of adult subjects with autism. Finally, we found significant increases in glial fibrillary acidic protein (GFAP) in vermis of both children and adults with autism when compared with controls. Taken together, our results provide further evidence that altered FMRP expression and increased mGluR5 protein production potentially leads to altered expression of GABAA receptors. PMID:21901840

E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

PubMed

Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C; Zhang, Zhen; Young, Randee E; Verheyden, Jamie M; Sun, Xin

2016-07-05

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.

The Influence of Corn Silk Polysaccharide on Signal Pathway of TGF-β1 in Type 2 Diabetic Mellitus Rat.

PubMed

Wen, Xianchun; Yue, Liling

2015-01-01

In prevention stage, comparing with normal control group, triglycerides, blood sugar (BG), 24-hour urinary protein and cholesterol (CHO) were higher in T2DM group, but weight and urea nitrogen (BUN) was less in it. 24-hour urinary protein and cholesterol (CHO) were higher in T2DM group than the intervention group. 24-hour urinary protein and BG in the intervention group were higher than normal control group, but BUN is less than normal control group; In the intervention group the weight of kidney and weight of rat were also higher than T2DM group, but CHO and 24-hour urinary protein were less than T2DM group. The expression of TGF-β1 in T2DM group were more than the other groups. In treatment stage, serum creatinine (Cr), weight, BG and CHO, TGand 24-hour urinary protein quantitative were significantly higher in the DN rats than those in the normal control rats (P>0.05). The expression level of TGF-β1 and triglyceride level in the corn silk dihydroxycorn silk3 treated group were obviously lower than those in the DN rats.

Intestinal alkaline phosphatase in the colonic mucosa of children with inflammatory bowel disease

PubMed Central

Molnár, Kriszta; Vannay, Ádám; Szebeni, Beáta; Bánki, Nóra Fanni; Sziksz, Erna; Cseh, Áron; Győrffy, Hajnalka; Lakatos, Péter László; Papp, Mária; Arató, András; Veres, Gábor

2012-01-01

AIM: To investigate intestinal alkaline phosphatase (iAP) in the intestinal mucosa of children with inflammatory bowel disease (IBD). METHODS: Colonic biopsy samples were taken from 15 newly diagnosed IBD patients and from 10 healthy controls. In IBD patients, specimens were obtained both from inflamed and non-inflamed areas. The iAP mRNA and protein expression was determined by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Tissue localization of iAP and Toll-like receptor (TLR) 4 was investigated by immunofluorescent staining. RESULTS: The iAP protein level in the inflamed mucosa of children with Crohn’s disease (CD) and ulcerative colitis (UC) was significantly decreased when compared with controls (both P < 0.05). Similarly, we found a significantly decreased level of iAP protein in the inflamed mucosa in CD compared with non-inflamed mucosa in CD (P < 0.05). In addition, the iAP protein level in inflamed colonic mucosa in patients with UC was decreased compared with non-inflamed mucosa in patients with CD (P < 0.05). iAP protein levels in the non-inflamed mucosa of patients with CD were similar to controls. iAP mRNA expression in inflamed colonic mucosa of children with CD and UC was not significantly different from that in non-inflamed colonic mucosa with CD. Expression of iAP mRNA in patients with non-inflamed mucosa and in controls were similar. Co-localization of iAP with TLR4 showed intense staining with a dotted-like pattern. iAP was present in the inflamed and non-inflamed mucosa of patients with CD, UC, and in control biopsy specimens, irrespective of whether it was present in the terminal ileum or in the colon. However, the fluorescent signal of TLR4 was more pronounced in the colon compared with the terminal ileum in all groups studied. CONCLUSION: Lower than normal iAP protein levels in inflamed mucosa of IBD patients may indicate a role for iAP in inflammatory lesions in IBD. Based on our results, administration of exogenous iAP enzyme to patients with the active form of IBD may be a therapeutic option. PMID:22783049

The BAX/BAK-like protein BOK is a prognostic marker in colorectal cancer.

PubMed

Carberry, Steven; D'Orsi, Beatrice; Monsefi, Naser; Salvucci, Manuela; Bacon, Orna; Fay, Joanna; Rehm, Markus; McNamara, Deborah; Kay, Elaine W; Prehn, Jochen H M

2018-01-26

The intrinsic or mitochondrial apoptosis pathway is controlled by the interaction of antiapoptotic and pro-apoptotic members of the BCL-2 protein family. Activation of this death pathway plays a crucial role in cancer progression and chemotherapy responses. The BCL-2-related ovarian killer (BOK) possesses three BCL-2 homology domains and has been proposed to act in a similar pro-apoptotic pathway as the pro-apoptotic proteins BAX and BAK. In this study, we showed that stage II and III colorectal cancer patients possessed decreased levels of BOK protein in their tumours compared to matched normal tissue. BOK protein levels in tumours were also prognostic of clinical outcome but increased BOK protein levels surprisingly associated with earlier disease recurrence and reduced overall survival. We found no significant association of BOK protein tumour levels with ER stress markers GRP78 or GRP94 or with cleaved caspase-3. In contrast, BOK protein levels correlated with Calreticulin. These data indicate BOK as a prognostic marker in colorectal cancer and suggest that different activities of BOK may contribute to cancer progression and prognosis.

Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

PubMed

Kashir, Junaid; Jones, Celine; Mounce, Ginny; Ramadan, Walaa M; Lemmon, Bernadette; Heindryckx, Bjorn; de Sutter, Petra; Parrington, John; Turner, Karen; Child, Tim; McVeigh, Enda; Coward, Kevin

2013-01-01

To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Laboratory study. University laboratory. Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Dysregulated post-synaptic density and endocytic zone in the amygdala of human heroin and cocaine abusers

PubMed Central

Ökvist, Anna; Fagergren, Pernilla; Whittard, John; Garcia-Osta, Ana; Drakenberg, Katarina; Horvath, Monika Cs.; Schmidt, Carl J.; Keller, Eva; Bannon, Michael J.; Hurd, Yasmin L.

2010-01-01

Background Glutamatergic transmission in the amygdala is hypothesized as an important mediator of stimulus-reward associations contributing to drug-seeking behavior and relapse. Insight is, however, lacking regarding the amygdala glutamatergic system in human drug abusers. Methods We examined glutamate receptors and scaffolding proteins associated with the post-synaptic density (PSD) of excitatory synapses in the human post-mortem amygdala. mRNA or protein levels were studied in a multi-drug (7 heroin, 8 cocaine, 7 heroin/cocaine and 7 control) or predominant heroin (29 heroin and 15 control) population of subjects. Results The amygdala of drug abusers was characterized by a striking positive correlation (r > 0.8) between AMPA GluA1 and post-synaptic protein-95 (PSD-95) mRNA levels, which was not evident in controls. Structural equation multi-group analysis of protein correlations also identified the relationship between GluA1 and PSD-95 protein levels as the distinguishing feature of abusers. In line with the GluA1—PSD-95 implications of enhanced synaptic plasticity, Homer 1b/c protein expression was significantly increased in both heroin and cocaine users as was its binding partner dynamin-3, localized to the endocytic zone. Furthermore, there was a positive relationship between Homer 1b/c and dynamin-3 in drug abusers that reflected an increase in the direct physical coupling between the proteins. A noted age-related decline of Homer 1b/c—dynamin-3 interactions, as well as GluA1 levels, was blunted in abusers. Conclusions Impairment of key components of the amygdala PSD and coupling to the endocytic zone, critical for the regulation of glutamate receptor cycling, may underlie heightened synaptic plasticity in human drug abusers. PMID:21126734

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

PubMed

Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

2002-09-01

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Expression of serum insulin-like growth factors, insulin-like growth factor-binding proteins, and the growth hormone-binding protein in heterozygote relatives of Ecuadorian growth hormone receptor deficient patients.

PubMed

Fielder, P J; Guevara-Aguirre, J; Rosenbloom, A L; Carlsson, L; Hintz, R L; Rosenfeld, R G

1992-04-01

Recently, an isolated population of apparent GH-receptor deficient (GHRD) patients has been identified in the Loja province of southern Ecuador. These individuals presented many of the physical and biochemical phenotypes characteristic of Laron-Syndrome and are believed to have a defect in the GH-receptor gene. In this study, we have compared the biochemical phenotypes between the affected individuals and their parents, considered to be obligate heterozygotes for the disorder. Serum GH, insulin-like growth factor I and II (IGF-I and IGF-II) levels were measured by RIA Insulin-like growth factor binding proteins. (IGFBPs) were measured by Western ligand blotting (WLB) of serum samples, following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and relative quantitation of serum IGFBPs was performed with a scanning laser densitometer. Serum GH-binding protein (GHBP) levels were measured with a ligand-mediated immunofunctional assay using a monoclonal antibody raised against the GHBP. These values were then compared to values obtained from normal, sex-matched adult Ecuadorian controls, to determine if the above parameters were abnormal in the heterozygotes. The serum IGF-I levels of the GHRD patients were less than 13% of control values for adults and 2% for children. However, the IGF-I levels of both the mothers and fathers were not significantly different from that of the control population. The serum IGF-II levels of the GHRD patients were approximately 20% of control values for adults and 12% for the children. The IGF-II levels of the mothers were reduced, but were not significantly different from that of the control population. However, IGF-II levels of the fathers were significantly lower than those of controls (64% of control male levels). WLB analysis of serum IGFBP levels of the affected subjects demonstrated increased IGFBP-2 and decreased IGFBP-3, suggesting an inverse relationship between these IGFBPs. The GHRD patients who had the lowest serum IGFBP-3 levels (as measured by WLB) demonstrated a serum protease activity that could proteolyze 125I-IGFBP-3. GHRD patients who had higher serum IGFBP-3 levels lacked this serum protease activity. There were no differences in the serum IGFBP profiles of the mothers or the fathers for either IGFBP-2 or IGFBP-3, and serum from both groups lacked the ability to significantly proteolyze 125I-IGFBP-3. While GHRD patients had very low levels of serum GHBP, some patients did have measurable GHBP levels.(ABSTRACT TRUNCATED AT 400 WORDS)

The RNA binding protein CsrA controls c-di-GMP metabolism by directly regulating the expression of GGDEF proteins

PubMed Central

Jonas, Kristina; Edwards, Adrianne N.; Simm, Roger; Romeo, Tony; Römling, Ute; Melefors, Öjar

2009-01-01

Summary The carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobility shift assays confirmed the direct and specific binding of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH resulted in a more than 20-fold increase in the cellular concentration of the second messenger c-di-GMP, implying that both proteins possess diguanylate cyclase activity. Phenotypic characterization revealed that both proteins are involved in the regulation of motility in a c-di-GMP dependent manner. CsrA was also found to regulate the expression of five additional GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels of c-di-GMP. All together, these data demonstrate a global role for CsrA in the regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins at the post-transcriptional level. PMID:18713317

A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

PubMed Central

Tian, Tian; Salis, Howard M.

2015-01-01

Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

An extensive program of periodic alternative splicing linked to cell cycle progression

PubMed Central

Dominguez, Daniel; Tsai, Yi-Hsuan; Weatheritt, Robert; Wang, Yang; Blencowe, Benjamin J; Wang, Zefeng

2016-01-01

Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control. DOI: http://dx.doi.org/10.7554/eLife.10288.001 PMID:27015110

Small heat shock proteins protect against {alpha}-synuclein-induced toxicity and aggregation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Outeiro, Tiago Fleming; Klucken, Jochen; Strathearn, Katherine E.

Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). {alpha}-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and {alpha}B-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messengermore » RNA levels are {approx}2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by {approx}80% in a culture model while {alpha}B-crystallin reduces toxicity by {approx}20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.« less

Thymosin Beta-4 Induces Mouse Hair Growth

PubMed Central

Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts. PMID:26083021

Thymosin Beta-4 Induces Mouse Hair Growth.

PubMed

Gao, Xiaoyu; Liang, Hao; Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues.

PubMed

Chou, Chao-Kai; Huang, Po-Jung; Tsou, Pei-Hsiang; Wei, Yongkun; Lee, Heng-Huan; Wang, Ying-Nai; Liu, Yen-Liang; Shi, Colin; Yeh, Hsin-Chih; Kameoka, Jun; Hung, Mien-Chie

2018-05-29

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R 2 ], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level. Copyright © 2018 Elsevier B.V. All rights reserved.

Decreased Serum Levels of Ghrelin and Brain-Derived Neurotrophic Factor in Premenopausal Women With Metabolic Syndrome.

PubMed

Jabbari, Masoumeh; Kheirouri, Sorayya; Alizadeh, Mohammad

2018-03-21

We aimed to investigate the association between serum levels of ghrelin and brain-derived neurotrophic factor (BDNF) with MetS and its components in premenopausal women. 43 patients with MetS and 43 healthy controls participated in this study. Participants' body mass index (BMI), waist circumference (WC), systolic and diastolic blood pressure (SBP and DBP) were measured. Serum levels of total cholesterol (TC), triglyceride (TG), low and high density lipoprotein cholesterol (LDL-C and HDL-C), fasting blood sugar (FBS), insulin, BDNF and ghrelin determined. Homeostasis model assessment insulin resistance index (HOMA-IR) was also calculated. Participants in MetS group had higher waist-to-hip ratios, elevated SBP and DBP, and higher serum levels of TG, FBS and insulin when compared with the control group. Serum ghrelin and BDNF levels were significantly lower in participants with MetS than in the healthier control subjects. There was a strong, positive correlation between serum ghrelin and BDNF levels. Both proteins negatively correlated with TG, FBS, HOMA-IR and positively with HDL-C. Furthermore, serum BDNF levels negatively associated with insulin levels. The findings indicate that variations occur in the circulating level of ghrelin and BDNF proteins in MetS patients. A strong correlation between serum ghrelin and BDNF suggests that production, release or practice of these 2 proteins might be related mechanically.

Australian Aboriginal Children with Otitis Media Have Reduced Antibody Titers to Specific Nontypeable Haemophilus influenzae Vaccine Antigens

PubMed Central

Kirkham, Lea-Ann S.; Corscadden, Karli J.; Wiertsema, Selma P.; Fuery, Angela; Jones, B. Jan; Coates, Harvey L.; Vijayasekaran, Shyan; Zhang, Guicheng; Keil, Anthony; Richmond, Peter C.

2017-01-01

ABSTRACT Indigenous populations experience high rates of otitis media (OM), with increased chronicity and severity, compared to those experienced by their nonindigenous counterparts. Data on immune responses to otopathogenic bacteria in these high-risk populations are lacking. Nontypeable Haemophilus influenzae (NTHi) is the predominant otopathogen in Australia. No vaccines are currently licensed to target NTHi; however, protein D (PD) from NTHi is included as a carrier protein in the 10-valent pneumococcal polysaccharide conjugate vaccine (PHiD10-CV), and other promising protein vaccine candidates exist, including outer membrane protein 4 (P4) and protein 6 (P6). We measured the levels of serum and salivary IgA and IgG against PD, P4, and P6 in Aboriginal and non-Aboriginal children with chronic OM who were undergoing surgery and compared the levels with those in healthy non-Aboriginal children (controls). We found that Aboriginal cases had lower serum IgG titers to all NTHi proteins assessed, particularly PD. In contrast, serum IgA and salivary IgA and IgG titers to each of these 3 proteins were equivalent to or higher than those in both non-Aboriginal cases and healthy controls. While serum antibody levels increased with age in healthy controls, no changes in titers were observed with age in non-Aboriginal cases, and a trend toward decreasing titers with age was observed in Aboriginal cases. This suggests that decreased serum IgG responses to NTHi outer membrane proteins may contribute to the development of chronic and severe OM in Australian Aboriginal children and other indigenous populations. These data are important for understanding the potential benefits of PHiD10-CV implementation and the development of NTHi protein-based vaccines for indigenous populations. PMID:28151410

Urinary L-FABP as a marker of vesicoureteral reflux in children: could it also have a protective effect on the kidney?

PubMed

Benzer, Meryem; Tekin Neijmann, Sebnem; Gültekin, Nazlı Dilay; Uluturk Tekin, Aslı

2017-01-01

Liver-type fatty acid-binding protein is a small cytoplasmic protein which is expressed in the human renal proximal tubular epithelium and synthesized in response to renal tubular injury. The aim of the present study was to investigate the importance of urinary liver-type fatty acid-binding protein levels in children who diagnosed with vesicoureteral reflux. Fifty-six patients with vesicoureteral reflux and 51 healthy controls were enrolled to the study. The cases were divided into three groups as follows: group A-the controls, group B-the patients who had renal parenchymal scarring and group C-the patients who had no scarring. Urinary liver-type fatty acid-binding protein was measured by enzyme-linked immunosorbent assay method. Creatinine was measured by modified Jaffe method, protein was measured by turbidimetric method, and urine density was determined by using the "falling drop" procedure. Urinary liver-type fatty acid-binding protein and urinary liver-type fatty acid-binding protein/creatinine levels were significantly higher in the whole patient group than in the controls (p = 0.016, 0.006). Significant differences were also determined by comparing the three groups (p = 0.015, 0.014), and those levels were found as significantly higher in group C. Urinary liver-type fatty acid-binding protein was considered to be helpful for the diagnosis of vesicoureteral reflux, and also it might contribute to understand the mechanisms causing scar tissue formation especially for the patients who had vesicoureteral reflux. Further clinical and experimental investigations are required to elucidate in detail the physiology of liver-type fatty acid-binding protein.

Comparison of Integrated Responses to Nonlethal and Lethal Hypothermal Stress in Milkfish (Chanos chanos): A Proteomics Study.

PubMed

Chang, Chia-Hao; Tang, Cheng-Hao; Kang, Chao-Kai; Lo, Wan-Yu; Lee, Tsung-Han

Milkfish is an important aquaculture species in Taiwan, and its high mortality during cold snaps in winter usually causes huge economic losses. To understand the effect of hypothermal stress and the corresponding compensatory stress response in milkfish, this study aimed to compare liver and gill protein levels between milkfish exposed to nonlethal (18°C), lethal (16°C), and control (28°C) temperatures. Using a proteomics approach based on two-dimensional electrophoresis and nano-LC-MS/MS analysis, this study identified thirty unique protein spots from milkfish livers and gills for which protein abundance was significantly different between nonlethal, lethal, and control temperature groups. Proteins identified in the liver were classified into three different categories according to their cellular function: (1) anti-oxidative stress, (2) apoptotic pathway, and (3) cytoskeleton. Similarly, proteins identified in the gill were sorted in five different functional categories: (1) cytoskeleton, (2) immune response, (3) protein quality control, (4) energy production, and (5) intracellular homeostasis. Based on functional information derived from the identified proteins, we assumed that different levels of hypothermal stress had a different effect and induced a different cellular response. Upon nonlethal hypothermal stress, the identified proteins were involved in anti-oxidative stress and anti-inflammation pathways, suggesting that milkfish had high levels of oxidative stress in the liver and exhibited inflammation response in the gill. Upon lethal hypothermal stress, however, identified proteins were associated with apoptosis in the liver and regulation of intracellular homeostasis in the gill. The present study provided evidence to illustrate different multi-physiological responses to nonlethal and lethal hypothermal stress in milkfish livers and gills.

Comparison of Integrated Responses to Nonlethal and Lethal Hypothermal Stress in Milkfish (Chanos chanos): A Proteomics Study

PubMed Central

Chang, Chia-Hao; Tang, Cheng-Hao; Kang, Chao-Kai; Lo, Wan-Yu; Lee, Tsung-Han

2016-01-01

Milkfish is an important aquaculture species in Taiwan, and its high mortality during cold snaps in winter usually causes huge economic losses. To understand the effect of hypothermal stress and the corresponding compensatory stress response in milkfish, this study aimed to compare liver and gill protein levels between milkfish exposed to nonlethal (18°C), lethal (16°C), and control (28°C) temperatures. Using a proteomics approach based on two-dimensional electrophoresis and nano-LC-MS/MS analysis, this study identified thirty unique protein spots from milkfish livers and gills for which protein abundance was significantly different between nonlethal, lethal, and control temperature groups. Proteins identified in the liver were classified into three different categories according to their cellular function: (1) anti-oxidative stress, (2) apoptotic pathway, and (3) cytoskeleton. Similarly, proteins identified in the gill were sorted in five different functional categories: (1) cytoskeleton, (2) immune response, (3) protein quality control, (4) energy production, and (5) intracellular homeostasis. Based on functional information derived from the identified proteins, we assumed that different levels of hypothermal stress had a different effect and induced a different cellular response. Upon nonlethal hypothermal stress, the identified proteins were involved in anti-oxidative stress and anti-inflammation pathways, suggesting that milkfish had high levels of oxidative stress in the liver and exhibited inflammation response in the gill. Upon lethal hypothermal stress, however, identified proteins were associated with apoptosis in the liver and regulation of intracellular homeostasis in the gill. The present study provided evidence to illustrate different multi-physiological responses to nonlethal and lethal hypothermal stress in milkfish livers and gills. PMID:27657931

Iron regulatory proteins and their role in controlling iron metabolism.

PubMed

Kühn, Lukas C

2015-02-01

Cellular iron homeostasis is regulated by post-transcriptional feedback mechanisms, which control the expression of proteins involved in iron uptake, release and storage. Two cytoplasmic proteins with mRNA-binding properties, iron regulatory proteins 1 and 2 (IRP1 and IRP2) play a central role in this regulation. Foremost, IRPs regulate ferritin H and ferritin L translation and thus iron storage, as well as transferrin receptor 1 (TfR1) mRNA stability, thereby adjusting receptor expression and iron uptake via receptor-mediated endocytosis of iron-loaded transferrin. In addition splice variants of iron transporters for import and export at the plasma-membrane, divalent metal transporter 1 (DMT1) and ferroportin are regulated by IRPs. These mechanisms have probably evolved to maintain the cytoplasmic labile iron pool (LIP) at an appropriate level. In certain tissues, the regulation exerted by IRPs influences iron homeostasis and utilization of the entire organism. In intestine, the control of ferritin expression limits intestinal iron absorption and, thus, whole body iron levels. In bone marrow, erythroid heme biosynthesis is coordinated with iron availability through IRP-mediated translational control of erythroid 5-aminolevulinate synthase mRNA. Moreover, the translational control of HIF2α mRNA in kidney by IRP1 coordinates erythropoietin synthesis with iron and oxygen supply. Besides IRPs, body iron absorption is negatively regulated by hepcidin. This peptide hormone, synthesized and secreted by the liver in response to high serum iron, downregulates ferroportin at the protein level and thereby limits iron absorption from the diet. Hepcidin will not be discussed in further detail here.

Comparison of salivary levels of mucin and amylase and their relation with clinical parameters obtained from patients with aggressive and chronic periodontal disease

PubMed Central

ACQUIER, Andrea Beatriz; PITA, Alejandra Karina De Couto; BUSCH, Lucila; SÁNCHEZ, Gabriel Antonio

2015-01-01

Objective Salivary mucin and amylase levels are increased in patients with chronic periodontitis (CP). Due to the fact that aggressive periodontitis (AgP) not only differs from chronic periodontitis in terms of its clinical manifestation, the aim of this study was to compare salivary mucin and amylase levels and their relation to the clinical parameters of patients with aggressive periodontitis with that of patients with chronic periodontitis. Material and Methods Eighty subjects were divided into two groups: 20 patients with AgP and their 20 matched controls and 20 patients with CP and their 20 matched controls, based on clinical attachment loss (CAL), probing pocket depth (PPD) and bleeding on probing (BOP). Whole unstimulated saliva was obtained and mucin, amylase and protein were determined by colorimetric methods. Pearson’s correlation analysis was used to determine the relationship between salivary mucin, amylase and protein levels and the clinical parameters. Results Salivary mucin, amylase and protein levels were increased in patients with AgP and CP but there were no differences between them or between control groups. Pearson’s correlation analysis, determined in the entire subjects studied, showed a positive and significant correlation of mucin, amylase and proteins with CAL and PPD and a negative correlation with the flow rate. When Pearson’s correlation analysis was carried out in each group separately, Fisher’s z transformation showed no significant difference between both groups. Conclusion Comparison of the salivary levels of mucin, amylase and protein and their relationship with clinical parameters of AgP patients with that of CP patients revealed no differences between both groups. PMID:26221923

Light-mediated control of DNA transcription in yeast

PubMed Central

Hughes, Robert M.; Bolger, Steven; Tapadia, Hersh; Tucker, Chandra L.

2012-01-01

A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light / cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems. PMID:22922268

A biomolecular proportional integral controller based on feedback regulations of protein level and activity

PubMed Central

2018-01-01

Homeostasis is the capacity of living organisms to keep internal conditions regulated at a constant level, despite environmental fluctuations. Integral feedback control is known to play a key role in this behaviour. Here, I show that a feedback system involving transcriptional and post-translational regulations of the same executor protein acts as a proportional integral (PI) controller, leading to enhanced transient performances in comparison with a classical integral loop. Such a biomolecular controller—which I call a level and activity-PI controller (LA-PI)—is involved in the regulation of ammonium uptake by Escherichia coli through the transporter AmtB. The PII molecules, which reflect the nitrogen status of the cell, inhibit both the production of AmtB and its activity (via the NtrB-NtrC system and the formation of a complex with GlnK, respectively). Other examples of LA-PI controller include copper and zinc transporters, and the redox regulation in photosynthesis. This scheme has thus emerged through evolution in many biological systems, surely because of the benefits it offers in terms of performances (rapid and perfect adaptation) and economy (protein production according to needs). PMID:29515895

Baseline Immunoglobulin E Levels as a Marker of Doxorubicin and Trastuzumab-Associated Cardiac Dysfunction

PubMed Central

Beer, Lynn A.; Kossenkov, Andrew V.; Liu, Qin; Luning Prak, Eline; Domchek, Susan; Speicher, David W.; Ky, Bonnie

2016-01-01

Rationale There is a critical need to develop robust, mechanistic strategies to identify patients at increased risk of cancer therapeutics-related cardiac dysfunction (CTRCD). Objective We aimed to discover new biomarkers associated with doxorubicin and trastuzumab-induced CTRCD using high-throughput proteomic profiling. Methods and Results Plasma, echocardiograms, and clinical outcomes were collected at standardized intervals in breast cancer patients undergoing doxorubicin and trastuzumab cancer therapy. Thirty-one longitudinal plasma samples from three cases with CTRCD and four age- and cancer-matched controls without CTRCD were processed and analyzed using label-free liquid chromatography- mass spectrometry (LC-MS). From these analyses, 862 proteins were identified from case/control pairs 1 and 2, and 1,360 proteins from case/control pair 3. Proteins with a greater than 1.5-fold change in cases compared to controls with a p<0.05 either at the time of CTRCD diagnosis or across all timepoints were considered candidate diagnostic or predictive biomarkers, respectively. The protein that demonstrated the largest differences between cases and controls was immunoglobulin E (IgE), with higher levels detected at baseline and across all timepoints in controls without CTRCD as compared to matched CTRCD cases (p<0.05). Similarly, in a validation study of 35 participants treated with doxorubicin and trastuzumab, high baseline IgE levels were associated with a significantly lower risk of CTRCD (p=0.018). Conclusions In patients receiving doxorubicin and trastuzumab, high baseline IgE levels are associated with a lower risk of CTRCD. These novel findings suggest a new paradigm in cardio-oncology, implicating the immune system as a potential mediator of doxorubicin and trastuzumab-induced cardiac dysfunction. PMID:27582370

Enhanced LPS-induced activation of IL-27 signalling in sarcoidosis.

PubMed

Ringkowski, Sabine; Loke, Joshua; Huang, Shuying; Ahmadzai, Hasib; Herth, Felix J F; Thomas, Paul S; Herbert, Cristan

2016-08-01

Granulomas in sarcoidosis have recently been described as containing Interleukin (IL)-27, one of the members of the IL-12 family of cytokines, which also includes IL-35. Levels of these cytokines and the IL-27 receptor subunits were hypothesised to differ between patients with sarcoidosis compared to healthy controls in peripheral blood. Using a cross-sectional study design, plasma and peripheral blood mononuclear cells (PBMC) were collected from patients and control subjects. Protein and mRNA (in PBMC) levels for IL-27 and IL-35 (IL27, EBI3, IL12A subunits) as well as IL-27 receptor (IL6ST and IL27RA subunits) were assessed spontaneously and following direct (LPS) and indirect (anti-CD3/28 activation beads) macrophage stimulation using RT- PCR, ELISA and flow cytometry. Following stimulation with LPS, PBMC of patients with sarcoidosis displayed significantly enhanced expression of IL27 and EBI3 mRNA (p = 0.020 and p = 0.037 respectively) compared to PBMCs from healthy controls. There was also significantly enhanced production of IL-27 by PBMC from patients with sarcoidosis compared to healthy controls in response to LPS stimulation (p = 0.027). IL6ST mRNA and IL6ST protein were significantly lower in patients with sarcoidosis (mRNA p = 0.0002; MFI p = 0.0015) whilst IL27RA protein levels were significantly higher in patients with sarcoidosis compared to healthy controls (MFI p < 0.0001). Plasma IL-35 protein levels did not differ between control and sarcoidosis subjects (p = 0.23). These results suggest there may be exaggerated activation of IL-27 signalling in response to LPS in sarcoidosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein Kinases in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

1999-09-01

studies identical at the amino acid level to calcium/calmodulin-dependent may provide insight into mechanisms of growth control and DNA protein kinase I...human homologues of these kinases(19, 20 ). Amino acid conservation in the coding region between mouse and human Hunk is greater than 90% identical. While...genes (13, 14). Over the past 4 years , several of the mRNA and protein levels (39-46). These findings clearly dem- these breast cancer susceptibility

Effects of microalgae Chlorella species crude extracts on intestinal adaptation in experimental short bowel syndrome.

PubMed

Kerem, Mustafa; Salman, Bulent; Pasaoglu, Hatice; Bedirli, Abdulkadir; Alper, Murat; Katircioglu, Hikmet; Atici, Tahir; Percin, E Ferda; Ofluoglu, Ebru

2008-07-28

To evaluate the effects of chlorella crude extract (CCE) on intestinal adaptation in rats subjected to short bowel syndrome (SBS). Wistar rats weighing 230-260 g were used in the study. After anesthesia a 75% small bowel resection was performed. Rats were randomized and divided into groups. Control group (n = 10): where 5% dextrose was given through a gastrostomy tube, Enteral nutrition (EN) group (n = 10): Isocaloric and isonitrogen EN (Alitraq, Abbott, USA), study group (n = 10): CCE was administrated through a gastrostomy tube. Rats were sacrificed on the fifteenth postoperative day and blood and tissue samples were taken. Histopathologic evaluation, intestinal mucosal protein and DNA levels, intestinal proliferation and apoptosis were determined in intestinal tissues, and total protein, albumin and citrulline levels in blood were studied. In rats receiving CCE, villus lengthening, crypt depth, mucosal DNA and protein levels, intestinal proliferation, and serum citrulline, protein and albumin levels were found to be significantly higher than those in control group. Apoptosis in CCE treated rats was significantly reduced when compared to EN group rats. CCE has beneficial effects on intestinal adaptation in experimental SBS.

Growth control of the eukaryote cell: a systems biology study in yeast.

PubMed

Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David Cj; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom Pj; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

2007-01-01

Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell.

Growth control of the eukaryote cell: a systems biology study in yeast

PubMed Central

Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David CJ; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom PJ; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

2007-01-01

Background Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Results Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. Conclusion This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell. PMID:17439666

Proteome Analyses of Staphylococcus aureus Biofilm at Elevated Levels of NaCl

PubMed Central

Islam, Nazrul; Ross, Julia M; Marten, Mark R

2016-01-01

Our studies demonstrate that sodium chloride (NaCl) induces changes in biofilm, mediated by increased production of polysaccharides intercellular adhesion (PIA). We identified 12 proteins that showed higher abundance in increased level of NaCl. This includes one important protein (IsaA) known to be associated with biofilm stability. In addition, we also found higher abundance of a cold shock protein, CspA, at higher NaCl. We have also identified several other proteins that are differentially expressed to the elevated levels of NaCl and mapped them in the regulatory pathways of PIA. The majority of proteins are involved with various aspects bacterial metabolic function. Our results demonstrated that NaCl influences gene regulatory networks controlling exopolysaccharide expression. PMID:26973848

Oxidative stress markers in aqueous humor of patients with senile cataracts.

PubMed

Sawada, Hideko; Fukuchi, Takeo; Abe, Haruki

2009-01-01

To investigate the levels of oxidative stress markers in human eyes with senile cataracts. We conducted a retrospective, case-controlled study of 57 patients with senile cataracts. To assess oxidative stress markers in the eye, we measured the enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) as well as the total protein levels in aqueous humor. In aqueous humor, SOD and CAT activity levels were 0.133 +/- 0.020 and 1.223 +/- 0.081 U/ml, respectively; protein levels were 2.372 +/- 0.166 mg/ml (means +/- SEM). We observed a significant increase in SOD activity and the protein level in progressed nuclear cataracts. No significant age-associated difference in antioxidant enzyme levels was detected. Significant increases in the levels of SOD activity and total protein correlated with the severity of the cataract but not with patient age, suggesting that progressed cataract is associated with molecules leaking from the lens capsule.

Identification of Glutathione S-Transferase Pi as a Protein Involved in Parkinson Disease Progression

PubMed Central

Shi, Min; Bradner, Joshua; Bammler, Theo K.; Eaton, David L.; Zhang, JianPeng; Ye, ZuCheng; Wilson, Angela M.; Montine, Thomas J.; Pan, Catherine; Zhang, Jing

2009-01-01

Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD. PMID:19498008

Genes associated with pro-apoptotic and protective mechanisms are affected differently on exposure of neuronal cell cultures to arsenite. No indication for endoplasmic reticulum stress despite activation of grp78 and gadd153 expression.

PubMed

Mengesdorf, Thorsten; Althausen, Sonja; Paschen, Wulf

2002-08-15

The effect of arsenite exposure on cell viability, protein synthesis, energy metabolism and the expression of genes coding for cytoplasmic (hsp70) and endoplasmic reticulum (ER; gadd153, grp78, grp94) stress proteins was investigated in primary neuronal cell cultures. Furthermore, signs of ER stress were evaluated by investigating xbp1 mRNA processing. Arsenite levels of 30 and 100 microM induced severe cell injury. Protein synthesis was reduced to below 20% of control in cultures exposed to 30 and 100 microM arsenite for 1 h, and it remained markedly suppressed until 24 h of exposure. Arsenite induced a transient inhibition of energy metabolism after 1 h of exposure, but energy state recovered completely after 3 h. Arsenite exposure affected the expression and translation of genes coding for HSP70 and GRP78, GRP94, GADD153 to different extents. While hsp70 mRNA levels rose drastically, approximally 550-fold after 6 h exposure, HSP70 protein levels did not change over the first 6 h. On the other hand, gadd153 mRNA levels rose only approximately 14-fold after 6 h exposure, while GADD153 protein levels were markedly increased after 3 and 6 h exposure. HSP70 protein levels were markedly increased and GADD153 protein levels decreased to almost control levels in cultures left in arsenite solution for 24 h, i.e. when only a small fraction of cells had escaped arsenite toxicity. Arsenite exposure of neurons thus induced an imbalance between pro-apoptotic and survival-activating pathways. Despite the marked increase in gadd153 mRNA levels, we did not observe signs of xbp1 processing in arsenite exposed cultures, indicating that arsenite did not produce ER stress.

Efficacy of DL-alpha-lipoic acid on methanol induced free radical changes, protein oxidative damages and hsp70 expression in folate deficient rat nervous tissue.

PubMed

Rajamani, Rathinam; Muthuvel, Arumugam; Manikandan, Sundaramahalingam; Srikumar, Ramasundaram; Sheeladevi, Rathinasamy

2007-05-01

DL-alpha-Lipoic acid (LPA) was reported to be effective in reducing free radicals generated by oxidative stress. The protective of effect of LPA on methanol (MeOH) induced free radical changes and oxidative damages in discrete regions of rat brain have been reported in this study. Folate deficient rat (FDD) model was used. The five animal groups (saline control, FDD control, FDD+MeOH, FDD+LPA+MeOH, LPA control) were used. The FDD+MeOH and FDD+LPA+MeOH animals were injected intraperitoneally with methanol (3gm/kg). After 24h, the level of free radical scavengers such as, superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione was estimated in six discrete regions of brain, retina and optic nerve. Level of protein thiol, protein carbonyl and lipid peroxidation was also estimated. Expression of heat shock protein 70 mRNA (hsp70) was studied in the cerebellum and hippocampus by reverse transcriptase PCR. All the samples showed elevation in the level of free radical scavenging enzymes and reduced level of glutathione in the FDD+MeOH group in relation to the other groups. hsp70 expression was more in FDD+MeOH group when compared to FDD+LPA+MeOH group. In conclusion, MeOH exposure leads to increased free radical generation and protein oxidative damages in the rat nervous tissue. Treatment with LPA prevents oxidative damage induced by MeOH exposure.

A casein diet added isoflavone-enriched soy protein favorably affects biomarkers of steatohepatitis in obese Zucker rats.

PubMed

Gudbrandsen, Oddrun Anita; Wergedahl, Hege; Berge, Rolf Kristian

2009-05-01

Dietary supplementation of a soy protein enriched with isoflavones (HDI) has been shown to improve fatty liver in obese rats. The main objective of this study was to investigate whether HDI would influence the inflammatory status in livers of obese rats with fatty liver. Male obese Zucker fa/fa rats were fed casein (controls) or casein supplemented with HDI (containing 4.00 g of genistein and 4.50 g of daidzein per kilogram of diet) for 6 wk. The HDI-fed rats had a markedly lower hepatic concentration of triacylglycerol when compared with controls. The decreased aspartate transaminase/alanine transaminase ratio in plasma, together with lower circulating levels of alkaline phosphatase and bile acids after HDI feeding, implied an improved hepatitis. This was supported by decreased plasma and hepatic mRNA levels of tumor necrosis factor-alpha, lower plasma levels of interleukin-1beta and monocyte chemoattractant protein-1, and an increased anti-inflammatory fatty acid index in plasma. HDI also seemed to protect the rats from oxidative damage, because the level of lipid peroxides in triacylglycerol-rich lipoproteins after in vitro copper oxidation was lower for HDI-fed rats when compared with controls. These results show that isoflavone-enriched soy protein favorably affects biomarkers of hepatic inflammation in obese Zucker fa/fa rats with fatty liver. Thus, dietary soy proteins enriched in isoflavones may be a promising agent to improve steatohepatitis in patients.

Changes in human parotid salivary protein and sialic acid levels during pregnancy.

PubMed

D'Alessandro, S; Curbelo, H M; Tumilasci, O R; Tessler, J A; Houssay, A B

1989-01-01

Saliva was collected with a Carlson-Crittenden device, under citric acid stimulation, in 107 pregnant women, 9 puerperal and 7 non-pregnant controls. No significant changes were found in salivary flow rate, pH and amylase levels. The total protein levels were decreased during pregnancy and the puerperium. The sialic acid levels decreased gradually but markedly during pregnancy, returning to normal levels in the puerperium. These changes in parotid saliva may be related to the hormonal changes of pregnancy.

Evaluation of plasma C-reactive protein levels in pregnant women with and without periodontal disease: A comparative study.

PubMed

Sharma, Anupriya; Ramesh, Amitha; Thomas, Biju

2009-09-01

Circulating C-reactive protein (CRP) levels are a marker of systemic inflammation and are associated with periodontal disease, a chronic bacterial infection associated with elevation of proinflammatory cytokines and prostaglandins. CRP has been associated with adverse pregnancy outcomes, including preterm delivery, preeclampsia, and intrauterine growth restriction. Furthermore, periodontal disease has been associated with increased risk of preterm low birth weight, low birth weight, and preterm birth. The present study was conducted to assess plasma CRP levels in pregnant women with and without periodontal disease; to evaluate the effect of periodontal therapy on the incidence of preterm delivery; and to compare the incidence of preterm delivery in pregnant women with and without periodontal disease. A total of 90 pregnant women aged between 18-35 years with gestational age between 12-28 weeks were recruited and divided into three equal groups (control group, study group, treatment group) of 30 each. Blood samples were taken for estimation of C-reactive protein levels from all groups at 12-20 weeks of gestation, determined using ultrasensitive turbidimetric immunoassay (QUANTIA-CRP US). The treatment group comprised plaque control, scaling, and root planning and daily rinsing with 0.2% chlorhexidine mouth before 28 weeks of gestation. The mean value of C-reactive protein levels in subjects with periodontal disease was higher compared to control group i.e., 1.20 +/- 0.247 mg/dl and 1.22 +/- 0.250 mg/dl, respectively, compared to 0.713 +/- 0.139 mg/ dl (P = 0.001). The mean value of CRP levels before treatment was greater than the mean value after treatment i.e., 1.22 +/- 0.25 compared to 0.84 +/- 0.189 (P < 0.001). The incidence of preterm delivery (< 37 weeks) was 31.7% in the periodontal disease group (study group) compared to 8.3% in the control group (P = 0.001). The incidence of preterm delivery in the treatment group was 15.0% compared to 31.7% in the nontreatment group (study group). The findings from the study suggest that periodontal disease in pregnant women is associated with increased C- reactive protein levels in early pregnancy, incidence of preterm delivery is higher in pregnant women with periodontal disease compared to healthy controls, periodontal therapy during pregnancy reduces plasma CRP levels and there is decrease in incidence of preterm delivery after periodontal therapy.

Application of sigma metrics for the assessment of quality control in clinical chemistry laboratory in Ghana: A pilot study.

PubMed

Afrifa, Justice; Gyekye, Seth A; Owiredu, William K B A; Ephraim, Richard K D; Essien-Baidoo, Samuel; Amoah, Samuel; Simpong, David L; Arthur, Aaron R

2015-01-01

Sigma metrics provide a uniquely defined scale with which we can assess the performance of a laboratory. The objective of this study was to assess the internal quality control (QC) in the clinical chemistry laboratory of the University of Cape Cost Hospital (UCC) using the six sigma metrics application. We used commercial control serum [normal (L1) and pathological (L2)] for validation of quality control. Metabolites (glucose, urea, and creatinine), lipids [triglycerides (TG), total cholesterol, high-density lipoprotein cholesterol (HDL-C)], enzymes [alkaline phosphatase (ALP), alanine aminotransferase (AST)], electrolytes (sodium, potassium, chloride) and total protein were assessed. Between-day imprecision (CVs), inaccuracy (Bias) and sigma values were calculated for each control level. Apart from sodium (2.40%, 3.83%), chloride (2.52% and 2.51%) for both L1 and L2 respectively, and glucose (4.82%), cholesterol (4.86%) for L2, CVs for all other parameters (both L1 and L2) were >5%. Four parameters (HDL-C, urea, creatinine and potassium) achieved sigma levels >1 for both controls. Chloride and sodium achieved sigma levels >1 for L1 but <1 for L2. In contrast, cholesterol, total protein and AST achieved sigma levels <1 for L1 but >1 for L2. Glucose and ALP achieved a sigma level >1 for both control levels whereas TG achieved a sigma level >2 for both control levels. Unsatisfactory sigma levels (<3) where achieved for all parameters using both control levels, this shows instability and low consistency of results. There is the need for detailed assessment of the analytical procedures and the strengthening of the laboratory control systems in order to achieve effective six sigma levels for the laboratory.

Application of sigma metrics for the assessment of quality control in clinical chemistry laboratory in Ghana: A pilot study

PubMed Central

Afrifa, Justice; Gyekye, Seth A.; Owiredu, William K. B. A.; Ephraim, Richard K. D.; Essien-Baidoo, Samuel; Amoah, Samuel; Simpong, David L.; Arthur, Aaron R.

2015-01-01

Background: Sigma metrics provide a uniquely defined scale with which we can assess the performance of a laboratory. The objective of this study was to assess the internal quality control (QC) in the clinical chemistry laboratory of the University of Cape Cost Hospital (UCC) using the six sigma metrics application. Materials and Methods: We used commercial control serum [normal (L1) and pathological (L2)] for validation of quality control. Metabolites (glucose, urea, and creatinine), lipids [triglycerides (TG), total cholesterol, high-density lipoprotein cholesterol (HDL-C)], enzymes [alkaline phosphatase (ALP), alanine aminotransferase (AST)], electrolytes (sodium, potassium, chloride) and total protein were assessed. Between-day imprecision (CVs), inaccuracy (Bias) and sigma values were calculated for each control level. Results: Apart from sodium (2.40%, 3.83%), chloride (2.52% and 2.51%) for both L1 and L2 respectively, and glucose (4.82%), cholesterol (4.86%) for L2, CVs for all other parameters (both L1 and L2) were >5%. Four parameters (HDL-C, urea, creatinine and potassium) achieved sigma levels >1 for both controls. Chloride and sodium achieved sigma levels >1 for L1 but <1 for L2. In contrast, cholesterol, total protein and AST achieved sigma levels <1 for L1 but >1 for L2. Glucose and ALP achieved a sigma level >1 for both control levels whereas TG achieved a sigma level >2 for both control levels. Conclusion: Unsatisfactory sigma levels (<3) where achieved for all parameters using both control levels, this shows instability and low consistency of results. There is the need for detailed assessment of the analytical procedures and the strengthening of the laboratory control systems in order to achieve effective six sigma levels for the laboratory. PMID:25657495

Genome Editing-Enabled HTS Assays Expand Drug Target Pathways for Charcot–Marie–Tooth Disease

PubMed Central

2015-01-01

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot–Marie–Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin. PMID:25188731

Salivary analytes in patients with oral squamous cell carcinoma.

PubMed

Fuchs, Petra Nola; Rogić, Dunja; Vidović-Juras, Danica; Susić, Mato; Milenović, Aleksandar; Brailo, Vlaho; Boras, Vanja Vucićević

2011-06-01

Literature data indicates that measurement of certain salivary constituents might serve as a useful diagnostic/prognostic tool in the patients with oral squamous cell carcinoma (OSCC). In 24 patients with OSCC (60 +/- 2.5 yrs) and in 24 controls (24 +/- 3.7 yrs) we have determined levels of salivary magnesium, calcium, copper, chloride, phosphate, potassium, sodium, total proteins and amylase. Sodium, potassium and chloride were determined by indirect potentiometry whereas copper, magnesium and phosphate were determined by atomic absorption spectrophotometry. Total proteins were determined by pyrogalol colorimetric method. Amylase levels were determined by continued colorimetric method. Statistical analysis was performed by use of chi2 test and Spearman's correlation test. The results of this study indicate that the concentrations of sodium and chloride were significantly elevated in patients with OSCC when compared to the controls. However, level of total protein was significantly decreased when compared to the healthy controls. Furthermore, there was a negative correlation between alcohol consumption and total protein concentration in patients with oral carcinoma. We might conclude that in patients with OSCC increased salivary sodium and chloride might reflect their overall dehydration status due to alcohol consumption rather than consequence of OSCC itself.

Generation of mammalian cells stably expressing multiple genes at predetermined levels.

PubMed

Liu, X; Constantinescu, S N; Sun, Y; Bogan, J S; Hirsch, D; Weinberg, R A; Lodish, H F

2000-04-10

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

The diagnostic role of serum inflammatory and soluble proteins on dementia subtypes: correlation with cognitive and functional decline.

PubMed

Oztürk, Candan; Ozge, Aynur; Yalin, Osman Ozgür; Yilmaz, I Arda; Delialioglu, Nuran; Yildiz, Cilem; Tesdelen, Bahar; Kudiaki, Cigdem

2007-01-01

In the past years, the possible involvement of inflammation in the pathogenesis of dementia has been the subject of several investigations. However there are restricted data about the profile of the inflammatory and soluble proteins in well evaluated Alzheimer's disease (AD), vascular dementia (VD), mild cognitive impairment (MCI) and healthy controls. There are also no reliable data regarding the relationship between the overlapping protein levels and cognitive or functional decline. We measured levels of IL-1beta, IL-2, IL-6, IL-18, TNF-alpha, beta-Amlyloid 1-40 and alpha1-antichymotrypsin levels in plasma in groups of total 82 subjects with AD, MCI, VD and controls using enzyme-linked immunosorbent assay (ELISA) method. Our study samples showed high levels of proinflammatory cytokine levels (especially IL-18) in all patient groups but only high levels of alpha1-antichymotrypsine in VD patients compared to controls. There is no significant correlation between the laboratory and clinical variables except for a link between IL-1beta and NPI scores of AD. In conclusion, this study yielded evidence of some shared mechanisms underlying AD and VD and thus motivates further studies of inflammatory markers in various types of dementia and MCI.

The Diagnostic Role of Serum Inflammatory and Soluble Proteins on Dementia Subtypes: Correlation with Cognitive and Functional Decline

PubMed Central

Öztürk, Candan; Özge, Aynur; Yalın, Osman Özgür; Yılmaz, İ. Arda; Delialioglu, Nuran; Yıldız, Çilem; Tesdelen, Bahar; Kudiaki, Cigdem

2007-01-01

In the past years, the possible involvement of inflammation in the pathogenesis of dementia has been the subject of several investigations. However there are restricted data about the profile of the inflammatory and soluble proteins in well evaluated Alzheimer’s disease (AD), vascular dementia (VD), mild cognitive impairment (MCI) and healthy controls. There are also no reliable data regarding the relationship between the overlapping protein levels and cognitive or functional decline. We measured levels of IL-1β, IL-2, IL-6, IL-18, TNF-α, β-Amlyloid 1–40 and α1-antichymotrypsin levels in plasma in groups of total 82 subjects with AD, MCI, VD and controls using enzyme-linked immunosorbent assay (ELISA) method. Our study samples showed high levels of proinflammatory cytokine levels (especially IL-18) in all patient groups but only high levels of α1-antichymotrypsine in VD patients compared to controls. There is no significant correlation between the laboratory and clinical variables except for a link between IL-1β and NPI scores of AD. In conclusion, this study yielded evidence of some shared mechanisms underlying AD and VD and thus motivates further studies of inflammatory markers in various types of dementia and MCI. PMID:18430978

Platelets derived from the bone marrow of diabetic animals show dysregulated endoplasmic reticulum stress proteins that contribute to increased thrombosis.

PubMed

Hernández Vera, Rodrigo; Vilahur, Gemma; Ferrer-Lorente, Raquel; Peña, Esther; Badimon, Lina

2012-09-01

Patients with diabetes mellitus have an increased risk of suffering atherothrombotic syndromes and are prone to clustering cardiovascular risk factors. However, despite their dysregulated glucose metabolism, intensive glycemic control has proven insufficient to reduce thrombotic complications. Therefore, we aimed to elucidate the determinants of thrombosis in a model of type 2 diabetes mellitus with cardiovascular risk factors clustering. Intravital microscopy was used to analyze thrombosis in vivo in Zucker diabetic fatty rats (ZD) and lean normoglycemic controls. Bone marrow (BM) transplants were performed to test the contribution of each compartment (blood or vessel wall) to thrombogenicity. ZD showed significantly increased thrombosis compared with lean normoglycemic controls. BM transplants demonstrated the key contribution of the hematopoietic compartment to increased thrombogenicity. Indeed, lean normoglycemic controls transplanted with ZD-BM showed increased thrombosis with normal glucose levels, whereas ZD transplanted with lean normoglycemic controls-BM showed reduced thrombosis despite presenting hyperglycemia. Significant alterations in megakaryopoiesis and platelet-endoplasmic reticulum stress proteins, protein disulfide isomerase and 78-kDa glucose-regulated protein, were detected in ZD, and increased tissue factor procoagulant activity was detected in plasma and whole blood of ZD. Our results indicate that diabetes mellitus with cardiovascular risk factor clustering favors BM production of hyperreactive platelets with altered protein disulfide isomerase and 78-kDa glucose-regulated protein expression that can contribute to increase thrombotic risk independently of blood glucose levels.

Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

PubMed Central

Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

2013-01-01

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

Natriuretic peptide receptor-C activation attenuates angiotensin II-induced enhanced oxidative stress and hyperproliferation of aortic vascular smooth muscle cells.

PubMed

Madiraju, Padma; Hossain, Ekhtear; Anand-Srivastava, Madhu B

2018-02-07

We showed previously that natriuretic peptide receptor-C (NPR-C) agonist, C-ANP 4-23 , attenuated the enhanced expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) through the inhibition of enhanced oxidative stress. Since the enhanced levels of endogenous angiotensin II (Ang II) contribute to the overexpression of Giα proteins and augmented oxidative stress in VSMC from SHR, the present study was undertaken to investigate if C-ANP 4-23 could also attenuate angiotensin II (Ang II)-induced oxidative stress and associated signaling. Ang II treatment of aortic VSMC augmented the levels of superoxide anion (O 2 - ), NADPH oxidase activity, and the expression of NADPH oxidase subunits and C-ANP 4-23 treatment attenuated all these to control levels. In addition, Ang II-induced enhanced levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyl content were also attenuated toward control levels by C-ANP 4-23 treatment. On the other hand, Ang II inhibited the levels of nitric oxide (NO) and augmented the levels of peroxynitrite (OONO - ) in VSMC which were restored to control levels by C-ANP 4-23 treatment. Furthermore, C-ANP 4-23 treatment attenuated Ang II-induced enhanced expression of Giα proteins, phosphorylation of p38, JNK, and ERK 1,2 as well as hyperproliferation of VSMC as determined by DNA synthesis, and metabolic activity. These results indicate that C-ANP 4-23 , via the activation of NPR-C, attenuates Ang II-induced enhanced nitroxidative stress, overexpression of Giα proteins, increased activation of the p38/JNK/ERK 1,2 signaling pathways, and hyperproliferation of VSMC. It may be suggested that C-ANP 4-23 could be used as a therapeutic agent in the treatment of vascular remodeling associated with hypertension and atherosclerosis.

Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response

PubMed Central

2014-01-01

While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

Protein Markers of Neurotransmitter Synthesis and Release in Postmortem Schizophrenia Substantia Nigra.

PubMed

Schoonover, Kirsten E; McCollum, Lesley A; Roberts, Rosalinda C

2017-01-01

The substantia nigra (SN) provides the largest dopaminergic input to the brain, projects to the striatum (the primary locus of action for antipsychotic medication), and receives GABAergic and glutamatergic inputs. This study used western blot analysis to compare protein levels of tyrosine hydroxylase (TH), glutamate decarboxylase (GAD67), and vesicular glutamate transporters (vGLUT1 and vGLUT2) in postmortem human SN in schizophrenia subjects (n=13) and matched controls (n=12). As a preliminary analysis, the schizophrenia group was subdivided by (1) treatment status: off medication (n=4) or on medication (n=9); or (2) treatment response: treatment resistant (n=5) or treatment responsive (n=4). The combined schizophrenia group had higher TH and GAD67 protein levels than controls (an increase of 69.6%, P=0.01 and 19.5%, P=0.004, respectively). When subdivided by medication status, these increases were found in the on-medication subjects (TH 88.3%, P=0.008; GAD67 40.6%, P=0.003). In contrast, unmedicated schizophrenia subjects had higher vGLUT2 levels than controls (an increase of 28.7%, P=0.041), but vGLUT2 levels were similar between medicated schizophrenia subjects and controls. Treatment-resistant subjects had significantly higher TH and GAD67 levels than controls (an increase of 121.0%, P=0.0003 and 58.7%, P=0.004, respectively). These data suggest increases in dopamine and GABA transmission in the SN in schizophrenia, with a potential relation to treatment and response.

The nitrative and oxidative stress in blood platelets isolated from breast cancer patients: the protectory action of aronia melanocarpa extract.

PubMed

Kedzierska, Magdalena; Olas, Beata; Wachowicz, Barbara; Stochmal, Anna; Oleszek, Wieslaw; Jeziorski, Arkadiusz; Piekarski, Janusz

2010-01-01

Since mechanisms involved in the relationship between oxidative stress and breast cancer are still unclear, the aim of our present study was to evaluate oxidative/nitrative modifications of blood platelet proteins by measuring the level of biomarkers of oxidative/nitrative stress such as carbonyl groups, thiol groups and 3-nitrotyrosine in proteins in patients with benign breast diseases and in patients with invasive breast cancer, and compare with the control group. Levels of carbonyl groups and 3-nitrotyrosine residues in platelet proteins were measured by ELISA and a competition ELISA, respectively. The method with 5,5′-dithio-bis(2-nitro-benzoic acid) has been used to analyse free thiol groups in platelet proteins. Patients were hospitalized in the Department of Oncological Surgery, Medical University of Lodz, Poland. Exogenous antioxidants reduce oxidative stress, therefore we also investigated in a model system in vitro the effects of a polyphenol rich extract of Aronia melanocarpa (Rosaceae, final concentration of 50 µg/ml, 5 min, 37°C) on modified blood platelet proteins as well from patients with breast cancer and from the healthy group. We demonstrated in platelet proteins from patients with invasive breast cancer a higher level of carbonyl groups than in the control healthy group (p < 0.02). The level of 3-nitrotyrosine in platelet proteins from patients with invasive breast cancer was also significantly higher than in the healthy subject group (p < 0.001). In contrast, the amount of thiol groups in platelet proteins from patients was significantly lower (about < 50%) than in control blood platelets. In a model system in vitro we also observed that the extract from berries of A. melanocarpa (50 µg/ml, 5 min, 37°C) due to antioxidant action, significantly reduced the oxidative/nitrative stress (measured by thiol groups and 3-nitrotyrosine) in platelets, not only from the healthy group, but also from patients with benign breast diseases and in patients with invasive breast cancer.

Principles for designing proteins with cavities formed by curved β sheets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcos, Enrique; Basanta, Benjamin; Chidyausiku, Tamuka M.

Active sites and ligand-binding cavities in native proteins are often formed by curved β sheets, and the ability to control β-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling β-sheet curvature by studying the geometry of β sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved β sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that β-sheetmore » curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites.« less

[The effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in rats exposed to lead].

PubMed

Feng, Chang; Fan, Guang-qin; Wu, Feng-yun; Lin, Fen; Li, Yan-shu; Chen, Ying

2012-07-01

To study the effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in hippocampus of rats exposed to lead. Male SD rats were divided into five groups. (1) control group, (2) group exposed to lead+2 by drinking water with 0.40 g/L lead acetate, (3) group exposed to methionine and choline (1:1, 400 mg/kg), (4) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 100 mg/kg), (5) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 400 mg/kg). In 8 weeks after exposure, all rats were killed. Then CREB mRNA and CaMK II mRNA expression levels in hippocampus were detected by real-time PCR, CREB and CaMK II protein expression levels in hippocampus were measured by western blot assay. The expression levels (0.743 ± 0.185 and 0.729 ± 0.199) of CaMKII mRNA and CREB mRNA in the hippocampus of lead group were significantly lower than those (0.950 ± 0.238 and 0.901 ± 0.232) of control group (P < 0.05), also the expression levels (0.271 ± 0.045 and 0.212 ± 0.058) of CREB protein and pCREB protein in the hippocampus of lead group were significantly lower than those (0.319 ± 0.058 and 0.506 ± 0.125) of control group (P < 0.05). The expression levels (1.014 ± 0.210 and 1.126 ± 0.379) of CaMKII mRNA and the expression levels (1.029 ± 0.335 and 0.932 ± 0.251) of CREB mRNA in the hippocampus of 2 groups exposed to lead acetate plus methionine and choline were significantly higher than those of lead group (P < 0.05). The expression levels (0.407 ± 0.951 and 0.563 ± 0.178) of CREB protein and pCREB protein in the hippocampus of group exposed to lead acetate plus 400 mg/kg methionine and choline were significantly higher than those of lead group (P < 0.05). Methionine and choline could decrease the inhibition effects of lead on the expression of CaMKII and CREB mRNA or CREB and pCREB proteins in the hippocampus of rats.

Correlation between decreased CSF α-synuclein and Aβ₁₋₄₂ in Parkinson disease.

PubMed

Buddhala, Chandana; Campbell, Meghan C; Perlmutter, Joel S; Kotzbauer, Paul T

2015-01-01

Accumulation of misfolded α-synuclein (α-syn) protein in Lewy bodies and neurites is the cardinal pathologic feature of Parkinson disease (PD), but abnormal deposition of other proteins may also play a role. Cerebrospinal fluid (CSF) levels of proteins known to accumulate in PD may provide insight into disease-associated changes in protein metabolism and their relationship to disease progression. We measured CSF α-syn, amyloid β₁₋₄₂ (Aβ₁₋₄₂), and tau from 77 nondemented PD and 30 control participants. CSF α-syn and Aβ₁₋₄₂ were significantly lower in PD compared with controls. In contrast with increased CSF tau in Alzheimer disease, CSF tau did not significantly differ between PD and controls. CSF protein levels did not significantly correlate with ratings of motor function or performance on neuropsychological testing. As expected, CSF Aβ₁₋₄₂ inversely correlated with [(11)C]-Pittsburgh compound B (PiB) mean cortical binding potential, with PiB(+) PD participants having lower CSF Aβ₁₋₄₂ compared with PiB(-) PD participants. Furthermore, CSF α-syn positively correlated with Aβ₁₋₄₂ in PD participants but not in controls, suggesting a pathophysiologic connection between the metabolisms of these proteins in PD. Copyright © 2015 Elsevier Inc. All rights reserved.

Short Communication: Transplacental Nucleoside Analogue Exposure and Mitochondrial Parameters in HIV-Uninfected Children

PubMed Central

Brogly, Susan B.; DiMauro, Salvatore; Van Dyke, Russell B.; Williams, Paige L.; Naini, Ali; Libutti, Daniel E.; Choi, Julia; Chung, Michelle

2011-01-01

Abstract Transplacental nucleoside analogue exposure can affect infant mitochondrial DNA (mtDNA). We evaluated mitochondria in peripheral blood mononuclear cells of children with and without clinical signs of mitochondrial dysfunction (MD) and antiretroviral (ARV) exposure. We previously identified 20 children with signs of MD (cases) among 1037 HIV-uninfected children born to HIV-infected women. We measured mtDNA copies/cell and oxidative phosphorylation (OXPHOS) NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) protein levels and enzyme activities, determined mtDNA haplogroups and deletions in 18 of 20 cases with stored samples and in sex- and age-matched HIV-uninfected children, both ARV exposed and unexposed, (1) within 18 months of birth and (2) at the time of presentation of signs of MD. In specimens drawn within 18 months of birth, mtDNA levels were higher and OXPHOS protein levels and enzyme activities lower in cases than controls. In contrast, at the time of MD presentation, cases and ARV-exposed controls had lower mtDNA levels, 214 and 215 copies/cell, respectively, than ARV-unexposed controls, 254 copies/cell. OXPHOS protein levels and enzyme activities were lower in cases than exposed controls, and higher in cases than unexposed controls, except for complex IV activity, which was higher in cases. Haplotype H was less frequent among cases (6%) than controls (31%). No deletions were found. The long-term significance of these small but potentially important alterations should continue to be studied as these children enter adolescence and adulthood. PMID:21142587

Potential proteins targeted by let-7f-5p in HeLa cells.

PubMed

Wang, Yu; Chen, Xiujuan; Zhang, Yi; Song, Jiandong

2017-07-24

MicroRNAs are a class of small, endogenous, non-coding RNAs mediating posttranscriptional gene silencing. The current authors hypothesized that let-7f-5p is likely involved in cell invasion and proliferation by regulating the expression of target genes. The current study combined let-7f-5p with iTRAQ to assess its effect on gene expression in HeLa cells. Results indicated that 164 proteins were expressed at different levels in HeLa cells overexpressing let-7f-5p and negative controls and that 172 proteins were expressed at different levels in let-7f-5p-silenced HeLa cells and negative controls. Results indicated that let-7f-5p may suppress insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in HeLa cells.

Dietary medium-chain triglycerides attenuate hepatic lipid deposition in growing rats with protein malnutrition.

PubMed

Kuwahata, Masashi; Kubota, Hiroyo; Amano, Saki; Yokoyama, Meiko; Shimamura, Yasuhiro; Ito, Shunsuke; Ogawa, Aki; Kobayashi, Yukiko; Miyamoto, Ken-ichi; Kido, Yasuhiro

2011-01-01

The objective of this study was to investigate the effects of dietary medium-chain triglycerides (MCT) on hepatic lipid accumulation in growing rats with protein malnutrition. Weaning rats were fed either a low-protein diet (3%, LP) or control protein diet (20%, CP), in combination with or without MCT. The four groups were as follows: CP-MCT, CP+MCT, LP-MCT, and LP+MCT. Rats in the CP-MCT, CP+MCT and LP+MCT groups were pair-fed their respective diets based on the amount of diet consumed by the LP-MCT group. Rats were fed each experimental diet for 30 d. Four weeks later, the respiratory quotient was higher in the LP-MCT group than those in the other groups during the fasting period. Hepatic triglyceride content increased in the LP groups compared with the CP groups. Hepatic triglyceride content in the LP+MCT group, however, was significantly decreased compared with that in the LP-MCT group. Levels of carnitine palmitoyltransferase (CPT) 1a mRNA and CPT2 mRNA were significantly decreased in the livers of the LP-MCT group, as compared with corresponding mRNA levels of the other groups. These results suggest that ingestion of a low-protein diet caused fatty liver in growing rats. However, when rats were fed the low-protein diet with MCT, hepatic triglyceride deposition was attenuated, and mRNA levels encoding CPT1a and CPT2 were preserved at the levels of rats fed control protein diets.

Pigeonpea Hybrid-Proline-Rich Protein (CcHyPRP) Confers Biotic and Abiotic Stress Tolerance in Transgenic Rice

PubMed Central

Mellacheruvu, Sunitha; Tamirisa, Srinath; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2016-01-01

In this study, we report the overexpression of Cajanus cajan hybrid-proline-rich protein encoding gene (CcHyPRP) in rice which resulted in increased tolerance to both abiotic and biotic stresses. Compared to the control plants, the transgenic rice lines, expressing CcHyPRP, exhibited high-level tolerance against major abiotic stresses, viz., drought, salinity, and heat, as evidenced by increased biomass, chlorophyll content, survival rate, root, and shoot growth. Further, transgenic rice lines showed increased panicle size and grain number compared to the control plants under different stress conditions. The CcHyPRP transgenics, as compared to the control, revealed enhanced activities of catalase and superoxide dismutase (SOD) enzymes and reduced malondialdehyde (MDA) levels. Expression pattern of CcHyPRP::GFP fusion-protein confirmed its predominant localization in cell walls. Moreover, the CcHyPRP transgenics, as compared to the control, exhibited increased resistance to the fungal pathogen Magnaporthe grisea which causes blast disease in rice. Higher levels of bZIP and endochitinase transcripts as well as endochitinase activity were observed in transgenic rice compared to the control plants. The overall results demonstrate the intrinsic role of CcHyPRP in conferring multiple stress tolerance at the whole-plant level. The multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants for enhanced tolerance/resistance to different stress factors. PMID:26834756

Effect of compound Maqin decoction on TGF-β1/Smad proteins and IL-10 and IL-17 content in lung tissue of asthmatic rats.

PubMed

Xie, Y H; Li, X P; Xu, Z X; Qian, P; Li, X L; Wang, Y Q

2016-09-02

In this research, compound Maqin decoction (CMD) has been shown to positively affect in airway inflammation of asthma models. We evaluated the effects of CMD on the expression of transforming growth factor (TGF)-β1/Smad proteins, interleukin (IL)-17, and IL-10 in lung tissue of asthmatic rats. Asthma was induced in a rat model using ovalbumin. After a 4-week treatment with CMD, rats were killed to evaluate the expression of TGF-β1 and Smad proteins in lung tissue. IL-10 and IL-17 levels in lung tissue homogenates were determined by ELISA. The expression of TGF-β1 and Smad3 protein increased, whereas expression of Smad7 protein decreased upon high-dose or low-dose treatment with CMD or by intervention with dexamethasone, compared to the control. There was a significant difference between treatment with a high dose CMD and the control treatment, but no significant difference was found between high-dose CMD treatment and dexamethasone intervention. The expression of TGF-β1 and Smad7 protein increased, whereas the expression of Smad3 protein decreased in the model group compared to other groups. In the CMD high-dose group, low-dose group, and dexamethasone intervention group, the IL-17 concentrations in lung tissue homogenates were decreased, while IL-10 levels were increased. Again, there was a significant difference between CMD high-dose and control treatment, but not between CMD high-dose treatment and dexamethasone intervention. Thus, positive effects of CMD against asthmatic airway remodeling may be due to its regulatory effect on TGF-β1, Smad3, and Smad7 protein levels and on cytokines such as IL-10 and IL-17.

Impact of Lipid and Protein Co-oxidation on Digestibility of Dairy Proteins in Oil-in-Water (O/W) Emulsions.

PubMed

Obando, Mónica; Papastergiadis, Antonios; Li, Shanshan; De Meulenaer, Bruno

2015-11-11

Enrichment of polyunsaturated fatty acids (PUFAs) is a growing trend in the food industry. However, PUFAs are known to be susceptible to lipid oxidation. It has been shown that oxidizing lipids react with proteins present in the food and that as a result polymeric protein complexes are produced. Therefore, the aim of this work was to investigate the impact of lipid and protein co-oxidation on protein digestibility. Casein and whey protein (6 mg/mL) based emulsions with 1% oil with different levels of PUFAs were subjected to respectively autoxidation and photo-oxidation. Upon autoxidation at 70 °C, protein digestibility of whey protein based emulsions containing fish oil decreased to 47.7 ± 0.8% after 48 h, whereas in the controls without oil 67.8 ± 0.7% was observed. Upon photo-oxidation at 4 °C during 30 days, mainly casein-based emulsions containing fish oil were affected: the digestibility amounted to 43.9 ± 1.2%, whereas in the control casein solutions without oil, 72.6 ± 0.2% of the proteins were digestible. Emulsions containing oils with high PUFA levels were more prone to lipid oxidation and thus upon progressive oxidation showed a higher impact on protein digestibility.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

PubMed Central

Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

2015-01-01

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

Monocyte Chemotactic Protein-1 (MCP-1) and Growth Factors Called into Question as Markers of Prolonged Psychosocial Stress

PubMed Central

Jonsdottir, Ingibjörg H.; Hägg, Daniel A.; Glise, Kristina; Ekman, Rolf

2009-01-01

Background Psychosocial stress is becoming a major contributor to increased mental ill-health and sick leave in many countries. Valid markers of chronic stress would be valuable for diagnostic and prognostic purposes. A recent study suggested monocyte chemotactic protein-1 (MCP-1), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as markers of chronic stress. We aimed to confirm these potential biomarkers of prolonged psychosocial stress in female patients. Methodology/Principal Findings Circulating levels of MCP-1, EGF and VEGF, along with several other cytokines, were measured in plasma from 42 female patients suffering from exhaustion due to prolonged psychosocial stress and 42 control subjects, using a protein biochip immunoassay. There were no significant differences between patients and controls in any of the cytokines or growth factors analyzed. Furthermore, when using a different protein bioassay and reanalyzing MCP-1 and VEGF in the same samples, markedly different levels were obtained. To further explore if inflammation is present in patients with exhaustion, the classical inflammatory marker C-reactive protein (CRP) was measured in another group of patients (n = 89) and controls (n = 88) showing a small but significant increase of CRP levels in the patients. Conclusions/Significance MCP-1, EGF and VEGF may not be suitable markers of prolonged psychosocial stress as previously suggested. Furthermore, significant differences were obtained when using two different protein assays measuring the same samples, indicating that comparing studies where different analytic techniques have been used might be difficult. Increased levels of CRP indicate that low-grade inflammation might be present in patients with exhaustion due to prolonged stress exposure but this inflammation does not seem to be reflected by increase in circulating MCP-1 or other cytokines measured. PMID:19888340

The structure of a cholesterol-trapping protein

Science.gov Websites

Date February 28, 2003 Date Berkeley Lab Science Beat Berkeley Lab Science Beat The structure of a Institute researchers determined the three-dimensional structure of a protein that controls cholesterol level in the bloodstream. Knowing the structure of the protein, a cellular receptor that ensnares

The drastic reduction of SMN protein in SMA I spinal cord motor neurons is not due to inefficient transcription.

PubMed

Mirabella, M; Servidei, S; Broccolini, A; Gandolfi, N; Ricci, E; Neri, G; Tonali, P; Brahe, C

1999-04-01

Spinal muscular atrophy (SMA) is caused by homozygous absence of the telomeric copy of the survival motor neuron (SMNt) gene. SMNt and its homologous centromeric copy (SMNc) encode the SMN protein, which is markedly reduced in SMA I patients. We have performed SMN transcript and protein studies on spinal cord sections of an SMA I patient using in situ hybridization and immunofluorescence. While the amount of protein was negligible, the level of transcripts was comparable with that of controls. These findings suggest that the reduced protein level is not caused by a deficient transcription of the SMNc gene.

Intrathecal inflammation precedes development of Alzheimer's disease

PubMed Central

Tarkowski, E; Andreasen, N; Tarkowski, A; Blennow, K

2003-01-01

Objectives: To analyse the cerebrospinal fluid (CSF) values of the proinflammatory cytokines, interleukin 1ß (IL1ß), tumour necrosis factor α (TNFα), GM-CSF, of the anti-inflammatory cytokine TGFß, of tau protein, a marker for neurodegeneration, and of ß amyloid (Aß), a protein involved in the formation of senile plaques, in prospectively followed up patients with mild cognitive impairment (MCI). Methods: Analyses of CSF levels of TNFα, IL1ß, GM-CSF, TGFß, ßa, and tau protein were performed using ELISA in 56 patients with MCI who were followed up prospectively and in 25 age matched, healthy controls. Results: Patients with MCI displayed significantly higher levels of TNFα and tau protein and significantly lower levels of TGFß and Aß compared with the healthy controls. After nine months of follow up, 25 patients still displayed MCI while the remaining 31 patients had progressed to Alzheimer's disease (AD). Only MCI patients who progressed to AD at follow up, showed significantly higher CSF levels of TNFα than controls. In addition, reduced CSF-Aß42 levels were only found in MCI patients that progressed to AD, further supporting the notion that disturbed metabolism of Aß is an early finding in AD. Conclusions: These results demonstrate increased production of the proinflammatory cytokine, TNFα and decreased production of the anti-inflammatory cytokine TGFß in patients with MCI at risk to develop AD, suggesting a propensity towards inflammation in this patient group and indicating that CNS inflammation is a early hallmark in the pathogenesis of AD. PMID:12933918

Narrow-leafed lupin (Lupinus angustifolius L.) β-conglutin proteins modulate the insulin signaling pathway as potential type 2 diabetes treatment and inflammatory-related disease amelioration.

PubMed

Lima-Cabello, Elena; Alche, Victor; Foley, Rhonda C; Andrikopoulos, Sofianos; Morahan, Grant; Singh, Karam B; Alche, Juan D; Jimenez-Lopez, Jose C

2017-05-01

We have investigated the potential use of β-conglutin protein isoforms from narrow-leafed lupin (Lupinus angustifolius L.) as a diabetes treatment. We produced purified recombinant β1-, β2-, β3-, β4-, and β6-conglutin proteins and showed that β1, β3, and β6 could bind to insulin. To assess β-conglutin proteins modulatory effect on insulin activation meditated kinases, whole blood and peripheral blood mononuclear cell cultures from type 2 diabetes (T2D) and healthy control subjects (C) were incubated with conglutin proteins. The treatment of peripheral blood mononuclear cells from T2D patients with β1, β3, and β6 proteins increased up to threefold mRNA and protein levels of genes important in insulin signaling pathways, namely insulin receptor substrate 1/p85/AKT/glucose transporter type 4. This was accompanied by a comparable fold-change decrease in the mRNA expression level of pro-inflammatory genes (iNOS and IL-1β) and proteins compared to healthy controls. The β2 and β4 isoforms had no effect on the insulin signaling pathway. However, these β-conglutin proteins elicited pro-inflammatory effects since levels of mRNA and proteins of inducible nitric oxide synthase and IL 1 beta were increased. Our results raise the possibility of using these particular β-conglutin proteins in the prevention and treatment of diabetes, as well as their potential as anti-inflammatory molecules. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assessment of test methods for evaluating effectiveness of cleaning flexible endoscopes.

PubMed

Washburn, Rebecca E; Pietsch, Jennifer J

2018-06-01

Strict adherence to each step of reprocessing is imperative to removing potentially infectious agents. Multiple methods for verifying proper reprocessing exist; however, each presents challenges and limitations, and best practice within the industry has not been established. Our goal was to evaluate endoscope cleaning verification tests with particular interest in the evaluation of the manual cleaning step. The results of the cleaning verification tests were compared with microbial culturing to see if a positive cleaning verification test would be predictive of microbial growth. This study was conducted at 2 high-volume endoscopy units within a multisite health care system. Each of the 90 endoscopes were tested for adenosine triphosphate, protein, microbial growth via agar plate, and rapid gram-negative culture via assay. The endoscopes were tested in 3 locations: the instrument channel, control knob, and elevator mechanism. This analysis showed substantial level of agreement between protein detection postmanual cleaning and protein detection post-high-level disinfection at the control head for scopes sampled sequentially. This study suggests that if protein is detected postmanual cleaning, there is a significant likelihood that protein will also be detected post-high-level disinfection. It also infers that a cleaning verification test is not predictive of microbial growth. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression

PubMed Central

Tonack, Sarah; Patel, Sabina; Jalali, Mehdi; Nedjadi, Taoufik; Jenkins, Rosalind E; Goldring, Christopher; Neoptolemos, John; Costello, Eithne

2011-01-01

AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised. RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable. PMID:21528072

Contribution of starter cultures to the proteolytic process of a fermented non-dried whole muscle ham product.

PubMed

Scannell, Amalia G M; Kenneally, Paul M; Arendt, Elke K

2004-06-01

Porcine longissimus dorsi muscles were cured by brine injection. Curing brine containing 15% (w/v) NaCl, 1.33% (w/v) glucose, 750 ppm sodium nitrite, and appropriate levels of either Lactobacillus sakei LAD, L. sakei LAD plus Kocuria varians FT4 (formally Micrococcus varians), L. sakei LAD plus papain and GDL (glucono-delta-lactone) plus K. varians FT4, was injected to the muscle at a pumping rate 15% w/v. The effect of these treatments on the proteolysis in the ham system was compared to a control ham, produced without starter culture and containing GDL acidulant to control pH and antibiotics to reduce the contribution of background microflora. Hydrolysis of sarcoplasmic and myofibrillar protein fractions was evaluated by SDS-PAGE and reverse phase-HPLC. Hams with different treatments were also investigated for differences in amino acid profile, protein and non-protein nitrogen level, colour, pH, water activity and moisture and microbiological evolution. There was no significant difference in the gross compositional analysis of any of the treatments compared to the control. There was no significant difference (p>0.05) in the protein content, non-protein nitrogen level, SDS-PAGE and free amino acid analysis between the control ham and ham inoculated with proteolytic starter culture. However, it was observed that hams containing starter cultures exhibited decreases in certain peptide fractions and corresponding increases in some free amino acids compared to the uninoculated control. It can be concluded that, while the principle mechanisms resulting in the proteolysis of this non-dried ham product involve the activity of endogeneous cathepsins, the addition of proteolytic starter cultures influence the amino acid profile thereby potentially enhancing the sensorial attributes of the ham. Copyright 2004 Elsevier B.V.

Excess copper induces anoxygenic photosynthesis in Anabaena doliolum: a homology based proteomic assessment of its survival strategy.

PubMed

Bhargava, Poonam; Mishra, Yogesh; Srivastava, Ashish Kumar; Narayan, Om Prakash; Rai, Lal Chand

2008-04-01

This study is the first to demonstrate operation of anoxygenic photosynthesis in copper acclimated Anabaena doliolum and to offer proteomic comparison with the control cells. The Cu-treated control strain showed a negative correlation in growth and intracellular Cu, partial inhibition of O(2)-evolution, PS II, PS I, whole chain, chlorophyll absorption, and nitrogenase activity. However, the acclimated strain growing in 250-fold excess Cu exhibited near normal growth, ATP content, PS I activity, carbon fixation, and almost complete inhibition of O(2)-evolution, PS II and chlorophyll absorption, but increased nitrogenase activity as compared to control. Proteomic decoding of the survival strategy of Cu-treated control and the acclimated strain using two-dimensional gel electrophoresis and MALDI-TOF MS analysis of proteins displaying significant and reproducible changes demonstrated involvement of transketolase, phycoerythrocyanin alpha-chain, iron superoxide dismutase (Fe-SOD), hypothetical protein alr 0803, manganese superoxide dismutase (Mn-SOD), phosphoribulokinase, and plastocyanin (PLC). Expression pattern of these proteins was attested at the transcriptional level using RT-PCR. Time course analysis of proteins of Cu-treated control strain revealed almost no change in PLC level, and a minor accumulation of transketolase, phycoerythrocyanin alpha-chain and both isoforms of SOD after 7 and recovery after 10 days. Acclimated strain under excess Cu, however, exhibited significant accumulation of both isoforms of SOD, plastocyanin, phosphoribulokinase and transketolase, which seem to counteract oxidative damage, serve as an alternate electron carrier from cytochrome b6/f complex to photosystem I and meet the NADPH and ATP requirements, respectively, under anoxygenic photosynthesis. In view of the kinetics of the hypothetical protein alr0803 (no change in expression level for 7, maximum after 10 and decline after 15 days) its involvement in metal homeostasis is suggested.

Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin.

PubMed

Wilson, Fiona A; Orellana, Renán A; Suryawan, Agus; Nguyen, Hanh V; Jeyapalan, Asumthia S; Frank, Jason; Davis, Teresa A

2008-07-01

Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.

Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin

PubMed Central

Wilson, Fiona A.; Orellana, Renán A.; Suryawan, Agus; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Frank, Jason; Davis, Teresa A.

2008-01-01

Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7–10 days of pST (150 μg·kg−1·day−1) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 μU/ml), 2) fed control (25 μU/ml), and 3) fed pST-treated (50 μU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1·eIF4E complex association and increased active eIF4E·eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation. PMID:18460595

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton.

PubMed

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton

PubMed Central

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels. PMID:28099508

Degradation of submandibular gland AQP5 by parasympathetic denervation of chorda tympani and its recovery by cevimeline, an M3 muscarinic receptor agonist.

PubMed

Li, Xuefei; Azlina, Ahmad; Karabasil, Mileva Ratko; Purwanti, Nunuk; Hasegawa, Takahiro; Yao, Chenjuan; Akamatsu, Tetsuya; Hosoi, Kazuo

2008-07-01

By chorda tympani denervation (CTD, parasympathectomy), the aquaporin 5 (AQP5), but not AQP1, protein level in the rat submandibular gland (SMG) was significantly decreased, dropping to 37% of that of the contralateral gland at 4 wk. The protein levels of AQP5 and AQP1 were not significantly affected by denervation of the cervical sympathetic trunk (sympathectomy). Administration of cevimeline hydrochloride, an M3 muscarinic receptor agonist (10 mg/kg for 7 days po), but not pilocarpine (0.3 mg/kg for 7 days po), recovered the AQP5 protein level reduced by CTD and increased the AQP1 protein level above the control one. The mRNA level of AQP5 was scarcely affected by CTD and cevimeline hydrochloride administration. Administration of chloroquine (50 mg/kg for 7 days po), a denaturant of lysosomes, increased the AQP5 protein level reduced by CTD. An extract obtained from the submandibular lysosomal fraction degraded the AQP5 protein in the total membrane fraction in vitro. These results suggest the possible regulation of the AQP5 protein level in the SMG by the parasympathetic nerves/M3 muscarinic receptor agonist and imply the involvement of lysosomal enzymes, but not a transcriptional mechanism, in this regulation.

Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates*

PubMed Central

Tennstaedt, Annette; Pöpsel, Simon; Truebestein, Linda; Hauske, Patrick; Brockmann, Anke; Schmidt, Nina; Irle, Inga; Sacca, Barbara; Niemeyer, Christof M.; Brandt, Roland; Ksiezak-Reding, Hanna; Tirniceriu, Anca Laura; Egensperger, Rupert; Baldi, Alfonso; Dehmelt, Leif; Kaiser, Markus; Huber, Robert; Clausen, Tim; Ehrmann, Michael

2012-01-01

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. PMID:22535953

Effects of rice bran fiber on heat-induced gel prepared with pork salt-soluble meat proteins in model system.

PubMed

Choi, Yun-Sang; Choi, Ji-Hun; Han, Doo-Jeong; Kim, Hack-Youn; Lee, Mi-Ai; Kim, Hyun-Wook; Jeong, Jong-Youn; Kim, Cheon-Jei

2011-05-01

The technological effects of rice bran fiber on pork salt-soluble meat proteins in a model system were investigated. Rice bran fiber at levels of 0% (control), 0.1%, 0.5%, 1%, and 2% was added at the same time as salt-soluble meat protein to maintain similar moisture levels in all samples. Samples with increasing amounts of added rice bran fiber had higher pH, yellowness, sarcoplasmic and total protein solubilities. The moisture content, myofibrillar protein solubility and water holding capacity were the highest in the treatments containing with 1% rice bran fiber. However, the lightness and redness, textural properties decreased with increasing rice bran fiber levels. SDS gel electrophoresis did not reveal any changes in proteins regardless different rice bran fiber levels. The apparent viscosity indicated that improvements in water holding capacity and decreased texture due to added rice bran fiber. Copyright © 2010 The American Meat Science Association. All rights reserved.

The pathogenesis of ethanol versus methionine and choline deficient diet-induced liver injury.

PubMed

Gyamfi, Maxwell Afari; Damjanov, Ivan; French, Samuel; Wan, Yu-Jui Yvonne

2008-02-15

The differences and similarities of the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) were examined. Mice (six/group) received one of four Lieber-Decarli liquid diets for 6 weeks: (1) paired-fed control diet; (2) control diet with ethanol (ethanol); (3) paired-fed methionine/choline deficient (MCD) diet; and (4) MCD plus ethanol (combination). Hepatotoxicity, histology, and gene expression changes were examined. Both MCD and ethanol induced macrovesicular steatosis. However, the combination diet produced massive steatosis with minor necrosis and inflammation. MCD and combination diets, but not ethanol, induced serum ALT levels by 1.6- and 10-fold, respectively. MCD diet, but not ethanol, also induced serum alkaline phosphatase levels suggesting bile duct injury. Ethanol increased liver fatty acid binding protein (L-FABP) mRNA and protein levels. In contrast, the combination diet decreased L-FABP mRNA and protein levels and increased hepatic free fatty acid and lipid peroxide levels. Ethanol, but not MCD, reduced hepatic S-adenosylmethionine (SAM) and GSH levels. Hepatic TNFalpha protein levels were increased in all treatment groups, however, IL-6, a hepatoprotective cytokine which promotes liver regeneration was increased in ethanol-fed mice (2-fold), but decreased in the combination diet-treated mice. In addition, the combination diet reduced phosphorylated STAT3 and Bcl-2 levels. While MCD diet might cause bile duct injury and cholestasis, ethanol preferentially interferes with the SAM-GSH oxidative stress pathway. The exacerbated liver injury induced by the combination diet might be explained by reduced L-FABP, increased free fatty acids, oxidative stress, and decreased IL-6 protein levels. The combination diet is an efficient model of steatohepatitis.

Postproduction Handling and Administration of Protein Pharmaceuticals and Potential Instability Issues.

PubMed

Nejadnik, M Reza; Randolph, Theodore W; Volkin, David B; Schöneich, Christian; Carpenter, John F; Crommelin, Daan J A; Jiskoot, Wim

2018-04-14

The safety and efficacy of protein pharmaceuticals depend not only on biological activity but also on purity levels. Impurities may be process related because of limitations in manufacturing or product related because of protein degradation occurring throughout the life history of a product. Although the pharmaceutical biotechnology industry has made great progress in improving bulk and drug product manufacturing as well as company-controlled storage and transportation conditions to minimize the level of degradation, there is less control over the many factors that may subsequently affect product quality after the protein pharmaceuticals are released and shipped by the manufacturer. Routine handling or unintentional mishandling of therapeutic protein products may cause protein degradation that remains unnoticed but can potentially compromise the clinical safety and efficacy of the product. In this commentary, we address some potential risks associated with (mis)handling of protein pharmaceuticals after release by the manufacturer. We summarize the environmental stress factors that have been shown to cause protein degradation and that may be encountered during typical handling procedures of protein pharmaceuticals in a hospital setting or during self-administration by patients. Moreover, we provide recommendations for improvements in product handling to help ensure the quality of protein pharmaceuticals during use. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant.

PubMed

Campo, G M; Squadrito, F; Ceccarelli, S; Calò, M; Avenoso, A; Campo, S; Squadrito, G; Altavilla, D

2001-04-01

Carbon tetrachloride (CCl4 )-induced hepatotoxicity is likely the result of a CCl4 -induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-alpha gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4 -induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH*), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-alpha, liver histology and hepatic mRNA levels for TNF-alpha were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4 = 404.61 +/- 10.33 U/L; Controls= 28.54 +/- 4.25 U/L), liver MAL (CCl4 = 0.67 +/- 0.16 nmol/mg protein; Controls= 0.13 +/- 0.06 nmol/mg protein), OH(7) levels assayed as 2,3-DHBA (CCl4 = 8.73 +/- 1.46 microM; Controls= 0.45 +/- 0.15 microM) and 2,5-DHBA (CCl4 = 24.61 +/- 3.32 microM; Controls= 2.75 +/- 0.93 microM), induced a severe depletion of GSH (CCl4 = 3.26 +/- 1.85 micromol/g protein; Controls= 17.82 +/- 3.13 micromol/g protein) and a marked decrease in VE levels (CCl4 = 5.67 +/- 1.22 nmol/g tissue; Controls= 13.47 +/- 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-alpha levels (CCl4 = 57.36 +/- 13.24 IU/ml; Controls= 7.26 +/- 2.31 IU/ml) and enhanced hepatic mRNA for TNF-alpha (CCl4 = 19.22 +/- 4.38 a.u.; Controls= 0.76 +/- 0.36 a.u.). IRFI 042 (100 mg/kg, 30 min after CCl4 injection) blunted liver MAL (0.32 +/- 0.17 nmol/mg protein), decreased the serum levels of ALT (128.71 +/- 13.23 U/L), and restored the hepatic concentrations of VE (9.52 +/- 3.21 nmol/g tissue), inhibited OH* production (2,3-DHBA= 3.54 +/- 1.31 microM; 2,5-DHBA= 7.37 +/- 2.46 microM), restored the endogenous antioxidant GSH (12.77 +/- 3.73 mmol/g protein) and improved histology. Furthermore IRFI 042 treatment suppressed plasma TNF-alpha concentrations (31.47 +/- 18.25 IU/ml) and hepatic TNF-alpha mRNA levels (11.65 +/- 3.21 a.u.). The acute treatment with vitamin E failed to exert any protective effect against CCl4 -induced hepatotoxicity. These investigations suggest that IRFI 042 treatment may be of benefit during free radical-mediated liver injury.

Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

NASA Astrophysics Data System (ADS)

Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

2015-02-01

The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

Intratracheal administration of p38α short-hairpin RNA plasmid ameliorates lung ischemia-reperfusion injury in rats.

PubMed

Lv, Xiangqi; Tan, Jing; Liu, Dongdong; Wu, Ping; Cui, Xiaoguang

2012-06-01

Lung ischemia-reperfusion injury (LIRI) remains a significant problem after lung transplantation. A crucial signaling enzyme involved in inflammation and apoptosis during LIRI is p38 mitogen-activated protein kinase (MAPK). Gene silencing of p38α by short hairpin RNA (shRNA) can downregulate p38α expression. The lungs have an extremely large surface area, which makes the absorption of shRNA highly effective. Therefore, we evaluated the therapeutic efficacy of p38α shRNA plasmids in a rat model of lung transplantation. The delivery of p38α shRNA plasmid was performed by intratracheal administration 48 hours before transplantation into donor rats. Control animals received scrambled shRNA plasmids. Reverse-transcription polymerase chain reaction and Western blots were used to assess gene silencing efficacy. The therapeutic effects of shRNA plasmids were evaluated by lung function tests. We determined the levels of inflammatory cytokines, the level of intercellular adhesion molecule 1 (ICAM-1), c-Myc mRNA expression, and ICAM-1 protein expression, and the presence of cell apoptosis. Rats administered p38α shRNA plasmids showed a significant downregulation in lung expression of p38α transcripts and protein levels. Compared with the control group, the p38α shRNA group showed a higher pulmonary vein oxygen level, lower wet weight-to-dry weight ratio, lower lung injury score, and lower serum levels of tumor necrosis factor-α, interleukin-6, and interleukin-8. Messenger RNA levels of ICAM-1 and c-Myc in the p38α shRNA group were dramatically lower than in the control group. Levels of ICAM-1 protein expression exhibited a similar trend. Cell apoptosis decreased in the p38α shRNA group vs the control group. Intratracheal administration of p38α shRNA plasmids provided therapeutic effects in a rat model of lung transplantation. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

The PARP1-Siah1 Axis Controls HIV-1 Transcription and Expression of Siah1 Substrates.

PubMed

Yu, Dan; Liu, Rongdiao; Yang, Geng; Zhou, Qiang

2018-06-26

Recent studies have revealed a key role of PARP1 that catalyzes the poly-ADP-ribosylation (PARylation) of substrates in regulating gene transcription. We show here that HIV-1 transcriptional activation also requires PARP1 activity. Because efficient HIV-1 transactivation is known to depend on the ELL2-containing super elongation complex (SEC), we investigated the functional relationship between PARP1 and ELL2-SEC in HIV-1 transcriptional control. We show that PARP1 elevates ELL2 protein levels to form more ELL2-SEC in cells. This effect is caused by PARP1's suppression of expression of Siah1, an E3 ubiquitin ligase for ELL2, at both mRNA and protein levels. At the mRNA level, PARP1 coordinates with the co-repressor NCoR to suppress Siah1 transcription. At the protein level, PARP1 promotes Siah1 proteolysis, likely through inducing PARylation-dependent ubiquitination (PARdU) of Siah1. Thus, a PARP1-Siah1 axis activates HIV-1 transcription and controls the expression of ELL2 and other Siah1 substrates. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Association between serum pregnancy-associated plasma protein-A and bicarbonate in hemodialysis patients.

PubMed

Bicik, Zerrin; Coskun, Abdurrahman; Serteser, Mustafa; Bulur, Atilla; Mese, Meral; Unsal, Ibrahim

2014-03-01

Acidosis is associated with protein-energy malnutrition, inflammation, and bone disease, and low bicarbonate levels have been implicated in higher mortality rates in chronic kidney disease. Recently, the concentration of serum pregnancy-associated plasma protein-A (PAPP-A) has become accepted as a prognostic marker in hemodialysis patients. This study determined the relationship between PAPP-A and bicarbonate levels in these patients. The study enrolled 65 hemodialysis patients (41 males, 24 females) and 26 control subjects (11 males, 15 females). Serum PAPP-A, intact parathormone (iPTH), calcium, phosphorus (P), and bicarbonate levels were measured. Correlations between PAPP-A and bicarbonate, iPTH, calcium, and phosphorus were evaluated. Median PAPP-A levels were significantly higher in hemodialysis patients [15.1 (<0.03-158.8) ng/ml] than in control subjects [6.6 (<0.03-16.4) ng/ml] (P < 0.05). There were statistically significant correlations between serum PAPP-A and bicarbonate, iPTH, and P in hemodialysis patients but not in control subjects. Elevation of serum PAPP-A has been found in hemodialysis patients and its significant correlation with bicarbonate suggests that it may be a prognostic factor. © 2014 Wiley Periodicals, Inc.

Abnormal mRNA Expression Levels of Telomere-Binding Proteins Represent Biomarkers in Myelodysplastic Syndromes: A Case-Control Study.

PubMed

Liu, Baoshan; Yan, Rongdi; Zhang, Jie; Wang, Bin; Sun, Hu; Cui, Xing

2017-08-02

As evidence was shown that abnormal shortening of telomeres begins to accumulate in myelodysplastic syndrome (MDS) patients, this study was conducted to determine the relationship between the mRNA expression levels of telomere-binding proteins (TRF1/TRF2/TIN2/TPP1/POT1/RAP1) and the risk level in MDS. There were 40 patients with MDS and 40 normal controls in this study. Methods including telomere content assays and quantitative reverse transcription-polymerase chain reaction were used to examine the mRNA levels of TRF1/TRF2/TIN2/TPP1/POT1/RAP1 in patients with MDS. Compared to the normal group used as a control, the mRNA expression levels of RAP1/POT1/TPP1 of the patients with MDS were decreased, whereas their levels of TRF1/TRF2 and TIN2 were increased. A positive correlation was found between the TRF1, TRF2, and TIN2 mRNA expression levels and the risk level of the International Prognostic Scoring System (IPSS) and the World Health Organization Prognostic Scoring System (WPSS) criteria; however, a negative correlation was found between RAP1/POT1/TPP1 mRNA expression levels and the risk levels of IPSS and WPSS criteria. Because the reduction of TRF1/TRF2/TIN2 mRNA expression and the increase of RAP1/POT1/TPP1 mRNA expression are closely related to the risk levels of the IPSS and WPSS criteria in MDS, it is thought that these telomere-binding proteins could lead to abnormal telomere length and function, which cause chromosomal abnormalities in MDS. With this evidence, we suggest that those proteins' mRNA expressions could be used as biomarkers for the assessment of the risk degree of MDS patients.

Immune recognition of salivary proteins from the cattle tick Rhipicephalus microplus differs according to the genotype of the bovine host.

PubMed

Garcia, Gustavo Rocha; Maruyama, Sandra Regina; Nelson, Kristina T; Ribeiro, José Marcos Chaves; Gardinassi, Luiz Gustavo; Maia, Antonio Augusto Mendes; Ferreira, Beatriz Rossetti; Kooyman, Frans N J; de Miranda Santos, Isabel K F

2017-03-14

Males of the cattle tick Rhipicephalus microplus produce salivary immunoglobulin-binding proteins and allotypic variations in IgG are associated with tick loads in bovines. These findings indicate that antibody responses may be essential to control tick infestations. Infestation loads with cattle ticks are heritable: some breeds carry high loads of reproductively successful ticks, in others, few ticks feed and they reproduce inefficiently. Different patterns of humoral immunity against tick salivary proteins may explain these phenotypes. We describe the profiles of humoral responses against tick salivary proteins elicited during repeated artificial infestations of bovines of a tick-resistant (Nelore) and a tick-susceptible (Holstein) breed. We measured serum levels of total IgG1, IgG2 and IgE immunoglobulins and of IgG1 and IgG2 antibodies specific for tick salivary proteins. With liquid chromatography followed by mass spectrometry we identified tick salivary proteins that were differentially recognized by serum antibodies from tick-resistant and tick-susceptible bovines in immunoblots of tick salivary proteins separated by two-dimensional electrophoresis. Baseline levels of total IgG1 and IgG2 were significantly higher in tick-susceptible Holsteins compared with resistant Nelores. Significant increases in levels of total IgG1, but not of IgG2 accompanied successive infestations in both breeds. Resistant Nelores presented with significantly higher levels of salivary-specific antibodies before and at the first challenge with tick larvae; however, by the third challenge, tick-susceptible Holsteins presented with significantly higher levels of IgG1 and IgG2 tick salivary protein-specific antibodies. Importantly, sera from tick-resistant Nelores reacted with 39 tick salivary proteins in immunoblots of salivary proteins separated in two dimensions by electrophoresis versus only 21 spots reacting with sera from tick-susceptible Holsteins. Levels of tick saliva-specific antibodies were not directly correlated with infestation phenotypes. However, in spite of receiving apparently lower amounts of tick saliva, tick-resistant bovines recognized more tick salivary proteins. These reactive salivary proteins are putatively involved in several functions of parasitism and blood-feeding. Our results indicate that neutralization by host antibodies of tick salivary proteins involved in parasitism is essential to control tick infestations.

The impact of nutrient density in terms of energy and/or protein on live performance, metabolism and carcass composition of female and male broiler chickens of two commercial broiler strains.

PubMed

Delezie, E; Bruggeman, V; Swennen, Q; Decuypere, E; Huyghebaert, G

2010-08-01

The objective of this study was to investigate the effects of diet composition on performance, slaughter yield and plasma metabolites, as different modern broiler strains show different responses to feed intake. Broilers of two commercial strains and of both sexes received one of three diets being different in energy and/or protein level [control diet, low energy/low protein diet (LM/LP) and low protein diet (LP)]. Low energy/low protein diet chickens were characterized by significantly lower body weights and feed intake compared with their LP and control counterparts. Broilers of the Cobb strain or broilers that were fed the control diet were most efficient in converting energy to body weight. No significant differences in plasma metabolites were detected due to diet composition or genotype. The diet with the lower energy and crude protein levels reached the lowest slaughter yield but the highest drumstick and wing percentages. The lowest mortality percentages were observed for broilers fed the LM/LP diet, and Cobb birds appeared to be more sensitive for metabolic disorders resulting in death. It is obvious from this study that different genotypes respond differently to changes in diet composition and therefore have adjusted nutritional requirements.

Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients.

PubMed

Cakir, Evrim; Ozbek, Mustafa; Sahin, Mustafa; Cakal, Erman; Gungunes, Askin; Ginis, Zeynep; Demirci, Taner; Delibasi, Tuncay

2012-12-18

Women with polycystic ovary syndrome (PCOS) have higher risk for cardiovascular disease (CVD). Heart type fatty acid binding protein (HFABP) has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductive-age PCOS women and 28 control subjects. We evaluated anthropometric and metabolic parameters, carotid intima media thickness and HFABP levels in both PCOS patients and control group. Mean fasting insulin, homeostasis model assessment insulin resistance index (HOMA-IR), triglyceride, total cholesterol, low density lipoprotein cholesterol, free testosterone, total testosterone, carotid intima media thickness (CIMT) levels were significantly higher in PCOS patients. Although HFABP levels were higher in PCOS patients, the difference did not reach statistically significant in early age groups. After adjustment for age and body mass index, HFABP level was positive correlated with hsCRP, free testosterone levels, CIMT and HOMA-IR. Heart type free fatty acid binding protein appeared to have an important role in metabolic response and subsequent development of atherosclerosis in insulin resistant, hyperandrogenemic PCOS patients.

Seasonal changes in amino acids, protein and total nitrogen in needles of fertilized Scots pine trees.

PubMed

Näsholm, T; Ericsson, A

1990-09-01

Seasonal changes in amino acids, protein and total nitrogen in needles of 30-year-old, fertilized Scots pine (Pinus sylvestris L.) trees growing in Northern Sweden were investigated over two years in field experiments. The studied plots had been fertilized annually for 17 years with (i) a high level of N, (ii) a medium level of N, or (iii) a medium level of N, P and K. Trees growing on unfertilized plots served as controls. In control trees, glutamine, glutamic acid, gamma-aminobutyric acid, aspartic acid and proline represented 50-70% of the total free amino acids determined. Arginine was present only in low concentrations in control trees throughout the year, but it was usually the most abundant amino acid in fertilized trees. Glutamine concentrations were high during the spring and summer in both years of study, whereas proline concentrations were high in the spring but otherwise low throughout the year. In the first year of study, glutamic acid concentrations were high during the spring and summer, whereas gamma-aminobutyric acid was present in high concentrations during the winter months. This pattern was less pronounced in the second year of investigation. The concentrations of most amino acids, except glutamic acid, increased in response to fertilization. Nitrogen fertilization increased the foliar concentration of arginine from < 1 micromol g(dw) (-1) in control trees to a maximum of 110 micromol g(dw) (-1). Trees fertilized with nitrogen, phosphorus and potassium had significantly lower arginine concentrations than trees fertilized with the same amount of nitrogen only. Protein concentrations were similar in all fertilized trees but higher than those in control trees. For all treatments, protein concentrations were high in winter and at a minimum in early spring. In summer, the protein concentration remained almost constant except for a temporary decrease which coincided with the expansion of new shoots. Apart from arginine, the amino acid composition of proteins was similar in all treatments.

Mechanisms of Translation Control Underlying Long-lasting Synaptic Plasticity and the Consolidation of Long-term Memory

PubMed Central

Santini, Emanuela; Huynh, Thu N.; Klann, Eric

2018-01-01

The complexity of memory formation and its persistence is a phenomenon that has been studied intensely for centuries. Memory exists in many forms and is stored in various brain regions. Generally speaking, memories are reorganized into broadly distributed cortical networks over time through systems level consolidation. At the cellular level, storage of information is believed to initially occur via altered synaptic strength by processes such as long-term potentiation (LTP). New protein synthesis is required for long-lasting synaptic plasticity as well as for the formation of long-term memory. The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cap-dependent protein synthesis and is required for numerous forms of long-lasting synaptic plasticity and long-term memory. As such, the study of mTORC1 and protein factors that control translation initiation and elongation have enhanced our understanding of how the process of protein synthesis is regulated during memory formation. Herein we will discuss the molecular mechanisms that regulate protein synthesis as well as pharmacological and genetic manipulations that demonstrate the requirement for proper translational control in long-lasting synaptic plasticity and long-term memory formation. PMID:24484700

THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS

PubMed Central

A, Güllü; S, Dede

2016-01-01

Background: TQ has been used as treatment and preventive agent for many diseases over the years. The goal of this study was to investigate the effects of TQ supplement on fractions of serum proteins. Materials and methods: Fourteen male Wistar-Albino rats (200-250 g weight) were used as material for two groups; (control (C) and thymoquinone (TQ) respectively. Each group contained seven rats. The control group had only corn oil, while the TQ group was dissolved in corn oil. 30 mg/kg/day were given by oral gavage for four weeks. The serum protein fractions were identified using cellulose acetate technique. Results: The total protein level and albumin, α-1, α-2 fractions and A/G ratio have showed no difference between groups (p>0.05). β-globulin fractions of TQ group were higher than control’s (p<0.05). In addition, it was observed that the γ-globulin levels of TQ group were lower than that of the control group’s (p<0.05). Conclusion: From the results, it was observed that the changes of these fractions may have originated from elevation or decline synthesis, or activities of containing proteins. PMID:28480357

Proteomic responses of fruits to environmental stresses

PubMed Central

Chan, Zhulong

2012-01-01

Fruits and vegetables are extremely susceptible to decay and easily lose commercial value after harvest. Different strategies have been developed to control postharvest decay and prevent quality deterioration during postharvest storage, including cold storage, controlled atmosphere (CA), and application of biotic and abiotic stimulus. In this review, mechanisms related to protein level responses of host side and pathogen side were characterized. Protein extraction protocols have been successfully developed for recalcitrant, low protein content fruit tissues. Comparative proteome profiling and functional analysis revealed that defense related proteins, energy metabolism, and antioxidant pathway played important roles in fruits in response to storage conditions and exogenous elicitor treatments. Secretome of pathogenic fungi has been well-investigated and the results indicated that hydrolytic enzymes were the key virulent factors for the pathogen infection. These protein level changes shed new light on interaction among fruits, pathogens, and environmental conditions. Potential postharvest strategies to reduce risk of fruit decay were further proposed based on currently available proteomic data. PMID:23335934

Proteomic analysis of high yield rice variety mutated from spaceflight

NASA Astrophysics Data System (ADS)

Ma, Y.; Cheng, Z.; Wang, W.; Sun, Y.

Seeds of pure rice varieties were flown on Chinese recoverable satellite, JB-1, for a 15-day flight in 1996. Many mutant rice varieties with various phenotypes were generated after continuous selection and breeding. Among the mutants, a variety 971-5 showed a significant increase in grain yield compared to its control (971ck). In this study, proteomic analysis of both mutant variety 971-5 and control variety 971ck were carried out to investigate the changes of protein expression level in their leaves at three different growth stages (early and middle stage of tillering, and booting stage). Results showed that (1) almost all differentially expressed proteins were down-regulated in 971-5 with only one exception, (2) the percentages of differentially expressed proteins were 3.1%, 2.1% and 3.1% at the three stages, respectively, and (3) one protein showed a significant alteration in its molecular weight (MW). These data demonstrated that the space environment can alter the expression level of rice proteins both quantitatively and qualitatively.

The regulated in development and DNA damage response 2 (REDD2) gene mediates human monocyte cell death through a reduction in thioredoxin-1 expression.

PubMed

Imen, Jguirim-Souissi; Billiet, Ludivine; Cuaz-Pérolin, Clarisse; Michaud, Nadège; Rouis, Mustapha

2009-05-15

In a previous study, we identified the regulated in development and DNA damage response 2 (REDD2) gene as a highly expressed gene in human atherosclerotic lesions in comparison to normal artery, as well as in cultured human macrophages, and showed its implication in oxidized low-density lipoprotein (LDL)-induced macrophage death sensitivity. In this article, we attempt to identify the mechanism by which REDD2 induces such a phenomenon. Transient transfection of U-937 monocytic cells with a pCI.CMV.REDD2 expression vector increased by approximately twofold the mRNA levels of REDD2 in comparison to control cells transfected with pCI.CMV.GFP. Reactive oxygen species (ROS) production was significantly induced in REDD2-transfected cells compared with control cells (157+/-48 and 100+/-8 arbitrary units/mg cell protein, respectively; p<0.05). Moreover, a significant increase in parameters known to reflect the oxidative modifications of LDL was observed. Among enzymes involved in ROS production or degradation, we found a specific reduction in thioredoxin-1 (Trx-1) mRNA ( approximately 52+/-7% decrease, p<0.01 vs control cells) and protein ( approximately 60+/-4% decrease, p<0.001 vs control cells) levels in cells overexpressing REDD2 in comparison to control cells. In contrast, transfection of U-937 cells with siRNA against REDD2 decreased the mRNA levels of REDD2 by approximately 60% and increased Trx-1 mRNA and protein levels. Moreover, we observed no or a moderate increase in Bax (proapoptotic) and a significant decrease in Bcl2 (antiapoptotic) gene expression in cells that overexpress REDD2 compared to control cells. In addition, we showed that Trx-1 mRNA and protein levels were increased at low H(2)O(2) doses and decreased at higher doses. Interestingly, macrophages isolated from human atherosclerotic lesions differentially express REDD2 and Trx-1. Indeed, in certain patients, levels of REDD2 mRNA were low and those of Trx-1 mRNA were high. In contrast, in other patients, levels of REDD2 were high and levels of Trx-1 mRNA were low.

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis

PubMed Central

Rojas-Rivera, D; Armisén, R; Colombo, A; Martínez, G; Eguiguren, A L; Díaz, A; Kiviluoto, S; Rodríguez, D; Patron, M; Rizzuto, R; Bultynck, G; Concha, M L; Sierralta, J; Stutzin, A; Hetz, C

2012-01-01

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane. PMID:22240901

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

PubMed

Rojas-Rivera, D; Armisén, R; Colombo, A; Martínez, G; Eguiguren, A L; Díaz, A; Kiviluoto, S; Rodríguez, D; Patron, M; Rizzuto, R; Bultynck, G; Concha, M L; Sierralta, J; Stutzin, A; Hetz, C

2012-06-01

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

Early Response of Protein Quality Control in Gills Is Associated with Survival of Hypertonic Shock in Mozambique tilapia

PubMed Central

Tang, Cheng-Hao; Lee, Tsung-Han

2013-01-01

The protein quality control (PQC) mechanism is essential for cell function and viability. PQC with proper biological function depends on molecular chaperones and proteases. The hypertonicity-induced protein damage and responses of PQC mechanism in aquatic organisms, however, are poorly understood. In this study, we examine the short-term effects of different hypertonic shocks on the levels of heat shock proteins (HSPs, e.g., HSP70 and HSP90), ubiquitin-conjugated proteins and protein aggregation in gills of the Mozambique tilapia (Oreochromis mossambicus). Following transfer from fresh water (FW) to 20‰ hypertonicity, all examined individuals survived to the end of experiment. Moreover, the levels of branchial HSPs and ubiquitin-conjugated proteins significantly increased at 3 and 24 h post-transfer, respectively. Up-regulation of HSPs and ubiquitin-conjugated proteins was sufficient to prevent the accumulation of aggregated proteins. However, the survival rate of tilapia dramatically declined at 5 h and all fish died within 7 h after direct transfer to 30‰ hypertonicity. We presumed that this result was due to the failed activation of gill PQC system, which resulted in elevating the levels of aggregated proteins at 3 and 4 h. Furthermore, in aggregated protein fractions, the amounts of gill Na+/K+-ATPase (NKA) remained relatively low when fish were transferred to 20‰ hypertonicity, whereas abundant NKA was found at 4 h post-transfer to 30‰ hypertonicity. This study demonstrated that the response of PQC in gills is earlier than observable changes in localization of ion-secreting transport proteins upon hypertonic challenge. To our knowledge, this is the first study to investigate the regulation of PQC mechanism in fish and characterize its important role in euryhaline teleost survival in response to hypertonic stress. PMID:23690986

Activated protein C plays no major roles in the inhibition of coagulation or increased fibrinolysis in acute coagulopathy of trauma-shock: a systematic review.

PubMed

Gando, Satoshi; Mayumi, Toshihiko; Ukai, Tomohiko

2018-01-01

The pathophysiological mechanisms of acute coagulopathy of trauma-shock (ACOTS) are reported to include activated protein C-mediated suppression of thrombin generation via the proteolytic inactivation of activated Factor V (FVa) and FVIIIa; an increased fibrinolysis via neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C. The aims of this study are to review the evidences for the role of activated protein C in thrombin generation and fibrinolysis and to validate the diagnosis of ACOTS based on the activated protein C dynamics. We conducted systematic literature search (2007-2017) using PubMed, the Cochrane Database of Systematic Reviews (CDSR), and the Cochrane Central Register of Controlled Trials (CENTRAL). Clinical studies on trauma that measured activated protein C or the circulating levels of activated protein C-related coagulation and fibrinolysis markers were included in our study. Out of 7613 studies, 17 clinical studies met the inclusion criteria. The levels of activated protein C in ACOTS were inconsistently decreased, showed no change, or were increased in comparison to the control groups. Irrespective of the activated protein C levels, thrombin generation was always preserved or highly elevated. There was no report on the activated protein C-mediated neutralization of PAI-1 with increased fibrinolysis. No included studies used unified diagnostic criteria to diagnose ACOTS and those studies also used different terms to refer to the condition known as ACOTS. None of the studies showed direct cause and effect relationships between activated protein C and the suppression of coagulation and increased fibrinolysis. No definitive diagnostic criteria or unified terminology have been established for ACOTS based on the activated protein C dynamics.

Changes of testicular phosphorylated proteins in response to restraint stress in male rats*

PubMed Central

Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Uabundit, Nongnut; Iamsaard, Sitthichai

2016-01-01

Objective: To investigate male reproductive parameters via changes of potential testicular protein markers in restraint-stress rats. Methods: Male Sprague-Dawley rats were divided into two groups (non-immobilized control and restraint-immobilized/stress groups, n=8 each group). The stress animals were immobilized (12 h/d) by a restraint cage for 7 consecutive days. All reproductive parameters, morphology and histology were observed and compared between groups. In addition, the expression of steroidogenic acute regulatory (StAR) and phosphotyrosine proteins (previously localized in Sertoli and late spermatid cells) in testicular lysate was assayed by immuno-Western blotting. Results: Testosterone level, sperm concentration and sperm head normality of stress rats were significantly decreased while the corticosterone level was increased as compared with the control (P<0.05). Histologically, stress rats showed low sperm mass in epididymal lumen and some atrophy of seminiferous tubules. Although the expression of testicular StAR protein was not significantly different between groups, changed patterns of the 131, 95, and 75 kDa testicular phosphorylated proteins were observed in the stress group compared with the control group. The intensity of a testicular 95-kDa phosphorylated protein was significantly decreased in stress rats. Conclusions: This study has demonstrated the alteration of testicular phosphorylated protein patterns, associated with adverse male reproductive parameters in stress rats. It could be an explanation of some infertility in stress males. PMID:26739523

Increased Circulating Levels of Vitamin D Binding Protein in MS Patients

PubMed Central

Rinaldi, Arturo Ottavio; Sanseverino, Isabella; Purificato, Cristina; Cortese, Antonio; Mechelli, Rosella; Francisci, Silvia; Salvetti, Marco; Millefiorini, Enrico; Gessani, Sandra; Gauzzi, Maria Cristina

2015-01-01

Vitamin D (vitD) low status is currently considered a main environmental factor in multiple sclerosis (MS) etiology and pathogenesis. VitD and its metabolites are highly hydrophobic and circulate mostly bound to the vitamin D binding protein (DBP) and with lower affinity to albumin, while less than 1% are in a free form. The aim of this study was to investigate whether the circulating levels of either of the two vitD plasma carriers and/or their relationship are altered in MS. We measured DBP and albumin plasma levels in 28 MS patients and 24 healthy controls. MS patients were found to have higher DBP levels than healthy subjects. Concomitant interferon beta therapy did not influence DBP concentration, and the difference with the control group was significant in both females and males. No significant correlation between DBP and albumin levels was observed either in healthy controls or in patients. These observations suggest the involvement of DBP in the patho-physiology of MS. PMID:25590278

Severe Protein-Calorie Malnutrition and Cognitive Development in Infancy and Early Childhood

ERIC Educational Resources Information Center

Brockman, Lois M.; Ricciuti, Henry N.

1971-01-01

Following nutritional recovery from severe protein-calorie deficiency, 20 young children evidenced a retarded level of categorization behavior compared to a control group of 19 adequately nourished children from similar socioeconomic background. (NH)

Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

PubMed

Moir, R D; Lynch, T; Bush, A I; Whyte, S; Henry, A; Portbury, S; Multhaup, G; Small, D H; Tanzi, R E; Beyreuther, K; Masters, C L

1998-02-27

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

The use of dissolved oxygen-controlled, fed-batch aerobic cultivation for recombinant protein subunit vaccine manufacturing.

PubMed

Farrell, Patrick; Sun, Jacob; Champagne, Paul-Philippe; Lau, Heron; Gao, Meg; Sun, Hong; Zeiser, Arno; D'Amore, Tony

2015-11-27

A simple "off-the-shelf" fed-batch approach to aerobic bacterial cultivation for recombinant protein subunit vaccine manufacturing is presented. In this approach, changes in the dissolved oxygen levels are used to adjust the nutrient feed rate (DO-stat), so that the desired dissolved oxygen level is maintained throughout cultivation. This enables high Escherichia coli cell densities and recombinant protein titers. When coupled to a kLa-matched scale-down model, process performance is shown to be consistent at the 2L, 20L, and 200L scales for two recombinant E. coli strains expressing different protein subunit vaccine candidates. Additionally, by mining historical DO-stat nutrient feeding data, a method to transition from DO-stat to a pre-determined feeding profile suitable for larger manufacturing scales without using feedback control is demonstrated at the 2L, 20L, and 200L scales. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of vinculin as a potential plasma marker for age-related macular degeneration.

PubMed

Kim, Hye-Jung; Woo, Se Joon; Suh, Eui Jin; Ahn, Jeeyun; Park, Ji Hyun; Hong, Hye Kyoung; Lee, Ji Eun; Ahn, Seong Joon; Hwang, Duck Jin; Kim, Ki Woong; Park, Kyu Hyung; Lee, Cheolju

2014-10-08

To identify plasma protein biomarkers for age-related macular degeneration (AMD) using a large-scale quantitative proteomic discovery procedure. Plasma proteomes from 20 exudative AMD patients and 20 healthy control patients were comparatively profiled by four-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins existing at statistically different levels were validated by enzyme-linked immunosorbent assay (ELISA) and Western blotting in 233 case-controlled samples. Newly discovered plasma biomarkers were further confirmed using in vivo and in vitro experiments. Out of 320 proteins identified, vinculin, protein S100A9, triosephosphate isomerase, protein S100A8, protein Z-dependent protease inhibitor, C-X-C motif chemokine 7, and tenascin X showed significantly differential expression in AMD patient plasma compared to control plasma. Among these, the area under the curve (AUC) for vinculin was 0.871 for discriminating between exudative AMD and controls (n = 201) and 0.879 for discriminating between AMD and controls (n = 233). A proteogenomic combination model using vinculin and two known risk genotypes in ARMS2 and CFH genes additionally provided excellent discrimination of AMD from controls (AUC = 0.916). The plasma level of vinculin was not associated with any confounding clinical variables, such as age, smoking, and other comorbidities. Additionally, vinculin was strongly expressed in retinal pigment epithelial cells of human eyes, and its expression was elevated when exposed to oxidative stress in vitro. Vinculin was identified as a potential plasma biomarker for AMD. The early detection of AMD using novel plasma biomarkers with genetic modeling may enable timely treatment and vision preservation in the elderly. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

S100β is associated with cognitive impairment in childhood-onset systemic lupus erythematosus patients.

PubMed

Lapa, A T; Postal, M; Sinicato, N A; Bellini, B S; Fernandes, P T; Marini, R; Appenzeller, S

2017-04-01

Objective To investigate serologic S100β protein levels in childhood-onset SLE patients (cSLE) and to elucidate their association with disease activity and neuropsychiatric (NP) manifestations. Methods We included 71 cSLE patients (67 females; median age 18 years; range 9-37 and 53 (47 females; median age of 20 years; range 6-29) age and sex matched healthy controls. Neurological manifestations were analysed according to the American College of Rheumatology (ACR) criteria. Cognitive evaluation was performed in all participants using Wechsler Intelligence Scale for Children (WISC-III) and Wechsler Adult Intelligence Scale (WAIS), according to age, and validated in Portuguese. SLE patients were further assessed for clinical and laboratory SLE manifestations, disease activity (SLE Disease Activity Index (SLEDAI)), damage (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI)) and current drug exposures. Sera S100β protein levels were measured by enzyme-linked immunosorbent assay using commercial kits. Results The median S100β protein level was 116.55 pg/mL (range 1.53-468.50) in cSLE and 54.98 pg/mL (range 0.69-181.00) in healthy controls ( p < 0.001). An association was observed between S100β protein and NP manifestations ( p = 0.03). The S100β protein levels was associated with cognitive impairment in cSLE patients ( p = 0.006). Conclusions S100β protein levels are increased in cSLE with cognitive impairment. S100β may be considered a potential biomarker that underlies central nervous system (CNS) dysfunction, especially cognitive impairment.

[The regulation mechanism of protein kinase Cδ on arsenic liver injury caused by coal-burning].

PubMed

Hu, Yong; Zhang, Ai-hua; Yao, Mao-lin; Tang, Xu-dong; Huang, Xiao-xin

2013-09-01

To investigate the effects of mRNA transcriptional and protein expressions of protein kinase Cδ (PKCδ) on the development of arsenic liver injury caused by coal-burning. Population study:133 arsenic exposures were selected as arsenic exposure groups including the ward non-patient group (25 cases) , no obvious hepatopathy group (38 cases) , mild (43 cases) and moderate to severe hepatopathy group (27 cases) from the area with endemic arsenism in Guizhou province. Another 34 healthy residents were selected as the control group in non-arsenic pollution village. The urine and peripheral blood were collected from the subjects. The arsenic contents in urine and mRNA expressions of PKCδ in peripheral blood were detected. Animal experiment study:thirty wistar rats were randomly by random number table divided into control group, drinking water arsenic poisoning group and coal-burning arsenic poisoning group (i.e., low, medium and high arsenic contaminated grain group) by random number table method, including 6 rats in each group. The control group was fed normally for 3 months, drinking water arsenic poisoning group and coal-burning arsenic poisoning groups were fed respectively with 10 mg/kg As2O3 solution and different concentrations (25, 50 and 100 mg/kg) of arsenic-containing feed which was persisted 3 months. The arsenic contents in urine, mRNA expression levels of PKCδ in peripheral blood and liver tissue and the protein expression levels of phosphorylated protein kinase Cδ(pPKCδ) in liver tissue were detected. The median(quartile) of arsenic contents in urine were 25.58 (18.62-40.73), 56.66 (38.93-76.77), 64.90 (39.55- 98.37) and 75.47 (41.30-109.70) µg/g Cr respectively for the non-patient group, no obvious hepatopathy group, mild and moderate to severe hepatopathy group. The levels were higher than that in the control group (23.34 (17.84-37.45) µg/g Cr) (P < 0.05), except for the ward non-patient group. The arsenic contents in rat urine were 2223.61 (472.98-3976.73), 701.16 (194.01-1300.27), 1060.94 (246.33-2585.47) and 3101.11 (1919.97-5407.07) µg/g Cr, respectively for the drinking water arsenic poisoning group, the low, medium and high dosage arsenic grain contamination groups, all higher than that in the control group (94.32 (22.65-195.25) µg/g Cr) (P < 0.05) . The protein expressions of pPKCδ in liver tissue were 324.83 ± 25.06, 278.50 ± 30.57, 308.83 ± 34.67 and 326.33 ± 35.09, which were significantly higher than that in the control group (240.17 ± 28.07) (P < 0.05) . The protein expression levels of pPKCδ in liver cell membrane were 0.49 ± 0.06,0.33 ± 0.05,0.37 ± 0.06 and 0.50 ± 0.08, which were significantly higher than that in the control group (0.28 ± 0.04) (P < 0.05) . The protein expression levels of pPKCδ in liver cell cytoplasm were 0.38 ± 0.06,0.31 ± 0.05, 0.35 ± 0.05 and 0.36 ± 0.05, which were significantly higher than that in the control group (0.24 ± 0.05) (P < 0.05). The arsenic may regulate protein expressions of pPKCδ and induce its membrane translocation, and cause the development of arsenic liver injury caused by coal-burning.

Light-regulated protein and mRNA synthesis in root caps of maize

NASA Technical Reports Server (NTRS)

Feldman, L. J.; Piechulla, B.; Sun, P. S.

1988-01-01

Illumination of maize roots initiates changes in mRNA levels and in the activities of proteins within the root cap. Using Northern analysis we showed a 5-6 fold increase in the levels of three specific mRNAs and a 14-fold increase in plastid mRNA. This increase is rapid, occurring within 30 minutes of illumination. With prolonged periods of darkness following illumination, messages return to levels observed in dark, control caps. For two species of mRNA illumination results in a reduction in message levels. Light-stimulated increases in the levels of specific mRNAs are proportionally greater than are increases in the activities of corresponding proteins. We suggest that the light-stimulated increase in protein activity in root caps may be preceded by and occur as a consequence of enhanced levels of mRNA. Our work suggests that photomorphogenesis in roots could involve changes in the levels of a wide variety of mRNAs within the root cap.

Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

PubMed

Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

2002-08-01

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Deteriorated glucose metabolism with a high-protein, low-carbohydrate diet in db mice, an animal model of type 2 diabetes, might be caused by insufficient insulin secretion.

PubMed

Arimura, Emi; Pulong, Wijang Pralampita; Marchianti, Ancah Caesarina Novi; Nakakuma, Miwa; Abe, Masaharu; Ushikai, Miharu; Horiuchi, Masahisa

2017-02-01

We previously showed the deleterious effects of increased dietary protein on renal manifestations and glucose metabolism in leptin receptor-deficient (db) mice. Here, we further examined its effects on glucose metabolism, including urinary C-peptide. We also orally administered mixtures corresponding to low- or high-protein diets to diabetic mice. In diet experiments, under pair-feeding (equivalent energy and fat) conditions using a metabolic cage, mice were fed diets with different protein content (L diet: 12 % protein, 71 % carbohydrate, 17 % fat; H diet: 24 % protein, 59 % carbohydrate, 17 % fat) for 15 days. In oral administration experiments, the respective mixtures (L mixture: 12 % proline, 71 % maltose or starch, 17 % linoleic acid; H mixture: 24 % proline, 59 % maltose or starch, 17 % linoleic acid) were supplied to mice. Biochemical parameters related to glucose metabolism were measured. The db-H diet mice showed significantly higher water intake, urinary volume, and glucose levels than db-L diet mice but similar levels of excreted urinary C-peptide. In contrast, control-H diet mice showed significantly higher C-peptide excretion than control-L diet mice. Both types of mice fed H diet excreted high levels of urinary albumin. When maltose mixtures were administered, db-L mixture mice showed significantly higher blood glucose after 30 min than db-H mixture mice. However, db mice administered starch-H mixture showed significantly higher blood glucose 120-300 min post-administration than db-L mixture mice, although both groups exhibited similar insulin levels. High-protein, low-carbohydrate diets deteriorated diabetic conditions and were associated with insufficient insulin secretion in db mice. Our findings may have implications for dietary management of diabetic symptoms in human patients.

Association of canine obesity with reduced serum levels of C-reactive protein.

PubMed

Veiga, Angela P M; Price, Christopher A; de Oliveira, Simone T; Dos Santos, Andréa P; Campos, Rómulo; Barbosa, Patricia R; González, Félix H D

2008-03-01

The prevalence of obesity is increasing in dogs as well as in humans. C-reactive protein (CRP) is an important tool for the detection of inflammation and/or early tissue damage and is linked to obesity in humans. The objective of the present study was to determine if serum CRP levels are altered in obese dogs. Fifteen lean (control group) and 16 overweight (obese group) dogs were examined. Blood samples were collected under fasted conditions for serum determination of CRP, glucose, insulin, cholesterol, triglyceride, and fructosamine. Results indicated that obese dogs were insulin resistant because serum insulin and insulin/glucose ratios were higher than in lean dogs (P < or = 0.05). Serum CRP concentrations were lower in obese dogs than in controls (P < or = 0.001). C-reactive protein was negatively correlated with insulin/glucose ratio (R = -0.42) and cholesterol (R = -0.39; P < or = 0.05). Furthermore, levels of cholesterol, triglycerides, and fructosamine were increased in the obese group compared with the control group. Based on these results, it can be postulated that CRP production is inhibited by obesity and insulin resistance in dogs.

Roux-en-Y gastric bypass surgery suppresses hypothalamic PTP1B protein level and alleviates leptin resistance in obese rats

PubMed Central

Liu, Jia-Yu; Mu, Song; Zhang, Shu-Ping; Guo, Wei; Li, Qi-Fu; Xiao, Xiao-Qiu; Zhang, Jun; Wang, Zhi-Hong

2017-01-01

The present study aimed to explore the effect of Roux-en-Y gastric bypass (RYGB) surgery on protein tyrosine phosphatase 1B (PTP1B) expression levels and leptin activity in hypothalami of obese rats. Obese rats induced by a high-fat diet (HFD) that underwent RYGB (n=11) or sham operation (SO, n=9), as well as an obese control cohort (Obese, n=10) and an additional normal-diet group (ND, n=10) were used. Food efficiency was measured at 8 weeks post-operation. Plasma leptin levels were evaluated and hypothalamic protein tyrosine phosphatase 1B (PTP1B) levels and leptin signaling activity were examined at the genetic and protein levels. The results indicated that food efficiency was typically lower in RYGB rats compared with that in the Obese and SO rats. In the RYGB group, leptin receptor expression and proopiomelanocortin was significantly higher, while Neuropeptide Y levels were lower than those in the Obese and SO groups. Furthermore, the gene and protein expression levels of PTP1B in the RYGB group were lower, while levels of phosphorylated signal transducer and activator of transcription 3 protein were much higher compared with those in the Obese and SO groups. In conclusion, RYGB surgery significantly suppressed hypothalamic PTP1B protein expression. PTP1B regulation may partially alleviate leptin resistance. PMID:28947917

Roux-en-Y gastric bypass surgery suppresses hypothalamic PTP1B protein level and alleviates leptin resistance in obese rats.

PubMed

Liu, Jia-Yu; Mu, Song; Zhang, Shu-Ping; Guo, Wei; Li, Qi-Fu; Xiao, Xiao-Qiu; Zhang, Jun; Wang, Zhi-Hong

2017-09-01

The present study aimed to explore the effect of Roux-en-Y gastric bypass (RYGB) surgery on protein tyrosine phosphatase 1B (PTP1B) expression levels and leptin activity in hypothalami of obese rats. Obese rats induced by a high-fat diet (HFD) that underwent RYGB (n=11) or sham operation (SO, n=9), as well as an obese control cohort (Obese, n=10) and an additional normal-diet group (ND, n=10) were used. Food efficiency was measured at 8 weeks post-operation. Plasma leptin levels were evaluated and hypothalamic protein tyrosine phosphatase 1B (PTP1B) levels and leptin signaling activity were examined at the genetic and protein levels. The results indicated that food efficiency was typically lower in RYGB rats compared with that in the Obese and SO rats. In the RYGB group, leptin receptor expression and proopiomelanocortin was significantly higher, while Neuropeptide Y levels were lower than those in the Obese and SO groups. Furthermore, the gene and protein expression levels of PTP1B in the RYGB group were lower, while levels of phosphorylated signal transducer and activator of transcription 3 protein were much higher compared with those in the Obese and SO groups. In conclusion, RYGB surgery significantly suppressed hypothalamic PTP1B protein expression. PTP1B regulation may partially alleviate leptin resistance.

Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule

PubMed Central

Cho, Ukrae; Zimmerman, Stephanie M.; Chen, Ling-chun; Owen, Elliot; Kim, Jesse V.; Kim, Stuart K.; Wandless, Thomas J.

2013-01-01

Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20–25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature. PMID:23991108

Cytotype Control of Drosophila Melanogaster P Element Transposition: Genomic Position Determines Maternal Repression

PubMed Central

Misra, S.; Buratowski, R. M.; Ohkawa, T.; Rio, D. C.

1993-01-01

P element transposition in Drosophila is controlled by the cytotype regulatory state: in P cytotype, transposition is repressed, whereas in M cytotype, transposition can occur. P cytotype is determined by a combination of maternally inherited factors and chromosomal P elements in the zygote. Transformant strains containing single elements that encoded the 66-kD P element protein zygotically repressed transposition, but did not display the maternal repression characteristic of P cytotype. Upon mobilization to new genomic positions, some of these repressor elements showed significant maternal repression of transposition in genetic assays, involving a true maternal effect. Thus, the genomic position of repressor elements can determine the maternal vs. zygotic inheritance of P cytotype. Immunoblotting experiments indicate that this genomic position effect does not operate solely by controlling the expression level of the 66-kD repressor protein during oogenesis. Likewise, P element derivatives containing the hsp26 maternal regulator sequence expressed high levels of the 66-kD protein during oogenesis, but showed no detectable maternal repression. These data suggest that the location of a repressor element in the genome may determine maternal inheritance of P cytotype by a mechanism involving more than the overall level of expression of the 66-kD protein in the ovary. PMID:8293979

Cryptochrome 2 expression level is critical for adrenocorticotropin stimulation of cortisol production in the capuchin monkey adrenal.

PubMed

Torres-Farfan, C; Abarzua-Catalan, L; Valenzuela, F J; Mendez, N; Richter, H G; Valenzuela, G J; Serón-Ferré, M

2009-06-01

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

Differential expression of a novel seven transmembrane domain protein in epididymal fat from aged and diabetic mice.

PubMed

Yang, H; Egan, J M; Rodgers, B D; Bernier, M; Montrose-Rafizadeh, C

1999-06-01

To identify novel seven transmembrane domain proteins from 3T3-L1 adipocytes, we used PCR to amplify 3T3-L1 adipocyte complementary DNA (cDNA) with primers homologous to the N- and C-termini of pancreatic glucagon-like peptide-1 (GLP-1) receptor. We screened a cDNA library prepared from fully differentiated 3T3-L1 adipocytes using a 500-bp cDNA PCR product probe. Herein describes the isolation and characterization of a 1.6-kb cDNA clone that encodes a novel 298-amino acid protein that we termed TPRA40 (transmembrane domain protein of 40 kDa regulated in adipocytes). TPRA40 has seven putative transmembrane domains and shows little homology with the known GLP-1 receptor or with other G protein-coupled receptors. The levels of TPRA40 mRNA and protein were higher in 3T3-L1 adipocytes than in 3T3-L1 fibroblasts. TPRA40 is present in a number of mouse and human tissues. Interestingly, TPRA40 mRNA levels were significantly increased by 2- to 3-fold in epididymal fat of 24-month-old mice vs. young controls as well as in db/db and ob/ob mice vs. nondiabetic control littermates. No difference in TPRA40 mRNA levels was observed in brain, heart, skeletal muscle, liver, or kidney. Furthermore, no difference in TPRA40 expression was detected in brown fat of ob/ob mice when compared with age-matched controls. Taken together, these data suggest that TPRA40 represents a novel membrane-associated protein whose expression in white adipose tissue is altered with aging and type 2 diabetes.

Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.

PubMed

Everman, Sarah; Meyer, Christian; Tran, Lee; Hoffman, Nyssa; Carroll, Chad C; Dedmon, William L; Katsanos, Christos S

2016-10-01

Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P < 0.01) whole body phenylalanine rate of appearance (μmol·kg -1 ·min -1 ), indicating suppression of muscle proteolysis, in both the control (1.02 ± 0.04 vs 0.76 ± 0.04) and the BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA. Copyright © 2016 the American Physiological Society.

Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans

PubMed Central

Everman, Sarah; Meyer, Christian; Tran, Lee; Hoffman, Nyssa; Carroll, Chad C.; Dedmon, William L.

2016-01-01

Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P < 0.01) whole body phenylalanine rate of appearance (μmol·kg−1·min−1), indicating suppression of muscle proteolysis, in both the control (1.02 ± 0.04 vs 0.76 ± 0.04) and the BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA. PMID:27530230

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

PubMed Central

Taylor, Shannon; Sunshine, Sara S.; McCarthy, Kathleen; Risher, Nicole; Newcomb, Tara; Weetall, Marla; Prior, Thomas W.; Swoboda, Kathryn J.; Chen, Karen S.; Paushkin, Sergey

2016-01-01

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy. PMID:26953792

Pregnancy-associated plasma protein-A (PAPP-A) levels in patients with severe allergic asthma are reduced by omalizumab.

PubMed

Bulut, Ismet; Ozseker, Zeynep F; Coskun, Abdurrahman; Serteser, Mustafa; Unsal, Ibrahim

2017-12-06

Remodeling is a crucial feature of severe asthma and may be associated with activation of the allergic cascade by immunoglobulin E (IgE). Omalizumab, an anti-IgE monoclonal antibody, effectively targets the severe allergic asthma phenotype. Pregnancy-associated plasma protein-A (PAPP-A) is an insulin-like growth factor binding protein-4 (IGFBP-4) protease, increasing local insulin-like growth factor (IGF)-1 concentrations, which in turn initiating a cascade involved in the regulation of cell growth, differentiation, and proliferation in various tissues. In the present study, we evaluated the effects of omalizumab on serum PAPP-A, IGFBP-4, and IGF-1 levels in subjects with severe allergic asthma. We studied 36 asthmatic subjects and 36 healthy controls. An ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit was used to measure serum PAPP-A levels, and routine commercial ELISA kits were employed to assess serum levels of IGF-1, IGFBP-4 in control subjects and asthmatic subjects before therapy (baseline) and after six months of omalizumab therapy in patients with severe asthma. Compared to control subjects, serum PAPP-A and IGFB-4 levels were significantly higher in asthmatic subjects (both p values < 0.001). However, the serum IGF-I levels of asthmatic subjects were similar to those of control subjects (p > 0.05). In asthma subjects, 6-month omalizumab treatment significantly decreased the serum PAPP-A (p < 0.001), IGF-I (p = 0.031), and IGFB4 (p = 0.025) levels. PAPP-A level may be a useful biomarker for predicting airway remodeling in patients with severe asthma receiving omalizumab, and may also reflect the response to treatment.

Comparison of serum vaspin levels and vaspin expression in adipose tissue and smooth muscle tissue in pregnant women with and without gestational diabetes.

PubMed

Tang, Yuping; Qiao, Ping; Qu, Xiaoxian; Bao, Yirong; Li, Yuhong; Liao, Yini; Ying, Hao

2017-10-01

Vaspin is associated with metabolic parameters and insulin resistance. However, the expression of vaspin in visceral adipose tissue (VAT) in pregnant women with gestational diabetes mellitus (GDM) has not been fully explored, and the contribution of vaspin to the biological mechanisms underlying GDM remains unclear. This study aimed to compare circulating vaspin levels and its expression in different insulin target tissues including subcutaneous adipose tissue (SAT), VAT and smooth muscle tissue (SMT) in pregnant women with and without GDM. A total of 37 women with GDM (GDM group) and 37 normal pregnant women (control group) were selected. Fasting plasma glucose (FPG), fasting insulin (FINS) and serum vaspin levels were quantified at term, and homeostasis model of assessment2-insulin resistance (HOMA2-IR) values were calculated. RT-qPCR and Western blotting were used to measure mRNA and protein levels of vaspin in VAT, SAT and SMT of 15 GDM women and normal pregnant women. In the GDM group, serum vaspin concentrations were significantly higher than in the control group. Serum vaspin levels were positively correlated with HOMA2-IR in the GDM group but not in the control group. In the GDM group, vaspin mRNA and protein expression levels in SAT and VAT were both significantly higher than in controls, but no difference was found in SMT. Moreover, relative mRNA but not protein expression levels of vaspin in SAT were highest among the three tissues in both groups. Circulating vaspin levels and expression of vaspin in SAT and VAT were higher in GDM women than in normal pregnant women. However, the specific role of vaspin from SAT and VAT in the pathogenesis of GDM needs further study. © 2017 John Wiley & Sons Ltd.

Peripheral leukocyte expression of the potential biomarker proteins Bdnf, Sirt1, and Psen1 is not regulated by promoter methylation in Alzheimer's disease patients.

PubMed

Carboni, Lucia; Lattanzio, Francesca; Candeletti, Sanzio; Porcellini, Elisa; Raschi, Elena; Licastro, Federico; Romualdi, Patrizia

2015-09-25

The identification of Alzheimer's disease (AD) biomarkers is crucial to support drug discovery. Within putative biomarkers, peripheral Bdnf levels correlate with cognitive decline and AD, although conflicting findings are reported. Sirtuin 1 (Sirt1) serum levels are lower in AD patients and Presenilin 1 (Psen1) is expressed by blood cells. DNA methylation is altered in AD patients, suggesting that epigenetic mechanisms play a role in AD pathophysiology. The objective of this study was to investigate promoter methylation levels of potential biomarkers in AD cases and controls. Peripheral blood DNA methylation levels were analysed by methylation-specific primer real-time PCR. Bdnf promoter methylation levels did not differ between AD patients and controls. Similarly, Sirt1 promoter revealed minimal levels of methylation which did not display significant differences between groups. No significant difference was revealed between AD patients and controls also in Psen1 methylation, showing a large variability of values among subjects. Although peripheral Bdnf expression is associated with differential promoter methylation in psychiatric and neurological disorders, our results suggest that different mechanisms take place in AD. The finding that the control of Sirt1 protein levels in blood is not exerted through the repression of mRNA expression by promoter hypermethylation is in agreement with previous data. In contrast, other studies reported that Psen1 methylation may be increased or decreased in AD patients, suggesting that additional studies are required. In conclusion, this study shows that peripheral levels of the potential AD biomarker proteins Bdnf, Sirt1, and Psen1 are not regulated by different promoter methylation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Dendritic cell immunotherapy followed by cART interruption during HIV-1 infection induces plasma protein markers of cellular immunity and neutrophil recruitment

PubMed Central

Cooper, Jason D.; Tomasik, Jakub; Bahn, Sabine; Aerts, Joeri L.; Osterhaus, Albert D. M. E.; Gruters, Rob A.; Andeweg, Arno C.

2018-01-01

Objectives To characterize the host response to dendritic cell-based immunotherapy and subsequent combined antiretroviral therapy (cART) interruption in HIV-1-infected individuals at the plasma protein level. Design An autologous dendritic cell (DC) therapeutic vaccine was administered to HIV-infected individuals, stable on cART. The effect of vaccination was evaluated at the plasma protein level during the period preceding cART interruption, during analytical therapy interruption and at viral reactivation. Healthy controls and post-exposure prophylactically treated healthy individuals were included as controls. Methods Plasma marker (‘analyte’) levels including cytokines, chemokines, growth factors, and hormones were measured in trial participants and control plasma samples using a multiplex immunoassay. Analyte levels were analysed using principle component analysis, cluster analysis and limma. Blood neutrophil counts were analysed using linear regression. Results Plasma analyte levels of HIV-infected individuals are markedly different from those of healthy controls and HIV-negative individuals receiving post-exposure prophylaxis. Viral reactivation following cART interruption also affects multiple analytes, but cART interruption itself only has only a minor effect. We find that Thyroxine-Binding Globulin (TBG) levels and late-stage neutrophil numbers correlate with the time off cART after DC vaccination. Furthermore, analysis shows that cART alters several regulators of blood glucose levels, including C-peptide, chromogranin-A and leptin. HIV reactivation is associated with the upregulation of CXCR3 ligands. Conclusions Chronic HIV infection leads to a change in multiple plasma analyte levels, as does virus reactivation after cART interruption. Furthermore, we find evidence for the involvement of TBG and neutrophils in the response to DC-vaccination in the setting of HIV-infection. PMID:29389978

Dendritic cell immunotherapy followed by cART interruption during HIV-1 infection induces plasma protein markers of cellular immunity and neutrophil recruitment.

PubMed

van den Ham, Henk-Jan; Cooper, Jason D; Tomasik, Jakub; Bahn, Sabine; Aerts, Joeri L; Osterhaus, Albert D M E; Gruters, Rob A; Andeweg, Arno C

2018-01-01

To characterize the host response to dendritic cell-based immunotherapy and subsequent combined antiretroviral therapy (cART) interruption in HIV-1-infected individuals at the plasma protein level. An autologous dendritic cell (DC) therapeutic vaccine was administered to HIV-infected individuals, stable on cART. The effect of vaccination was evaluated at the plasma protein level during the period preceding cART interruption, during analytical therapy interruption and at viral reactivation. Healthy controls and post-exposure prophylactically treated healthy individuals were included as controls. Plasma marker ('analyte') levels including cytokines, chemokines, growth factors, and hormones were measured in trial participants and control plasma samples using a multiplex immunoassay. Analyte levels were analysed using principle component analysis, cluster analysis and limma. Blood neutrophil counts were analysed using linear regression. Plasma analyte levels of HIV-infected individuals are markedly different from those of healthy controls and HIV-negative individuals receiving post-exposure prophylaxis. Viral reactivation following cART interruption also affects multiple analytes, but cART interruption itself only has only a minor effect. We find that Thyroxine-Binding Globulin (TBG) levels and late-stage neutrophil numbers correlate with the time off cART after DC vaccination. Furthermore, analysis shows that cART alters several regulators of blood glucose levels, including C-peptide, chromogranin-A and leptin. HIV reactivation is associated with the upregulation of CXCR3 ligands. Chronic HIV infection leads to a change in multiple plasma analyte levels, as does virus reactivation after cART interruption. Furthermore, we find evidence for the involvement of TBG and neutrophils in the response to DC-vaccination in the setting of HIV-infection.

Opsin1-2, G(q)α and arrestin levels at Limulus rhabdoms are controlled by diurnal light and a circadian clock.

PubMed

Battelle, Barbara-Anne; Kempler, Karen E; Parker, Alexander K; Gaddie, Cristina D

2013-05-15

Dark and light adaptation in photoreceptors involve multiple processes including those that change protein concentrations at photosensitive membranes. Light- and dark-adaptive changes in protein levels at rhabdoms have been described in detail in white-eyed Drosophila maintained under artificial light. Here we tested whether protein levels at rhabdoms change significantly in the highly pigmented lateral eyes of wild-caught Limulus polyphemus maintained in natural diurnal illumination and whether these changes are under circadian control. We found that rhabdomeral levels of opsins (Ops1-2), the G protein activated by rhodopsin (G(q)α) and arrestin change significantly from day to night and that nighttime levels of each protein at rhabdoms are significantly influenced by signals from the animal's central circadian clock. Clock input at night increases Ops1-2 and G(q)α and decreases arrestin levels at rhabdoms. Clock input is also required for a rapid decrease in rhabdomeral Ops1-2 beginning at sunrise. We found further that dark adaptation during the day and the night are not equivalent. During daytime dark adaptation, when clock input is silent, the increase of Ops1-2 at rhabdoms is small and G(q)α levels do not increase. However, increases in Ops1-2 and G(q)α at rhabdoms are enhanced during daytime dark adaptation by treatments that elevate cAMP in photoreceptors, suggesting that the clock influences dark-adaptive increases in Ops1-2 and G(q)α at Limulus rhabdoms by activating cAMP-dependent processes. The circadian regulation of Ops1-2 and G(q)α levels at rhabdoms probably has a dual role: to increase retinal sensitivity at night and to protect photoreceptors from light damage during the day.

Sex steroid-induced changes in circulating monocyte chemoattractant protein-1 levels may contribute to metabolic dysfunction in obese men.

PubMed

Ruige, Johannes B; Bekaert, Marlies; Lapauw, Bruno; Fiers, Tom; Lehr, Stefan; Hartwig, Sonja; Herzfeld de Wiza, Daniella; Schiller, Martina; Passlack, Waltraud; Van Nieuwenhove, Yves; Pattyn, Piet; Cuvelier, Claude; Taes, Youri E; Sell, Henrike; Eckel, Juergen; Kaufman, Jean-Marc; Ouwens, D Margriet

2012-07-01

Low testosterone accompanied by elevated estradiol associates with the development of metabolic dysfunction in men. The aim of the study was to explore the hypothesis that alterations in sex steroid levels induce metabolic dysfunction through adipokines. Circulating levels of sex steroids and 28 adipokines were determined in a cross-sectional study of morbidly obese men and aged-matched controls, as well as in a randomized clinical trial with healthy young men in which obesity-related alterations in sex steroid levels were mimicked by treatment with an aromatase inhibitor plus estradiol patches. Morbidly obese men had lower testosterone levels than normal-weight controls. Estradiol levels were increased in morbidly obese men (without DM2) as compared to normal-weight controls. Circulating levels of multiple proinflammatory cytokines, including IL-1Ra, IL-5, IL-6, IL-10, leptin, monocyte chemoattractant protein 1 (MCP1), and macrophage inflammatory protein 1α, positively associated with estradiol and negatively with testosterone. The associations with estradiol, but not with testosterone, remained significant after adjusting for adipocyte cell size. In a separate clinical trial, the direct adverse effects of lowering testosterone and raising estradiol on MCP1 were substantiated in vivo. Initial alterations in sex steroid levels may contribute to metabolic dysfunction through adverse effects on adipokine levels in obese men. The direct adverse effects on MCP1, a chemokine highly linked to the development of metabolic dysfunction, were substantiated in a trial mimicking obesity-related alterations of sex steroid levels in healthy young males.

Myelin protein zero and its antibody in serum as biomarkers of n-hexane-induced peripheral neuropathy and neurotoxicity effects.

PubMed

Jia, Xiaowei; Liu, Qingjun; Zhang, Yanshu; Dai, Yufei; Duan, Huawei; Bin, Ping; Niu, Yong; Liu, Jie; Zhong, Liuzhen; Guo, Jisheng; Liu, Xiaofeng; Zheng, Yuxin

2014-01-01

Chronic exposure to n-hexane can lead to peripheral neuropathy that no effective treatment regimen could be applied presently. This study investigated whether myelin protein zero (P0) protein and its antibody could be used to distinguish n-hexane intoxication and protect workers from peripheral neuropathy. We compared P0 protein and its antibody among three levels of n-hexane-exposed groups, which included 18 patients with n-hexane-induced peripheral neuropathy as case group, 120 n-hexane-exposed workers as n-hexaneexposed control group, and 147 non-hexane-exposed participants used as control group. ELISA method was applied to detect P0 protein and its antibody. P0 protein in serum was significantly higher in the case group and n-hexane-exposed control group in comparison with the control group (P < 0.01). Compared with the n-hexane-exposed control group, the case group also had significant increase of P0 protein (P < 0.01). After 6 months therapy, P0 protein was observed to decrease significantly in the case group (P < 0.01). The P0 antibody in serum was significantly higher in the n-hexane-exposed control group than in the control group (P < 0.01), but not significantly different between cases and controls. P0 antibodies in serum may be a short-term effect biomarker for n-hexane exposure. P0 protein in serum may be an early effective biomarker for peripheral nerve neuropathy and its biological limit value needs investigation in the future study.

Nuclear AgNOR protein enhancement in nucleoplasms of peripheral blood lymphocytes of babies/children with Down syndrome.

PubMed

Imamoglu, Nalan; Eroz, Recep; Canatan, Halit; Demirtas, Halil; Saatci, Çetin

2016-03-01

Down syndrome (DS) is one of the most common chromosomal disorders. The factors contributing to the mental retardation together with other defects in this syndrome have not been fully explained. Individuals with DS have extra rRNA gene family since they carry an extra chromosome 21. The few reports available are on the relationship between the nucleolus organizer regions (NORs) and DS phenotype. The in vivo regulation of NORs expression on the extra chromosome 21 is not completely understood. Previous studies have shown that nucleoli of lymphocytes from infants (mostly neonates) with DS contain more in vivo and in vitro nucleolar AgNOR proteins when compared with healthy infants. The objective of this study is to compare the in vivo nuclear AgNOR protein level in nucleoplasms (also called as karyoplasm) of nonstimulated peripheral blood lymphocytes from babies/children with DS and healthy controls. Peripheral blood samples obtained from 20 babies/children with DS and 20 matched healthy controls were smeared on clean glass slides and then AgNOR staining was performed. The AgNOR protein level in nucleoplasms of lymphocytes from both groups was calculated using a computer program. Nearly 100 interphase nuclei per individual were analysed. Average nuclear AgNOR protein levels in nucleoplasms of lymphocytes from babies/children with DS were found to be significantly higher than those of the controls (P < 0.001). On the basis of our present results, we propose that the increase of nuclear AgNOR protein in in vivo conditions may contribute to the formation of DS phenotypes. © 2016 Wiley Periodicals, Inc.

Nitrosative stress in human skeletal muscle attenuated by exercise countermeasure after chronic disuse.

PubMed

Salanova, Michele; Schiffl, Gudrun; Gutsmann, Martina; Felsenberg, Dieter; Furlan, Sandra; Volpe, Pompeo; Clarke, Andrew; Blottner, Dieter

2013-01-01

Activity-induced nitric oxide (NO) imbalance and "nitrosative stress" are proposed mechanisms of disrupted Ca(2+) homeostasis in atrophic skeletal muscle. We thus mapped S-nitrosylated (SNO) functional muscle proteins in healthy male subjects in a long-term bed rest study (BBR2-2 Study) without and with exercise as countermeasure in order to assess (i) the negative effects of chronic muscle disuse by nitrosative stress, (ii) to test for possible attenuation by exercise countermeasure in bed rest and (iii) to identify new NO target proteins. Muscle biopsies from calf soleus and hip vastus lateralis were harvested at start (Pre) and at end (End) from a bed rest disuse control group (CTR, n=9) and two bed rest resistive exercise groups either without (RE, n=7) or with superimposed vibration stimuli (RVE, n=7). At subcellular compartments, strong anti-SNO-Cys immunofluorescence patterns in control muscle fibers after bed rest returned to baseline following vibration exercise. Total SNO-protein levels, Nrf-2 gene expression and nucleocytoplasmic shuttling were changed to varying degrees in all groups. Excess SNO-protein levels of specific calcium release/uptake proteins (SNO-RyR1, -SERCA1 and -PMCA) and of contractile myosin heavy chains seen in biopsy samples of chronically disused skeletal muscle were largely reduced by vibration exercise. We also identified NOS1 as a novel NO target in human skeletal muscle controlled by activity driven auto-nitrosylation mechanisms. Our findings suggest that aberrant levels of functional SNO-proteins represent signatures of uncontrolled nitrosative stress management in disused human skeletal muscle that can be offset by exercise as countermeasure.

Acute-phase proteins in relation to neuropsychiatric symptoms and use of psychotropic medication in Huntington's disease.

PubMed

Bouwens, J A; Hubers, A A M; van Duijn, E; Cobbaert, C M; Roos, R A C; van der Mast, R C; Giltay, E J

2014-08-01

Activation of the innate immune system has been postulated in the pathogenesis of Huntington's disease (HD). We studied serum concentrations of C-reactive protein (CRP) and low albumin as positive and negative acute-phase proteins in HD. Multivariate linear and logistic regression was used to study the association between acute-phase protein levels in relation to clinical, neuropsychiatric, cognitive, and psychotropic use characteristics in a cohort consisting of 122 HD mutation carriers and 42 controls at first biomarker measurement, and 85 HD mutation carriers and 32 controls at second biomarker measurement. Significant associations were found between acute-phase protein levels and Total Functioning Capacity (TFC) score, severity of apathy, cognitive impairment, and the use of antipsychotics. Interestingly, all significant results with neuropsychiatric symptoms disappeared after additional adjusting for antipsychotic use. High sensitivity CRP levels were highest and albumin levels were lowest in mutation carriers who continuously used antipsychotics during follow-up versus those that had never used antipsychotics (mean difference for CRP 1.4 SE mg/L; P=0.04; mean difference for albumin 3 SE g/L; P<0.001). The associations found between acute-phase proteins and TFC score, apathy, and cognitive impairment could mainly be attributed to the use of antipsychotics. This study provides evidence that HD mutation carriers who use antipsychotics are prone to develop an acute-phase response. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

Glucose ingestion stimulates atherothrombotic inflammation in polycystic ovary syndrome

PubMed Central

Kirwan, John P.; Rote, Neal S.; Minium, Judi

2013-01-01

Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation that can increase the risk of atherothrombosis. We performed a cross-sectional study to examine the effect of glucose ingestion on markers of atherothrombotic inflammation in mononuclear cells (MNC) of 16 women with PCOS (8 lean, 8 obese) and 16 weight-matched controls. Activator protein-1 (AP-1) activation and the protein content of early growth response-1 (EGR-1), matrix matalloproteinases-2 (MMP2), and tissue factor (TF) were quantified from MNC obtained from blood drawn fasting and 2 h after glucose ingestion. Plasma MMP9 and C-reactive protein (CRP) were measured from fasting blood samples. Truncal fat was determined by DEXA. Lean women with PCOS exhibited greater AP-1 activation and MMP2 protein content after glucose ingestion and higher plasma MMP9 and CRP levels than lean controls. Obese women with PCOS exhibited greater EGR-1 and TF protein content after glucose ingestion, and plasma CRP levels were even higher compared with lean subjects regardless of PCOS status. Truncal fat correlated with MMP9 and CRP levels and glucose-stimulated increases in AP-1 activation and EGR-1 and TF protein content. Testosterone correlated with glucose-stimulated AP-1 activation, and androstenedione correlated with MMP9 and CRP levels and glucose-stimulated AP-1 activation. Thus, both PCOS and obesity contribute to an atherothrombotic state in which excess abdominal adiposity and hyperandrogenism may be specific risk factors for developing atherothrombosis. PMID:23249695

Influence of a fermented protein-fortified dairy product on serum insulin-like growth factor-I in women with anorexia nervosa: A randomized controlled trial.

PubMed

Trombetti, Andrea; Carrier, Edouard; Perroud, Alain; Lang, François; Herrmann, François R; Rizzoli, René

2016-10-01

Patients with anorexia nervosa (AN) have low serum IGF-I levels that may contribute to a lower bone mineral mass. We investigated the effects of a fermented, protein-fortified, dairy product on serum IGF-I levels in patients with AN during an in-hospital refeeding program. In this multicenter, randomized, double-blind, placebo-controlled, clinical trial conducted at 3 university hospitals and 3 private clinics in France and Switzerland, 62 women recently admitted with confirmed AN and with a baseline low serum IGF-I level were randomized to 2 daily isocaloric fresh cheese pots containing either 15 g/150 g or 3 g/150 g (controls) of protein for 4 weeks. The primary outcome was the change in IGF-I levels. In the primary intention-to-treat analysis, mean serum IGF-I levels increased during the intervention phase from 22.9 ± 1.5 to 28.6 ± 1.3 nmol/L (means ± SEM) (+20.2%) in the intervention group and from 20.2 ± 1.2 to 25.7 ± 1.5 nmol/L (+16.8%) in controls. In a preplanned analysis of covariance with repeated measures, the between-group difference was close to statistical significance (P = 0.071). In a post-hoc mixed-regression model analysis, the difference was statistically significant (4.9 nmol/l increase; P = 0.003), as was the change of the ratio IGF-I/IGF-BP3 (P=0.004). There was no between-group difference in biochemical markers of bone turnover (osteocalcin, P1NP, CTX) or in serum parathyroid hormone level. Serum calcium levels slightly increased during the intervention phase in the higher protein group (P = 0.02). IGF-BP2 decreased significantly more in the intervention group during the follow up period at week 4 after supplements cessation (P = 0.019). Intake of a fermented, protein-fortified, isocaloric dairy product during 4 weeks may slightly increase serum IGF-I levels in women with AN, without significant changes in bone turnover markers. NCT01823822 (www.clinicaltrials.gov). Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Evaluation of plasma C-reactive protein levels in pregnant women with and without periodontal disease: A comparative study

PubMed Central

Sharma, Anupriya; Ramesh, Amitha; Thomas, Biju

2009-01-01

Background and Objectives: Circulating C-reactive protein (CRP) levels are a marker of systemic inflammation and are associated with periodontal disease, a chronic bacterial infection associated with elevation of proinflammatory cytokines and prostaglandins. CRP has been associated with adverse pregnancy outcomes, including preterm delivery, preeclampsia, and intrauterine growth restriction. Furthermore, periodontal disease has been associated with increased risk of preterm low birth weight, low birth weight, and preterm birth. The present study was conducted to assess plasma CRP levels in pregnant women with and without periodontal disease; to evaluate the effect of periodontal therapy on the incidence of preterm delivery; and to compare the incidence of preterm delivery in pregnant women with and without periodontal disease. Materials and Methods: A total of 90 pregnant women aged between 18-35 years with gestational age between 12-28 weeks were recruited and divided into three equal groups (control group, study group, treatment group) of 30 each. Blood samples were taken for estimation of C-reactive protein levels from all groups at 12-20 weeks of gestation, determined using ultrasensitive turbidimetric immunoassay (QUANTIA-CRP US). The treatment group comprised plaque control, scaling, and root planning and daily rinsing with 0.2% chlorhexidine mouth before 28 weeks of gestation. Results: The mean value of C-reactive protein levels in subjects with periodontal disease was higher compared to control group i.e., 1.20 ± 0.247 mg/dl and 1.22 ± 0.250 mg/dl, respectively, compared to 0.713 ± 0.139 mg/ dl (P = 0.001). The mean value of CRP levels before treatment was greater than the mean value after treatment i.e., 1.22 ± 0.25 compared to 0.84 ± 0.189 (P < 0.001). The incidence of preterm delivery (< 37 weeks) was 31.7% in the periodontal disease group (study group) compared to 8.3% in the control group (P = 0.001). The incidence of preterm delivery in the treatment group was 15.0% compared to 31.7% in the nontreatment group (study group). Conclusion: The findings from the study suggest that periodontal disease in pregnant women is associated with increased C- reactive protein levels in early pregnancy, incidence of preterm delivery is higher in pregnant women with periodontal disease compared to healthy controls, periodontal therapy during pregnancy reduces plasma CRP levels and there is decrease in incidence of preterm delivery after periodontal therapy. PMID:20379412

C-terminal Src kinase (Csk) regulates the tricellular junction protein Gliotactin independent of Src

PubMed Central

Samarasekera, G. D. N. Gayathri; Auld, Vanessa Jane

2018-01-01

Tricellular junctions (TCJs) are uniquely placed permeability barriers formed at the corners of polarized epithelia where tight junctions in vertebrates or septate junctions (SJ) in invertebrates from three cells converge. Gliotactin is a Drosophila TCJ protein, and loss of Gliotactin results in SJ and TCJ breakdown and permeability barrier loss. When overexpressed, Gliotactin spreads away from the TCJs, resulting in disrupted epithelial architecture, including overproliferation, cell delamination, and migration. Gliotactin levels are tightly controlled at the mRNA level and at the protein level through endocytosis and degradation triggered by tyrosine phosphorylation. We identified C-terminal Src kinase (Csk) as a tyrosine kinase responsible for regulating Gliotactin endocytosis. Increased Csk suppresses the Gliotactin overexpression phenotypes by increasing endocytosis. Loss of Csk causes Gliotactin to spread away from the TCJ. Although Csk is known as a negative regulator of Src kinases, the effects of Csk on Gliotactin are independent of Src and likely occur through an adherens junction associated complex. Overall, we identified a new Src-independent role for Csk in the control of Gliotactin, a key tricellular junction protein. PMID:29167383

Congenitally learned helpless rats show abnormalities in intracellular signaling.

PubMed

Kohen, Ruth; Neumaier, John F; Hamblin, Mark W; Edwards, Emmeline

2003-03-15

Affective disorders and the drugs used to treat them lead to changes in intracellular signaling. We used a genetic animal model to investigate to what extent changes in intracellular signal transduction confer a vulnerability to mood or anxiety disorders. Levels of gene expression in a selectively bred strain of rats with a high vulnerability to develop congenitally learned helplessness (cLH), a strain highly resistant to the same behavior (cNLH) and outbred Sprague-Dawley (SD) control animals were compared using quantitative reverse transcription polymerase chain reaction. Congenitally learned helpless animals had a 24%-30% reduced expression of the cyclic adenosine monophosphate response element binding protein messenger ribonucleic acid (mRNA) in the hippocampus and a 40%-41% increased level of the antiapoptotic protein bcl-2 mRNA in the prefrontal cortex compared to cNLH and SD rats. Other significant changes included changes in the expression levels of the alpha catalytic subunit of protein kinase A, glycogen synthase kinase 3beta, and protein kinase C epsilon. Congenitally learned helpless animals show evidence of altered signal transduction and regulation of apoptosis compared to cNLH and SD control animals.

Evaluation of proteome alterations induced by cadmium stress in sunflower (Helianthus annuus L.) cultures.

PubMed

Lopes Júnior, Cícero Alves; Barbosa, Herbert de Sousa; Moretto Galazzi, Rodrigo; Ferreira Koolen, Hector Henrique; Gozzo, Fábio Cesar; Arruda, Marco Aurélio Zezzi

2015-09-01

The present study evaluates, at a proteomic level, changes in protein abundance in sunflower leaves in the absence or presence (at 50 or 700mg) of cadmium (as CdCl2). At the end of the cultivation period (45 days), proteins are extracted from leaves with phenol, separated by two-dimensional difference gel electrophoresis (2-D DIGE), and excised from the gels. The differential protein abundances (for proteins differing by more than 1.8 fold, which corresponds to 90% variation) are characterized using nESI-LC-MS/MS. The protein content decreases by approximately 41% in plants treated with 700mg Cd compared with control plants. By comparing all groups of plants evaluated in this study (Control vs. Cd-lower, Control vs. Cd-higher and Cd-lower vs. Cd-higher), 39 proteins are found differential and 18 accurately identified; the control vs. Cd-higher treatment is that presenting the most differential proteins. From identified proteins, those involved in energy and disease/defense (including stress), are the ribulose bisphosphate carboxylase large chain, transketolase, and heat shock proteins are the most differential abundant proteins. Thus, at the present study, photosynthesis is the main process affected by Cd in sunflowers, although these plants are highly tolerant to Cd. Copyright © 2015 Elsevier Inc. All rights reserved.

Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

PubMed

Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

2017-03-01

The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mulberry (Morus alba L.) Fruit Extract Containing Anthocyanins Improves Glycemic Control and Insulin Sensitivity via Activation of AMP-Activated Protein Kinase in Diabetic C57BL/Ksj-db/db Mice.

PubMed

Choi, Kyung Ha; Lee, Hyun Ah; Park, Mi Hwa; Han, Ji-Sook

2016-08-01

The effect of mulberry (Morus alba L.) fruit extract (MFE) on hyperglycemia and insulin sensitivity in an animal model of type 2 diabetes was evaluated. C57BL/Ksj-diabetic db/db mice were divided into three groups: diabetic control, rosiglitazone, and MFE groups. Blood glucose, plasma insulin, and intraperitoneal glucose were measured, and an insulin tolerance test was performed after MFE supplementation in db/db mice. In addition, the protein levels of various targets of insulin signaling were measured by western blotting. The blood levels of glucose and HbA1c were significantly lower in the MFE-supplemented group than in the diabetic control group. Moreover, glucose and insulin tolerance tests showed that MFE treatment increased insulin sensitivity. The homeostatic index of insulin resistance significantly decreased in the MFE-supplemented group relative to the diabetic control group. MFE supplementation significantly stimulated the levels of phosphorylated (p)-AMP-activated protein kinase (pAMPK) and p-Akt substrate of 160 kDa (pAS160) and enhanced the level of plasma membrane-glucose transporter 4 (GLUT4) in skeletal muscles. Further, dietary MFE significantly increased pAMPK and decreased the levels of glucose 6-phosphatase and phosphoenolpyruvate carboxykinase in the liver. MFE may improve hyperglycemia and insulin sensitivity via activation of AMPK and AS160 in skeletal muscles and inhibition of gluconeogenesis in the liver.

[Association between serum aluminium level and methylation of amyloid precursor protein gene in workers engaged in aluminium electrolysis].

PubMed

Yang, X J; Yuan, Y Z; Niu, Q

2016-04-20

To investigate the association between serum aluminium level and methylation of the promoter region of amyloid precursor protein (APP)gene in workers engaged in aluminium electrolysis. In 2012, 366 electrolysis workers in an aluminium factory were enrolled as exposure group (working years >10 and age >40 years)and divided into low-exposure group and high-exposure group based on the median serum aluminium level. Meanwhile, 102 workers in a cement plant not exposed to aluminium were enrolled as control group. Graphite furnace atomic absorption spectrometry was used to measure serum aluminium level, methylation specific PCR was used to measure the methylation rate of the promoter region of APP gene, and ELI-SA was used to measure the protein expression of APP in lymphocytes in peripheral blood. The exposure group had a significantly higher serum aluminium level than the control group (45.07 μg/L vs 30.51 μg/L, P< 0.01). The exposure group had a significantly lower methylation rate of the promoter region of APP gene than the control group (18.85% vs 25.49%, P=0.025), and the high-exposure group had a significantly lower methylation rate of the promoter region of APP gene than the low-exposure group (15.84% vs 21.85%, P<0.05). The exposure group had a significantly higher protein expression of APP in lymphocytes in peripheral blood than the control group (66.73 ng/ml vs 54.17 ng/ml, P<0.05); compared with the low-exposure group (65.39 ng/ml), the high-exposure group showed an increase in the protein expression of APP in lymphocytes in peripheral blood (67.22 ng/ml), but there was no significant difference between these two groups (P>0.05). The multivariate logistic regression analysis showed that with reference to the control group, low aluminium exposure (OR=1.86, 95% CI 1.67~3.52)and high aluminium exposure (OR=2.98, 95% CI 1.97~4.15)were risk factors for a reduced methylation rate of the promoter region of APP gene. Reduced methylation of the promoter region of APP gene may be associated with increased serum aluminium level, and downregulated methylation of the promoter region of APP gene may accelerate APP gene transcription.

SCF(KMD) controls cytokinin signaling by regulating the degradation of type-B response regulators.

PubMed

Kim, Hyo Jung; Chiang, Yi-Hsuan; Kieber, Joseph J; Schaller, G Eric

2013-06-11

Cytokinins are plant hormones that play critical roles in growth and development. In Arabidopsis, the transcriptional response to cytokinin is regulated by action of type-B Arabidopsis response regulators (ARRs). Although central elements in the cytokinin signal transduction pathway have been identified, mechanisms controlling output remain to be elucidated. Here we demonstrate that a family of F-box proteins, called the kiss me deadly (KMD) family, targets type-B ARR proteins for degradation. KMD proteins form an S-phase kinase-associated PROTEIN1 (SKP1)/Cullin/F-box protein (SCF) E3 ubiquitin ligase complex and directly interact with type-B ARR proteins. Loss-of-function KMD mutants stabilize type-B ARRs and exhibit an enhanced cytokinin response. In contrast, plants with elevated KMD expression destabilize type-B ARR proteins leading to cytokinin insensitivity. Our results support a model in which an SCF(KMD) complex negatively regulates cytokinin responses by controlling levels of a key family of transcription factors.

Apolipoprotein C-II Is a Potential Serum Biomarker as a Prognostic Factor of Locally Advanced Cervical Cancer After Chemoradiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harima, Yoko, E-mail: harima@takii.kmu.ac.jp; Ikeda, Koshi; Utsunomiya, Keita

Purpose: To determine pretreatment serum protein levels for generally applicable measurement to predict chemoradiation treatment outcomes in patients with locally advanced squamous cell cervical carcinoma (CC). Methods and Materials: In a screening study, measurements were conducted twice. At first, 6 serum samples from CC patients (3 with no evidence of disease [NED] and 3 with cancer-caused death [CD]) and 2 from healthy controls were tested. Next, 12 serum samples from different CC patients (8 NED, 4 CD) and 4 from healthy controls were examined. Subsequently, 28 different CC patients (18 NED, 10 CD) and 9 controls were analyzed in themore » validation study. Protein chips were treated with the sample sera, and the serum protein pattern was detected by surface-enhanced laser desorption and ionization–time-of-flight mass spectrometry (SELDI-TOF MS). Then, single MS-based peptide mass fingerprinting (PMF) and tandem MS (MS/MS)-based peptide/protein identification methods, were used to identify protein corresponding to the detected peak. And then, turbidimetric assay was used to measure the levels of a protein that indicated the best match with this peptide peak. Results: The same peak 8918 m/z was identified in both screening studies. Neither the screening study nor the validation study had significant differences in the appearance of this peak in the controls and NED. However, the intensity of the peak in CD was significantly lower than that of controls and NED in both pilot studies (P=.02, P=.04) and validation study (P=.01, P=.001). The protein indicated the best match with this peptide peak at 8918 m/z was identified as apolipoprotein C-II (ApoC-II) using PMF and MS/MS methods. Turbidimetric assay showed that the mean serum levels of ApoC-II tended to decrease in CD group when compared with NED group (P=.078). Conclusion: ApoC-II could be used as a biomarker for detection in predicting and estimating the radiation treatment outcome of patients with CC.« less

Controlling nuclear RNA levels.

PubMed

Schmid, Manfred; Jensen, Torben Heick

2018-05-10

RNA turnover is an integral part of cellular RNA homeostasis and gene expression regulation. Whereas the cytoplasmic control of protein-coding mRNA is often the focus of study, we discuss here the less appreciated role of nuclear RNA decay systems in controlling RNA polymerase II (RNAPII)-derived transcripts. Historically, nuclear RNA degradation was found to be essential for the functionalization of transcripts through their proper maturation. Later, it was discovered to also be an important caretaker of nuclear hygiene by removing aberrant and unwanted transcripts. Recent years have now seen a set of new protein complexes handling a variety of new substrates, revealing functions beyond RNA processing and the decay of non-functional transcripts. This includes an active contribution of nuclear RNA metabolism to the overall cellular control of RNA levels, with mechanistic implications during cellular transitions.

Low doses of the pesticide lindane induce protein release by the fat body of female cockroach Blaberus craniifer (Dictyoptera).

PubMed

Goudey-Perrière, Françoise; Lemonnier, François; Bergougnoux, Véronique; Perrière, Claude

2007-11-01

In the ovoviviparous cockroach Blaberus craniifer, low doses of the pesticide lindane (1-6 microg/g of body mass) have been implicated in the enhancement of ovarian growth and vitellogenesis onset in headless female ovaries. In order to investigate lindane effects on protein release by the fat body, we used antibodies raised against egg proteins to quantify protein levels in fat body, hemolymph and ovaries of treated-fed or -decapitated females 3- or 5-days -old. In vitro assays used fat body in Grace's medium to quantify the protein amount released in the medium. Individual data for each treatment were related to their corresponding control in paired series. In vivo, ovarian enhanced protein content was linked to an enhanced protein secretion by the fat body. This was ascertained in vitro by high levels of released protein in the medium containing lindane (1 microM) by fat body from females, but not from males. This effect was inhibited by EDTA, a calcium chelator. The present results confirmed that low doses of lindane (about 3 microg/g of body mass) acted as a juvenile hormone analogue, at the level of the ovaries, by enhancing protein uptake, and also at the level of the fat body, by triggering protein release. This property is calcium-dependent.

Quality control in the secretory assembly line.

PubMed Central

Helenius, A

2001-01-01

As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway. PMID:11260794

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

PubMed

Balachandran, Manasi; Giannone, Richard J; Bemis, David A; Kania, Stephen A

2017-01-01

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

PubMed Central

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

2017-01-01

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models. PMID:28859130

Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma.

PubMed

Yang, Yang; Hui, Lv; Yuqin, Che; Jie, Li; Shuai, Hou; Tiezhu, Zhou; Wei, Wang

2014-08-01

Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 10 4 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; P<0.001). In addition, the two cell lines had a higher expression level of p53 protein in the experimental group as compared with the control group (t=40.810; P<0.001). Protein expression levels of Bcl-xL decreased significantly in the experimental group as compared with the control group (t=19.640; P=0.000). Therefore, saw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction.

Study of telomere length and different markers of oxidative stress in patients with Parkinson's disease.

PubMed

Watfa, G; Dragonas, C; Brosche, T; Dittrich, R; Sieber, C C; Alecu, C; Benetos, A; Nzietchueng, R

2011-04-01

Many studies have shown that short telomere length (TL) is associated with high oxidative stress and various age-related diseases. Parkinson's disease (PD) is an age-related disease, and although its pathogenic mechanism is uncertain, oxidative stress is believed to be implicated in this pathology. The aim of this case-control study was to assess both TL and the different markers of oxidative stress in elderly patients with PD compared to age control subjects. 20 PD patients and 15 age-matched controls, >65 years were studied. TL was measured by Southern blotting from DNA samples extracted from white blood cells. Superoxide dismutase (SOD) activity and plasma levels of total glutathione and protein carbonyls were determined. There was a trend for lower TL in PD patients: 6.06 ± 0.81 kb in PD versus 6.45 ± 0.73 kb in controls (p = 0.08). No significant difference was found between the two groups in terms of oxidative stress markers. In controls, age was the main determinant of telomere shortening (r = -0.547; p = 0.03) whereas, in PD patients, telomere shortening was mainly dependent on plasmatic concentrations of carbonyl proteins (r= -0.544; p=0.044). In PD patients, a negative association was observed between plasma carbonyl protein levels and SOD activity (r= -0.622, p=0.004). In PD, TL is shorter in presence of high oxidative stress as measured by carbonyl protein levels. The absence of telomere attrition with age among patients with PD could reflect a telomere regulation by mechanisms other than age.

The effect of balneotherapy on C-reactive protein, serum cholesterol, triglyceride, total antioxidant status and HSP-60 levels.

PubMed

Oláh, Mihály; Koncz, Agnes; Fehér, Judit; Kálmánczhey, Judit; Oláh, Csaba; Balogh, Sándor; Nagy, György; Bender, Tamás

2010-05-01

An increasing body of evidence substantiating the effectiveness of balneotherapy has accumulated during recent decades. In the present study, 42 ambulatory patients (23 males and 19 females, mean age 59.5 years) with degenerative musculoskeletal disease were randomised into one of two groups-bathing in tap water or in mineral water at the same temperature-and subjected to 30-min balneotherapy sessions on 15 occasions. Study parameters comprised serum levels of sensitised C-reactive protein (CRP), plasma lipids, heat shock protein (HSP-60) and total antioxidant status (TAS). In both groups, CRP levels followed a decreasing tendency, which still persisted 3 months later. At 3 months after balneotherapy, serum cholesterol levels were still decreasing in patients who had used medicinal water, but exhibited a trend towards an increase in the control group. Triglyceride levels followed a decreasing trend in both patient groups. TAS showed a declining tendency in both groups. No changes of HSP-60 levels were observed in either group. Balneotherapy with the thermal water from Hajdúszoboszló spa had a more pronounced physiological effect compared to that seen in the control group treated with tap water in a 3 month period.

Increased levels of the 14-3-3 η and γ proteins in the synovial fluid of dogs with unilateral cranial cruciate ligament rupture.

PubMed

Sardari, Kamran; Chavez-Muñoz, Claudia; Kilani, Ruhangiz T; Schiller, Terri; Ghahary, Aziz

2011-10-01

The present study investigated whether the 14-3-3 η and γ proteins, which are potent matrix metalloprotease (MMP) stimulators, are detectable in the synovial fluid of dogs with cranial cruciate ligament rupture (CCLR). Synovial fluid samples from 7 dogs with unilateral CCLR and control samples from 4 dogs without a history of any joint inflammation or any other abnormalities underwent Western blot analysis for the 14-3-3 η, γ, and σ proteins as well as MMP-1 and MMP-3. Craniocaudal and lateral radiographic projections of the stifle joint were evaluated for the presence and severity of 13 specific radiographic markers of osteoarthritis and graded numerically. The Spearman method was used to detect any correlation between the 14-3-3-η level in the synovial fluid and the radiograph-based grade. The η isoform was present only in the samples from the dogs with CCLR. The levels of 14-3-3-γ, MMP-1, and MMP-3 were significantly higher in the samples from the dogs with CCLR than in the control samples (P < 0.05). However, there was no significant difference between the CCLR and control samples in the level of the σ isoform. The Spearman method showed a significant correlation between the 14-3-3-η level in the synovial fluid and the presence of either patellar osteophytes or lateral or medial (or both) condylar periarticular osteophytes (P < 0.05). The MMP stimulatory effect of the 14-3-3 η and γ isoforms may be the reason for the high levels of MMP-1 and MMP-3 observed. Thus, 14-3-3 proteins, especially the η isoform, may be important markers of osteoarthritis caused by CCLR.

Increased levels of the 14-3-3 η and γ proteins in the synovial fluid of dogs with unilateral cranial cruciate ligament rupture

PubMed Central

Sardari, Kamran; Chavez-Muñoz, Claudia; Kilani, Ruhangiz T.; Schiller, Terri; Ghahary, Aziz

2011-01-01

The present study investigated whether the 14-3-3 η and γ proteins, which are potent matrix metalloprotease (MMP) stimulators, are detectable in the synovial fluid of dogs with cranial cruciate ligament rupture (CCLR). Synovial fluid samples from 7 dogs with unilateral CCLR and control samples from 4 dogs without a history of any joint inflammation or any other abnormalities underwent Western blot analysis for the 14-3-3 η, γ, and σ proteins as well as MMP-1 and MMP-3. Craniocaudal and lateral radiographic projections of the stifle joint were evaluated for the presence and severity of 13 specific radiographic markers of osteoarthritis and graded numerically. The Spearman method was used to detect any correlation between the 14-3-3-η level in the synovial fluid and the radiograph-based grade. The η isoform was present only in the samples from the dogs with CCLR. The levels of 14-3-3-γ, MMP-1, and MMP-3 were significantly higher in the samples from the dogs with CCLR than in the control samples (P < 0.05). However, there was no significant difference between the CCLR and control samples in the level of the σ isoform. The Spearman method showed a significant correlation between the 14-3-3-η level in the synovial fluid and the presence of either patellar osteophytes or lateral or medial (or both) condylar periarticular osteophytes (P < 0.05). The MMP stimulatory effect of the 14-3-3 η and γ isoforms may be the reason for the high levels of MMP-1 and MMP-3 observed. Thus, 14-3-3 proteins, especially the η isoform, may be important markers of osteoarthritis caused by CCLR. PMID:22468024

[Effects of n-hexane exposure on human serum myelin basic proteins].

PubMed

Zhou, Wei; Yi, Juan; Huang, Hui-Ping; Xiang, Ying-Ping; He, Jia-Xi; Liu, Qing-Jun; Huang, Xian-Qing

2011-06-01

To explore the effects of n-hexane on expression of serum myelin proteins (MBP) in workers occupationally exposed to n-hexane. In this study, 269 workers exposed to n-hexane for more than one year and 104 subjects not exposed to n-hexane served as the exposure group and the control group, respectively. The urinary 2,5-hexanedione levels in all subjects were detected. On the basis of urinary 2,5-hexanedione levels, the exposure group was divided into the high exposure sub-group and low exposure sub-group. The serum myelin basic protein (MBP) levels were measured by ELISA kit. The mean concentration of urinary 2,5-hexanedione in the exposed group was (3.10 +/- 1.35) mg/L. The concentration of urinary 2,5-hexanedione in the control group was undetectable. The levels of serum MBP in the high exposure sub-group and low exposure sub-group were (2.43 +/- 0.24) and (1.62 +/- 0.23) microg/L, respectively, which were significantly higher than that (0.78 +/- 0.12) microg/L in the controls (P < 0.01). Pearson correlation analysis showed the positive correlation between serum MBP levels and urinary 2,5-hexanedione levels (r = 0.781, P < 0.01). The results of present study showed that the serum MBP levels of workers occupationally exposed to n-hexane significantly elevated, and the serum MBP can serve as the effective biomarker of n-hexane exposure.

Conversion of high and low pollen protein diets into protein in worker honey bees (Hymenoptera: Apidae).

PubMed

Basualdo, M; Barragán, S; Vanagas, L; García, C; Solana, H; Rodríguez, E; Bedascarrasbure, E

2013-08-01

Adequate protein levels are necessary to maintain strong honey bee [Apis mellifera (L.)] colonies. The aim of this study was to quantify how pollens with different crude protein contents influence protein stores within individual honey bees. Caged bees were fed one of three diets, consisting of high-protein-content pollen, low-protein-content pollen, or protein-free diet as control; measurements were made based on protein content in hemolymph and fat body, fat body weight, and body weight. Vitellogenin in hemolymph was also measured. Bees fed with high crude protein diet had significantly higher levels of protein in hemolymph and fat bodies. Caged bees did not increase pollen consumption to compensate for the lower protein in the diet, and ingesting approximately 4 mg of protein per bee could achieve levels of 20 microg/microl protein in hemolymph. Worker bees fed with low crude protein diet took more time in reaching similar protein content of the bees that were fed with high crude protein diet. The data showed that fat bodies and body weight were not efficient methods of measuring the protein status of bees. The determination of total protein or vitellogenin concentration in the hemolymph from 13-d-old bees and protein concentration of fat bodies from 9-d-old bees could be good indicators of nutritional status of honey bees.

Increased Tumor Necrosis Factor (TNF)-α and Its Promoter Polymorphisms Correlate with Disease Progression and Higher Susceptibility towards Vitiligo

PubMed Central

Laddha, Naresh C.; Dwivedi, Mitesh; Begum, Rasheedunnisa

2012-01-01

Abstract Tumor Necrosis Factor (TNF)-α, is a paracrine inhibitor of melanocytes, which plays a critical role in the pathogenesis of several autoimmune diseases including vitiligo, as abnormal immune responses have frequently been observed in vitiligo patients. Moreover, vitiligo patients show higher lesion levels of TNF-α. Genetic polymorphisms in the promoter region of TNF-α are involved in the regulation of its expression. The present study explores TNF-α promoter polymorphisms and correlates them with TNF-α transcript and protein levels in vitiligo patients and controls of Gujarat along with its effect on disease onset and progression. PCR-RFLP technique was used for genotyping of these polymorphisms in 977 vitiligo patients and 990 controls. TNF-α transcript and protein levels were measured by Real time PCR and ELISA respectively. The genotype and allele frequencies for the investigated polymorphisms were significantly associated with vitiligo patients. The study revealed significant increase in TNF-α transcript and protein levels in vitiligo patients compared to controls. In particular, haplotypes: AATCC, AACCT, AGTCT, GATCT, GATCC and AGCCT were found to increase the TNF-α levels in vitiligo patients. Analysis of TNF-α levels based on the gender and disease progression suggests that female patients and patients with active vitiligo had higher levels of TNF-α. Also, the TNF-α levels were high in patients with generalized vitiligo as compared to localized vitiligo. Age of onset analysis of the disease suggests that the haplotypes: AACAT, AACCT, AATCC and AATCT had a profound effect in the early onset of the disease. Moreover, the analysis suggests that female patients had an early onset of vitiligo. Overall, our results suggest that TNF-α promoter polymorphisms may be genetic risk factors for susceptibility and progression of the disease. The up-regulation of TNF-α transcript and protein levels in individuals with susceptible haplotypes advocates the crucial role of TNF-α in autoimmune pathogenesis of vitiligo. PMID:23284977

Nitrofen interferes with trophoblastic expression of retinol-binding protein and transthyretin during lung morphogenesis in the nitrofen-induced congenital diaphragmatic hernia model.

PubMed

Kutasy, Balazs; Gosemann, Jan H; Doi, Takashi; Fujiwara, Naho; Friedmacher, Florian; Puri, Prem

2012-02-01

Retinoids play a key role in lung development. Retinoid signaling pathway has been shown to be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH) but the exact mechanism is not clearly understood. Retinol-binding protein (RBP) and transthyretin (TTR) are transport proteins for delivery of retinol to the tissues via circulation. Previous studies have shown that pulmonary retinol levels are decreased during lung morphogenesis in the nitrofen CDH model. In human newborns with CDH, both retinol and RBP levels are decreased. It has been reported that maternal RBP does not cross the placenta and the fetus produces its own RBP by trophoblast. RBP and TTR synthesized in the fetus are essential for retinol transport to the developing organs including lung morphogenesis. We hypothesized that nitrofen interferes with the trophoblastic expression of RBP and TTR during lung morphogenesis and designed this study to examine the trophoblastic expression of RBP and TTR, and the total level of RBP and TTR in the lung in the nitrofen model of CDH. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs and placenta harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Total lung RBP and TTR levels using protein extraction were compared with enzyme linked immunoassay (ELISA). Immunohistochemistry was performed to evaluate trophoblastic RBP and TTR expression. Total protein levels of lung RBP and TTR were significantly lower in CDH (0.26 ± 0.003 and 6.4 ± 0.5 μg/mL) compared with controls (0.4 ± 0.001 and 9.9 ± 1.6 μg/mL, p < 0.05). In the control group, immunohistochemical staining showed strong immunoreactivity of RBP and TTR in the trophoblast compared to CDH group. Decreased trophoblast expression of retinol transport proteins suggest that nitrofen may interfere with the fetal retinol transport resulting in reduced pulmonary RBP and TTR levels and causing pulmonary hypoplasia in CDH.

Expression of vasoactive proteins in gastric antral mucosa reflects vascular dysfunction in patients with cirrhosis and portal hypertension.

PubMed

Trebicka, Jonel; Wix, Cyrus; von Heydebrand, Matthias; Hittatiya, Kanishka; Reiberger, Thomas; Klein, Sabine; Schierwagen, Robert; Kristiansen, Glen; Peck-Radosavljevic, Markus; Fischer, Hans-Peter; Møller, Søren; Bendtsen, Flemming; Krag, Aleksander; Sauerbruch, Tilman

2015-04-01

Patients with cirrhosis display hypocontractility of splanchnic vessels because of dysregulation of vasoactive proteins, such as decreased effect of RhoA/ROCK and increased activity of β-Arrestin-2 and eNOS. However, it is unknown whether the dysregulation of vasoactive proteins is displayed in other vessels. We investigated whether expression of vasoactive proteins can be evaluated in gastric mucosa vessels. Biopsies from the gastric mucosa of 111 patients with cirrhosis were collected at three different centres and from 13 controls. Forty-nine patients had received TIPS. Portal pressure gradient was measured in 49 patients with TIPS and in 16 patients without TIPS. Biopsies from the antrum were conserved in formaldehyde for immunohistochemistry or shock-frozen for PCR and Western blot. The mucosal transcription of vascular markers (αSMA, CD31) was higher in cirrhotic patients than controls, which was confirmed by immunohistochemistry. On average, relative mucosal levels of RhoA and ROCK were lower, while β-Arrestin-2 levels were higher in cirrhotic patients compared to controls. Transcriptional levels of eNOS increased with presence of ascites and grade of oesophageal varices. Patients with TIPS showed less pronounced markers of vascular dysfunction in gastric mucosa. This is the first evidence that the expression of vasoactive proteins in mucosa from the gastric antrum of patients with cirrhosis reflects their vascular dysfunction and possibly changes after therapeutic interventions. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Proteomic Profiling of Recombinant Escherichia coli in High-Cell- Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

PubMed Central

Aldor, Ilana S.; Krawitz, Denise C.; Forrest, William; Chen, Christina; Nishihara, Julie C.; Joly, John C.; Champion, Kathleen M.

2005-01-01

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield. PMID:15811994

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

PubMed

Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

2015-11-01

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate.

PubMed

Lu, Xin; Sun, Jibin; Nimtz, Manfred; Wissing, Josef; Zeng, An-Ping; Rinas, Ursula

2010-04-20

The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS. The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures. The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures.

The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate

PubMed Central

2010-01-01

Background The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS. Results The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures. Conclusions The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures. PMID:20406453

Increased levels of glutamate in the central nervous system are associated with behavioral symptoms in experimental malaria.

PubMed

Miranda, A S; Vieira, L B; Lacerda-Queiroz, N; Souza, A H; Rodrigues, D H; Vilela, M C; Gomez, M V; Machado, F S; Rachid, M A; Teixeira, A L

2010-12-01

Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has been associated with cognitive, behavioral and motor dysfunctions, seizures and coma. The underlying mechanisms of CM are incompletely understood. Glutamate and other metabolites such as lactate have been implicated in its pathogenesis. In the present study, we investigated the involvement of glutamate in the behavioral symptoms of CM. Seventeen female C57BL/6 mice (20-25 g) aged 6-8 weeks were infected with P. berghei ANKA by the intraperitoneal route using a standardized inoculation of 10⁶ parasitized red blood cells suspended in 0.2 mL PBS. Control animals (N = 17) received the same volume of PBS. Behavioral and neurological symptoms were analyzed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. Glutamate release was measured in the cerebral cortex and cerebrospinal fluid of infected and control mice by fluorimetric assay. All functional categories of the SHIRPA battery were significantly altered in the infected mice at 6 days post-infection (dpi) (P ≤ 0.05). In parallel to CM symptoms, we found a significant increase in glutamate levels in the cerebral cortex (mean ± SEM; control: 11.62 ± 0.90 nmol/mg protein; infected at 3 dpi: 10.36 ± 1.17 nmol/mg protein; infected at 6 dpi: 26.65 ± 0.73 nmol/mg protein; with EGTA, control: 5.60 ± 1.92 nmol/mg protein; infected at 3 dpi: 6.24 ± 1.87 nmol/mg protein; infected at 6 dpi: 14.14 ± 0.84 nmol/mg protein) and in the cerebrospinal fluid (control: 128 ± 51.23 pmol/mg protein; infected: 301.4 ± 22.52 pmol/mg protein) of infected mice (P ≤ 0.05). These findings suggest a role of glutamate in the central nervous system dysfunction found in CM.

Proteomic changes in female rat hippocampus following exposure to a terrified sound stress.

PubMed

Yang, Juan; Hu, Lili; Song, Tusheng; Liu, Yong; Wu, Qiuhua; Zhao, Lingyu; Liu, Liying; Zhao, Xiaoge; Zhang, Dianzeng; Huang, Chen

2014-06-01

Stress plays a profound role in the onset of affective disorders, including an elevation in risk factors for depression and anxiety. Women are twice as vulnerable to stress as men because of greater sensitivity to a substance produced during times of anxiety. To better define the abnormal proteins implicated in cognitive deficits and other stress-induced dysfunction, female rats were exposed to terrified sound stress, and two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) were utilized to determine the differential protein expression in the hippocampus in sound-stressed female rats compared with controls. Quantitative differences were found in 44 protein spots which were differentially expressed between the stressed and control groups (fold change of >2; p < 0.01). Eighteen protein spots were downregulated, and 26 protein spots were upregulated in the stressed group. The seven most differentially expressed proteins were identified and validated as follows: dihydropyrimidinase-related protein 2 (DRP-2), creatine kinase B type, dynamin-1 protein, alpha-internexin, glial fibrillary acidic protein beta, gamma-enolase, and peptidyl-prolyl cis-trans isomerase A. Changes in protein levels were detected in the hippocampus of female rats subjected to terrified sound stress. The findings herein may open new opportunities for further investigations on the modulation induced in the hippocampus by stress at the molecular level, especially with respect to females stress.

Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism.

PubMed

Ralphe, J Carter; Bedell, Kurt; Segar, Jeffrey L; Scholz, Thomas D

2005-05-01

In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P < 0.05) and decreased in PTU-treated (2.0 +/- 0. 3 vs. 5.2 +/- 1; P < 0.05) rats. Aralar1 mRNA levels were unchanged in T3-treated and somewhat decreased in PTU-treated (7.1 +/- 1.0 vs. 9.3 +/- 0.1, P < 0.05) rats. Citrin mRNA levels were decreased in T3-treated and unchanged in PTU-treated rats. EAAT1 protein levels (arbitrary units) in T3-treated cardiac mitochondria were increased compared with controls (8.9 +/- 0.4 vs. 5.9 +/- 0.6; P < 0.005) and unchanged in PTU-treated mitochondria. No difference in malate/aspartate shuttle capacity was found between PTU-treated and control cardiac mitochondria. Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.

Elevated blood pressure, race/ethnicity, and C-reactive protein levels in children and adolescents.

PubMed

Lande, Marc B; Pearson, Thomas A; Vermilion, Roger P; Auinger, Peggy; Fernandez, Isabel D

2008-12-01

Adult hypertension is independently associated with elevated C-reactive protein levels, after controlling for obesity and other cardiovascular risk factors. The objective of this study was to determine, with a nationally representative sample of children, whether the relationship between elevated blood pressure and C-reactive protein levels may be evident before adulthood. Cross-sectional data for children 8 to 17 years of age who participated in the National Health and Nutrition Examination Survey between 1999 and 2004 were analyzed. Bivariate analyses compared children with C-reactive protein levels of >3 mg/L versus or=95th percentile and 1.3% had diastolic blood pressure of >or=95th percentile. Children with C-reactive protein levels of >3 mg/L had higher systolic blood pressure, compared with children with C-reactive protein levels of or=95th percentile was independently associated with C-reactive protein levels in boys but not girls. Subset analyses according to race/ethnicity demonstrated that the independent association of elevated systolic blood pressure with C-reactive protein levels was largely limited to black boys. These data indicate that there is interplay between race/ethnicity, elevated systolic blood pressure, obesity, and inflammation in children, a finding that has potential implications for disparities in cardiovascular disease later in life.

Acute mercury exposition of virgin, pregnant, and lactating rats: Histopathological kidney and liver evaluations.

PubMed

Oliveira, Vitor Antunes; Favero, Gaia; Stacchiotti, Alessandra; Giugno, Lorena; Buffoli, Barbara; de Oliveira, Claudia Sirlene; Lavazza, Antonio; Albanese, Massimo; Rodella, Luigi Fabrizio; Pereira, Maria Ester; Rezzani, Rita

2017-05-01

This work investigated the effects of mercury chloride (HgCl 2 ) acute exposure on virgin, pregnant and lactating rats by determination of renal and hepatic morphological and ultrastructural parameters and the expression of oxidative stress and stress tolerance markers, due to kidney and liver are the organs that more accumulate inorganic mercury. Adult Wistar rats virgin (90 days old), pregnant (18 th gestation day) and lactating (7 th lactation day) were injected once with HgCl 2 (5 mg/kg) or saline (controls). We observed that HgCl 2 exposure of virgin rats caused significant inflammatory infiltration and severe morphological variations, like glomeruli atrophy, dilatation of Bowman's capsule, tubular degeneration and hepatocytes alteration. Moreover, virgin rats presented mitochondrial modification, important oxidative stress and increase in stress tolerance proteins at both kidney and liver level, compared with virgin controls. In detail, virgin rats exposed to HgCl 2 presented significantly elevated level of inducible nitric oxide synthase, heat shock protein 27 and glucose regulated proteins 75 expressions at both renal tubular and hepatocytes level, respect untreated virgin rats. Interestingly, pregnant and lactating rats exposed to HgCl 2 presented weak renal and liver morphological alterations, showing weak inflammatory infiltration and no significant difference in structural mitochondrial transmembrane protein, oxidative stress markers and stress tolerance proteins expressions respect controls (virgin, pregnant and lactating rats). Although, both control and HgCl 2 -exposed pregnant and lactating rats showed renal glomeruli greater in diameter respect virgin rats. In conclusion, we believe that virgin rats are more sensitive to HgCl 2 toxicity respect pregnant and lactating rats. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1500-1512, 2017. © 2016 Wiley Periodicals, Inc.

Abnormal subcellular localization of AQP5 and downregulated AQP5 protein in parotid glands of streptozotocin-induced diabetic rats.

PubMed

Wang, Di; Yuan, Zhenfang; Inoue, Noriko; Cho, Gota; Shono, Masayuki; Ishikawa, Yasuko

2011-05-01

The mechanisms underlying diabetic xerostomia have not been clarified in relation with aquaporin-5 (AQP5) subcellular localization in salivary glands. Western blotting, real-time PCR, and immunocytochemistry were used to analyse AQP5 protein levels and mRNA expression. AQP5 protein levels were measured in the apical plasma membrane (APM) and detergent-insoluble fraction prepared from streptozotocin-diabetic rat parotid glands. Despite an increase in AQP5 mRNA, AQP5 protein levels were decreased in diabetic parotid glands compared with controls. Immunohistochemical studies indicated that AQP5, under unstimulated conditions, colocalised with flotillin-2 and GM1 with a diffuse pattern in the apical cytoplasm of acinar and duct cells in both control and diabetic rats. Ten minutes after intravenous injection of muscarinic agonist cevimeline, AQP5 was dramatically increased together with flotillin-2 and GM1 in the APM of parotid acinar and duct cells of control but not diabetic rats. Sixty minutes after injection, AQP5 was located in a diffuse pattern in the apical cytoplasm in both rats. Treatment of the parotid tissues with cevimeline for 10min increased the Triton X-100 solubility of AQP5 in control but not diabetic rats. Administration of insulin to diabetic rats tended to restore the cevimeline-induced translocation of AQP5. Lack of AQP5 translocation in the salivary gland in response to a muscarinic agonist and downregulation of AQP5 protein might lead to diabetic xerostomia. Cevimeline is useful to cure diabetic xerostomia under insulin administration. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Containerless protein crystal growth method

NASA Technical Reports Server (NTRS)

Rhim, Won-Kyu; Chung, Sang K.

1991-01-01

A method of growing protein crystals from levitated drops is introduced and unique features of containerless approach in 1-g and micro-G laboratories are discussed. Electrostatic multidrop levitation system which is capable of simultaneous four drop levitation is described. A method of controlling protein saturation level in a programmed way is introduced and discussed. Finally, some of the unique features of containerless approach of protein crystal growth in space are discussed and summarized.

Plasma Protein Pentosidine and Carboxymethyllysine, Biomarkers for Age-related Macular Degeneration*

PubMed Central

Ni, Jiaqian; Yuan, Xianglin; Gu, Jiayin; Yue, Xiuzhen; Gu, Xiaorong; Nagaraj, Ram H.; Crabb, John W.

2009-01-01

Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein Nε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors. PMID:19435712

Effect of Inhaling Bergamot Oil on Depression-Related Behaviors in Chronic Stressed Rats.

PubMed

Saiyudthong, Somrudee; Mekseepralard, Chantana

2015-10-01

Bergamot essential oil (BEO) possesses sedation and anxiolytic properties similar to diazepam. After long period of exposure to stressors, including restrained stress, depressive-like behavior can be produced. BEO has been suggested to reduce depression. However, there is no scientific evidence supporting this property. To investigate the effect of BEO in chronic stressed rats on: 1) behavior related depressive disorder, 2) hypothalamic pituitary adrenal (HPA) axis response, and iii) brain-derived neurotrophic factor (BDNF) protein levels in hippocampus. Male Wistar rats, weighing 200 to 250 g, were induced chronic restrained stress 15 minutes dailyfor two weeks. For the next two weeks, these rats were divided intofour groups, control-i.p., fluoxetine-i.p., control-inhale, and BEO-inhale. Fluoxetine (10 mg/kg i.p.) or saline was intraperitoneally administered daily while 2.5% BEO or saline was inhaled daily. At the end of the treatment, rats were assessed for depressive-like behavior using the forced swimming test (FST). After the behavioral test, the animals were immediately decapitated and trunk blood samples were collected for the measurement ofcorticosterone and adrenocorticotropic hormone (ACTH) levels and hippocampus was dissected and stored in afreezer at -80 °C until assay for BDNF protein. BEO andfluoxetine significantly decreased the immobility time in the FST (p < 0.05). Fluoxetine tended to decrease serum corticosterone and significantly (p < 0.05) decreased serum ACTH while BEO had no effect on these two stress hormones. For BDNF protein determination, neither BEO norfluoxetine had any effect on BDNF protein levels in hippocampus compared to their controls. The inhalation ofBEO decrease behavior related depressive disorder similar tofluoxetine but has no effect on HPA axis response and BDNF protein levels in chronic restrained stress.

Effects of altered gravity on a distribution of rDNA and nucleolar proteins and the expression of nucleolar proteins in plants

NASA Astrophysics Data System (ADS)

Sobol, Margaryta; Kordyum, Elizabeth; Medina, Francisco Javier

The nucleolus is an inner nuclear organelle originated from the activity of hundreds of rRNA genes, typically spanning several megabases. It morphologically reflects the functional events leading to ribosome biogenesis, from the transcription of rDNA through the processing of nascent pre-rRNA to the assembly of pre-ribosomes. A typical nucleolus consists of three major elements, namely fibrillar centers (FCs), the dense fibrillar component (DFC), and granular component (GC). The rate of ribosome biosynthesis and the subnucleolar structure are reliable monitors of the general level of cell metabolism and, consequently, of the rate of cellular growth, being influenced with many external factors, among which altered gravity could be included. Thus, we can hypothesize that the structural organization of the nucleolar subcomponents and the level, distribution and quantitative/qualitative characteristics of the nucleolar proteins would be changed under conditions of altered gravity. To confirm our hypothesis, we applied parallel procedures, such as cytochemistry, immunofluorescence, confocal laser microscopy, immunogold electron microscopy, monoand bi-dimensional electrophoresis and immunoblotting in root meristematic cells from two-day cress seedlings grown under slow horizontal clinorotation (2 rpm) and in stationary control. The complex model of the ultrastructural organization and functions of the nucleolus was created based on the location of rDNA and the nucleolar proteins fibrillarin, NhL90 and NhL68, these latter being cress nucleolin homologues. The principal stages of ribosome biogenesis, namely ribosomal gene activation, rDNA transcription and pre-rRNA processing were reflected in this model. Compared to the pattern shown in control ground gravity conditions, we found firstly a redistribution of both rDNA and nucleolar proteins in nucleolar subcomponents, induced by clinorotation. Under the conditions of altered gravity, nucleolar DNA concentrated predominantly in FCs in the form of condensed chromatin inclusions and internal non condensed fibrils, redistributing from the DFC and the transition zone between FCs and the DFC, recognized as the site of rDNA transcription. Regarding nucleolar proteins, a general decrease in the levels of fibrillarin and the nucleolin homologues, evaluated by estimating the density of immunogold labeling on the nucleolus, was recorded firstly in clinorotated samples, compared to controls. Furthermore, the intranucleolar location of the investigated proteins was also observed to change in response to the growth in altered gravity conditions. In particular, a decrease in the quantity of these proteins in the transition zone FCs-DFC as well as in the bulk of the DFC was observed in the experimental samples, compared to controls, whereas the content of the proteins was much higher in the inner space of FCs. Concerning the two-dimensional nuclear proteome, we revealed a decrease in the isoelectric point (pI) range of soluble proteins, which are known to be actively engaged in RNA (including rRNA) metabolism, and a shortening in the molecular weight range of them under clinorotation. Besides, minor and major protein spots in clinorotated samples showed decreased optical densities in comparison to control ones. Moreover, we showed the shortening of both the pI and the molecular weight ranges of the spots corresponding to the major nucleolin homologue NhL90 (detected by cross-reaction with anti-onion NopA100) in the fraction of soluble proteins in altered gravity. Based on these data, an effect of altered gravity in lowering the level of rDNA transcription as well as rRNA processing, that could be the evidence of a decrease in the level of nucleolar functional activity, is suggested.

A high-fat, high-protein diet attenuates the negative impact of casein-induced chronic inflammation on testicular steroidogenesis and sperm parameters in adult mice.

PubMed

Zhao, Jing-Lu; Zhao, Yu-Yun; Zhu, Wei-Jie

2017-10-01

The interaction between obesity and chronic inflammation has been studied. Diet-induced obesity or chronic inflammation could reduce the testicular functions of males. However, the mechanism underlying the reproductive effects of fattening foods in males with or without chronic inflammation still needs further discussion. This study was aimed to investigate the effects of high-fat, high-protein diet on testicular steroidogenesis and sperm parameters in adult mice under physiological and chronic inflammatory conditions. Because casein can trigger a non-infectious systemic inflammatory response, we used casein injection to induce chronic inflammation in male adult Kunming mice. Twenty-four mice were randomly and equally divided into four groups: (i) normal diet+saline (Control); (ii) normal diet+casein (ND+CS); (iii) high-fat, high-protein diet+saline (HFPD+SI); (iv) high-fat, high-protein diet+casein (HFPD+CS). After 8weeks, there was a significant increase in body weight for groups HFPD+SI and HFPD+CS and a decrease in group ND+CS compared with the control. The serum levels of tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10) and lipid profiles were increased markedly in groups ND+CS, HFPD+SI and HFPD+CS compared with the control. A remarkable reduction of serum adiponectin level occurred in group HFPD+CS compared with group ND+CS. Sperm parameters (sperm count, viability and abnormality) were also adversely affected in groups ND+CS and HFPD+SI. Groups ND+CS and HFPD+SI showed severe pathological changes in testicular tissues. Semiquantitative RT-PCR, Western blot and immunohistochemical staining also showed significant reductions in both testicular mRNA and protein levels of steroidogenic acute regulatory (StAR) and cytochrome P450scc (CYP11A1) in groups HFPD+SI and HFPD+CS compared with the control, whereas testicular mRNA and protein levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) in groups HFPD+SI and HFPD+CS significantly increased. The mRNA and protein levels of the StAR and 3β-HSD in group HFPD+CS were both higher than those of in group ND+CS. These results indicated that Kunming male mice with high-fat, high-protein diet and casein injection for 8weeks can be used to establish a diet-induced obesity and chronic systemic inflammation. The sperm parameters in groups ND+CS and HFPD+SI decreased accompanied by pathological changes of testicular tissue. This resultant effect of reduced serum testosterone levels was associated with the overproduction of TNF-α and IL-10 and down-regulation of StAR and CYP11A1. Under the same casein-induced chronic inflammation condition, the mice with high-fat, high-protein diet had better testicular steroidogenesis activity and sperm parameters compared with the mice in normal diet, indicating that the mice with casein-induced inflammatory injury consuming a high-fat, high-protein diet gained weight normally, reduced serum adiponectin level and increased testosterone production by an upregulation of 3β-HSD expression. High-fat, high-protein diet attenuated the negative impact of casein-induced chronic inflammation on testicular steroidogenesis and sperm parameters. Copyright © 2017 Elsevier Inc. All rights reserved.

How to talk about protein-level false discovery rates in shotgun proteomics.

PubMed

The, Matthew; Tasnim, Ayesha; Käll, Lukas

2016-09-01

A frequently sought output from a shotgun proteomics experiment is a list of proteins that we believe to have been present in the analyzed sample before proteolytic digestion. The standard technique to control for errors in such lists is to enforce a preset threshold for the false discovery rate (FDR). Many consider protein-level FDRs a difficult and vague concept, as the measurement entities, spectra, are manifestations of peptides and not proteins. Here, we argue that this confusion is unnecessary and provide a framework on how to think about protein-level FDRs, starting from its basic principle: the null hypothesis. Specifically, we point out that two competing null hypotheses are used concurrently in today's protein inference methods, which has gone unnoticed by many. Using simulations of a shotgun proteomics experiment, we show how confusing one null hypothesis for the other can lead to serious discrepancies in the FDR. Furthermore, we demonstrate how the same simulations can be used to verify FDR estimates of protein inference methods. In particular, we show that, for a simple protein inference method, decoy models can be used to accurately estimate protein-level FDRs for both competing null hypotheses. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hsp 70, hsCRP and oxidative stress in patients with acute coronary syndromes.

PubMed

Amanvermez, Ramazan; Acar, Ethem; Günay, Murat; Baydın, Ahmet; Yardan, Türker; Bek, Yüksel

2012-05-01

Acute coronary syndromes (ACS) like unstable angina (UA) and acute myocardial infarction (AMI) can lead to the morbidity and mortality. The diagnosis and management of patients with ACS in the earliest times after symptom onset are considerably important in the emergency service. Study aimed to investigate the serum levels of heat shock protein 70 (Hsp 70), high sensitivity C-reactive protein (hsCRP), total creatine kinase (CK) activity, creatine kinase MB (CK-MB), cardiac troponin I (cTnI), leukocyte count (WBCs) and markers of oxidative stress in the first hours of ACS and to view their diagnostic values. 70 patients with ACS after admission and 20 sex-matched healthy controls were included in this study. Serum Hsp 70, hsCRP, CK, CK-MB, cTnI, protein carbonyls, malondialdehyde as well as whole blood WBCs were measured. The level of hsCRP was statistically higher in patients with AMI and UA than that of control group (p<0.001). WBCs and oxidized protein levels were higher in AMI than in UA and control groups. cTnI was related to CK-MB in AMI and UA groups (r=0.731, r=0.806, p<0.001, respectively) and also related with hsCRP in UA group (r=0.824, p<0.001). The mean Hsp 70 level was higher by 32.2% in AMI and 12.7% in UA patients compared to control subjects. hsCRP may have a role in the inflammatory response after ACS. In addition to cTnI and CK-MB, WBCs and hsCRP may be useful as a marker for the identification of ACS patients with chest pain in early diagnosing.

Decreased placental and muscular expression of the fibroblast growth factor 19 in gestational diabetes mellitus.

PubMed

Wang, Dongyu; Xu, Shuqia; Ding, Wenjing; Zhu, Caixia; Deng, Songqing; Qiu, Xiwen; Wang, Zilian

2018-05-07

Fibroblast growth factor (FGF) 19 has been shown to improve glycaemic homeostasis and lipid metabolism in animal models. In humans, decreased FGF19 level has been described in diabetes. This study aimed to investigate the expression of FGF19 in gestational diabetes mellitus (GDM). Samples for measurement were obtained from 20 GDM women and 25 healthy controls. The mRNA and protein expression levels of FGF19, FGF21 and co-receptor β-klotho (KLB) in placenta, rectus muscle and subcutaneous fat tissues were quantified by real-time quantitative polymerase chain reaction, western-blot and immunohistochemistry, respectively. Women with GDM had significantly lower mRNA and protein expressions of FGF19 than control women had in placenta (mRNA: 0.33 ± 0.05 vs. 0.72 ± 0.09; protein: 0.34 ± 0.13 vs. 0.85 ± 0.20) and rectus muscle (mRNA: 0.83 ± 0.11 vs. 1.28 ± 0.19; protein: 0.78 ± 0.24 vs. 1.23 ± 0.39). However, there were no significant differences between GDM women and controls with respect to the expression levels of FGF21 and KLB in placenta and rectus muscle. There were almost no detectable FGF19 and FGF21 expressions in subcutaneous fat tissue. Moreover, KLB expression levels were not different between GDM and control group in subcutaneous fat. FGF19expressions are decreased in GDM women's placenta and rectus muscle. This may contribute to the pathophysiology or development of GDM. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Significance of phosphatase and tensin homologue (PTEN), O(6)-methylguanine-DNA methyltransferase (MGMT), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein expression in gynaecomastia.

PubMed

Zhu, L; Liu, Z; Yang, J; Cai, J

2009-01-01

This study was designed to investigate the pathogenesis of gynaecomastia by measuring phosphatase and tensin homologue (PTEN), O(6)-methylguanine-DNA methyltransferase (MGMT) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein in breast tissue specimens from 68 patients with gynaecomastia and 24 normal male controls using immunohistochemical staining. The gynaecomastia cases were divided into three different histological types: florid, intermediate and fibrous. The PTEN, MGMT and DNA-PKcs proteins were detected in both gynaecomastia and normal breast tissue, but the levels of immunohistochemical staining of each protein were significantly lower in gynaecomastia breast tissue than in normal breast tissue. There were also significant differences in the levels of immunohistochemical staining for the three proteins according to gynaecomastia histological type. These results suggest that abnormally low levels of PTEN, MGMT and DNA-PKcs protein in gynaecomastia breast tissue may play a role in the development of gynaecomastia. Further research is required to elucidate fully their individual roles in the pathophysiology of gynaecomastia.

One-plasmid tunable coexpression for mycobacterial protein–protein interaction studies

PubMed Central

Chang, Yong; Mead, David; Dhodda, Vinay; Brumm, Phil; Fox, Brian G

2009-01-01

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA Δ9 desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications. PMID:19760663

Protective effect of Euterpe oleracea Mart (açaí) extract on programmed changes in the adult rat offspring caused by maternal protein restriction during pregnancy.

PubMed

de Bem, Graziele Freitas; da Costa, Cristiane Aguiar; de Oliveira, Paola Raquel Braz; Cordeiro, Viviane Silva Cristino; Santos, Izabelle Barcellos; de Carvalho, Lenize Costa Reis Marins; Souza, Marcelo Augusto Vieira; Ognibene, Dayane Texeira; Daleprane, Julio Beltrame; Sousa, Pergentino José Cunha; Resende, Angela Castro; de Moura, Roberto Soares

2014-09-01

This study examined the effect of açaí (Euterpe oleracea Mart.) seed extract (ASE) on cardiovascular and renal alterations in adult offspring, whose mothers were fed a low-protein (LP) diet during pregnancy. Four groups of rats were fed: control diet (20% protein); ASE (200 mg/kg per day); and LP (6% protein); LP + ASE (6% protein + ASE) during pregnancy. After weaning, all male offspring were fed a control diet and sacrificed at 4 months old. We evaluated the blood pressure, vascular function, serum and urinary parameters, plasma and kidney oxidative damage, and antioxidant activity and renal structural changes. Hypertension and the reduced acetylcholine-induced vasodilation in the LP group were prevented by ASE. Serum levels of urea, creatinine and fractional excretion of sodium were increased in LP and reduced in LP + ASE. ASE improved nitrite levels and the superoxide dismutase and glutathione peroxidase activity in LP, with a corresponding decrease of malondialdehyde and protein carbonyl levels. Kidney volume and glomeruli number were reduced and glomerular volume was increased in LP. These renal alterations were prevented by ASE. Treatment of protein-restricted dams with ASE provides protection from later-life hypertension, oxidative stress, renal functional and structural changes, probably through a vasodilator and antioxidant activity. © 2014 Royal Pharmaceutical Society.

[Effects of Electroacupunctrue Combined with Dietary Control on Peroxisome Proliferator-activa- ted Receptor-α, and Liver Fatty Acid-binding Protein Levels in Non-alcoholic Fatty Liver Disease Rats].

PubMed

Zhang, Yi; Tang, Cheng-lin; Tian, Yuan; Yuan, Hai-zhou; Yang, Hui; Tang, Nian-zhen; Gao, Rui-qi; Cao, Jing

2015-10-01

To observe the effect of electroacupunctrue (EA) intervention or EA combined with dietary control on peroxisome proliferator-activated receptor (PPAR)-α, and liver fatty acid-binding protein (L-FABP) levels in non-alcoholic fatty liver disease (NAFLD) rats, so as to reveal its mechanism underlying improvement of NAFLD. Sixty SD male rats were randomly divided into common diet (control) group (n = 10) and high-fat diet group (n = 45). The NAFLD model was established by feeding the animals with high-fat forage (HFF, including cholesterol, sodium cholate, propylthiouracil, sucrose, lard and common forage) for 5 weeks. Forty NAFLD rats were then randomized into model, EA + HFF, low-fat forage (LFF) and EA+ LFF groups (n = 10 rats in each group). EA (4 Hz/20 Hz, 3 mA) was applied to ipsilateral "Zusanli" (ST 36),"Sanyinjiao" (SP 6) and "Taichong" (LR 3) for 20 min, once daily for 4 weeks. The pathologic changes of the hepatic tissue were detected by H. E. staining. Serum total cholesterol (TC) and triglyceride (TG) contents were determined by using enzymatic methods, serum free fat acids (FFA) content was detected by colorimetry. The expression levels of PPAR-α and L-FABP protein and gene of the liver tissue were determined by Western blot and RT-PCR, respectively. H. E. staining showed that the hepatocytes presented moderate or severe bullous adipose degeneration in rats of the model group, vesicular steatosis in the EA + HFF and LFF groups, turned to almost normal but with small amount of lipid droplets in the EA + LFF group. The contents of serum TC, TG and FFA were significantly higher in the model group than in the control group (P < 0.05), and were obviously decreased in the EA + HFF, LFF and EA + LFF groups in comparison with the model group (P < 0.05). Compared to the control group, hepatic PPAR-α protein and mRNA were markedly down-regulated in the model group, and hepatic L-FABP protein and mRNA considerably up-regulated in the model group (P < 0.05). Following EA intervention and low fat diet feeding, PPAR-α protein and mRNA were markedly up-regulated while L-FABP protein and mRNA remarkably down-regulated in the EA + HFF, LFF and EA + LFF groups (P < 0.05). EA intervention or EA combined with dietary control can relieve hepatic pathological changes in NAFLD rats, which may be related to its effects in down-regulating blood lipid level and hepatic L-FABP protein and mRNA expression and in up-regulating PPAR-α protein and mRNA expression, and the effect of combined treatment was relatively better than simple EA or LFF treatment.

Resveratrol ameliorates diet-induced dysregulation of lipid metabolism in zebrafish (Danio rerio)

PubMed Central

Li, Lin; Yan, Qiaoqiao; Yi, Weijie; Ying, Chenjiang; Wu, Hongmei

2017-01-01

Defective lipid metabolism is associated with increased risk of various chronic diseases, such as obesity, cardiovascular diseases, and diabetes. Resveratrol (RSV), a natural polyphenol, has been shown the potential of ameliorating disregulations of lipid metabolism. The objective of this study was to investigate the effects of feed intake and RSV on lipid metabolism in zebrafish (Danio rerio). The adult males were randomly allocated to 6 groups: control (Con, 8 mg cysts/fish/day), control with 20 μmol/L RSV (Con+RSV), calorie restriction (CR, 5 mg cysts/fish/day), calorie restriction with RSV (CR+RSV), overfeed (OF, 60 mg cysts/fish/day), and overfeed with RSV (OF+RSV) groups. The treatment period was 8 weeks. Results showed that CR reduced body length, body weight, and condition factor of zebrafish. CR reduced levels of plasma triglyceride (TG) and induced protein expression of phosphorylated AMP-activated protein kinase-α (pAMPKα), silent information regulator 2 homolog 1 (Sirt1), and peroxisome proliferator activated receptor gamma coactivator-1α (PGC1α). RSV attenuated CR-induced pAMPKα/AMPKαincreases. RSV increased levels of Sirt1 protein in the OF zebrafish, and decreased OF-induced increase in peroxisome proliferator-activated receptor-γ (PPARγ) protein level. Additionally, RSV down-regulated caveolin-1 and up-regulated microtubule-associated protein 1 light chain 3 -II (LC3-II) protein levels in OF zebrafish. In conclusion, these results suggest that 1) CR reduces plasma TG level through activation of the AMPKα-Sirt1- PGC1α pathway; 2) under different dietary stress conditions RSV might regulate AMPK phosphorylation bi-directionally; 3) RSV might regulate lipid metabolism through the AMPKα-Sirt1-PPARγ pathway in OF zebrafish. PMID:28686680

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis

NASA Technical Reports Server (NTRS)

Gruppi, C. M.; Wolgemuth, D. J.

1993-01-01

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

Ocular surface inflammation, and nerve growth factor level in tears in active thyroid-associated ophthalmopathy.

PubMed

Yoon, Jin Sook; Choi, Soo Hyun; Lee, Joon H; Lee, Sung Jun; Lee, Sang Yeul

2010-02-01

To measure tear nerve growth factor (NGF) concentrations in cases of active thyroid-associated ophthalmopathy (TAO) before and after glucocorticoid treatment, and to correlate NGF levels with disease inflammatory activity and thyroid autoantibody concentration. The study involved 20 patients with active TAO and 20 age- and gender-matched controls. Tear break-up time (BUT) was obtained, the Schirmer test was performed, and tear NGF/total protein ratio was measured in control subjects and patients with active TAO before, and 2 and 4 weeks after, steroid treatment. Tear BUT and Schirmer values significantly increased after 2 and 4 weeks of steroid treatment (p < 0.001 and p = 0.004 respectively). Baseline tear NGF/total protein ratio was higher in patients with active TAO than in control subjects, and the ratio significantly decreased after 2 and 4 weeks of steroid treatment (p < 0.001). Tear NGF/total protein ratio did not correlate with inflammatory activity score, exophthalmos value and thyroid binding inhibiting immunoglobulin (TBII) level (p > 0.05). Tear NGF may have a specific role in ocular surface inflammation, which protects against ocular surface damage in patients with active TAO. Anti-inflammatory treatment significantly reduced the level of NGF in tears, increased tear film stability and production, and decreased congestive symptoms.

Oral supplementation of Lithospermum erythrorhizon prevents the development of atopic dermatitis with reducing ceramide degradation in the epidermis of NC/Nga mice.

PubMed

Kim, JungMin; Kim, YoungRan; Seo, DaeBang; Kim, SungHan; Lee, SangJun; Cho, Yunhi

2009-09-01

Lithospermum erythrorhizon Sieb. et Zucc. (LE) is widely used in the treatment of abnormal skin conditions, but its systemic efficacy, especially in atopic dermatitis (AD), is not clear. To examine the systemic efficacy of LE on the clinical manifestation of AD-like skin lesions, NC/Nga mice, a murine model of AD, were fed a control diet (group CA: atopic control) or a diet with a 70% ethanol extract from 5% LE (group LE) for 10 weeks. In group LE, the clinical manifestation of AD-like skin lesions was prevented as the level of serum IgE, epidermal hyperproliferation, and the number and duration of scratching episodes, which were greater in group CA, were significantly reduced to a similar level of the normal control group of BALB/c mice (group C). In addition, the level of ceramides, the major lipid maintaining the epidermal barrier, in the epidermis of group LE was increased, and was inversely associated with a decreased protein level of ceramidase, an enzyme of ceramide degradation. However, the mRNA and the protein levels of serine palmitoyl transferase (enzyme for de novo ceramide synthesis) in groups C, CA and LE did not differ. It was demonstrated that oral supplementation with LE extract prevented the development of atopic dermatitis with reducing ceramide degradation coupled with a low expression of ceramidase protein.

Is there a role for antioxidants in prevention of pulmonary hypoplasia in nitrofen-induced rat model of congenital diaphragmatic hernia?

PubMed

Cigdem, Murat Kemal; Kizil, Goksel; Onen, Abdurrahman; Kizil, Murat; Nergiz, Yusuf; Celik, Yusuf

2010-04-01

Many studies suggest a role for antioxidants in the prevention of lung hypoplasia in nitrofen-induced rat models with congenital diaphragmatic hernia (CDH). This study investigates the oxidative status and the histological outcome of prenatal administration of vitamins E and C with synergistic effect, and effect of N-acetylcysteine (NAC) to improve lung maturation of nitrofen-induced rats. CDH was induced by maternal administration of a single oral dose of nitrofen on day 9.5 of gestation, and the Sprague-Dawley rats were randomly divided into five groups: nitrofen (N), nitrofen + vitamin C (NC), nitrofen + vitamin E (NE), nitrofen + vitamin C + vitamin E (NCE) and nitrofen + NAC (NNAC). A control group in which only vehicle was administered was included. Cesarean section was performed on day 21. Body weight (BW) and total lung weight (LW) of all fetuses with CDH were recorded; lung histological evaluation was performed, and protein content of lungs, determination of thiobarbituric acid reactive substances, and the protein carbonyls in tissue samples were determined. A total of 133 rat fetuses with CDH were investigated. The body weight and the lung weight of fetuses of all groups that were exposed to nitrofen were significantly decreased than of the control group (P < 0.05). The animals exposed to nitrofen with different antioxidants showed increased protein levels in lung tissue. However, in the NCE and the NNAC groups, protein levels were significantly increased than in the others. Malondialdehyde levels significantly decreased in the NCE and the NNAC groups when compared with the NC and the NE groups. In addition, the NCE and NNAC groups decreased protein oxidation to control levels, and no significant difference was observed between control and these two antioxidants groups. The N, NC, NE and NNAC groups showed minimal improvement in lung histology; the NCE groups showed the most improvement in lung histology when compared with the other nitrofen plus antioxidant groups. Prenatal administration of NAC and vitamin E in combination with vitamin C represented the best effects to avoid oxidative damage and protein content of the lungs in rat pups with CDH at birth.

Association of high sensitivity C-reactive protein and abdominal aortic aneurysm: a meta-analysis and systematic review.

PubMed

Wang, Yunpeng; Shen, Guanghui; Wang, Haiyang; Yao, Ye; Sun, Qingfeng; Jing, Bao; Liu, Gaoyan; Wu, Jia; Yuan, Chao; Liu, Siqi; Liu, Xinyu; Li, Shiyong; Li, Haocheng

2017-12-01

To evaluate the association of high sensitivity C-reactive protein (hsCRP) with the presence of abdominal aortic aneurysm (AAA). Medline, Cochrane, Embase, and Google Scholar databases were searched until 22 June 2016 using the keywords predictive factors, biomarkers, abdominal aortic aneurysm, prediction, high sensitivity C-reactive protein, and hsCRP. Prospective studies, retrospective studies, and cohort studies were included. Twelve case-control studies were included in the meta-analysis with a total of 8345 patients (1977 in the AAA group and 6368 in the control group). The pooled results showed that AAA patients had higher hsCRP value than the control group (difference in means = 1.827, 95% CI = 0.010 to 3.645, p = .049). Subgroup analysis found AAA patients with medium or small aortic diameter (<50 mm) had higher hsCRP plasma levels than the control group (difference in means = 1.301, 95% CI = 0.821 to 1.781, p < .001). In patients with large aortic diameter (≥50 mm), no difference was observed in hsCRP levels between the AAA and control groups (difference in means = 1.769, 95% CI = -1.387 to 4.925, p = .272). Multi-regression analysis found the difference in means of hsCRP plasma levels between AAA and control groups decreased as aortic diameter increased (slope = -0.04, p < .001), suggesting that hsCRP levels may be inversely associated with increasing aneurysm size. Our findings suggest that hsCRP levels may possibly be used as a diagnostic biomarker for AAA patients with medium or small aortic diameter but not for AAA patients with large aortic diameter. The correlation between serum hsCRP level and AAA aneurysm is not conclusive due to the small number of included articles and between-study heterogeneity.

AMPKα, hs-CRP and FcγR in diabetic nephropathy and drug intervention.

PubMed

Sun, Ping; Lu, Lei; Chen, Jun; Liu, Xiao Dan; Zhang, Qing; Wang, Xu

2018-06-01

The aim of this study was to investigate the roles of AMP-activated protein kinase α subunit (AMPKα), hypersensitive C-reactive protein (hs-CRP) and Fcγ receptor (FcγR) in diabetic nephropathy and drug intervention effects. Sixty Sprague Dawley male rats were randomly divided into the control (n=30) and observation (n=30) groups. The model of type 2 diabetic nephropathy was established by high-fat and high-glucose diet and streptozotocin injection. The rats in the observation group were treated with baicalein and the rats in control group did not receive any drug intervention. The pathological changes of kidneys were observed by hematoxylin and eosin (H&E) staining. The expression of AMPKα mRNA in renal tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR). The levels of hs-CRP and FcγR were measured by enzyme-linked immunosorbent assay (ELISA) at 1, 4, 6 and 8 weeks after drug intervention and blood urea nitrogen (BUN) and the 24 h urinary micro-albumin (U-ALB) levels were compared at 1, 4, 6 and 8 weeks after intervention. After 8 weeks of drug intervention, the pathological changes of kidneys in the observation group were significantly lower than those in the control group (p<0.05), while the relative expression levels of AMPKα mRNA and protein in the control group were higher than those in the observation group (p<0.05). The levels of hs-CRP, BUN and 24 h U-ALB in the control group were significantly higher than those in the observation group at different time-points after drug intervention and the level of FcγR in the control group was significantly lower than that in the observation group (p<0.05). Baicalein may protect renal function by inhibiting the expression of AMPKα and inflammatory reaction, and can also decrease BUN and 24 h U-ALB levels and improve the pathological changes of the kidney.

Protein Dynamics Associated with Failed and Rescued Learning in the Ts65Dn Mouse Model of Down Syndrome

PubMed Central

Ahmed, Md. Mahiuddin; Dhanasekaran, A. Ranjitha; Block, Aaron; Tong, Suhong; Costa, Alberto C. S.; Stasko, Melissa; Gardiner, Katheleen J.

2015-01-01

Down syndrome (DS) is caused by an extra copy of human chromosome 21 (Hsa21). Although it is the most common genetic cause of intellectual disability (ID), there are, as yet, no effective pharmacotherapies. The Ts65Dn mouse model of DS is trisomic for orthologs of ∼55% of Hsa21 classical protein coding genes. These mice display many features relevant to those seen in DS, including deficits in learning and memory (L/M) tasks requiring a functional hippocampus. Recently, the N-methyl-D-aspartate (NMDA) receptor antagonist, memantine, was shown to rescue performance of the Ts65Dn in several L/M tasks. These studies, however, have not been accompanied by molecular analyses. In previous work, we described changes in protein expression induced in hippocampus and cortex in control mice after exposure to context fear conditioning (CFC), with and without memantine treatment. Here, we extend this analysis to Ts65Dn mice, measuring levels of 85 proteins/protein modifications, including components of MAP kinase and MTOR pathways, and subunits of NMDA receptors, in cortex and hippocampus of Ts65Dn mice after failed learning in CFC and after learning was rescued by memantine. We show that, compared with wild type littermate controls, (i) of the dynamic responses seen in control mice in normal learning, >40% also occur in Ts65Dn in failed learning or are compensated by baseline abnormalities, and thus are considered necessary but not sufficient for successful learning, and (ii) treatment with memantine does not in general normalize the initial protein levels but instead induces direct and indirect responses in approximately half the proteins measured and results in normalization of the endpoint protein levels. Together, these datasets provide a first view of the complexities associated with pharmacological rescue of learning in the Ts65Dn. Extending such studies to additional drugs and mouse models of DS will aid in identifying pharmacotherapies for effective clinical trials. PMID:25793384

Elevated S100A8/A9 and S100A12 Serum Levels Reflect Intraocular Inflammation in Juvenile Idiopathic Arthritis-Associated Uveitis: Results From a Pilot Study.

PubMed

Walscheid, Karoline; Heiligenhaus, Arnd; Holzinger, Dirk; Roth, Johannes; Heinz, Carsten; Tappeiner, Christoph; Kasper, Maren; Foell, Dirk

2015-12-01

Juvenile idiopathic arthritis-associated uveitis (JIAU) is the most common uveitis entity in childhood. As S100A8/A9 and S100A12 proteins are valuable biomarkers in childhood arthritis, we investigated the occurrence of these proteins in childhood uveitis. Serum samples from patients with JIAU (n = 79) or idiopathic anterior uveitis (IAU, n = 24), as well as from nonuveitic controls (n = 24), were collected. Furthermore, aqueous humor samples (JIAU n = 17, nonuveitic controls n = 16, IAU n = 12) were obtained. Samples were analyzed for S100A8/A9 and S100A12 protein levels by ELISA. Intergroup comparisons were performed, involving patient data, clinical data, and S100 levels. S100A8/A9 and S100A12 serum levels were elevated in IAU and JIAU patients as compared to nonuveitic controls (all P < 0.05). S100 serum levels in JIAU patients were higher in active arthritis (not significant; P = 0.289 for S100A8/A9 and P = 0.196 for S100A12) and active uveitis (P = 0.010 for S100A8/A9 and P = 0.026 for S100A12) than in controls. No significant differences in S100 levels were found in a subgroup analysis for sex, antinuclear antibody (ANA) status, disease duration, or presence of uveitis complications. In JIAU patients, S100 serum levels correlated with age and age at onset of uveitis. A longitudinal analysis in JIAU patients showed a correlation of serum S100A8/A9 and S100A12 levels with uveitis activity (both P = 0.03). S100A8/A9 levels in aqueous humor of patients with JIAU (P = 0.001) and IAU (P = 0.0002) were increased as compared to nonuveitic controls. Increased S100A8/A9 and S100A12 levels are found in the serum and aqueous humor of patients with autoimmune uveitis. Serum levels reflect activity of joint and eye disease.

PTB and TIAR binding to insulin mRNA 3'- and 5'UTRs; implications for insulin biosynthesis and messenger stability.

PubMed

Fred, Rikard G; Mehrabi, Syrina; Adams, Christopher M; Welsh, Nils

2016-09-01

Insulin expression is highly controlled on the posttranscriptional level. The RNA binding proteins (RBPs) responsible for this result are still largely unknown. To identify RBPs that bind to insulin mRNA we performed mass spectrometry analysis on proteins that bound synthetic oligonucloetides mimicing the 5'- and the 3'-untranslated regions (UTRs) of rat and human insulin mRNA in vitro . We observed that the RBPs heterogeneous nuclear ribonucleoprotein (hnRNP) U, polypyrimidine tract binding protein (PTB), hnRNP L and T-cell restricted intracellular antigen 1-related protein (TIA-1-related protein; TIAR) bind to insulin mRNA sequences, and that the in vitro binding affinity of these RBPs changed when INS-1 cells were exposed to glucose, 3-isobutyl-1-methylxanthine (IBMX) or nitric oxide. High glucose exposure resulted in a modest increase in PTB and TIAR binding to an insulin mRNA sequence. The inducer of nitrosative stress DETAnonoate increased markedly hnRNP U and TIAR mRNA binding. An increased PTB to TIAR binding ratio in vitro correlated with higher insulin mRNA levels and insulin biosynthesis rates in INS-1 cells. To further investigate the importance of RNA-binding proteins for insulin mRNA stability, we decreased INS-1 and EndoC-βH1 cell levels of PTB and TIAR by RNAi. In both cell lines, decreased levels of PTB resulted in lowered insulin mRNA levels while decreased levels of TIAR resulted in increased insulin mRNA levels. Thapsigargin-induced stress granule formation was associated with a redistribution of TIAR from the cytosol to stress granules. These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.

The role of the postsynaptic density in the pathology of the fragile X syndrome.

PubMed

Kindler, Stefan; Kreienkamp, Hans-Jürgen

2012-01-01

The protein repertoire of excitatory synapses controls dendritic spine morphology, synaptic plasticity and higher brain functions. In brain neurons, the RNA-associated fragile X mental retardation protein (FMRP) binds in vivo to various transcripts encoding key postsynaptic components and may thereby substantially regulate the molecular composition of dendritic spines. In agreement with this notion functional loss of FMRP in patients affected by the fragile X syndrome (FXS) causes cognitive impairment. Here we address our current understanding of the functional role of individual postsynaptic proteins. We discuss how FMRP controls the abundance of select proteins at postsynaptic sites, which signaling pathways regulate the local activity of FMRP at synapses, and how altered levels of postsynaptic proteins may contribute to FXS pathology.

Proximate nutritional composition of CELSS crops grown at different CO2 partial pressures

NASA Technical Reports Server (NTRS)

Wheeler, R. M.; Mackowiak, C. L.; Sager, J. C.; Knott, W. M.; Berry, W. L.

1994-01-01

Two Controlled Ecological Life Support System (CELSS) candidate crops, soybean (Glycine max) and potato (Solanum tuberosum), were grown hydroponically in controlled environments maintained at carbon dioxide (CO2) partial pressures ranging from 0.05 to 1.00 kPa (500 to 10,000 ppm at 101 kPa atmospheric pressure). Plants were harvested at maturity (90 days for soybean and 105 days for potato) and all tissues analyzed for proximate nutritional composition (i.e. protein, fat, carbohydrate, crude fiber, and ash content). Soybean seed ash and crude fiber were higher and carbohydrate was lower than values reported for field-grown seed. Potato tubers showed little difference from field-grown tubers. Crude fiber of soybean stems and leaves increased with increased CO2, as did soybean leaf protein (total nitrogen). Potato leaf and stem (combined) protein levels also increased with increased CO2, while leaf and stem carbohydrates decreased. Values for leaf and stem protein and ash were higher than values generally reported for field-grown plants for both species. Results suggest that CO2 partial pressure should have little influence on proximate composition of potato tubers or soybean seed, but that high ash and protein levels might be expected from leaves and stems of crops grown in controlled environments of a CELSS.

Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

PubMed

Beilin, Orit; Karussis, Dimitrios M; Korczyn, Amos D; Gurwitz, David; Aronovich, Ramona; Mizrachi-Kol, Rachel; Chapman, Joab

2007-04-16

Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

The localization and concentration of the PDE2-encoded high-affinity cAMP phosphodiesterase is regulated by cAMP-dependent protein kinase A in the yeast Saccharomyces cerevisiae.

PubMed

Hu, Yun; Liu, Enkai; Bai, Xiaojia; Zhang, Aili

2010-03-01

The genome of the yeast Saccharomyces cerevisiae encodes two cyclic AMP (cAMP) phosphodiesterases, a low-affinity one, Pde1, and a high-affinity one, Pde2. Pde1 has been ascribed a function for downregulating agonist-induced cAMP accumulation in a protein kinase A (PKA)-governed negative feedback loop, whereas Pde2 controls the basal cAMP level in the cell. Here we show that PKA regulates the localization and protein concentration of Pde2. Pde2 is accumulated in the nucleus in wild-type cells growing on glucose, or in strains with hyperactive PKA. In contrast, in derepressed wild-type cells or cells with attenuated PKA activity, Pde2 is distributed over the nucleus and cytoplasm. We also show evidence indicating that the Pde2 protein level is positively correlated with PKA activity. The increase in the Pde2 protein level in high-PKA strains and in cells growing on glucose was due to its increased half-life. These results suggest that, like its low-affinity counterpart, the high-affinity phosphodiesterase may also play an important role in the PKA-controlled feedback inhibition of intracellular cAMP.

Mycobacterium leprae upregulates IRGM expression in monocytes and monocyte-derived macrophages.

PubMed

Yang, Degang; Chen, Jia; Zhang, Linglin; Cha, Zhanshan; Han, Song; Shi, Weiwei; Ding, Ru; Ma, Lan; Xiao, Hong; Shi, Chao; Jing, Zhichun; Song, Ningjing

2014-08-01

Leprosy is caused by the infection of Mycobacterium leprae, which evokes a strong inflammatory response and leads to nerve damage. Immunity-related GTPase family M protein (IRGM) plays critical roles in controlling inflammation. The objective of the study was to investigate whether IRGM is involved in the infection of M. leprae. Levels of IRGM were assessed in M. leprae-infected CD4(+) T cells, monocytes, and monocyte-derived macrophages. Data revealed that both protein and mRNA levels of IRGM were increased in monocytes after M. leprae infection. Interestingly, monocyte-derived macrophages showed more prominent IRGM expression with M. leprae infection, whereas the bacteria did not affect IRGM in CD4(+) T cells. Furthermore, we assessed levels of IRGM in CD4(+) T cells and monocytes from 78 leprosy patients and 40 healthy controls, and observed upregulated protein level of IRGM in the monocytes from leprosy patients. Also, IRGM expression was inversely correlated with the severity of the disease. These findings suggested a close involvement of IRGM in M. leprae infection and indicated a potential mechanism of defending M. leprae infection.

Redox control of protein-DNA interactions: from molecular mechanisms to significance in signal transduction, gene expression, and DNA replication.

PubMed

Shlomai, Joseph

2010-11-01

Protein-DNA interactions play a key role in the regulation of major cellular metabolic pathways, including gene expression, genome replication, and genomic stability. They are mediated through the interactions of regulatory proteins with their specific DNA-binding sites at promoters, enhancers, and replication origins in the genome. Redox signaling regulates these protein-DNA interactions using reactive oxygen species and reactive nitrogen species that interact with cysteine residues at target proteins and their regulators. This review describes the redox-mediated regulation of several master regulators of gene expression that control the induction and suppression of hundreds of genes in the genome, regulating multiple metabolic pathways, which are involved in cell growth, development, differentiation, and survival, as well as in the function of the immune system and cellular response to intracellular and extracellular stimuli. It also discusses the role of redox signaling in protein-DNA interactions that regulate DNA replication. Specificity of redox regulation is discussed, as well as the mechanisms providing several levels of redox-mediated regulation, from direct control of DNA-binding domains through the indirect control, mediated by release of negative regulators, regulation of redox-sensitive protein kinases, intracellular trafficking, and chromatin remodeling.

β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

PubMed

Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

2012-08-15

Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

Altered expression of BDNF, BDNF pro-peptide and their precursor proBDNF in brain and liver tissues from psychiatric disorders: rethinking the brain-liver axis.

PubMed

Yang, B; Ren, Q; Zhang, J-C; Chen, Q-X; Hashimoto, K

2017-05-16

Brain-derived neurotrophic factor (BDNF) has a role in the pathophysiology of psychiatric disorders. The precursor proBDNF is converted to mature BDNF and BDNF pro-peptide, the N-terminal fragment of proBDNF; however, the precise function of these proteins in psychiatric disorders is unknown. We sought to determine whether expression of these proteins is altered in the brain and peripheral tissues from patients with psychiatric disorders. We measured protein expression of proBDNF, mature BDNF and BDNF pro-peptide in the parietal cortex, cerebellum, liver and spleen from control, major depressive disorder (MDD), schizophrenia (SZ) and bipolar disorder (BD) groups. The levels of mature BDNF in the parietal cortex from MDD, SZ and BD groups were significantly lower than the control group, whereas the levels of BDNF pro-peptide in this area were significantly higher than controls. In contrast, the levels of proBDNF and BDNF pro-peptide in the cerebellum of MDD, SZ and BD groups were significantly lower than controls. Moreover, the levels of mature BDNF from the livers of MDD, SZ and BD groups were significantly higher than the control group. The levels of mature BDNF in the spleen did not differ among the four groups. Interestingly, there was a negative correlation between mature BDNF in the parietal cortex and mature BDNF in the liver in all the subjects. These findings suggest that abnormalities in the production of mature BDNF and BDNF pro-peptide in the brain and liver might have a role in the pathophysiology of psychiatric disorders, indicating a brain-liver axis in psychiatric disorders.

The pink-eyed dilution locus controls the biogenesis of melanosomes and levels of melanosomal proteins in the eye.

PubMed

Orlow, S J; Brilliant, M H

1999-02-01

The pink-eyed dilution (p) locus is known to control the quantity of melanin pigment made within melanocytes and retinal pigment epithelium (RPE) in the eye. We have examined the effects of several mutant allele combinations at the murine p locus on the number and morphology of melanosomes in choroidal melanocytes and RPE cells as well as on the levels of four proteins known to be present within melanosomes: tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) and lysosome-associated membrane protein-1 (LAMP-1). By electron microscopy, we observed a modest diminution in the size and number of choroidal melanosomes in pbs/pJ mice but a more dramatic decrease in the RPE in comparison with wild-type P/P mice. By contrast, a drastic reduction in melanosome size and number was present in the choroid and RPE of pun/pun and p6H/pcp mice, and in the RPE of p6H/pcp mice, melanosomes were essentially undetectable. In wild-type mice, levels of tyrosinase, TRP-1 and TRP-2 were high at birth and showed a second peak of expression at 10-14 days of age, declining to undetectable levels by 42 days. All three mutant allele combinations reduced the levels of these melanosomal proteins with the relative severity of effects being p6H/pcp>pun/pun>pbs/pJ. In the null p6H/pcp mice, levels of these proteins were extremely low at birth, no postnatal peak was observed, and levels declined to undetectable by 14 days. Levels of LAMP-1 in wild-type mice rose initially and then declined whereas in the mutant mice, levels decreased gradually from birth. Higher levels of LAMP-1 were observed in each of the mutants than in the wild-type mice at 21 days of age. Our results demonstrate that mutations at the p locus affect the size, number, shape and contents of melanosomes, implicating the p gene product in the normal biogenesis of this organelle. Copyright 1999 Academic Press.

Proteomic analysis reveals the enhancement of human serum apolipoprotein A-1(APO A-1) in individuals infected with multiple dengue virus serotypes.

PubMed

Manchala, Nageswar Reddy; Dungdung, Ranjeet; Pilankatta, Rajendra

2017-10-01

Human serum protein profiling of the individual infected with multiple dengue virus serotypes for identifying the potential biomarkers and to investigate the cause for the severity of dengue virus infection. Dengue virus NS1-positive serum samples were pooled into two groups (S2 and S3) based on the molecular serotyping and number of heterotypic infections. The pooled serum samples were subjected to two-dimensional gel electrophoresis (2DGE) to identify the differentially expressed proteins. The peptide masses of upregulated protein were detected by matrix-assisted laser desorption-ionisation time-of-flight MALDI-TOF mass spectrometry and analysed by MASCOT search engine. The results were compared with the control group (S1). The commonly upregulated protein was validated by quantitative ELISA and compared with control as well as single serotypic infected samples. Based on 2DGE, total thirteen proteins were differentially upregulated in S2 and S3 groups as compared to control. Some of the upregulated proteins were involved in mediating the complement activation of immune response. The apolipoprotein A-1 (APO A-1) was upregulated in S2 and S3 groups. Upon validation, APO A-1 levels were increased in line with the number of heterotypic infection of dengue viruses. Heterotypic infection of dengue viruses upregulate the serum proteins involved in the complement pathway in the early phase of infection. There was a significant increase in the level of APO A-1 in three different serotypic infections of dengue virus as compared to control. Further, the role of APO-A1 can be explored in elucidating the mechanism of dengue pathogenesis. © 2017 John Wiley & Sons Ltd.

Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: non-label methods comparison, q-values and LOWESS curve fitting

PubMed Central

Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

2009-01-01

Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic “two-state” experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error. PMID:19337574

Hypersensitivity linked to exposure of broad bean protein(s) in allergic patients and BALB/c mice.

PubMed

Kumar, Dinesh; Kumar, Sandeep; Verma, Alok K; Sharma, Akanksha; Tripathi, Anurag; Chaudhari, Bhushan P; Kant, Surya; Das, Mukul; Jain, Swatantra K; Dwivedi, Premendra D

2014-01-01

Broad bean (Vicia faba L.), a common vegetable, belongs to the family Fabaceae and is consumed worldwide. Limited studies have been done on allergenicity of broad beans. The aim of this study was to determine if broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins. Simulated gastric fluid (SGF) assay and immunoglobulin E (IgE) immunoblotting were carried out to identify pepsin-resistant and IgE-binding proteins. The allergenicity of broad beans was assessed in allergic patients, BALB/c mice, splenocytes, and RBL-2H3 cells. Eight broad bean proteins of approximate molecular weight 70, 60, 48, 32, 23, 19, 15, and 10 kDa that remained undigested in SGF, showed IgE-binding capacity as well. Of 127 allergic patients studied, broad bean allergy was evident in 16 (12%). Mice sensitized with broad bean showed increased levels of histamine, total and specific IgE, and severe signs of systemic anaphylaxis compared with controls. Enhanced levels of histamine, prostaglandin D2, cysteinyl leukotriene, and β-hexosaminidase release were observed in the primed RBL-2H3 cells following broad bean exposure. The levels of interleukin IL-4, IL-5, IL-13 and regulated on activation, normal T-cell expressed and secreted were found enhanced in broad bean-treated splenocytes culture supernatant compared with controls. This study inferred that broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli.

PubMed

Pesavento, Christina; Becker, Gisela; Sommerfeldt, Nicole; Possling, Alexandra; Tschowri, Natalia; Mehlis, Anika; Hengge, Regine

2008-09-01

During the transition from post-exponential to stationary phase, Escherichia coli changes from the motile-planktonic to the adhesive-sedentary "lifestyle." We demonstrate this transition to be controlled by mutual inhibition of the FlhDC/motility and sigma(S)/adhesion control cascades at two distinct hierarchical levels. At the top level, motility gene expression and the general stress response are inversely coordinated by sigma(70)/sigma(FliA)/sigma(S) competition for core RNA polymerase and the FlhDC-controlled FliZ protein acting as a sigma(S) inhibitor. At a lower level, the signaling molecule bis-(3'-5')-cyclic-diguanosine monophosphate (c-di-GMP) reduces flagellar activity and stimulates transcription of csgD, which encodes an essential activator of adhesive curli fimbriae expression. This c-di-GMP is antagonistically controlled by sigma(S)-regulated GGDEF proteins (mainly YegE) and YhjH, an EAL protein and c-di-GMP phosphodiesterase under FlhDC/FliA control. The switch from motility-based foraging to the general stress response and curli expression requires sigma(S)-modulated down-regulation of expression of the flagellar regulatory cascade as well as proteolysis of the flagellar master regulator FlhDC. Control of YhjH by FlhDC and of YegE by sigma(S) produces a fine-tuned checkpoint system that "unlocks" curli expression only after down-regulation of flagellar gene expression. In summary, these data reveal the logic and sequence of molecular events underlying the motile-to-adhesive "lifestyle" switch in E. coli.

Proximal tubule proteins are significantly elevated in bladder urine of patients with ureteropelvic junction obstruction and may represent novel biomarkers: A pilot study.

PubMed

Gerber, Claire; Harel, Miriam; Lynch, Miranda L; Herbst, Katherine W; Ferrer, Fernando A; Shapiro, Linda H

2016-04-01

Ureteropelvic junction obstruction (UPJO) is the major cause of hydronephrosis in children and may lead to renal injury and early renal dysfunction. However, diagnosis of the degree of obstruction and severity of renal injury relies on invasive and often inconclusive renal scans. Biomarkers from voided urine that detect early renal injury are highly desirable because of their noninvasive collection and their potential to assist in earlier and more reliable diagnosis of the severity of obstruction. Early in response to UPJO, increased intrarenal pressure directly impacts the proximal tubule brush border. We hypothesize that single-pass, apically expressed proximal tubule brush border proteins will be shed into the urine early and rapidly and will be reliable noninvasive urinary biomarkers, providing the tools for a more reliable stratification of UPJO patients. We performed a prospective cohort study at Connecticut Children's Medical Center. Bladder urine samples from 12 UPJO patients were obtained prior to surgical intervention. Control urine samples were collected from healthy pediatric patients presenting with primary nocturnal enuresis. We determined levels of NGAL, KIM-1 (previously identified biomarkers), CD10, CD13, and CD26 (potentially novel biomarkers) by ELISA in control and experimental urine samples. Urinary creatinine levels were used to normalize the urinary protein levels measured by ELISA. Each of the proximal tubule proteins outperformed the previously published biomarkers. No differences in urinary NGAL and KIM-1 levels were observed between control and obstructed patients (p = 0.932 and p = 0.799, respectively). However, levels of CD10, CD13, and CD26 were significantly higher in the voided urine of obstructed individuals when compared with controls (p = 0.002, p = 0.024, and p = 0.007, respectively) (Figure). Targeted identification of reliable, noninvasive biomarkers of renal injury is critical to aid in diagnosing patients at risk, guiding therapeutic decisions and monitoring treatment efficacy. Proximal tubule brush border proteins are reliably detected in the urine of obstructed patients and may be more effective at predicting UPJO. Copyright © 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion*

PubMed Central

Hung, Chien-Wen; Klein, Tobias; Cassidy, Liam; Linke, Dennis; Lange, Sabrina; Anders, Uwe; Bureik, Matthias; Heinzle, Elmar; Schneider, Konstantin; Tholey, Andreas

2016-01-01

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. PMID:27477394

A novel amino acids oral supplementation in hemodialysis patients: a pilot study.

PubMed

Bolasco, Piergiorgio; Caria, Stefania; Cupisti, Adamasco; Secci, Romina; Saverio Dioguardi, Francesco

2011-01-01

Protein malnutrition and lowering serum albumin is frequent in hemodialysis patients. A special amino acid formulation has recently been used with favorable effects in elderly people but no data exist in renal patients. To assess the effects of this novel amino acid formulation in stable hemodialysis patients with reduced albumin levels. Thirty stable hemodialysis patients with serum albumin levels <3.5 g/dL, normalized protein nitrogen appearance (nPNA) <1.1 g/kg/d, and body mass index (BMI) >20 kg/m(2) were selected: 15 patients were randomized to oral amino acid supplementation (4 g thrice a day) for 3 months and 15 patients comparable for age, gender, and dialysis durations formed the control group. Biochemistry and bioimpedentiometry parameters were measured at baseline and at the end of treatment. No difference was observed between study group and control group at baseline. At the end of the study period, no change occurred in the studied parameters in the control group, whereas increase in serum albumin (3.1 ± 0.3 vs. 3.6 ± 0.2 g/dL, p < 0.001) and in total proteins (5.7 ± 0.4 vs. 6.4 ± 0.7 g/dL, p < 0.001) occurred in the study group. Hemoglobin rose from 10.7 ± 0.9 to 11.7 ± 0.8 g/dL (p < 0.05) at the same erythropoiesis-stimulating agent (ESA) dosage. C-Reactive protein (CRP) levels decreased in the study group (8.7 ± 7.3 vs. 3.8 ± 3.1 mg/L, p < 0.01). Increase of body weight and of equilibrated protein catabolic rate (ePCR) was observed in the study group. Oral amino acids supplementation was able to improve albumin and total protein in hypoalbuminemia hemodialysis patients. This effect was associated with reduction of CRP levels that is with lowering of pro-inflammatory status and anemia improvement.

Autoantibodies to nervous system-specific proteins are elevated in sera of flight crew members: biomarkers for nervous system injury.

PubMed

Abou-Donia, Mohamed B; Abou-Donia, Martha M; ElMasry, Eman M; Monro, Jean A; Mulder, Michel F A

2013-01-01

This descriptive study reports the results of assays performed to detect circulating autoantibodies in a panel of 7 proteins associated with the nervous system (NS) in sera of 12 healthy controls and a group of 34 flight crew members including both pilots and attendants who experienced adverse effects after exposure to air emissions sourced to the ventilation system in their aircrafts and subsequently sought medical attention. The proteins selected represent various types of proteins present in nerve cells that are affected by neuronal degeneration. In the sera samples from flight crew members and healthy controls, immunoglobin (IgG) was measured using Western blotting against neurofilament triplet proteins (NFP), tubulin, microtubule-associated tau proteins (tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and glial S100B protein. Significant elevation in levels of circulating IgG-class autoantibodies in flight crew members was found. A symptom-free pilot was sampled before symptoms and then again afterward. This pilot developed clinical problems after flying for 45 h in 10 d. Significant increases in autoantibodies were noted to most of the tested proteins in the serum of this pilot after exposure to air emissions. The levels of autoantibodies rose with worsening of his condition compared to the serum sample collected prior to exposure. After cessation of flying for a year, this pilot's clinical condition improved, and eventually he recovered and his serum autoantibodies against nervous system proteins decreased. The case study with this pilot demonstrates a temporal relationship between exposure to air emissions, clinical condition, and level of serum autoantibodies to nervous system-specific proteins. Overall, these results suggest the possible development of neuronal injury and gliosis in flight crew members anecdotally exposed to cabin air emissions containing organophosphates. Thus, increased circulating serum autoantibodies resulting from neuronal damage may be used as biomarkers for chemical-induced CNS injury.

Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Jinmei; Wang Xuefeng; Xi Zhiqin

2006-10-06

Purpose: TRAP220 (thyroid hormone receptor-associated protein) functions as a coactivator for nuclear receptors and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. Thus, TRAP220 enhances the function of thyroid/steroid hormone receptors such as thyroid hormone and oestrogen receptors. This study investigated the expression of TRAP220 mRNA and protein level in epileptic brains comparing with human control. Methods: We examined the expression of TRAP220 mRNA and protein levels in temporal lobes from patients with chronic pharmacoresistant epilepsy who have undergone surgery. Results: Expression of TRAP220 mRNA and protein was shown to be decreased significantly in themore » temporal cortex of the patients with epilepsy. Conclusions: Our work showed that a decrease in TRAP220 mRNA and protein levels may be involved in the pathophysiology of epilepsy and may be associated with impairment of the brain caused by frequent seizures.« less

Pervasive, Coordinated Protein-Level Changes Driven by Transcript Isoform Switching during Meiosis.

PubMed

Cheng, Ze; Otto, George Maxwell; Powers, Emily Nicole; Keskin, Abdurrahman; Mertins, Philipp; Carr, Steven Alfred; Jovanovic, Marko; Brar, Gloria Ann

2018-02-22

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous genome-wide measurements of mRNA, translation, and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases, or over 8% of all measured genes, involves temporally regulated switching between production of a canonical, translatable transcript and a 5' extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether or not an mRNA is induced but rather on the type of transcript produced. Copyright © 2018 Elsevier Inc. All rights reserved.

Laser Trabeculoplasty Induces Changes in the Trabecular Meshwork Glycoproteome: A pilot study

PubMed Central

Amelinckx, Adriana; Castello, Maria; Arrieta-Quintero, Esdras; Lee, Tinthu; Salas, Nelson; Hernandez, Eleut; Lee, Richard K.; Bhattacharya, Sanjoy K.; Parel, Jean-Marie A

2009-01-01

Laser trabeculoplasty (LT) is a commonly used modality of treatment for glaucoma. The mechanism by which LT lowers the intraocular pressure (IOP) is unknown. Using cat eyes, selective laser trabeculoplasty (SLT) with a Q-switched frequency doubled Nd:YAG laser was used to treat the trabecular meshwork (TM). Laser treated TM was then subjected to proteomic analysis for detection of molecular changes and histological analysis for the detection of structural and protein expression patterns. In addition, the protein glycosylation patterns of laser treated and non-treated TM was assessed and differentially glycosylated proteins were proteomically identified. SLT laser treatment to the TM resulted in elevated glycosylation levels compared to non-lasered TM. TM laser treatment also resulted in protein expression levels changes of several proteins. Elevated levels of biglycan, keratocan and prolargin were detected in laser treated TM compared to non-lasered controls. Further investigation is anticipated to provide insight into how glycosylation changes affect TM proteins and TM regulation of aqueous outflow in response to laser trabeculoplasty. PMID:19432485

Laser trabeculoplasty induces changes in the trabecular meshwork glycoproteome: a pilot study.

PubMed

Amelinckx, Adriana; Castello, Maria; Arrieta-Quintero, Esdras; Lee, Tinthu; Salas, Nelson; Hernandez, Eleut; Lee, Richard K; Bhattacharya, Sanjoy K; Parel, Jean-Marie A

2009-07-01

Laser trabeculoplasty (LT) is a commonly used modality of treatment for glaucoma. The mechanism by which LT lowers the intraocular pressure (IOP) is unknown. With the use of cat eyes, selective laser trabeculoplasty (SLT) with a Q-switched frequency doubled Nd:YAG laser was used to treat the trabecular meshwork (TM). Laser treated TM was then subjected to proteomic analysis for detection of molecular changes and histological analysis for the detection of structural and protein expression patterns. In addition, the protein glycosylation patterns of laser treated and nontreated TM was assessed and differentially glycosylated proteins were proteomically identified. SLT laser treatment to the TM resulted in elevated glycosylation levels compared to nonlasered TM. TM laser treatment also resulted in protein expression levels changes of several proteins. Elevated levels of biglycan, keratocan and prolargin were detected in laser treated TM compared to nonlasered controls. Further investigation is anticipated to provide insight into how glycosylation changes affect TM proteins and TM regulation of aqueous outflow in response to laser trabeculoplasty.

The burden of trisomy 21 disrupts the proteostasis network in Down syndrome

PubMed Central

Rauniyar, Abhishek K.; Jiang, Hua; Liggett, L. Alexander; Maclean, Kenneth N.

2017-01-01

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction. PMID:28430800

Ethanol extract of Moringa oliefera prevents in vitro glucose induced cataract on isolated goat eye lens.

PubMed

Kurmi, Raghvendra; Ganeshpurkar, Aditya; Bansal, Divya; Agnihotri, Abhishek; Dubey, Nazneen

2014-02-01

The aim of current work was to evaluate in vitro anticataract potential of Moringa oliefera extract. Goat eye lenses were divided into 4 groups; Group served as control, Group II as toxic control, Group III and Group IV were incubated in extract (250 μg/ml and 500 μg/ml of extract of M. oliefera) Group II, III and IV were incubated in 55 mM glucose in artificial aqueous humor to induce lens opacification. Estimation of total, water soluble protein, catalase, glutathione and malondialdehyde along with photographic evaluation of lens was done. Group II (toxic control) lenses showed high amount of MDA (Malondialdehyde), soluble, insoluble protein, decreased catalase and glutathione levels, while lenses treated with Moringa oliefera extract (Group III and Group IV) showed significant (FNx01 P < 0.05) reduction in MDA and increased level of catalase, glutathione, total and soluble protein. Results of present findings suggest protective effect of Moringa oliefera in prevention of in vitro glucose induced cataract.

Protein-based materials, toward a new level of structural control.

PubMed

van Hest, J C; Tirrell, D A

2001-10-07

Through billions of years of evolution nature has created and refined structural proteins for a wide variety of specific purposes. Amino acid sequences and their associated folding patterns combine to create elastic, rigid or tough materials. In many respects, nature's intricately designed products provide challenging examples for materials scientists, but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes. An increasingly important approach to this problem has been to use biological systems for production of materials. Through protein engineering, artificial genes can be developed that encode protein-based materials with desired features. Structural elements found in nature, such as beta-sheets and alpha-helices, can be combined with great flexibility, and can be outfitted with functional elements such as cell binding sites or enzymatic domains. The possibility of incorporating non-natural amino acids increases the versatility of protein engineering still further. It is expected that such methods will have large impact in the field of materials science, and especially in biomedical materials science, in the future.

Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

PubMed

Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

2017-09-15

Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

Calpeptin is neuroprotective against acrylamide-induced neuropathy in rats.

PubMed

Su, Benyu; Guan, Qiangdong; Wang, Miaomiao; Liu, Ning; Wei, Xiaomin; Wang, Shue; Yang, Xiwei; Jiang, Wenchong; Xu, Mengmeng; Yu, Sufang

2018-05-01

The aim of this study is to explore the potent neuroprotective effect of calpeptin (CP) on neuron damage induced by acrylamide (ACR) and its mechanism. Behavioural indicators such as hind limb splay, rota-rod performance, and gait analysis were assessed weekly to evaluate neurobehavioural changes after ACR and/or CP administration. The histopathological alterations and the changes of μ-calpain, m-calpain, microtubule-associated protein 2 (MAP2), and α-tubulin and β-tubulin protein levels in spinal cord were determined. Results showed that after administration of 30 mg/kg ACR, decreased body weight, attenuated neurobehavioural function, injury of motor neuron, increased protein levels of m-calpain and β-tubulin, suppressed MAP2 protein level, and no significant changes of μ-calpain and α-tubulin protein levels were observed compared with the control group rats. After administration of 200 μg/kg CP, partially restored body weight and neurobehavioural function, improvement of motor neuron injury, decreased protein levels of m- calpain and β-tubulin, and reversed effects of MAP2 protein level were observed compared with the ACR group rats. Our results suggested that CP alleviates neuropathy induced by ACR in rats. The calpain's overactivation causes the degrading of MAP2 and eventually leads to the destruction of microtubules (MTs), which may be one of the mechanisms of cytoskeletal damage induced by ACR. Copyright © 2018 Elsevier B.V. All rights reserved.

Chemical Synthesis of Proteins

PubMed Central

Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

2010-01-01

Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

Recovery of skeletal muscle after 3 mo of hindlimb immobilization in rats

NASA Technical Reports Server (NTRS)

Booth, F. W.; Seider, M. J.

1979-01-01

During immobilization, skeletal muscle undergoes decreases in size and strength with concomitant atrophic and degenerative changes in slow-twitch muscle fibers. Currently there are no objective data in slow-twitch muscle demonstrating recovery of biochemical or physiological indices following termination of immobilization. The purpose of this study was to determine whether the soleus, a slow-twitch muscle, could recover normal biochemical or physiological levels following termination of immobilization. Adenosine triphosphate, glycogen, and protein concentration (mg/g wet wt) all significantly decreased following 90 days of hindlimb immobilization, but these three values returned to control levels by the 60th recovery day. Similarly, soleus muscle wet weight and protein content (mg protein/muscle) returned to control levels by the 14th recovery day. In contrast, maximal isometric tension did not return to normal until the 120th day. These results indicate that following muscular atrophy, which was achieved through 90 days of hindlimb immobilization, several biochemical and physiological values in skeletal muscle are recovered at various times after the end of immobilization.

Recovery of insulin sensitivity and optimal body composition after rapid weight loss in obese dogs fed a high-protein medium-carbohydrate diet.

PubMed

André, A; Leriche, I; Chaix, G; Thorin, C; Burger, M; Nguyen, P

2017-06-01

This study investigated the effects of an experimental high-protein medium-carbohydrate diet (protein level, 46% metabolizable energy, ME). First, postprandial plasma glucose and insulin kinetics were determined in steady-state overweight/obese Beagle dogs (28%-41% excess body weight) for an experimental high-protein medium-carbohydrate diet (protein level, 46% ME) and a commercial high-carbohydrate medium-protein diet (protein level, 24%ME) in obese dogs. Secondly, all the dogs were included in a weight loss programme. They were fed the high-protein medium-carbohydrate diet, and the energy allocation was gradually reduced until they reached their optimal body weight. Insulin sensitivity and body composition were evaluated before and after weight loss using a euglycaemic-hyperinsulinaemic clamp and the deuterium oxide dilution technique respectively. For statistical analysis, linear mixed effect models were used with a significance level of 5%. Postprandial plasma glucose and insulin concentrations were substantially lower with the high-protein medium-carbohydrate diet than the high-carbohydrate medium-protein diet. These differences can be explained mainly by the difference in carbohydrate content between the two diets. Energy restriction (35% lower energy intake than in the obese state) resulted in a 2.23 ± 0.05% loss in body weight/week, and the dogs reached their optimal body weight in 12-16 weeks. Weight loss was associated with a significant increase in insulin sensitivity. The high-protein medium-carbohydrate diet allowed fat-free mass preservation despite a relatively high rate of weekly weight loss. The increase in insulin sensitivity indicated improved control of carbohydrate metabolism, possible due to weight loss and to the nature of the diet. Thus, a high-protein medium-carbohydrate diet is a good nutritional solution for managing the weight of overweight dogs. This diet may improve glycaemic control, which could be beneficial for preventing or managing impaired glucose tolerance in obese dogs and for safe and successful weight loss while preserving lean body mass. Journal of Animal Physiology and Animal Nutrition © 2017 Blackwell Verlag GmbH.

Tick capillary feeding for the study of proteins involved in tick-pathogen interactions as potential antigens for the control of tick infestation and pathogen infection

PubMed Central

2014-01-01

Background Ticks represent a significant health risk to animals and humans due to the variety of pathogens they can transmit during feeding. The traditional use of chemicals to control ticks has serious drawbacks, including the selection of acaricide-resistant ticks and environmental contamination with chemical residues. Vaccination with the tick midgut antigen BM86 was shown to be a good alternative for cattle tick control. However, results vary considerably between tick species and geographic location. Therefore, new antigens are required for the development of vaccines controlling both tick infestations and pathogen infection/transmission. Tick proteins involved in tick-pathogen interactions may provide good candidate protective antigens for these vaccines, but appropriate screening procedures are needed to select the best candidates. Methods In this study, we selected proteins involved in tick-Anaplasma (Subolesin and SILK) and tick-Babesia (TROSPA) interactions and used in vitro capillary feeding to characterize their potential as antigens for the control of cattle tick infestations and infection with Anaplasma marginale and Babesia bigemina. Purified rabbit polyclonal antibodies were generated against recombinant SUB, SILK and TROSPA and added to uninfected or infected bovine blood to capillary-feed female Rhipicephalus (Boophilus) microplus ticks. Tick weight, oviposition and pathogen DNA levels were determined in treated and control ticks. Results The specificity of purified rabbit polyclonal antibodies against tick recombinant proteins was confirmed by Western blot and against native proteins in tick cell lines and tick tissues using immunofluorescence. Capillary-fed ticks ingested antibodies added to the blood meal and the effect of these antibodies on tick weight and oviposition was shown. However, no effect was observed on pathogen DNA levels. Conclusions These results highlighted the advantages and some of the disadvantages of in vitro tick capillary feeding for the characterization of candidate tick protective antigens. While an effect on tick weight and oviposition was observed, the effect on pathogen levels was not evident probably due to high tick-to-tick variations among other factors. Nevertheless, these results together with previous results of RNA interference functional studies suggest that these proteins are good candidate vaccine antigens for the control of R. microplus infestations and infection with A. marginale and B. bigemina. PMID:24450836

Host cell proteins in biotechnology-derived products: A risk assessment framework.

PubMed

de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

2015-11-01

To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

Molecular imaging of drug-modulated protein-protein interactions in living subjects.

PubMed

Paulmurugan, Ramasamy; Massoud, Tarik F; Huang, Jing; Gambhir, Sanjiv S

2004-03-15

Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor kappaB promoter/enhancer elements using tumor necrosis factor alpha, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes.

PubMed

Bruzzoni-Giovanelli, Heriberto; Fernandez, Plinio; Veiga, Lucía; Podgorniak, Marie-Pierre; Powell, Darren J; Candeias, Marco M; Mourah, Samia; Calvo, Fabien; Marín, Mónica

2010-02-09

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

Perturbations in the apoptotic pathway and mitochondrial network dynamics in peripheral blood mononuclear cells from bipolar disorder patients

PubMed Central

Scaini, G; Fries, G R; Valvassori, S S; Zeni, C P; Zunta-Soares, G; Berk, M; Soares, J C; Quevedo, J

2017-01-01

Bipolar disorder (BD) is a severe psychiatric disorder characterized by phasic changes of mood and can be associated with progressive structural brain change and cognitive decline. The numbers and sizes of glia and neurons are reduced in several brain areas, suggesting the involvement of apoptosis in the pathophysiology of BD. Because the changes in mitochondrial dynamics are closely related with the early process of apoptosis and the specific processes of apoptosis and mitochondrial dynamics in BD have not been fully elucidated, we measured the apoptotic pathway and the expression of mitochondrial fission/fusion proteins from BD patients and healthy controls. We recruited 16 patients with BD type I and sixteen well-matched healthy controls and investigated protein levels of several pro-apoptotic and anti-apoptotic factors, as well as the expression of mitochondrial fission/fusion proteins in peripheral blood mononuclear cells (PBMCs). Our results showed that the levels of the anti-apoptotic proteins Bcl-xL, survivin and Bcl-xL/Bak dimer were significantly decreased, while active caspase-3 protein levels were significantly increased in PBMCs from BD patients. Moreover, we observed the downregulation of the mitochondrial fusion-related proteins Mfn2 and Opa1 and the upregulation of the fission protein Fis1 in PBMCs from BD patients, both in terms of gene expression and protein levels. We also showed a significantly decrease in the citrate synthase activity. Finally, we found a positive correlation between Mfn2 and Opa1 with mitochondrial content markers, as well as a negative correlation between mitochondrial fission/fusion proteins and apoptotic markers. Overall, data reported here are consistent with the working hypothesis that apoptosis may contribute to cellular dysfunction, brain volume loss and progressive cognitive in BD. Moreover, we show an important relationship between mitochondrial dynamics and the cell death pathway activation in BD patients, supporting the link between mitochondrial dysfunction and the pathophysiology of BD. PMID:28463235

Insight into mechanism of oxidative DNA damage in angiomyolipomas from TSC patients

PubMed Central

Habib, Samy L

2009-01-01

Background The tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors, both angiomyolipomas and renal cell carcinomas. Loss of heterozygosity at the 8-oxoG-DNA glycosylase (OGG1) allele is found in human kidney clear cell carcinoma identifying loss of OGG1 function as a possible contributor to tumorigenesis in the kidney. Tuberin regulates OGG1 through the transcription factor NF-YA in cultured cells. The purpose of this study is to determine the effect of tuberin-deficiency on OGG1 protein and mRNA levels as well as on 8-oxodG levels in kidney tumors from patients with TSC. In addition we evaluated the phophorylation level of downstream targets of mTOR, phospho-S70K, in kidney tumor tissue from TSC patients. Results Kidney angiomyolipoma tissue from TSC patients expresses significant levels of phopho-tuberin and low levels of tuberin compared to control kidney tissue. The increase in tuberin phosphorylation and the decrease tuberin expression are associated with decrease in OGG1 protein and mRNA levels in tumor samples compared to normal kidney samples. The decrease OGG1 expression is also associated with significant decrease in the transcription factor, NF-YA, expression in tumor samples compared to normal tissues. In addition, the levels of 8-oxodG are 4-fold higher in tumors compared to control samples. The significant increase of phospho-tuberin expression is associated with increase phosphorylation of S6K in tumor samples compared to controls. Cyclin D1 expression is also 3-fold higher in increase in the tumor tissues compared to normal kidney tissues. Conclusion These data indicate that tuberin deficiency in angiomyolipoma enhances mTOR activation by phosphorylation of S6K and downregulation of protein and mRNA expression of OGG1 resulted in accumulation of oxidized DNA in patients with TSC. These data suggest that tuberin and OGG1 are important proteins in the pathogenesis of angiomyolipoma in TSC patients. PMID:19265534

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

PubMed

Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P; Norris, Steven J; Stevenson, Brian

2006-07-01

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Proteomic analysis of mouse islets after multiple low-dose streptozotocin injection.

PubMed

Xie, Xiaolei; Li, Shuai; Liu, Siyu; Lu, Yan; Shen, Pingping; Ji, Jianguo

2008-02-01

The islets of Langerhans are scattered throughout the pancreas and play a major role in the control of metabolic fuel homeostasis. To get a better understanding of the mechanisms underlying type 1 diabetes mellitus, we have generated a mouse model by injections of multiple low-dose streptozotocin. The islets in the mouse pancreas were handpicked and proteins from the islets were then isolated and separated by two-dimensional gel electrophoresis. Seven proteins were found to be altered significantly at expression level. Among the seven proteins, four were up-regulated and three were down-regulated in diabetic mice as compared with controls. These proteins were successfully identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and the changes of selected protein expression were further validated by quantitative real time PCR and Western blotting. Voltage-dependent anion-selective channel protein 1 and peroxiredoxin-4 were found for the first time to be associated with type 1 diabetes mellitus in mouse islets in the current study. These results suggest that glucose transport, beta cell proliferation/death, and oxidative stress play important roles in maintaining the balance of glucose level. Our study also provides novel insight into the mechanism of type 1 diabetes mellitus.

Whey protein enhances normal inflammatory responses during cutaneous wound healing in diabetic rats

PubMed Central

2011-01-01

Background Prolonged wound healing is a complication of diabetes that contributes to mortality. Impaired wound healing occurs as a consequence of excessive reactive oxygen species (ROS) production. Whey protein (WP) is able to reduce the oxygen radicals and increase the levels of the antioxidant glutathione. Thus, the aim of this study was to determine whether dietary supplementation with WP could enhance normal inflammatory responses during wound healing in diabetic rats. Animals were assigned into a wounded control group (WN), a wounded diabetic group (WD) and a wounded diabetic group orally supplemented with whey protein (WDWP) at a dose of 100 mg/kg body weight. Results Whey protein was found to significantly decrease the levels of malondialdehyde (MDA), nitric oxide (NO) and ROS. A significant restoration of the glutathione level was observed in WDWP rats. During the early wound healing stage, IL-1β, TNF-α, IL-6, IL-4 and neutrophil infiltration were significantly decreased in WD mice. WP supplementation was found to restore the levels of these inflammatory markers to the levels observed in control animals. In addition, the time required for wound healing was significantly prolonged in diabetic rats. WP was found to significantly decrease the time required for wound healing in WDWP rats. Conclusion In conclusion, dietary supplementation with WP enhances the normal inflammatory responses during wound healing in diabetic mice by restoring the levels of oxidative stress and inflammatory cytokines. PMID:22168406

Dietary soya protein improves intra-myocardial lipid deposition and altered glucose metabolism in a hypertensive, dyslipidaemic, insulin-resistant rat model.

PubMed

Oliva, María E; Creus, Agustina; Ferreira, María R; Chicco, Adriana; Lombardo, Yolanda B

2018-01-01

This study investigates the effects of replacing dietary casein by soya protein on the underlying mechanisms involved in the impaired metabolic fate of glucose and lipid metabolisms in the heart of dyslipidaemic rats chronically fed (8 months) a sucrose-rich (62·5 %) diet (SRD). To test this hypothesis, Wistar rats were fed an SRD for 4 months. From months 4 to 8, half the animals continued with the SRD and the other half were fed an SRD in which casein was substituted by soya. The control group received a diet with maize starch as the carbohydrate source. Compared with the SRD-fed group, the following results were obtained. First, soya protein significantly (P<0·001) reduced the plasma NEFA levels and normalised dyslipidaemia and glucose homoeostasis, improving insulin resistance. The protein levels of fatty acid translocase at basal state and under insulin stimulation and the protein levels and activity of muscle-type carnitine palmitoyltransferase 1 were normalised. Second, a significant (P<0·001) reduction of TAG, long-chain acyl CoA and diacylglycerol levels was observed in the heart muscle. Third, soya protein significantly increased (P<0·01) GLUT4 protein level under insulin stimulation and normalised glucose phosphorylation and oxidation. A reduction of phosphorylated AMP protein kinase protein level was recorded without changes in uncoupling protein 2 and PPARα. Fourth, hydroxyproline concentration decreased in the left ventricle and hypertension was normalised. The new information provided shows the beneficial effects of soya protein upon the altered pathways of glucose and lipid metabolism in the heart muscle of this rat model.

Using blood cytokine measures to define high inflammatory biotype of schizophrenia and schizoaffective disorder.

PubMed

Boerrigter, Danny; Weickert, Thomas W; Lenroot, Rhoshel; O'Donnell, Maryanne; Galletly, Cherrie; Liu, Dennis; Burgess, Martin; Cadiz, Roxanne; Jacomb, Isabella; Catts, Vibeke S; Fillman, Stu G; Weickert, Cynthia Shannon

2017-09-18

Increases in pro-inflammatory cytokines are found in the brain and blood of people with schizophrenia. However, increased cytokines are not evident in all people with schizophrenia, but are found in a subset. The cytokine changes that best define this subset, termed the "elevated inflammatory biotype", are still being identified. Using quantitative RT-PCR, we measured five cytokine mRNAs (IL-1β, IL-2 IL-6, IL-8 and IL-18) from peripheral blood of healthy controls and of people with schizophrenia or schizoaffective disorder (n = 165). We used a cluster analysis of the transcript levels to define those with low and those with elevated levels of cytokine expression. From the same cohort, eight cytokine proteins (IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IFNγ and TNFα) were measured in serum and plasma using a Luminex Magpix-based assay. We compared peripheral mRNA and protein levels across diagnostic groups and between those with low and elevated levels of cytokine expression according to our transcription-based cluster analysis. We found an overall decrease in the anti-inflammatory IL-2 mRNA (p = 0.006) and an increase in three serum cytokines, IL-6 (p = 0.010), IL-8 (p = 0.024) and TNFα (p < 0.001) in people with schizophrenia compared to healthy controls. A greater percentage of people with schizophrenia (48%) were categorised into the elevated inflammatory biotype compared to healthy controls (33%). The magnitude of increase in IL-1β, IL-6, IL-8 and IL-10 mRNAs in people in the elevated inflammation biotype ranged from 100 to 220% of those in the non-elevated inflammatory biotype and was comparable between control and schizophrenia groups. Blood cytokine protein levels did not correlate with cytokine mRNA levels, and plasma levels of only two cytokines distinguished the elevated and low inflammatory biotypes, with IL-1β significantly increased in the elevated cytokine control group and IL-8 significantly increased in the elevated cytokine schizophrenia group. Our results confirm that individuals with schizophrenia are more likely to have elevated levels of inflammation compared to controls. We suggest that efforts to define inflammatory status based on peripheral measures need to consider both mRNA and protein measures as each have distinct advantages and disadvantages and can yield different results.

Cerebrospinal Fluid Biomarkers of Neurodegeneration Are Decreased or Normal in Narcolepsy.

PubMed

Jørgen Jennum, Poul; Østergaard Pedersen, Lars; Czarna Bahl, Justyna Maria; Modvig, Signe; Fog, Karina; Holm, Anja; Rahbek Kornum, Birgitte; Gammeltoft, Steen

2017-01-01

To investigate whether cerebrospinal fluid (CSF) biomarkers of neurodegeneration are altered in narcolepsy in order to evaluate whether the hypocretin deficiency and abnormal sleep-wake pattern in narcolepsy leads to neurodegeneration. Twenty-one patients with central hypersomnia (10 type 1 narcolepsy, 5 type 2 narcolepsy, and 6 idiopathic hypersomnia cases), aged 33 years on average and with a disease duration of 2-29 years, and 12 healthy controls underwent CSF analyses of the levels of β-amyloid, total tau protein (T-tau), phosphorylated tau protein (P-tau181), α-synuclein, neurofilament light chain (NF-L), and chitinase 3-like protein-1 (CHI3L1). Levels of β-amyloid were lower in patients with type 1 narcolepsy (375.4 ± 143.5 pg/mL) and type 2 narcolepsy (455.9 ± 65.0 pg/mL) compared to controls (697.9 ± 167.3 pg/mL, p < .05). Furthermore, in patients with type 1 narcolepsy, levels of T-tau (79.0 ± 27.5 pg/mL) and P-tau181 (19.1 ± 4.3 pg/mL) were lower than in controls (162.2 ± 49.9 pg/mL and 33.8 ± 9.2 pg/mL, p < .05). Levels of α-synuclein, NF-L, and CHI3L1 in CSF from narcolepsy patients were similar to those of healthy individuals. Six CSF biomarkers of neurodegeneration were decreased or normal in narcolepsy indicating that taupathy, synucleinopathy, and immunopathy are not prevalent in narcolepsy patients with a disease duration of 2-29 years. Lower CSF levels of β-amyloid, T-tau protein, and P-tau181 in narcolepsy may indicate that hypocretin deficiency and an abnormal sleep-wake pattern alter the turnover of these proteins in the central nervous system. © Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

Effects of pomegranate juice in circulating parameters, cytokines, and oxidative stress markers in endurance-based athletes: A randomized controlled trial.

PubMed

Fuster-Muñoz, E; Roche, E; Funes, L; Martínez-Peinado, P; Sempere, J M; Vicente-Salar, N

2016-05-01

The aim of the present study was to assess the effects of pomegranate juice on the level of oxidative stress in the blood of endurance-based athletes. Pomegranate juice is rich in polyphenols, conferring it a higher antioxidant capacity than other beverages with polyphenolic antioxidants. A randomized double-blind, multicenter trial was performed in athletes from three different sport clubs located in southeastern of Spain. Plasma oxidative stress markers (protein carbonyls and malondialdehyde [MDA]) as well as C-reactive protein and sE-selectin were measured. Thirty-one athletes participated in the study. Participants were divided into three groups. The first group was supplemented with 200 mL/d pomegranate juice (PJ; n = 10) over a 21-d period, the second with 200 mL/d pomegranate juice diluted 1:1 with water (PJD; n = 11), and a control group that did not consume pomegranate juice (C; n = 10). Nine athletes were excluded due to protocol violations (n = 4 in the PJ group and n = 5 in the PJD group) because they did not observe the 24 h of rest before the last blood test. The control group increased levels of carbonyls (+0.7 ± 0.3 nmols/mg protein) and MDA (+3.2 ± 1.0 nmols/g protein), whereas the PJ and PJD groups maintained or decreased their levels, respectively. On the other hand, lactate levels increased in the PJ group (from 10.3 at day 0 to 21.2 mg/dL at day 22). A nonsignificant decrease was detected in sE-selectin and C-reactive protein in the groups consuming pomegranate juice. Consumption of pomegranate juice over a 21-d period improved MDA levels and carbonyls, and thus decreased the oxidative damage caused by exercise. Copyright © 2016 Elsevier Inc. All rights reserved.

Aerobic exercise training rescues cardiac protein quality control and blunts endoplasmic reticulum stress in heart failure rats.

PubMed

Bozi, Luiz H M; Jannig, Paulo R; Rolim, Natale; Voltarelli, Vanessa A; Dourado, Paulo M M; Wisløff, Ulrik; Brum, Patricia C

2016-11-01

Cardiac endoplasmic reticulum (ER) stress through accumulation of misfolded proteins plays a pivotal role in cardiovascular diseases. In an attempt to reestablish ER homoeostasis, the unfolded protein response (UPR) is activated. However, if ER stress persists, sustained UPR activation leads to apoptosis. There is no available therapy for ER stress relief. Considering that aerobic exercise training (AET) attenuates oxidative stress, mitochondrial dysfunction and calcium imbalance, it may be a potential strategy to reestablish cardiac ER homoeostasis. We test the hypothesis that AET would attenuate impaired cardiac ER stress after myocardial infarction (MI). Wistar rats underwent to either MI or sham surgeries. Four weeks later, rats underwent to 8 weeks of moderate-intensity AET. Myocardial infarction rats displayed cardiac dysfunction and lung oedema, suggesting heart failure. Cardiac dysfunction in MI rats was paralleled by increased protein levels of UPR markers (GRP78, DERLIN-1 and CHOP), accumulation of misfolded and polyubiquitinated proteins, and reduced chymotrypsin-like proteasome activity. These results suggest an impaired cardiac protein quality control. Aerobic exercise training improved exercise capacity and cardiac function of MI animals. Interestingly, AET blunted MI-induced ER stress by reducing protein levels of UPR markers, and accumulation of both misfolded and polyubiquinated proteins, which was associated with restored proteasome activity. Taken together, our study provide evidence for AET attenuation of ER stress through the reestablishment of cardiac protein quality control, which contributes to better cardiac function in post-MI heart failure rats. These results reinforce the importance of AET as primary non-pharmacological therapy to cardiovascular disease. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Effect of lipoic acid on paraoxonase-1 and paraoxonase-3 protein levels, mRNA expression and arylesterase activity in liver hepatoma cells.

PubMed

Ozgun, Eray; Sayilan Ozgun, Gulben; Tabakcioglu, Kiymet; Suer Gokmen, Selma; Sut, Necdet; Eskiocak, Sevgi

2017-10-01

Paraoxonase-1 (PON1) and PON3 (PON3) are anti-atherosclerotic enzymes, synthesized primarily in liver and bound to HDL in circulation. The aim of the present study was to investigate the effects of therapeutic doses of lipoic acid on PON1 and PON3 protein levels, mRNA expression and arylesterase activity in liver. We treated HepG2 cells with 10, 40 and 200 μM lipoic acid for 72 h. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. PON1 and PON3 protein levels were measured by Western blotting, their mRNA expression was measured by quantitative PCR and arylesterase activity was measured spectrophotometrically. 200 µM lipoic acid caused a significant increase on PON1 and PON3 protein levels and arylesterase activity as compared with control, 10 µM and 40 µM lipoic acid-treated cells. 200 µM lipoic acid also caused a significant decrease on PON1 mRNA expression whereas on a significant increase PON3 mRNA expression as compared with control, 10 µM and 40 µM lipoic acid-treated cells. Our study showed that although lipoic acid up-regulates PON3 but down-regulates PON1 mRNA expression, it increases both PON1 and PON3 protein levels and arylesterase activity in HepG2 cells. We can report that lipoic acid may be useful for preventing atherosclerosis at therapeutic doses.

Regulation of Nur77 protein turnover through acetylation and deacetylation induced by p300 and HDAC1.

PubMed

Kang, Shin-Ae; Na, Hyelin; Kang, Hyun-Jin; Kim, Sung-Hye; Lee, Min-Ho; Lee, Mi-Ock

2010-09-15

Although the roles of Nur77, an orphan member of the nuclear hormone receptor superfamily, in the control of cellular proliferation, apoptosis, inflammation, and glucose metabolism, are well recognized, the molecular mechanism regulating the activity and expression of Nur77 is not fully understood. Acetylation of transcription factors has emerged recently as a major post-translational modification that regulates protein stability and transcriptional activity. Here, we examined whether Nur77 is acetylated, and we characterized potential associated factors. First, Nur77 was found to be an acetylated protein when examined by immunoprecipitation and western blotting using acetyl protein-specific antibodies. Second, expression of p300, which possesses histone acetyltransferase activity, enhanced the acetylation and protein stability of Nur77. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, also increased Nur77 acetylation. Among the several types of HDACs, HDAC1 was found as the major enzyme affecting protein level of Nur77. HDAC1 decreased the acetylation level, protein level, and transcriptional activity of Nur77. Interestingly, overexpression of Nur77 induced expression of both p300 and HDAC1. Finally, the expression of Nur77 increased along with that of p300, but decreased with induction of HDAC1 after treatment with epithelial growth factor, nerve growth factor, or 6-mercaptopurine, suggesting that the self-control of the acetylation status contributes to the transient induction of Nur77 protein. Taken together, these results demonstrate that acetylation of Nur77 is modulated by p300 and HDAC1, and suggest that acetylation is an important post-translational modification for the rapid turnover of Nur77 protein. Copyright 2010 Elsevier Inc. All rights reserved.

Cytokines in relation to autoantibodies before onset of symptoms for systemic lupus erythematosus.

PubMed

Eriksson, C; Rantapää-Dahlqvist, S

2014-06-01

A number of cytokines and chemokines were analysed and related to autoantibodies in blood samples pre-dating the onset of symptoms of systemic lupus erythematosus. Thirty-five patients with systemic lupus erythematosus (American College of Rheumatology criteria) were identified as having donated blood samples, prior to symptom onset, to the Biobank of northern Sweden. Altogether, 140 age- and sex-matched controls were also identified. The concentrations of interferon-α, interleukin-4, interleukin-9, interleukin-10, interferon inducible protein-10 and monocyte chemotactic protein-1 were analysed using multiplex technology and related to autoantibodies (ANA, ENA, anti-dsDNA and anti-histone antibodies) analysed from the same blood sample. The interferon-γ inducible protein-10 levels were higher in the pre-symptomatic individuals than in controls (p < 0.05) and correlated with interferon-α (p < 0.01). The interferon-γ inducible protein-10 and interferon-α concentrations were significantly increased in individuals positive for autoantibodies: interferon-γ inducible protein-10 for ANA; anti-SSA/Ro and anti-Jo-1 antibodies; interferon-α with anti-SSB/La antibodies. The levels of interleukin-10, interferon-γ inducible protein-10 and monocyte chemotactic protein-1 increased significantly from the pre-symptomatic individuals to after onset of systemic lupus erythematosus. An increased concentration of interferon-γ inducible protein-10 pre-dated the onset of systemic lupus erythematosus and was related to autoantibodies before the onset of disease. The levels of interferon-γ inducible protein-10 and interferon-α were correlated. These findings support the proposal that the interferon system is important early in the pathogenesis of systemic lupus erythematosus and autoantibody formation. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Morphology and Molecular Mechanisms of Hepatic Injury in Rats under Simulated Weightlessness and the Protective Effects of Resistance Training.

PubMed

Du, Fang; Ding, Ye; Zou, Jun; Li, Zhili; Tian, Jijing; She, Ruiping; Wang, Desheng; Wang, Huijuan; Lv, Dongqiang; Chang, Lingling

2015-01-01

This study investigated the effects of long-term simulated weightlessness on liver morphology, enzymes, glycogen, and apoptosis related proteins by using two-month rat-tail suspension model (TS), and liver injury improvement by rat-tail suspension with resistance training model (TS&RT). Microscopically the livers of TS rats showed massive granular degeneration, chronic inflammation, and portal fibrosis. Mitochondrial and endoplasmic reticulum swelling and loss of membrane integrity were observed by transmission electron microscopy (TEM). The similar, but milder, morphological changes were observed in the livers of TS&RT rats. Serum biochemistry analysis revealed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher (p<0.05) in TS rats than in controls. The levels of ALT and AST in TS&RT rats were slightly lower than in RT rats, but they were insignificantly higher than in controls. However, both TS and TS&RT rats had significantly lower levels (p<0.05) of serum glucose and hepatic glycogen than in controls. Immunohistochemistry demonstrated that the expressions of Bax, Bcl-2, and active caspase-3 were higher in TS rats than in TS&RT and control rats. Real-time polymerase chain reaction (real-time PCR) showed that TS rats had higher mRNA levels (P < 0.05) of glucose-regulated protein 78 (GRP78) and caspase-12 transcription than in control rats; whereas mRNA expressions of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) were slightly higher in TS rats. TS&RT rats showed no significant differences of above 4 mRNAs compared with the control group. Our results demonstrated that long-term weightlessness caused hepatic injury, and may trigger hepatic apoptosis. Resistance training slightly improved hepatic damage.

Morphology and Molecular Mechanisms of Hepatic Injury in Rats under Simulated Weightlessness and the Protective Effects of Resistance Training

PubMed Central

Zou, Jun; Li, Zhili; Tian, Jijing; She, Ruiping; Wang, Desheng; Wang, Huijuan; Lv, Dongqiang; Chang, Lingling

2015-01-01

This study investigated the effects of long-term simulated weightlessness on liver morphology, enzymes, glycogen, and apoptosis related proteins by using two-month rat-tail suspension model (TS), and liver injury improvement by rat-tail suspension with resistance training model (TS&RT). Microscopically the livers of TS rats showed massive granular degeneration, chronic inflammation, and portal fibrosis. Mitochondrial and endoplasmic reticulum swelling and loss of membrane integrity were observed by transmission electron microscopy (TEM). The similar, but milder, morphological changes were observed in the livers of TS&RT rats. Serum biochemistry analysis revealed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher (p<0.05) in TS rats than in controls. The levels of ALT and AST in TS&RT rats were slightly lower than in RT rats, but they were insignificantly higher than in controls. However, both TS and TS&RT rats had significantly lower levels (p<0.05) of serum glucose and hepatic glycogen than in controls. Immunohistochemistry demonstrated that the expressions of Bax, Bcl-2, and active caspase-3 were higher in TS rats than in TS&RT and control rats. Real-time polymerase chain reaction (real-time PCR) showed that TS rats had higher mRNA levels (P < 0.05) of glucose-regulated protein 78 (GRP78) and caspase-12 transcription than in control rats; whereas mRNA expressions of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) were slightly higher in TS rats. TS&RT rats showed no significant differences of above 4 mRNAs compared with the control group. Our results demonstrated that long-term weightlessness caused hepatic injury, and may trigger hepatic apoptosis. Resistance training slightly improved hepatic damage. PMID:26000905

Improvement of glucose and lipid metabolism via mung bean protein consumption: clinical trials of GLUCODIA™ isolated mung bean protein in the USA and Canada.

PubMed

Kohno, Mitsutaka; Sugano, Hideo; Shigihara, Yuhko; Shiraishi, Yoshiaki; Motoyama, Takayasu

2018-01-01

The aim of the present study was to confirm the effects of a commercially available mung bean protein isolate (GLUCODIA™) on glucose and lipid metabolism. The main component of GLUCODIA™ is 8S globulin, which constitutes 80 % of the total protein. The overall structure of this protein closely resembles soyabean β-conglycinin, which accounts for 20 % of total soya protein (soya protein isolate; SPI). Many physiological beneficial effects of β-conglycinin have been reported. GLUCODIA™ is expected to produce beneficial effects with fewer intakes than SPI. We conducted two independent double-blind, placebo-controlled clinical studies. In the first (preliminary dose decision trial) study, mung bean protein was shown to exert physiological beneficial effects when 3·0 g were ingested per d. In the second (main clinical trial) study, mung bean protein isolate did not lower plasma glucose levels, although the mean insulin level decreased with consumption of mung bean protein. The homeostatic model assessment of insulin resistance (HOMA-IR) values significantly decreased with mung bean protein. The mean TAG level significantly decreased with consumption of mung bean protein isolate. A significant increase in serum adiponectin levels and improvement in liver function enzymes were observed. These findings suggest that GLUCODIA™ could be useful in the prevention of insulin resistance and visceral fat accumulation, which are known to trigger the metabolic syndrome, and in the prevention of liver function decline.

Interaction of maternal protein and carbon monoxide on pup mortality in mice: implications for global infant mortality.

PubMed

Singh, Jarnail

2006-06-01

The United States Surgeon General declared 2005 as the "Year of Healthy Child." To improve the health of all children, we need to start before pregnancy, with their mothers. Unfortunately, protein deficiency in the diets of poor pregnant mothers in developing countries is widespread. Carbon monoxide (CO) pollution is serious public health problem in developed and developing countries. A two-way factorial experimental design was used. Mice were maintained on 27%, 16%, 8%, or 4% protein diets. Dams were exposed to 0 ppm (control), 65 ppm, or 125 ppm CO in air, in environmental chambers for 6 hr/day during the first 2 weeks of pregnancy. Controls were also subjected to environmental chamber conditions. Food and water were available at all times. Animals were allowed to deliver, and data on pup mortality was recorded. Litter size was not affected by CO exposure, but was directly related to the dietary protein levels. Pup weight was inversely related to the CO exposure level, and directly related to the dietary protein levels. Pup mortality on date of birth was increased by CO exposure and was inversely related to the dietary protein levels. Pup mortality at 1 week of age was increased by CO exposure and 55% of all pups died in 125 ppm CO exposed group. Pup mortality at 1 week of age was inversely related to dietary protein levels. All pups in the 4% dietary protein and in all concentrations of CO died. All pups in the 8% protein group and in all CO concentrations died except in 125 ppm CO group. Pup mortality in the 16% dietary protein group ranged from 14.8% in 0 ppm to 36.8% in 65 ppm CO groups. Pup mortality in the 27% dietary protein group ranged from 14.3% in the 0 ppm to 41.1% in the 125 ppm CO groups. DATA suggest that protein deficiency and CO exposure enhance pup mortality. The protein and CO also interact to increase pup mortality in 16% and 27% protein groups. Carbon monoxide exposure, along with protein deficiency during gestation, may be contributing factors for high rates of infant mortality in developing countries. The results of the study also suggest that un-vented combustion for heating and cooking, ambient pollution, and biomass smoke may have a major impact on the health of children worldwide; and may explain the causes of high infant mortality in poor countries and some sections of the United States population.

Prediction of Cortical Defect Using C-Reactive Protein and Urine Sodium to Potassium Ratio in Infants with Febrile Urinary Tract Infection

PubMed Central

Jung, Su Jin

2016-01-01

Purpose We investigated whether C-reactive protein (CRP) levels, urine protein-creatinine ratio (uProt/Cr), and urine electrolytes can be useful for discriminating acute pyelonephritis (APN) from other febrile illnesses or the presence of a cortical defect on 99mTc dimercaptosuccinic acid (DMSA) scanning (true APN) from its absence in infants with febrile urinary tract infection (UTI). Materials and Methods We examined 150 infants experiencing their first febrile UTI and 100 controls with other febrile illnesses consecutively admitted to our hospital from January 2010 to December 2012. Blood (CRP, electrolytes, Cr) and urine tests [uProt/Cr, electrolytes, and sodium-potassium ratio (uNa/K)] were performed upon admission. All infants with UTI underwent DMSA scans during admission. All data were compared between infants with UTI and controls and between infants with or without a cortical defect on DMSA scans. Using multiple logistic regression analysis, the ability of the parameters to predict true APN was analyzed. Results CRP levels and uProt/Cr were significantly higher in infants with true APN than in controls. uNa levels and uNa/K were significantly lower in infants with true APN than in controls. CRP levels and uNa/K were relevant factors for predicting true APN. The method using CRP levels, u-Prot/Cr, u-Na levels, and uNa/K had a sensitivity of 94%, specificity of 65%, positive predictive value of 60%, and negative predictive value of 95% for predicting true APN. Conclusion We conclude that these parameters are useful for discriminating APN from other febrile illnesses or discriminating true APN in infants with febrile UTI. PMID:26632389

Dysfunction of protein kinase FA/GSK-3 alpha in lymphocytes of patients with schizophrenic disorder.

PubMed

Yang, S D; Yu, J S; Lee, T T; Yang, C C; Ni, M H; Yang, Y Y

1995-09-01

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP.Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK-3 alpha. Immunoblotting and kinase activity analysis in an anti-kinase FA/GSK-3 alpha immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK-3 alpha in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK-3 alpha activity in the high levels of 14.8 +/- 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK-3 alpha activity in the low levels of 2.8 +/- 1.6 units/mg, indicating that the different levels of kinase FA/GSK-3 alpha activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide initial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK-3 alpha, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes.

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

PubMed Central

Culver, J N; Lehto, K; Close, S M; Hilf, M E; Dawson, W O

1993-01-01

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes. Images Fig. 2 Fig. 3 Fig. 4 PMID:8446627

Biomarkers and bacterial pneumonia risk in patients with treated HIV infection: a case-control study.

PubMed

Bjerk, Sonja M; Baker, Jason V; Emery, Sean; Neuhaus, Jacqueline; Angus, Brian; Gordin, Fred M; Pett, Sarah L; Stephan, Christoph; Kunisaki, Ken M

2013-01-01

Despite advances in HIV treatment, bacterial pneumonia continues to cause considerable morbidity and mortality in patients with HIV infection. Studies of biomarker associations with bacterial pneumonia risk in treated HIV-infected patients do not currently exist. We performed a nested, matched, case-control study among participants randomized to continuous combination antiretroviral therapy (cART) in the Strategies for Management of Antiretroviral Therapy trial. Patients who developed bacterial pneumonia (cases) and patients without bacterial pneumonia (controls) were matched 1∶1 on clinical center, smoking status, age, and baseline cART use. Baseline levels of Club Cell Secretory Protein 16 (CC16), Surfactant Protein D (SP-D), C-reactive protein (hsCRP), interleukin-6 (IL-6), and d-dimer were compared between cases and controls. Cases (n = 72) and controls (n = 72) were 25.7% female, 51.4% black, 65.3% current smokers, 9.7% diabetic, 36.1% co-infected with Hepatitis B/C, and 75.0% were on cART at baseline. Median (IQR) age was 45 (41, 51) years with CD4+ count of 553 (436, 690) cells/mm(3). Baseline CC16 and SP-D were similar between cases and controls, but hsCRP was significantly higher in cases than controls (2.94 µg/mL in cases vs. 1.93 µg/mL in controls; p = 0.02). IL-6 and d-dimer levels were also higher in cases compared to controls, though differences were not statistically significant (p-value 0.06 and 0.10, respectively). In patients with cART-treated HIV infection, higher levels of systemic inflammatory markers were associated with increased bacterial pneumonia risk, while two pulmonary-specific inflammatory biomarkers, CC16 and SP-D, were not associated with bacterial pneumonia risk.

Effects of yak-activated protein on hematopoiesis and related cytokines in radiation-induced injury in mice.

PubMed

Duan, Yabin; Yao, Xingchen; Zhu, Junbo; Li, Yongping; Zhang, Juanling; Zhou, Xuejiao; Qiao, Yijie; Yang, Meng; Li, Xiangyang

2017-12-01

The aim of the present study was to investigate the protective effects of yak-activated protein on hematopoiesis and cytokine function in radiation-induced injury in mice. A total of 180 Kunming mice were randomly divided into three groups (A, B and C). Of these, 60 were randomly divided into a normal control group, a radiation model group, a positive control group and 3 yak-activated protein groups (high, medium and low dose groups; 10, 5 and 2.5 mg/kg, respectively). The other 120 mice were used for the subsequent experiments on days 7 and 14 following radiation. Yak-activated protein was administered orally to mice in the treatment groups and an equal volume of saline was administered orally to mice in the normal control and radiation model groups for 14 days. The positive control group received amifostine (150 mg/kg) via intraperitoneal injection. With the exception of the control group, the groups of mice received a 5 Gy quantity of X-radiation evenly over their whole body once. Changes in the peripheral hemogram, thymus and spleen indices, DNA content in the bone marrow, interleukin (IL)-2 and IL-6 levels, and the expression levels of B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) following irradiation were assessed. The low dose of yak-activated protein significantly increased Spleen indices in mice 14 days after irradiation and the high and middle dose of yak-activated protein significantly increased Thymus indices in mice 14 days after irradiation (P<0.05) compared with the control group. In addition, hemogram results increased gradually in the low-yak-activated protein dose group and were significantly higher 7 days after irradiation compared with the radiation model group (P<0.05). The DNA content in the bone marrow was markedly increased in the yak-activated protein groups, and increased significantly in the low dose group at 7 days post-irradiation compared with the radiation model group (P<0.05). The IL-2 content was significantly increased in the yak-activated protein groups (P<0.05). Furthermore, Bcl-2 expression was increased and Bax expression was decreased (P<0.05). These results suggest that yak-activated protein exerts protective effects against radiation-induced injury in mice. The optimal effects of yak-activated protein were observed in the medium dose group 14 days after irradiation.

Effects of yak-activated protein on hematopoiesis and related cytokines in radiation-induced injury in mice

PubMed Central

Duan, Yabin; Yao, Xingchen; Zhu, Junbo; Li, Yongping; Zhang, Juanling; Zhou, Xuejiao; Qiao, Yijie; Yang, Meng; Li, Xiangyang

2017-01-01

The aim of the present study was to investigate the protective effects of yak-activated protein on hematopoiesis and cytokine function in radiation-induced injury in mice. A total of 180 Kunming mice were randomly divided into three groups (A, B and C). Of these, 60 were randomly divided into a normal control group, a radiation model group, a positive control group and 3 yak-activated protein groups (high, medium and low dose groups; 10, 5 and 2.5 mg/kg, respectively). The other 120 mice were used for the subsequent experiments on days 7 and 14 following radiation. Yak-activated protein was administered orally to mice in the treatment groups and an equal volume of saline was administered orally to mice in the normal control and radiation model groups for 14 days. The positive control group received amifostine (150 mg/kg) via intraperitoneal injection. With the exception of the control group, the groups of mice received a 5 Gy quantity of X-radiation evenly over their whole body once. Changes in the peripheral hemogram, thymus and spleen indices, DNA content in the bone marrow, interleukin (IL)-2 and IL-6 levels, and the expression levels of B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) following irradiation were assessed. The low dose of yak-activated protein significantly increased Spleen indices in mice 14 days after irradiation and the high and middle dose of yak-activated protein significantly increased Thymus indices in mice 14 days after irradiation (P<0.05) compared with the control group. In addition, hemogram results increased gradually in the low-yak-activated protein dose group and were significantly higher 7 days after irradiation compared with the radiation model group (P<0.05). The DNA content in the bone marrow was markedly increased in the yak-activated protein groups, and increased significantly in the low dose group at 7 days post-irradiation compared with the radiation model group (P<0.05). The IL-2 content was significantly increased in the yak-activated protein groups (P<0.05). Furthermore, Bcl-2 expression was increased and Bax expression was decreased (P<0.05). These results suggest that yak-activated protein exerts protective effects against radiation-induced injury in mice. The optimal effects of yak-activated protein were observed in the medium dose group 14 days after irradiation. PMID:29285056

Circulating levels of carbamylated protein and neutrophil extracellular traps are associated with periodontitis severity in patients with rheumatoid arthritis: A pilot case-control study.

PubMed

Kaneko, Chihiro; Kobayashi, Tetsuo; Ito, Satoshi; Sugita, Noriko; Murasawa, Akira; Nakazono, Kiyoshi; Yoshie, Hiromasa

2018-01-01

An interrelationship between rheumatoid arthritis (RA) and periodontitis has been suggested due to their common pathogenic mechanisms. Protein carbamylation and neutrophil extracellular traps (NETs) formation have been shown to be related to autoimmune conditions, including RA, but their association with periodontitis has not been elucidated. Therefore, we assessed whether or not circulating levels of carbamylated protein (CarP) and NETs are associated with periodontitis severity and influenced by periodontal treatment. We conducted a retrospective case-control study that included 40 patients with RA and periodontitis, 30 patients with periodontitis, and 43 systemically and periodontally healthy controls to assess the circulating levels of CarP and NETs and rheumatologic and periodontal conditions. The same assessments were also performed in 22 patients with RA and periodontitis after 2 months of periodontal treatment, including oral hygiene instruction and full-mouth supragingival scaling. Patients with RA and periodontitis showed significantly higher serum levels of CarP and NETs than the control group (P = 0.04 and P < 0.001, respectively). The serum levels of CarP and NETs were significantly correlated positively with the mean values of probing depth (P = 0.01 and P = 0.007, respectively) and clinical attachment level (P = 0.007 and P = 0.001, respectively) in the 40 patients with RA and periodontitis. Multiple logistic regression analyses also revealed significantly positive associations between the serum levels of CarP and NETs and moderate to severe periodontitis (P = 0.03 and P = 0.001, respectively). Furthermore, periodontal treatment significantly decreased the serum levels of CarP and NETs in patients with RA and periodontitis (P = 0.03 and P = 0.02). The circulating levels of CarP and NETs are associated with periodontitis severity and influenced by periodontal treatment in patients with RA.

Circulating levels of carbamylated protein and neutrophil extracellular traps are associated with periodontitis severity in patients with rheumatoid arthritis: A pilot case-control study

PubMed Central

Kaneko, Chihiro; Ito, Satoshi; Sugita, Noriko; Murasawa, Akira; Nakazono, Kiyoshi; Yoshie, Hiromasa

2018-01-01

Objectives An interrelationship between rheumatoid arthritis (RA) and periodontitis has been suggested due to their common pathogenic mechanisms. Protein carbamylation and neutrophil extracellular traps (NETs) formation have been shown to be related to autoimmune conditions, including RA, but their association with periodontitis has not been elucidated. Therefore, we assessed whether or not circulating levels of carbamylated protein (CarP) and NETs are associated with periodontitis severity and influenced by periodontal treatment. Methods We conducted a retrospective case-control study that included 40 patients with RA and periodontitis, 30 patients with periodontitis, and 43 systemically and periodontally healthy controls to assess the circulating levels of CarP and NETs and rheumatologic and periodontal conditions. The same assessments were also performed in 22 patients with RA and periodontitis after 2 months of periodontal treatment, including oral hygiene instruction and full-mouth supragingival scaling. Results Patients with RA and periodontitis showed significantly higher serum levels of CarP and NETs than the control group (P = 0.04 and P < 0.001, respectively). The serum levels of CarP and NETs were significantly correlated positively with the mean values of probing depth (P = 0.01 and P = 0.007, respectively) and clinical attachment level (P = 0.007 and P = 0.001, respectively) in the 40 patients with RA and periodontitis. Multiple logistic regression analyses also revealed significantly positive associations between the serum levels of CarP and NETs and moderate to severe periodontitis (P = 0.03 and P = 0.001, respectively). Furthermore, periodontal treatment significantly decreased the serum levels of CarP and NETs in patients with RA and periodontitis (P = 0.03 and P = 0.02). Conclusion The circulating levels of CarP and NETs are associated with periodontitis severity and influenced by periodontal treatment in patients with RA. PMID:29394286

Decreased expression of Freud-1/CC2D1A, a transcriptional repressor of the 5-HT1A receptor, in the prefrontal cortex of subjects with major depression.

PubMed

Szewczyk, Bernadeta; Albert, Paul R; Rogaeva, Anastasia; Fitzgibbon, Heidi; May, Warren L; Rajkowska, Grazyna; Miguel-Hidalgo, Jose J; Stockmeier, Craig A; Woolverton, William L; Kyle, Patrick B; Wang, Zhixia; Austin, Mark C

2010-09-01

Serotonin1A (5-HT(1A)) receptors are reported altered in the brain of subjects with major depressive disorder (MDD). Recent studies have identified transcriptional regulators of the 5-HT(1A) receptor and have documented gender-specific alterations in 5-HT(1A) transcription factor and 5-HT(1A) receptors in female MDD subjects. The 5' repressor element under dual repression binding protein-1 (Freud-1) is a calcium-regulated repressor that negatively regulates the 5-HT(1A) receptor gene. This study documented the cellular expression of Freud-1 in the human prefrontal cortex (PFC) and quantified Freud-1 protein in the PFC of MDD and control subjects as well as in the PFC of rhesus monkeys chronically treated with fluoxetine. Freud-1 immunoreactivity was present in neurons and glia and was co-localized with 5-HT(1A) receptors. Freud-1 protein level was significantly decreased in the PFC of male MDD subjects (37%, p=0.02) relative to gender-matched control subjects. Freud-1 protein was also reduced in the PFC of female MDD subjects (36%, p=0.18) but was not statistically significant. When the data was combined across genders and analysed by age, the decrease in Freud-1 protein level was greater in the younger MDD subjects (48%, p=0.01) relative to age-matched controls as opposed to older depressed subjects. Similarly, 5-HT(1A) receptor protein was significantly reduced in the PFC of the younger MDD subjects (48%, p=0.01) relative to age-matched controls. Adult male rhesus monkeys administered fluoxetine daily for 39 wk revealed no significant change in cortical Freud-1 or 5-HT(1A) receptor proteins compared to vehicle-treated control monkeys. Reduced protein expression of Freud-1 in MDD subjects may reflect dysregulation of this transcription factor, which may contribute to the altered regulation of 5-HT(1A) receptors observed in subjects with MDD. These data may also suggest that reductions in Freud-1 protein expression in the PFC may be associated with early onset of MDD.

Effects of ration level on immune functions in chinook salmon (Oncorhynchus tshawytscha)

USGS Publications Warehouse

Alcorn, S.W.; Pascho, R.J.; Murray, A.L.; Shearer, K.D.

2003-01-01

The relationship between nutritional status and disease resistance in cultured salmonids can be affected by dietary manipulations. Careful attention to feeding levels may be important to avoid imbalances in nutrient levels that could ultimately impair a fish's ability to resist infectious microorganisms. In the current study, fish in three feed-level groups were fed an experimental diet either to satiation, 64% of satiation or 40% of satiation. A fourth group of fish were fed a commercial diet at the 64% of satiation level and served as controls. To evaluate certain indices of disease resistance in the test and control fish, a panel of assays was employed to measure humoral and cellular immune functions 30, 39 and 54 weeks after starting the dietary treatments. The panel included measures of blood hematocrit and leucocrit levels, plasma protein concentration and serum lysozyme and complement activity. Cellular analyses included differential blood leucocyte counts, NBT reduction and phagocytosis by pronephros macrophages and myeloperoxidase activity of pronephros neutrophils. No differences were observed in those indices between fish tested from the control-diet group (commercial diet fed at the 64% rate) and fish tested from the 64% feed-level group, except that fish fed the commercial diet had a greater concentration of plasma protein. Leucocrit values and plasma protein concentrations tended to increase among the experimental feed groups as the ration increased from 40% to satiation. More importantly, phagocytic activity by anterior kidney leucocytes was found to be inversely proportional to the feed level. Whereas the results of this study provide evidence that the salmonid immune system may be fairly robust with regard to available metabolic energy, the significant changes observed in phagocytic cell activity suggest that some cellular immune functions may be affected by the feed level.

Elevated Serum Levels of the Antiapoptotic Protein Decoy-Receptor 3 Are Associated with Advanced Liver Disease.

PubMed

Bamias, Giorgos; Gizis, Michalis; Delladetsima, Ioanna; Laoudi, Eyfrosyni; Siakavellas, Spyros I; Koutsounas, Ioannis; Kaltsa, Garyfallia; Vlachogiannakos, John; Vafiadis-Zouboulis, Irene; Daikos, George L; Papatheodoridis, George V; Ladas, Spiros D

2016-01-01

Background. Decoy-receptor 3 (DcR3) exerts antiapoptotic and immunomodulatory function and is overexpressed in neoplastic and inflammatory conditions. Serum DcR3 (sDcR3) levels during the chronic hepatitis/cirrhosis/hepatocellular carcinoma (HCC) sequence have not been explored. Objective. To assess the levels and significance of sDcR3 protein in various stages of chronic liver disease. Methods. We compared sDcR3 levels between healthy controls and patients with chronic viral hepatitis (CVH), decompensated cirrhosis (DC), and HCC. Correlations between sDcR3 levels and various patient- and disease-related factors were analyzed. Results. sDcR3 levels were significantly higher in patients with CVH than in controls (P < 0.01). sDcR3 levels were elevated in DC and HCC, being significantly higher compared not only to controls (P < 0.001 for both) but to CVH patients as well (P < 0.001 for both). In addition, DcR3 protein was detected in large quantities in the ascitic fluid of cirrhotics. In patients with CVH, sDcR3 significantly correlated to fibrosis severity, as estimated by Ishak score (P = 0.019) or by liver stiffness measured with elastography (Spearman r = 0.698, P < 0.001). In cirrhotic patients, significant positive correlations were observed between sDcR3 levels and markers of severity of hepatic impairment, including MELD score (r = 0.653, P < 0.001). Conclusions. Circulating levels of DcR3 are elevated during chronic liver disease and correlate with severity of liver damage. sDcR3 may serve as marker for liver fibrosis severity and progression to end-stage liver disease.

Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1{alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Weiping, E-mail: weiping.qin@mssm.edu; Department of Medicine, Mount Sinai School of Medicine, NY; Pan, Jiangping

Research highlights: {yields} In rat gastrocnemius muscle, dexamethasone reduced PGC-1{alpha} cellular and nuclear levels without altering mRNA levels for this factor. {yields} Dexamethasone reduced phosphorylating of p38 MAPK, which stabilizes PGC-1{alpha} and promotes its nuclear entry. {yields} Co-administration of testosterone with dexamethasone increased cellular and nuclear levels of PGC-1{alpha} protein without changing its mRNA levels. {yields} Co-administration of testosterone restored p38 MAPK levels to those of controls. -- Abstract: Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis,more » REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1{alpha} has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1{alpha}, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1{alpha} protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1{alpha} levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius muscle. Regulation of FOXO1, PGC-1{alpha} and p38 MAPK by testosterone may represent a novel mechanism by which this agent protects against dexamethasone-induced muscle atrophy.« less

Effect of Fat-based versus Carbohydrate-based Enteral Feeding on Glycemic Control in Critically Ill Patients: A Randomized Clinical Trial.

PubMed

Nourmohammadi, Mahdieh; Moghadam, Omid Moradi; Lahiji, Mohammad Niakan; Hatamian, Sevak; Shariatpanahi, Zahra Vahdat

2017-08-01

The aim of this study was to evaluate the preventive effects of high-fat enteral feeding on glycemic control and clinical outcomes in critically ill patients: a randomized clinical trial. This study was done on 42 normoglycemic patients admitted to Intensive Care Unit (ICU). Patients were randomly classified into three groups of 14 each. Control group (A) received carbohydrate-based diet (protein: 20%, fat: 30%, and carbohydrate: 50%), study groups received two types of high-fat diet; Group B (protein: 20%, fat: 45% including half of olive oil and half sunflower oil, and carbohydrate: 35%); and Group C (protein: 20%, fat: 45% including sunflower oil, and carbohydrate: 35%) in the first 48 h of admission. Basal characteristics of participants were the same. After the feeding trial, there was no difference between the groups in mean plasma and capillary glucose levels and insulin requirements. Serum high density lipoprotein (HDL)-cholesterol level was increased significantly in Group B on day 10 compared to admission level (40.75 ± 5.58 vs. 43.56 ± 2.25, P = 0.05). We did not find any difference in organ failure involvement and mortality rate between groups. The number of ICU free days was significantly more in Group B compared to the control group ( P = 0.04). High-fat diets have no preventive effect on stress hyperglycemia. High monounsaturated fat diet may increase serum HDL-cholesterol level and decrease the length of stay in ICU.

Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma

PubMed Central

YANG, YANG; HUI, LV; YUQIN, CHE; JIE, LI; SHUAI, HOU; TIEZHU, ZHOU; WEI, WANG

2014-01-01

Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 104 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; P<0.001). In addition, the two cell lines had a higher expression level of p53 protein in the experimental group as compared with the control group (t=40.810; P<0.001). Protein expression levels of Bcl-xL decreased significantly in the experimental group as compared with the control group (t=19.640; P=0.000). Therefore, saw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction. PMID:25009620

Fish oil supplementation of rats during pregnancy reduces adult disease risks in their offspring.

PubMed

Joshi, Sadhana; Rao, Shobha; Golwilkar, Ajit; Patwardhan, Manisha; Bhonde, Ramesh

2003-10-01

Metabolic programming in utero due to maternal undernutrition is considered to increase the risk of adult diseases in offspring. It is therefore of relevance to investigate how dietary supplementation of specific nutrients can ameliorate the negative effects of maternal malnutrition. We examined the effects of supplementing fish oil or folic acid, both of which are conventional supplements in maternal intervention, on risk factors in the offspring as adults. Pregnant female rats from 4 groups (n = 6/group) were fed casein diets with 18 g/100 g protein (control diet), 12 g/100 g protein supplemented with 8 mg folic acid/kg diet (0.08 mg/kg diet) (FAS), 12 g/100 g protein without folic acid (FAD) or 12 g/100 g protein supplemented with 7 g/100 g fish oil (FOIL). Pups were weaned to a standard laboratory diet with 18 g/100 g protein. Serum glucose, insulin and cholesterol and plasma homocysteine levels were measured in the offspring at 6 and 11 mo of age. Serum glucose in 11-mo-old male and female pups was greater (P < 0.05) in both the FAS (males 2.46 +/- 0.51, females 2.49 +/- 0.29 mmol/L) and FAD groups (2.48 +/- 0.28 and 2.67 +/- 0.41 mmol/L) than in controls (2.03 +/- 0.15 and 2.02 +/- 0.18 mmol/L). Serum insulin concentrations were higher (P < 0.05) in the FAD group (males 1476 +/- 317, females 1441 +/- 220 pmol/L) but were lower in males from the FAS group (483 +/- 165 pmol/L) compared with controls (males 917 +/- 373, females 981 +/- 264 pmol/L). Glucose and insulin concentrations did not differ between the control and FOIL groups. Plasma homocysteine levels were lower (P < 0.05) only in 11-mo-old folate-deficient males; none of the other groups differed from the controls. Maternal supplementation of fish oil to a diet containing marginal protein was beneficial in maintaining circulating glucose, insulin, cholesterol and homocysteine levels in the offspring as adults.

Salivary gland dysfunction markers in type 2 diabetes mellitus patients.

PubMed

Aitken-Saavedra, Juan; Rojas-Alcayaga, Gonzalo; Maturana-Ramírez, Andrea; Escobar-Álvarez, Alejandro; Cortes-Coloma, Andrea; Reyes-Rojas, Montserrat; Viera-Sapiain, Valentina; Villablanca-Martínez, Claudia; Morales-Bozo, Irene

2015-10-01

Diabetes mellitus (DM) is a chronic disease of the carbohydrate metabolism that, when not rigorously controlled, compromises systemic and organ integrity, thereby causing renal diseases, blindness, neuropathy, arteriosclerosis, infections, and glandular dysfunction, including the salivary glands. The aim of this study was to determine the relationship between the qualitative and quantitative parameters of salivary alteration, which are indicators of salivary gland dysfunction, and the level of metabolic control of type 2 diabetes patients. A convenience sample of 74 voluntary patients with type 2 DM was selected, each of whom donated a sample of unstimulated saliva. Salivary parameters such as salivary flow rate, protein concentration, pH, and xerostomia were studied. There is a positive relationship between the level of metabolic control measured with HbA1 and the protein concentration in saliva (Spearman rho = 0.329 and p = 0.004). The same assay showed an inverse correlation between HbA1 and pH (Spearman rho = -0.225 and p = 0.05). The protein concentration in saliva and, to a lesser extent, the pH may be useful as glandular dysfunction indicators in DM2 patients. Saliva, type 2 diabetes mellitus, pH, protein concentration, xerostomia.

Dairy cow responses to graded levels of rapeseed and soya bean expeller supplementation on a red clover/grass silage-based diet.

PubMed

Rinne, M; Kuoppala, K; Ahvenjärvi, S; Vanhatalo, A

2015-12-01

The effects of rapeseed and soya bean expeller (SBE) supplementation on digestion and milk production responses in dairy cows were investigated in an incomplete Latin square design using five cows and four 3-week periods. The experimental diets consisted of five concentrate treatments fed at a rate of 9 kg/day: a mixture of barley and oats, which was replaced with rapeseed or SBE at two levels (CP concentration (g/kg dry matter (DM)) of 130 for the control concentrate and 180 and 230 for the two protein supplemented levels). A mixture of grass and red clover silage (1:1) was fed ad libitum and it had a CP concentration of 157 g/kg DM. Supply of nutrients to the lower tract was measured using the omasal canal sampling technique, and total digestion from total faecal collection. Protein supplementation increased omasal canal amino acid (AA) flows and plasma concentrations of AA, and was also reflected as increased milk production. However, N use efficiency (NUE) decreased with increased protein supplementation. Rapeseed expeller (RSE) tended to increase silage DM intake and elicited higher milk production responses compared with SBE and also resulted in a higher NUE. The differences between the protein supplements in nitrogen metabolism were relatively small, for example, there were no differences in the efficiency of microbial protein synthesis or omasal canal flows of nitrogenous components between them, but plasma methionine concentration was lower for soya bean-fed cows at the high CP level in particular. The lower milk protein production responses to SBE than to RSE supplementation were at least partly caused by increased silage DM and by the lower methionine supply, which may further have been amplified by the use of red clover in the basal diet. Although feed intake, diet digestion, AA supply and milk production were all consistently improved by protein supplementation, there was a simultaneous decrease in NUE. In the current study, the milk protein production increased only 9% and energy-corrected milk production by 7% when high level of protein supplementation (on average 2.9 kg DM/day) was compared with the control diet without protein supplementation showing that dairy production could be sustained at a high level even without external protein supplements, at least in the short term. The economic and environmental aspects need to be carefully evaluated when decisions about protein supplementation for dairy cows are taken.

Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus.

PubMed

Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young-Hwan; Yu, Jae-Hyuk

2013-07-11

FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus. Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion mutant and 8 proteins differentially secreted in wild type and the flbA mutant. Based on proteomic analyses, we further examined the role of FlbA and found that FlbA down-regulates gliT expression and SOD activity. Our results proposed that FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and exogenous gliotoxin in A. fumigatus. Copyright © 2013 Elsevier B.V. All rights reserved.

Cellular cholesterol regulates ubiquitination and degradation of the cholesterol export proteins ABCA1 and ABCG1.

PubMed

Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C; Brown, Andrew J; Sandoval, Cecilia; Hallab, Jeannette C; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-03-14

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

PubMed Central

Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-01-01

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

[Protein and ribonucleic acid metabolism in the central nervous system of rats during space flight in the "Kosmos-605" satellite].

PubMed

Gazenko, O G; Demin, N N; Panov, A N; Rubinskaia, N L; Tigranian, R A

1976-01-01

On the 2nd postflight day the activity of neutral protamine peptide hydrolase of different compartments of the rat brain did not differ from the control level. With respect to the protein and RNA content and concentration motoneurons of anterior horns of the spinal cord and their glial-cells-satellites of rats exposed to the ground-based synchronous experiment did not differ from those of vivarium controls, except cells of the supraoptic nucleus. That was found on the 2nd and 27th postflight days. On the 2nd postflight day the protein and RNA concentration in neurons decreased and the protein concentration and content in gliocytes lowered; the RNA concentration and content in Purkinje cells of the cerebellum became reduced. On the 27th postflight day the RNA concentration in neurons of the supraoptic nucleus remained diminished whereas the protein content increased; in spinal motoneurons the protein concentration decreased and in adjacent gliocytes the protein concentration and content lowered. All the above changes made no more than 15% of the control values.

The Effect of a High-Protein Diet and Exercise on Cardiac AQP7 and GLUT4 Gene Expression.

PubMed

Palabiyik, Orkide; Karaca, Aziz; Taştekin, Ebru; Yamasan, Bilge Eren; Tokuç, Burcu; Sipahi, Tammam; Vardar, Selma Arzu

2016-10-01

High-protein (HP) diets are commonly consumed by athletes despite their potential health hazard, which is postulated to enforce a negative effect on bone and renal health. However, its effects on heart have not been known yet. Aquaporin-7 (AQP7) is an aquaglyceroporin that facilitates glycerol and water transport. Glycerol is an important cardiac energy production substrate, especially during exercise, in conjunction with fatty acids and glucose. Glucose transporter 4 (GLUT4) is an insulin-sensitive glucose transporter in heart. We aimed to investigate the effect of HPD on AQP7 and GLUT4 levels in the rat heart subjected to exercise. Male Sprague-Dawley rats were divided into control (n = 12), exercise (E) training (n = 10), HPD (n = 12), and HPD-E training (n = 9) groups. The HPD groups were fed a 45 % protein-containing diet 5 weeks. The HPD-E and E groups were performed the treadmill exercise during the 5-week study period. Real-time polymerase chain reaction and immunohistochemistry techniques were used to determine the gene expression and localization of AQP7 and GLUT4 in heart tissue. Results of relative gene expression were calculated by the 'Pfaffl' mathematical method using the REST program. Differences in AQP7 and GLUT4 gene expression were expressed as fold change compared to the control group. Heart weight/tibia ratio and ventricular wall thickness were evaluated as markers of cardiac hypertrophy. Further, serum glucose, glycerol, and insulin levels were also measured. AQP7 gene expression was found to be increased in the E (3.47-fold, p < 0.001), HPD (5.59-fold, p < 0.001), and HPD-E (3.87-fold, p < 0.001) groups compared to the control group. AQP7 protein expression was also increased in the HPD and HPD-E groups (p < 0.001). Additionally, cardiac mRNA expression levels of GLUT4 showed a significant increase in the E (2.16-fold, p < 0.003), HPD (7.14-fold, p < 0.001), and HPD-E (3.43-fold, p < 0.001) groups compared to the control group. GLUT4 protein expression was significantly increased in the E, HPD, and HPD-E groups compared to the control group (p = 0.024, p < 0.001, and p < 0.001, respectively). Furthermore, Serum glucose levels were significantly different between groups (p < 0.005). This difference was observed between the HPD groups and normal-protein diet groups (C and E). Serum insulin levels were higher for HPD groups compared with the normal-protein diet groups (p < 0.001), whereas no differences were observed between the exercise and sedentary groups (p = 0.111). Serum glycerol levels were significantly increased in the HPD groups compared with control and E groups (p < 0.05 and p < 0.05, respectively). Consumption of HPD supplementation caused the increased effects on AQP7 and GLUT4 expression in rat heart.

Artificially Engineered Protein Polymers.

PubMed

Yang, Yun Jung; Holmberg, Angela L; Olsen, Bradley D

2017-06-07

Modern polymer science increasingly requires precise control over macromolecular structure and properties for engineering advanced materials and biomedical systems. The application of biological processes to design and synthesize artificial protein polymers offers a means for furthering macromolecular tunability, enabling polymers with dispersities of ∼1.0 and monomer-level sequence control. Taking inspiration from materials evolved in nature, scientists have created modular building blocks with simplified monomer sequences that replicate the function of natural systems. The corresponding protein engineering toolbox has enabled the systematic development of complex functional polymeric materials across areas as diverse as adhesives, responsive polymers, and medical materials. This review discusses the natural proteins that have inspired the development of key building blocks for protein polymer engineering and the function of these elements in material design. The prospects and progress for scalable commercialization of protein polymers are reviewed, discussing both technology needs and opportunities.

ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders

PubMed Central

Nam, Hyeyoung; Wang, Chia-Yu; Zhang, Lin; Zhang, Wei; Hojyo, Shintaro; Fukada, Toshiyuki; Knutson, Mitchell D.

2013-01-01

The liver, pancreas, and heart are particularly susceptible to iron-related disorders. These tissues take up plasma iron from transferrin or non-transferrin-bound iron, which appears during iron overload. Here, we assessed the effect of iron status on the levels of the transmembrane transporters, ZRT/IRT-like protein 14 and divalent metal-ion transporter-1, which have both been implicated in transferrin- and non-transferrin-bound iron uptake. Weanling male rats (n=6/group) were fed an iron-deficient, iron-adequate, or iron-overloaded diet for 3 weeks. ZRT/IRT-like protein 14, divalent metal-ion transporter-1 protein and mRNA levels in liver, pancreas, and heart were determined by using immunoblotting and quantitative reverse transcriptase polymerase chain reaction analysis. Confocal immunofluorescence microscopy was used to localize ZRT/IRT-like protein 14 in the liver and pancreas. ZRT/IRT-like protein 14 and divalent metal-ion transporter-1 protein levels were also determined in hypotransferrinemic mice with genetic iron overload. Hepatic ZRT/IRT-like protein 14 levels were found to be 100% higher in iron-loaded rats than in iron-adequate controls. By contrast, hepatic divalent metal-ion transporter-1 protein levels were 70% lower in iron-overloaded animals and nearly 3-fold higher in iron-deficient ones. In the pancreas, ZRT/IRT-like protein 14 levels were 50% higher in iron-overloaded rats, and in the heart, divalent metal-ion transporter-1 protein levels were 4-fold higher in iron-deficient animals. At the mRNA level, ZRT/IRT-like protein 14 expression did not vary with iron status, whereas divalent metal-ion transporter-1 expression was found to be elevated in iron-deficient livers. Immunofluorescence staining localized ZRT/IRT-like protein 14 to the basolateral membrane of hepatocytes and to acinar cells of the pancreas. Hepatic ZRT/IRT-like protein 14, but not divalent metal-ion transporter-1, protein levels were elevated in iron-loaded hypotransferrinemic mice. In conclusion, ZRT/IRT-like protein 14 protein levels are up-regulated in iron-loaded rat liver and pancreas and in hypotransferrinemic mouse liver. Divalent metal-ion transporter-1 protein levels are down-regulated in iron-loaded rat liver, and up-regulated in iron-deficient liver and heart. Our results provide insight into the potential contributions of these transporters to tissue iron uptake during iron deficiency and overload. PMID:23349308

Neonatal hyperthyroidism on rat heart: interrelation with nitric oxide and sex.

PubMed

Rodríguez, L; Detomaso, F; Braga, P; Prendes, M; Perosi, F; Cernadas, G; Balaszczuk, A; Fellet, A

2015-06-01

To clarify the mechanism mediating the effect of hyperthyroidism on cardiac function during the second month of life in rats. Male and female Sprague-Dawley rats were assigned to a control or to a triiodothyronine (T3)-treated group. Treatment of each group was started on the third day after birth. Control rats (Eut) received 0.9 NaCl [0.1 ml/100 g body weight (BW)] every second day during 60 days and T3-treated rats (Hyper) received subcutaneous (SC) T3 injections every second day during 60 days. Hyperthyroidism decreased left ventricle volume only in male rats. Female euthyroid rats presented higher atrial nitric oxide synthase (NOS) activity than male rats and hormonal treatment decreased this enzyme's activity in both sexes. Euthyroid male and female rats had similar atrial NOS protein levels, but females had higher caveolin (cav) 3 protein levels. T3 treatment increased this protein only in males. Female rats had lower ventricular NOS activity than male rats; hyperthyroidism increased NOS activity in both sexes but this effect was associated with lower cav 3 protein levels. Hyperthyroidism did not change cav 1 protein levels in both male and female rats. The results of this study demonstrating clinically relevant sex-related differences in the pathophysiology of the hyperthyroid heart have raised new questions regarding the mechanisms responsible for the observed differences. This study suggests that sex-related intrinsic factors such as nitric oxide may modulate the response to hyperthyroidism that leads to cardiovascular dysfunction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keith, Dove; Finlay, Liam; Butler, Judy

Highlights: • 24 month old rats were supplemented with 0.2% lipoic acid in the diet for 2 weeks. • Lipoic acid shifts phase of core circadian clock proteins. • Lipoic acid corrects age-induced desynchronized lipid metabolism rhythms. - Abstract: It is well established that lipid metabolism is controlled, in part, by circadian clocks. However, circadian clocks lose temporal precision with age and correlates with elevated incidence in dyslipidemia and metabolic syndrome in older adults. Because our lab has shown that lipoic acid (LA) improves lipid homeostasis in aged animals, we hypothesized that LA affects the circadian clock to achieve thesemore » results. We fed 24 month old male F344 rats a diet supplemented with 0.2% (w/w) LA for 2 weeks prior to sacrifice and quantified hepatic circadian clock protein levels and clock-controlled lipid metabolic enzymes. LA treatment caused a significant phase-shift in the expression patterns of the circadian clock proteins Period (Per) 2, Brain and Muscle Arnt-Like1 (BMAL1), and Reverse Erythroblastosis virus (Rev-erb) β without altering the amplitude of protein levels during the light phase of the day. LA also significantly altered the oscillatory patterns of clock-controlled proteins associated with lipid metabolism. The level of peroxisome proliferator-activated receptor (PPAR) α was significantly increased and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were both significantly reduced, suggesting that the LA-supplemented aged animals are in a catabolic state. We conclude that LA remediates some of the dyslipidemic processes associated with advanced age, and this mechanism may be at least partially through entrainment of circadian clocks.« less

Expression of apoptosis related proteins: RAIDD, ZIP kinase, Bim/BOD, p21, Bax, Bcl-2 and NF-kappaB in brains of patients with Down syndrome.

PubMed

Engidawork, E; Gulesserian, T; Seidl, R; Cairns, N; Lubec, G

2001-01-01

Down syndrome (DS) is a genetic disease that exhibits significant neuropathological parallels with Alzheimer's disease (AD). One of the features of DS, neuronal loss, has been hypothesized to occur as a result of apoptosis. An increasing number of proteins are implicated in apoptosis and several of them were shown to be altered in AD, however, the knowledge in DS is far from complete. To further substantiate the hypothesis that apoptosis is the underlying mechanism for neuronal loss and contribute towards the current knowledge of apoptosis in DS, we analyzed the expression of apoptosis related proteins in frontal cortex and cerebellum of DS by western blot and ELISA techniques. Quantitative analysis revealed a significant increase in DS frontal (P < 0.0001) and cerebellar (P < 0.05) Bim/BOD (Bcl-2 interacting mediator of cell death/Bcl-2 related ovarian death gene), cerebellar Bcl-2 (P < 0.01) as well as p21 (P < 0.05) levels compared to controls. No significant change was detected in Bax, RAIDD (receptor interacting protein (RIP)-associated ICH-1/CED-3-homologus protein with death domain), ZIP (Zipper interacting protein) kinase and NF-kappaB p65 levels in both regions, although frontal cortex levels of RAIDD, Bcl-2 and p21 levels tended to increase. In addition, a 45 kDa truncated form of NF-kappaB p65 displayed a significant elevation (P < 0.05) in DS cerebellum. No significant correlation had been obtained between postmortem interval and level of the proteins analyzed. With regard to age, it was only NF-kappaB p65 that showed significant correlation (r = -0.8964, P = 0.0155, n = 9) in frontal cortex of controls. These findings provide further evidence that apoptosis indeed accounts for the neuronal loss in DS but Bax and RAIDD do not appear to take part in this process.

Comprehensive Identification of Glycated Peptides and Their Glycation Motifs in Plasma and Erythrocytes of Control and Diabetic Subjects

PubMed Central

Zhang, Qibin; Monroe, Matthew E.; Schepmoes, Athena A.; Clauss, Therese R. W.; Gritsenko, Marina A.; Meng, Da; Petyuk, Vladislav A.; Smith, Richard D.; Metz, Thomas O.

2011-01-01

Non-enzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semi-quantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies. PMID:21612289

Impact of a nutritional formula enriched in fish oil and micronutrients on pressure ulcers in critical care patients.

PubMed

Theilla, Miriam; Schwartz, Betty; Cohen, Jonathan; Shapiro, Haim; Anbar, Ronit; Singer, Pierre

2012-07-01

Pressure ulcers are an important source of morbidity and suffering for patients and a formidable burden on caregivers. To assess the impact of a feeding formula enriched with fish oil on healing of preexisting pressure ulcers and serum levels of C-reactive protein in critical care patients. Adult patients with pressure ulcers grade II or higher were randomly allocated to receive either a formula enriched with fish oil or an isocaloric control formula. Wound healing was assessed by using the Pressure Ulcer Scale for Healing tool on days 7, 14, and 28. Blood levels of C-reactive protein were measured on days 0, 7, and 14. Baseline demographics did not differ between the study (n = 20) and the control (n = 20) groups. The mean score on the ulcer healing tool increased significantly (P = .02) from day 0 to day 28 in the control group (from 9.25 [SD, 2.12] to 10.75 [SD, 3.41]) compared with the study group (from 9.10 [SD, 2.84] to 9.40 [SD, 3.72]). Mean levels of C-reactive protein decreased significantly (P= .02) from day 0 to day 14 in the study group (from 191 [SD, 104.4] mg/L to 111.7 [SD, 97.8] mg/L) compared with the control group (from 145 [SD, 90] mg/L to 139 [SD, 62] mg/L). Administration of a feeding formula enriched with fish oil was associated with decreased progression of pressure ulcers and a decrease in blood concentrations of C-reactive protein.

Extracellular matrix metalloproteinase inducer expression in the baboon endometrium: menstrual cycle and endometriosis

PubMed Central

Braundmeier, A G; Fazleabas, A T; Nowak, R A

2016-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN; BSG) regulates tissue remodeling through matrix metalloproteinases (MMPs). In human and non-human primates, endometrial remodeling is important for menstruation and the pathogenesis of endometriosis. We hypothesized that as in humans, BSG and MMPs are expressed in the endometrium of cycling baboons, and their expression is hormonally regulated by ovarian hormones, but endometriosis disrupts this regulation. BSG expression was evaluated in the baboon endometrium by q-PCR and immunohistochemistry. In the endometrium of control cycling animals, BSG mRNA levels were highest in late secretory stage tissue. BSG protein localized to glandular epithelial cells during the proliferative phase; whereas, secretory stage tissues expressed BSG in glandular and luminal epithelia with weak stromal staining. Several MMPs were differentially expressed throughout the menstrual cycle with the highest levels found during menstruation. In ovariectomized animals, BSG endometrial mRNA levels were highest with treatment of both estrogen and progesterone than that with only estrogen. Estrogen alone resulted in BSG protein localization primarily in the endometrial glandular epithelia, while estrogen and progesterone treatment displayed BSG protein localization in both the glandular and stromal cells. Exogenous hormone treatment resulted in differential expression patterns of all MMPs compared with the control cycling animals. In the eutopic endometrium of endometriotic animals, BSG mRNA levels and protein were elevated early but decreased later in disease progression. Endometriosis elevated the expression of all MMPs except MMP7 compared with the control animals. In baboons, BSG and MMP endometrial expression is regulated by both ovarian hormones, and their expression patterns are dysregulated in endometriotic animals. PMID:20841363

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

PubMed

Lakatos, Eszter; Salehi-Reyhani, Ali; Barclay, Michael; Stumpf, Michael P H; Klug, David R

2017-01-01

We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

DOE PAGES

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.; ...

2017-08-31

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/SSB in salivary gland epithelial cells.

PubMed

Katsiougiannis, S; Tenta, R; Skopouli, F N

2015-08-01

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren's syndrome (SS) autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/Sjögren's syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens. © 2015 British Society for Immunology.

Soluble thrombomodulin levels in plasma of multiple sclerosis patients and their implication.

PubMed

Festoff, Barry W; Li, Chaoyang; Woodhams, Barry; Lynch, Sharon

2012-12-15

Thrombomodulin (TM) on the cell-surface of cerebrovascular endothelial cells (CECs) is released into blood upon CEC damage. TM promotes activation of protein C (APC), an anticoagulant, anti-inflammatory, neuroprotective molecule that protects CECs and impedes inflammatory cell migration across the blood-brain barrier (BBB). Multiple sclerosis (MS) is associated with CEC damage and BBB dysfunction. We evaluated soluble TM (sTM) levels as a biomarker of BBB integrity and whether glatiramer acetate (GA) influenced sTM levels in MS patients. sTM levels quantified by 2-site ELISA from sera of healthy controls and systemic lupus erythematosus (SLE) patients (CEC-damage positive control) were compared with levels from patients with relapsing-remitting (RRMS) or secondary-progressive MS (SPMS), stratified as: RRMS/GA/no relapse, RRMS/GA/in relapse, RRMS no GA/no relapse, RRMS/no GA/in relapse; and SPMS/no GA. Additionally, soluble endothelial protein C receptor (sEPCR) levels were assessed in the non-stratified MS group, SLE patients, and controls. sTM levels were highest in RRMS patients taking GA with or without relapse, followed in decreasing order by SLE, RRMS/no GA/in relapse, SPMS, RRMS/no GA/no relapse, healthy controls. sEPCR levels were highest in MS patients, then SLE, then controls. sTM may be a useful biomarker of BBB integrity in RRMS patients. Further evaluation of sEPCR is needed. The finding that the highest sTM levels were in RRMS patients taking GA is interesting and warrants further investigation. Published by Elsevier B.V.

Epidermal Hydration Is Improved by Enhanced Ceramide Metabolism in Aged C57BL/6J Mice After Dietary Supplementation of Royal Jelly.

PubMed

Jeon, Sanghun; Cho, Yunhi

2015-09-01

Epidermal hydration is maintained by the epidermal lipid barrier, of which ceramide (Cer) is the major constituent. We examined the dietary effect of royal jelly (RJ) on epidermal hydration in aged mice. Altered Cer metabolism was further determined by measuring epidermal levels of individual Cer, glucosylceramide (GC), and sphingomyelin (SM) species, and of Cer-metabolizing enzymes. Aged C57BL/6J mice were fed a control diet (group AGED) or diets with 1% RJ harvested from two different areas (groups AGED+RJ1:AGED + RJ2) for 16 weeks. Aged C57BL/6J mice with no dietary intervention (the control group: group C) represented the onset of aging. In group AGED, epidermal levels of hydration, Cer1/2/5/6/7, GC-A/B/C/D, SM1/2/3, and β-glucocerebrosidase (GCase) protein, an enzyme of GC hydrolysis for Cer generation, were lower than in group C; these levels, as well as those of Cer3/4 and acidic sphingomyelinase (aSMase) protein, an enzyme of SM hydrolysis for Cer generation, were higher in group AGED + RJ1 than in group AGED. Despite increases in GC-B, SM1/2/3, and serine palmitoyltransferase2 protein, an enzyme of de novo Cer synthesis, in group AGED + RJ2 to levels higher than in group AGED, epidermal levels of hydration, Cer1-7, GC-A/C/D, GCase, and aSMase proteins were similar in these two groups. Expression of GCase and aSMase mRNAs, and of Cer synthase3 and ceramidase proteins, enzymes of de novo Cer synthesis and degradation, did not differ among groups. Dietary RJ1 improved epidermal hydration by enhancing Cer metabolism with increased levels of all Cer, GC, and SM species, and of GCase and aSMase proteins.

Effects of different levels of protein intake and physical training on growth and nutritional status of young rats.

PubMed

Ribeiro, Sandra Maria Lima; Rogero, Marcelo Macedo; Bacurau, Reury Frank Pereira; de Campos, Patrícia Lopes; Luz, Silmara dos Santos; Lancha, Antonio Herber; Tirapegui, Julio

2010-01-01

This study aimed to investigate the effects of physical training, and different levels of protein intake in the diet, on the growth and nutritional status of growing rats. Newly-weaned Wistar rats (n=48) were distributed into six experimental groups; three of them were subjected to physical swim training (1 h per day, 5 d per week, for 4 wk, after 2 wk of familiarization) and the other three were considered as controls (non-trained). Each pair of groups, trained and non-trained, received diets with a different level of protein in their composition: 14%, 21% or 28%. The animals were euthanized at the end of the training period and the following analyses were performed: proteoglycan synthesis as a biomarker of bone and cartilage growth, IGF-I (insulin-like growth factor-I) assay as a biomarker of growth and nutritional status, total RNA and protein concentration and protein synthesis measured in vivo using a large-dose phenylalanine method. As a main finding, increased dietary protein, combined with physical training, was able to improve neither tissue protein synthesis nor muscle growth. In addition, cartilage and bone growth seem to be deteriorated by the lower and the higher levels of protein intake. Our data allow us to conclude that protein enhancement in the diet, combined with physical exercise, does not stimulate tissue protein synthesis or muscle mass growth. Furthermore, physical training, combined with low protein intake, was not favorable to bone development in growing animals.

DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning

PubMed Central

Rodriguez, Kevin; Perales, Mariano; Snipes, Stephen; Yadav, Ram Kishor; Diaz-Mendoza, Mercedes; Reddy, G. Venugopala

2016-01-01

The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana. WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity. PMID:27671631

Molecular Signatures and Diagnostic Biomarkers of Cumulative Blast-Graded Mild TBI

DTIC Science & Technology

2014-12-01

100 ± 9% 191 ± 20%* Values are mean ± SE of 4 rats/ group expressed as percentage of control . The level of the averaged control for each...protein is arbitrarily set to 100 with SE adjusted proportionally with remaining groups normalized to the level in control . *Significantly increased...not significant vs sham; **- pɘ.05, and ***-pɘ.01 Rat number in each group ranged 4-7. Mean, pg/ml for GFAP and UCHL1 and AU in % control

Direct, rapid effects of 25-hydroxyvitamin D3 on isolated intestinal cells.

PubMed

Phadnis, Ruta; Nemere, Ilka

2003-10-01

Scattered reports in the literature have suggested that the metabolite 25-hydroxyvitamin D(3) [25(OH)D(3)] has biological activity. In the present work, perfusion of isolated duodenal loops of normal chickens with 100 nM 25(OH)D(3) resulted in enhanced transport of (45)Ca within 2 min relative to the vehicle controls. We then tested the effect of a range of 25(OH)D(3) concentrations on (45)Ca handling by isolated intestinal cells in time course studies. Following a basal uptake period, cell suspensions from 7-week old chicks were treated either with 25, 100, or 300 nM 25(OH)D(3), or the vehicle ethanol (0.01%, final concentration). Both 25 and 100 nM 25(OH)D(3) resulted in a significant (P < 0.05) reduction in (45)Ca levels, relative to controls, between 1-10 min after treatment, while 300 nM 25(OH)D(3) resulted in a significant increase in (45)Ca levels, relative to controls, after 10 min of incubation. The effect of 100 nM 25(OH)D(3) (a physiological level) on cell calcium was abolished by the presence of 6.5 nM 24,25-dihydroxyvitamin D(3). In cell preparations from 14- or 28-week old birds 100nM 25(OH)D(3) had no effect, relative to vehicle controls. Incubation of cells with 2 microM BAY K8644, a calcium channel activator, stimulated (45)Ca uptake within 3 min relative to vehicle controls (P < 0.05), while addition of either 20 microM forskolin or 100 nM phorbol ester (stimulators of the PKA and PKC pathways, respectively) resulted in enhanced radionuclide levels after 10 min of incubation (P < 0.05, relative to corresponding controls). Finally, cells were treated with 100 nM 25(OH)D(3) or vehicle and samples taken at various times for analyses of protein kinase C and A activities. No effect of 25(OH)D(3) on protein kinase C activity was observed, while protein kinase A activity was stimulated to nearly 200% of controls at 1 min after 25(OH)D(3) addition (P < 0.05, relative to corresponding controls) and began declining at 3 min, returning to control levels 5 min after additions. We conclude that 25(OH)D(3) has a direct effect on calcium handling in enterocytes of young animals that may in part be mediated by the protein kinase A signal transduction pathway. Copyright 2003 Wiley-Liss, Inc.

Visual-spatial learning impairments are associated with hippocampal PSD-95 protein dysregulation in a mouse model of fragile X syndrome.

PubMed

Gandhi, Réno M; Kogan, Cary S; Messier, Claude; Macleod, Lindsey S

2014-03-05

Fragile X syndrome is the most common cause of inherited intellectual disability and is caused by the lack of fragile X mental retardation protein (FMRP) expression. In-vitro findings in mice and post-mortem autopsies in humans are characterized by dendritic spine abnormalities in the absence of Fmrp/FMRP. Biochemical and electrophysiological studies have identified postsynaptic density protein (PSD)-95 as having an established role in dendritic morphology as well as a molecular target of Fmrp. How Fmrp affects the expression of PSD-95 following behavioral learning is unknown. In the current study, wild type controls and Fmr1 knockout mice were trained in a subset of the Hebb-Williams (H-W) mazes. Dorsal hippocampal PSD-95 protein levels relative to a stable cytoskeleton protein (β-tubulin) were measured. We report a significant upregulation of PSD-95 protein levels in wild type mice, whereas training-related protein increases were blunted in Fmr1 knockout mice. In addition, there was a significant negative correlation between mean total errors on the mazes and PSD-95 protein levels. The coefficient of determination indicated that the mean total errors on the H-W mazes accounted for 35% of the variance in PSD-95 protein levels. These novel findings suggest that reduced PSD-95-associated postsynaptic plasticity may contribute to the learning and memory deficits observed in human fragile X syndrome patients.

Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress

PubMed Central

Mustafa, Ghazala; Komatsu, Setsuko

2014-01-01

Flooding stress has a negative impact on soybean cultivation because it severely impairs growth and development. To understand the flooding responsive mechanism in early stage soybeans, a glycoproteomic technique was used. Two-day-old soybeans were treated with flooding for 2 days and roots were collected. Globally, the accumulation level of glycoproteins, as revealed by cross-reaction with concanavalin A decreased by 2 days of flooding stress. Glycoproteins were enriched from total protein extracts using concanavalin A lectin resin and analyzed using a gel-free proteomic technique. One-hundred eleven and 69 glycoproteins were identified without and with 2 days of flooding stress, respectively. Functional categorization of these identified glycoproteins indicated that the accumulation level of proteins related to protein degradation, cell wall, and glycolysis increased, while stress-related proteins decreased under flooding stress. Also the accumulation level of glycoproteins localized in the secretory pathway decreased under flooding stress. Out of 23 common glycoproteins between control and flooding conditions, peroxidases and glycosyl hydrolases were decreased by 2 days of flooding stress. mRNA expression levels of proteins in the endoplasmic reticulum and N-glycosylation related proteins were downregulated by flooding stress. These results suggest that flooding might negatively affect the process of N-glycosylation of proteins related to stress and protein degradation; however glycoproteins involved in glycolysis are activated. PMID:25477889

Relationship between C-Reactive Protein Level and Diabetic Retinopathy: A Systematic Review and Meta-Analysis

PubMed Central

Chen, Song; Duan, Hongtao; Kong, Jiahui; Li, Zedong

2015-01-01

Objectives To date, the relationship between C-reactive protein (CRP) level and diabetic retinopathy (DR) remains controversial. Therefore, a systematic review and meta-analysis was used to reveal the potential relationship between CRP level and DR. Methods A systematic search of PubMed, Embase.com, and Web of Science was performed to identify all comparative studies that compared the CRP level of two groups (case group and control group). We defined that diabetic patients without retinopathy and /or matched healthy persons constituted the control group, and patients with DR were the case group. Results Two cross sectional studies and twenty case control studies including a total of 3679 participants were identified. After pooling the data from all 22 studies, obvious heterogeneity existed between the studies, so a subgroup analysis and sensitivity analysis were performed. Removing the sensitivity studies, the blood CRP levels in the case group were observed to be higher than those in the control group [SMD = 0.22, 95% confidence interval (CI), 0.11–0.34], and the blood CRP levels in the proliferative diabetic retinopathy (PDR) group were also higher than those in the non-proliferative diabetic retinopathy (NPDR) group [SMD = 0.50, 95% CI, 0.30–0.70]. Conclusions The results from this current meta-analysis indicate that the CRP level might be used as a biomarker to determine the severity of DR. PMID:26636823

Influence of exercise on bone remodeling-related hormones and cytokines in ovariectomized rats: a model of postmenopausal osteoporosis.

PubMed

Li, Lihui; Chen, Xi; Lv, Shuang; Dong, Miaomiao; Zhang, Li; Tu, Jiaheng; Yang, Jie; Zhang, Lingli; Song, Yinan; Xu, Leiting; Zou, Jun

2014-01-01

This study aims to explore the effects of exercise on postmenopausal osteoporosis and the mechanisms by which exercise affects bone remodeling. Sixty-three Wistar female rats were randomly divided into five groups: (1) control group, (2) sham-operated group, (3) OVX (Ovariectomy) group, (4) DES-OVX (Diethylstilbestrol-OVX) group, and (5) Ex-OVX (Exercise-OVX) group. The rat osteoporosis model was established through ovariectomy. The Ex-OVX rats were made to run 251.2 meters every day, 6 d/wk for 3 months in a running wheel. Trabecular bone volume (TBV%), total resorption surface (TRS%), trabecular formation surface (TFS%), mineralization rate (MAR), bone cortex mineralization rate (mAR), and osteoid seam width (OSW) were determined by bone histomorphometry. The mRNA and protein levels of interleukin-1β (IL-1β2), interleukin-6 (IL-6), and cyclooxygenase-2 (Cox-2) were determined by in situ hybridization and immunohistochemistry, respectively. Serum levels of estrogen estradiol (E2), calcitonin (CT), osteocalcin (BGP), and parathyroid hormone (PTH) were determined by ELISA assays. The investigation revealed that compared to the control and the sham-operated groups, the OVX group showed significantly lower levels of TBV%, E2, and CT, but much higher levels of TRS%, TFS%, MAR, OSW, BGP, and PTH. The Ex-OVX group showed increased TBV% and serum levels of E2 and CT compared to the OVX group. Ovariectomy also led to a significant increase in IL-1β mRNA and protein levels in the bone marrow and IL-6 and Cox-2 protein levels in tibias. In addition, the Ex-OVX group showed lower levels of IL-1 mRNA and protein, IL-6 mRNA, and Cox-2 mRNA and protein than those in the OVX group. The upshot of the study suggests that exercise can significantly increase bone mass in postmenopausal osteoporosis rat models by inhibiting bone resorption and increasing bone formation, especially in trabecular bones.

Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

PubMed Central

Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

2015-01-01

Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

Effects of heat shock on neuroblastoma (N1E 115) cell proliferation and differentiation.

PubMed

Stoklosinski, A; Kruse, H; Richter-Landsberg, C; Rensing, L

1992-05-01

Heat shock (44 degrees C) applied for only 15 min induced the development of neurites in neuroblastoma cells 3-6 days later. During the first day after heat shock a transient increase in the rate of cytokinesis together with a synchronizing effect was observed, which led to waves of cytokinesis 14.5 h apart. Individual cell cycles were determined and showed a lengthening in the minimal cell cycle duration and a decrease in the cell cycle variance after shock. Two to 3 days after heat shock the proliferation rate decreased and then recovered. During the 6 days after heat shock, total protein synthesis was lower compared to the untreated cultures. The synthesis of heat shock proteins (100, 90, 84, 70, 68 kDa and some of lower MW) reached a maximum 6 h after heat shock. Parallel changes in the phosphorylation state of proteins were observed in an in vitro assay. Four proteins (100, 89, 67, and 15 kDa) increased and two proteins (97, 73 kDa) decreased their phosphorylation state significantly. Six days after heat shock two proteins (89, 55 kDa) increased their phosphorylation state; the 55-kDa phosphoprotein was identified as tubulin. The effect of heat shock on the intracellular calcium level was determined by measuring Fura 2 fluorescence. Six hours after shock, the Ca2+ level increased to a maximum (about three times the control value) and then dropped during the following days below the control values. We conclude from these results that a decrease in the calcium level may be causally involved in the differentiation process. The calcium effect is probably mediated by changes in the activity of different kinases. This assumption is compatible with the results of experiments with cyclic nucleotides when 10(-5) M cAMP and cGMP were added to in vitro assays of protein phosphorylation. They had different stimulating effects in heat-shocked, differentiating, and growing (control) cells.

A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

PubMed Central

Bergua, María; Zwart, Mark P.; El-Mohtar, Choaa; Shilts, Turksen; Elena, Santiago F.

2014-01-01

ABSTRACT Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels—the cellular and the whole-organism levels—by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems. PMID:25031351

Multilevel Control of Arabidopsis 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase by Protein Phosphatase 2A[W

PubMed Central

Leivar, Pablo; Antolín-Llovera, Meritxell; Ferrero, Sergi; Closa, Marta; Arró, Montserrat; Ferrer, Albert; Boronat, Albert; Campos, Narciso

2011-01-01

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B′′ regulatory subunits of protein phosphatase 2A (PP2A), designated B′′α and B′′β, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B′′α and B′′β are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B′′α and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B′′β. Our data indicate that PP2A exerts multilevel control on HMGR through the five-member B′′ protein family during normal development and in response to a variety of stress conditions. PMID:21478440

Minocycline restores cognitive-relative altered proteins in young bile duct-ligated rat prefrontal cortex.

PubMed

Li, Shih-Wen; Chen, Yu-Chieh; Sheen, Jiunn-Ming; Hsu, Mei-Hsin; Tain, You-Lin; Chang, Kow-Aung; Huang, Li-Tung

2017-07-01

Bile duct ligation (BDL) model is used to study hepatic encephalopathy accompanied by cognitive impairment. We employed the proteomic analysis approach to evaluate cognition-related proteins in the prefrontal cortex of young BDL rats and analyzed the effect of minocycline on these proteins and spatial memory. BDL was induced in young rats at postnatal day 17. Minocycline as a slow-release pellet was implanted into the peritoneum. Morris water maze test and two-dimensional liquid chromatography-tandem mass spectrometry were used to evaluate spatial memory and prefrontal cortex protein expression, respectively. We used 2D/LC-MS/MS to analyze for affected proteins in the prefrontal cortex of young BDL rats. Results were verified with Western blotting, immunohistochemistry, and quantitative real-time PCR. The effect of minocycline in BDL rats was assessed. BDL induced spatial deficits, while minocycline rescued it. Collapsin response mediator protein 2 (CRMP2) and manganese-dependent superoxide dismutase (MnSOD) were upregulated and nucleoside diphosphate kinase B (NME2) was downregulated in young BDL rats. BDL rats exhibited decreased levels of brain-derived neurotrophic factor (BDNF) mRNA as compared with those by the control. However, minocycline treatment restored CRMP2 and NME2 protein expression, BDNF mRNA level, and MnSOD activity to control levels. We demonstrated that BDL altered the expression of CRMP2, NME2, MnSOD, and BDNF in the prefrontal cortex of young BDL rats. However, minocycline treatment restored the expression of the affected mediators that are implicated in cognition. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative evaluation of levels of C-reactive protein and PMN in periodontitis patients related to cardiovascular disease

PubMed Central

Anitha, G.; Nagaraj, M.; Jayashree, A.

2013-01-01

Background: Numerous cross-sectional studies have suggested that chronic periodontitis is a risk factor for cardiovascular diseases. There is evidence that periodontitis and cardiovascular diseases are linked by inflammatory factors including C-reactive protein. The purpose of the study was to investigate the levels of CRP and PNM cells as a marker of inflammatory host response in the serum of chronic periodontitis patients and in patients with CVD. Materials and Methods: Study population included 75 patients; both male and female above 35 years were included for the study. The patients were divided into three groups of 25 each – Group I: Chronic periodontitis patients with CVD, Group II: Chronic periodontitis patients without CVD and Group III: Control subjects (without chronic periodontitis and CVD). Patients with chronic periodontitis had ≥8 teeth involved with probing depth (PD) ≥5 mm involved. The control group had PD ≤ 3 mm and no CVD. Venous blood was collected from the patients and C-reactive protein levels were analyzed by immunoturbidimetry. PMN was recorded by differential count method. Results: On comparison, OHI-S Index, GI, mean PD, CRP and PMN values showed significant difference from Group I to III. CRP level was highly significant in Group I when compared with Group II and Group III. PMN level was highly significant in Group I when compared with Group III PMN level which was not significant. Conclusion: This study indicated that periodontitis may add the inflammation burden of the individual and may result in increased levels of CVD based on serum CRP levels. Thus, controlled prospective trials with large sample size should be carried out to know the true nature of the relationship if indeed one exists. PMID:24049333

Comparative evaluation of levels of C-reactive protein and PMN in periodontitis patients related to cardiovascular disease.

PubMed

Anitha, G; Nagaraj, M; Jayashree, A

2013-05-01

Numerous cross-sectional studies have suggested that chronic periodontitis is a risk factor for cardiovascular diseases. There is evidence that periodontitis and cardiovascular diseases are linked by inflammatory factors including C-reactive protein. The purpose of the study was to investigate the levels of CRP and PNM cells as a marker of inflammatory host response in the serum of chronic periodontitis patients and in patients with CVD. Study population included 75 patients; both male and female above 35 years were included for the study. The patients were divided into three groups of 25 each - Group I: Chronic periodontitis patients with CVD, Group II: Chronic periodontitis patients without CVD and Group III: Control subjects (without chronic periodontitis and CVD). Patients with chronic periodontitis had ≥8 teeth involved with probing depth (PD) ≥5 mm involved. The control group had PD ≤ 3 mm and no CVD. Venous blood was collected from the patients and C-reactive protein levels were analyzed by immunoturbidimetry. PMN was recorded by differential count method. On comparison, OHI-S Index, GI, mean PD, CRP and PMN values showed significant difference from Group I to III. CRP level was highly significant in Group I when compared with Group II and Group III. PMN level was highly significant in Group I when compared with Group III PMN level which was not significant. This study indicated that periodontitis may add the inflammation burden of the individual and may result in increased levels of CVD based on serum CRP levels. Thus, controlled prospective trials with large sample size should be carried out to know the true nature of the relationship if indeed one exists.

Lower weight gain and hepatic lipid content in hamsters fed high fat diets supplemented with white rice protein, brown rice protein, soy protein, and their hydrolysates.

PubMed

Zhang, Huijuan; Bartley, Glenn E; Mitchell, Cheryl R; Zhang, Hui; Yokoyama, Wallace

2011-10-26

The physiological effects of the hydrolysates of white rice protein (WRP), brown rice protein (BRP), and soy protein (SP) hydrolyzed by the food grade enzyme, alcalase2.4 L, were compared to the original protein source. Male Syrian Golden hamsters were fed high-fat diets containing either 20% casein (control) or 20% extracted proteins or their hydrolysates as the protein source for 3 weeks. The brown rice protein hydrolysate (BRPH) diet group reduced weight gain 76% compared with the control. Animals fed the BRPH supplemented diet also had lower final body weight, liver weight, very low density lipoprotein cholesterol (VLDL-C), and liver cholesterol, and higher fecal fat and bile acid excretion than the control. Expression levels of hepatic genes for lipid oxidation, PPARα, ACOX1, and CPT1, were highest for hamsters fed the BRPH supplemented diet. Expression of CYP7A1, the gene regulating bile acid synthesis, was higher in all test groups. Expression of CYP51, a gene coding for an enzyme involved in cholesterol synthesis, was highest in the BRPH diet group. The results suggest that BRPH includes unique peptides that reduce weight gain and hepatic cholesterol synthesis.

Neurochemical aftermath of amateur boxing.

PubMed

Zetterberg, Henrik; Hietala, M Albert; Jonsson, Michael; Andreasen, Niels; Styrud, Ewa; Karlsson, Ingvar; Edman, Ake; Popa, Cornel; Rasulzada, Abdullah; Wahlund, Lars-Olof; Mehta, Pankaj D; Rosengren, Lars; Blennow, Kaj; Wallin, Anders

2006-09-01

Little solid information is available on the possible risks for neuronal injury in amateur boxing. To determine whether amateur boxing and severity of hits are associated with elevated levels of biochemical markers for neuronal injury in cerebrospinal fluid. Longitudinal study. Referral center specializing in evaluation of neurodegenerative disorders. Fourteen amateur boxers (11 men and 3 women) and 10 healthy male nonathletic control subjects. The boxers underwent lumbar puncture 7 to 10 days and 3 months after a bout. The control subjects underwent LP once. Neurofilament light protein, total tau, glial fibrillary acidic protein, phosphorylated tau, and beta-amyloid protein 1-40 (Abeta([1-40])) and 1-42 (Abeta([1-42])) concentrations in cerebrospinal fluid were measured. Increased levels after a bout compared with after 3 months of rest from boxing were found for 2 markers for neuronal and axonal injury, neurofilament light protein (mean +/- SD, 845 +/- 1140 ng/L vs 208 +/- 108 ng/L; P = .008) and total tau (mean +/- SD, 449 +/- 176 ng/L vs 306 +/- 78 ng/L; P = .006), and for the astroglial injury marker glial fibrillary acidic protein (mean +/- SD, 541 +/- 199 ng/L vs 405 +/- 138 ng/L; P = .003). The increase was significantly higher among boxers who had received many hits (>15) or high-impact hits to the head compared with boxers who reported few hits. In the boxers, concentrations of neurofilament light protein and glial fibrillary acidic protein, but not total tau, were significantly elevated after a bout compared with the nonathletic control subjects. With the exception of neurofilament light protein, there were no significant differences between boxers after 3 months of rest from boxing and the nonathletic control subjects. Amateur boxing is associated with acute neuronal and astroglial injury. If verified in longitudinal studies with extensive follow-up regarding the clinical outcome, analyses of cerebrospinal fluid may provide a scientific basis for medical counseling of athletes after boxing or head injury.

Mechanism for the decrease in the FIP1L1-PDGFRalpha protein level in EoL-1 cells by histone deacetylase inhibitors.

PubMed

Ishihara, Kenji; Kaneko, Motoko; Kitamura, Hajime; Takahashi, Aki; Hong, Jang Ja; Seyama, Toshio; Iida, Koji; Wada, Hiroshi; Hirasawa, Noriyasu; Ohuchi, Kazuo

2008-01-01

Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate. EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein. When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days. The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA. Copyright 2008 S. Karger AG, Basel.

Cytokine responses in young and old rhesus monkeys: effect of caloric restriction.

PubMed

Mascarucci, Paolo; Taub, Dennis; Saccani, Simona; Paloma, Marjorie A; Dawson, Harry; Roth, George S; Lane, Mark A; Ingram, Donald K

2002-05-01

Caloric restriction (CR) is the only known intervention demonstrated to retard a great variety of aging processes, extend median and maximum life-span, and decrease the incidence of age-associated diseases in mammals. Paralleling findings from rodent studies, studies in rhesus monkeys (Macaca mulatta) suggest that CR may retard many age-sensitive parameters in primates. A recent study in rhesus monkeys showed age-related dysregulation of cytokine levels. Specifically, age-related increases in interleukin-10 (IL-10) and IL-6 proteins were observed in supernatants from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs), and interferon-gamma (IFN-gamma) protein exhibited an age-related decrease in phytohemagglutinin (PHA)-stimulated PBMCs. To investigate effects of CR on age-related changes in cytokine production, we obtained PBMCs from control and CR rhesus monkeys aged 6-7 and 22-25 years. We evaluated IL-10 and IL-6 protein and gene expression after exposure to LPS and IFN-gamma protein and gene expression after PHA stimulation. The results revealed significantly higher levels of IFN-gamma protein and gene expression in aged monkeys on CR for 2 years compared with controls. No significant CR effects were observed on IL-10 and IL-6 protein levels. IFN-gamma plays an important role in the initial defense mechanism against viral and microbial disease and cancer. Altered regulation of IFN-gamma in old CR rhesus monkeys may be a key factor in reducing cancer incidence and other age-associated diseases.

Effects of crocin and voluntary exercise, alone or combined, on heart VEGF-A and HOMA-IR of HFD/STZ induced type 2 diabetic rats.

PubMed

Ghorbanzadeh, V; Mohammadi, M; Dariushnejad, H; Chodari, L; Mohaddes, G

2016-10-01

Hyperglycemia is the main risk factor for microvascular complications in type 2 diabetes. Crocin and voluntary exercise have anti-hyperglycemic effects in diabetes. In this research, we evaluated the effects of crocin and voluntary exercise alone or combined on glycemia control and heart level of VEGF-A. Animals were divided into eight groups as: control (con), diabetes (Dia), crocin (Cro), voluntary exercise (Exe), crocin and voluntary exercise (Cro-Exe), diabetic-crocin (Dia-Cro), diabetic-voluntary exercise (Dia-Exe), diabetic-crocin-voluntary exercise (Dia-Cro-Exe). Type 2 diabetes was induced by a high-fat diet (4 weeks) and injection of streptozotocin (STZ) (i.p, 35 mg/kg). Animals received oral administration of crocin (50 mg/kg) or performed voluntary exercise alone or together for 8 weeks. Oral glucose tolerance test (OGTT) was performed on overnight fasted control, diabetic and treated rats after 8 weeks of treatment. Then, serum insulin and heart VEGF-A protein levels were measured. Crocin combined with voluntary exercise significantly decreased blood glucose levels (p < 0.001) and insulin resistance (HOMA-IR) (p < 0.001) compared to diabetic group. VEGF-A level was significantly (p < 0.01) lower in Dia group compared to control group. The combination of crocin and voluntary exercise significantly enhanced VEGF-A protein levels in Dia-Cro-Exe and Cro-Exe group compared to diabetic and control groups, respectively; p < 0.001 and p < 0.05. Crocin combined with voluntary exercise improved insulin resistance (HOMA-IR) and reduced glucose levels in diabetic rats. Since both crocin and voluntary exercise can increase VEGF-A protein expression in heart tissue, they probably are able to increase angiogenesis in diabetic animals.

Protein levels and colony development of Africanized and European honey bees fed natural and artificial diets.

PubMed

Morais, M M; Turcatto, A P; Pereira, R A; Francoy, T M; Guidugli-Lazzarini, K R; Gonçalves, L S; de Almeida, J M V; Ellis, J D; De Jong, D

2013-12-19

Pollen substitute diets are a valuable resource for maintaining strong and health honey bee colonies. Specific diets may be useful in one region or country and inadequate or economically unviable in others. We compared two artificial protein diets that had been formulated from locally-available ingredients in Brazil with bee bread and a non-protein sucrose diet. Groups of 100 newly-emerged, adult workers of Africanized honey bees in Brazil and European honey bees in the USA were confined in small cages and fed on one of four diets for seven days. The artificial diets included a high protein diet made of soy milk powder and albumin, and a lower protein level diet consisting of soy milk powder, brewer's yeast and rice bran. The initial protein levels in newly emerged bees were approximately 18-21 µg/µL hemolymph. After feeding on the diets for seven days, the protein levels in the hemolymph were similar among the protein diet groups (~37-49 µg/µL after seven days), although Africanized bees acquired higher protein levels, increasing 145 and 100% on diets D1 and D2, respectively, versus 83 and 60% in the European bees. All the protein diets resulted in significantly higher levels of protein than sucrose solution alone. In the field, the two pollen substitute diets were tested during periods of low pollen availability in the field in two regions of Brazil. Food consumption, population development, colony weight, and honey production were evaluated to determine the impact of the diets on colony strength parameters. The colonies fed artificial diets had a significant improvement in all parameters, while control colonies dwindled during the dearth period. We conclude that these two artificial protein diets have good potential as pollen substitutes during dearth periods and that Africanized bees more efficiently utilize artificial protein diets than do European honey bees.

Proteomic analysis of soybean hypocotyl during recovery after flooding stress.

PubMed

Khan, Mudassar Nawaz; Sakata, Katsumi; Komatsu, Setsuko

2015-05-21

Soybean is a nutritionally important crop, but exhibits reduced growth and yields under flooding stress. To investigate soybean responses during post-flooding recovery, a gel-free proteomic technique was used to examine the protein profile in the hypocotyl. Two-day-old soybeans were flooded for 2 days and hypocotyl was collected under flooding and during the post-flooding recovery period. A total of 498 and 70 proteins were significantly changed in control and post-flooding recovering soybeans, respectively. Based on proteomic and clustering analyses, three proteins were selected for mRNA expression and enzyme activity assays. Pyruvate kinase was increased under flooding, but gradually decreased during post-flooding recovery period at protein abundance, mRNA, and enzyme activity levels. Nucleotidylyl transferase was decreased under flooding and increased during post-flooding recovery at both mRNA expression and enzyme activity levels. Beta-ketoacyl reductase 1 was increased under flooding and decreased during recovery at protein abundance and mRNA expression levels, but its enzyme activity gradually increased during the post-flooding recovery period. These results suggest that pyruvate kinase, nucleotidylyl transferase, and beta-ketoacyl reductase play key roles in post-flooding recovery in soybean hypocotyl by promoting glycolysis for the generation of ATP and regulation of secondary metabolic pathways. This study analyzed post-flooding recovery response mechanisms in soybean hypocotyl, which is a model organ for studying secondary growth, using a gel-free proteomic technique. Mass spectrometry analysis of proteins extracted from soybean hypocotyls identified 20 common proteins between control and flooding-stressed soybeans that changed significantly in abundance over time. The hypocotyl proteins that changed during post-flooding recovery were assigned to protein, development, secondary metabolism, and glycolysis categories. The analysis revealed that three proteins, pyruvate kinase, nucleotidylyl transferase, and beta-ketoacyl reductase, were increased in hypocotyl under flooding conditions and during post-flooding recovery. The proteins are involved in glycolysis, nucleotide synthesis and amino acid activation, and complex fatty acid biosynthesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Chronic exposure to nitric oxide alters the free iron pool in endothelial cells: Role of mitochondrial respiratory complexes and heat shock proteins

PubMed Central

Ramachandran, Anup; Ceaser, Erin; Darley-Usmar, Victor M.

2004-01-01

The mechanisms of nitric oxide (NO) signaling include binding to the iron centers in soluble guanylate cyclase and cytochrome c oxidase and posttranslational modification of proteins by S-nitrosation. Low levels of NO control mitochondrial number in cells, but little is known of the impact of chronic exposure to high levels of NO on mitochondrial function in endothelial cells. The focus of this study is the interaction of NO with mitochondrial respiratory complexes in cell culture and the effect this has on iron homeostasis. We demonstrate that chronic exposure of endothelial cells to NO decreased activity and protein levels of complexes I, II, and IV, whereas citrate synthase and ATP synthase were unaffected. Inhibition of these respiratory complexes was accompanied by an increase in cellular S-nitrosothiol levels, modification of cysteines residues, and an increase in the labile iron pool. The NO-dependent increase in the free iron pool and inhibition of complex II was prevented by inhibition of mitochondrial protein synthesis, consistent with a major contribution of the organelle to iron homeostasis. In addition, inhibition of mitochondrial protein synthesis was associated with an increase in heat shock protein 60 levels, which may be an additional mechanism leading to preservation of complex II activity. PMID:14691259

Dysregulation of fragile X mental retardation protein and metabotropic glutamate receptor 5 in superior frontal cortex of individuals with autism: a postmortem brain study

PubMed Central

2011-01-01

Background Fragile X syndrome is caused by loss of function of the fragile X mental retardation 1 (FMR1) gene and shares multiple phenotypes with autism. We have previously found reduced expression of the protein product of FMR1 (FMRP) in vermis of adults with autism. Methods In the current study, we have investigated levels of FMRP in the superior frontal cortex of people with autism and matched controls using Western blot analysis. Because FMRP regulates the translation of multiple genes, we also measured protein levels for downstream molecules metabotropic glutamate receptor 5 (mGluR5) and γ-aminobutyric acid (GABA) A receptor β3 (GABRβ3), as well as glial fibrillary acidic protein (GFAP). Results We observed significantly reduced levels of protein for FMRP in adults with autism, significantly increased levels of protein for mGluR5 in children with autism and significantly increased levels of GFAP in adults and children with autism. We found no change in expression of GABRβ3. Our results for FMRP, mGluR5 and GFAP confirm our previous work in the cerebellar vermis of people with autism. Conclusion These changes may be responsible for cognitive deficits and seizure disorder in people with autism. PMID:21548960

Light Moderates the Induction of Phosphoenolpyruvate Carboxylase by NaCl and Abscisic Acid in Mesembryanthemum crystallinum 1

PubMed Central

McElwain, Elizabeth F.; Bohnert, Hans J.; Thomas, John C.

1992-01-01

In Mesembryanthemum crystallinum, phosphoenolpyruvate carboxylase is synthesized de novo in response to osmotic stress, as part of the switch from C3-photosynthesis to Crassulacean acid metabolism. To better understand the environmental signals involved in this pathway, we have investigated the effects of light on the induced expression of phosphoenolpyruvate carboxylase mRNA and protein in response to stress by 400 millimolar NaCl or 10 micromolar abscisic acid in hydroponically grown plants. When plants were grown in high-intensity fluorescent or incandescent light (850 microeinsteins per square meter per second), NaCl and abscisic acid induced approximately an eightfold accumulation of phosphoenolpyruvate carboxylase mRNA when compared to untreated controls. Levels of phosphoenolpyruvate carboxylase protein were high in these abscisic acid- and NaCl-treated plants, and detectable in the unstressed control. Growth in high-intensity incandescent (red) light resulted in approximately twofold higher levels of phosphoenolpyruvate carboxylase mRNA in the untreated plants when compared to control plants grown in high-intensity fluorescent light. In low light (300 microeinsteins per square meter per second fluorescent), only NaCl induced mRNA levels significantly above the untreated controls. Low light grown abscisic acid- and NaCl-treated plants contained a small amount of phosphoenolpyruvate carboxylase protein, whereas the (untreated) control plants did not contain detectable amounts of phosphoenolpyruvate carboxylase. Environmental stimuli, such as light and osmotic stress, exert a combined effect on gene expression in this facultative halophyte. ImagesFigure 1Figure 2 PMID:16668999

Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

PubMed

Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

2013-01-01

To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

Ingestion of soy-whey blended protein augments sports performance and ameliorates exercise-induced fatigue in a rat exercise model.

PubMed

Ren, Guangxu; Yi, Suqing; Zhang, Hongru; Wang, Jing

2017-02-22

This study sought to determine the effects of soy-whey blended protein supplementation on sports performance and related biochemical parameters after long-term training. After a week of adaptation, eighteen 6-week-old male Wistar rats were randomly assigned to 3 groups: the standard chow diet plus whey protein (Whey) group, the standard chow diet plus soy-whey blended protein (BP) group and the standard chow diet only (control) group. Each group included 6 rats for the seven-week experiment. Before the experiment, the baseline values of body weight, grasping force and time to exhaustion due to the loaded-swimming test were recorded for each group. During the experimental period, all rats performed the loaded-swimming test until exhaustion five days each week. The results showed that the mean maximum grasping force of the BP group significantly increased between the 5 th and the 7 th week (p < 0.05) compared with the other groups. The ingestion of blended protein for 7 weeks significantly increased the mean time to exhaustion due to swimming by 1.5-fold and 1.2-fold compared with the control and Whey groups, respectively. The plasma levels of leucine, isoleucine and valine were significantly higher at 60 min after the blended protein intervention compared with the Whey and control interventions (p < 0.05). Furthermore, the ingestion of soy-whey blended protein enhanced the activities of lactate dehydrogenase and superoxide dismutase and decreased the levels of malondialdehyde in serum. These results collectively suggest that soy-whey blended protein ingestion with resistance exercise can improve sports performance and ameliorate exercise-induced fatigue in rats.

Hepatitis C virus core protein triggers abnormal porphyrin metabolism in human hepatocellular carcinoma cells.

PubMed

Nakano, Takafumi; Moriya, Kyoji; Koike, Kazuhiko; Horie, Toshiharu

2018-01-01

Porphyria cutanea tarda (PCT), the most common of the human porphyrias, arises from a deficiency of uroporphyrinogen decarboxylase. Studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with PCT. While these observations implicate HCV infection as a risk factor for PCT pathogenesis, the mechanism of interaction between the virus and porphyrin metabolism is unknown. This study aimed to assess the effect of HCV core protein on intracellular porphyrin metabolism to elucidate the link between HCV infection and PCT. The accumulation and excretion of porphyrins after treatment with 5-aminolevulinic acid, a porphyrin precursor, were compared between cells stably expressing HCV core protein and controls. Cells expressing HCV core protein had lower amounts of intracellular protoporphyrin IX and heme and had higher amounts of excreted coproporphyrin III, the oxidized form of coproporphyrinogen III, compared with controls. These observations suggest that HCV core protein affects porphyrin metabolism and facilitates the export of excess coproporphyrinogen III and/or coproporphyrin III, possibly via porphyrin transporters. Real-time PCR analysis revealed that the presence of HCV core protein increased the mRNA expression of porphyrin exporters ABCG2 and FLVCR1. Western blot analysis showed a higher expression level of FLVCR1, but not ABCG2, as well as a higher expression level of mature ALAS1, which is the rate-limiting enzyme in the heme synthesis pathway, in HCV core protein-expressing cells compared with controls. The data indicate that HCV core protein induced abnormal intracellular porphyrin metabolism, with an over-excretion of coproporphyrin III. These findings may partially account for the susceptibility of HCV-infected individuals to PCT development.

Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia

PubMed Central

Catts, Vibeke Sørensen; Derminio, Dominique Suzanne; Hahn, Chang-Gyu; Weickert, Cynthia Shannon

2015-01-01

Background: There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. Aims: To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. Methods: Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. Results: We found a 20% decrease in NR1 protein (t(66)=−2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=−2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. Conclusions: Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient. PMID:27336043

Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia.

PubMed

Catts, Vibeke Sørensen; Derminio, Dominique Suzanne; Hahn, Chang-Gyu; Weickert, Cynthia Shannon

2015-01-01

There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. We found a 20% decrease in NR1 protein (t(66)=-2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=-2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient.

IgA Antibodies in Rett Syndrome

ERIC Educational Resources Information Center

Reichelt, K. L.; Skjeldal, O.

2006-01-01

The level of IgA antibodies to gluten and gliadin proteins found in grains and to casein found in milk, as well as the level of IgG to gluten and gliadin, have been examined in 23 girls with Rett syndrome and 53 controls. Highly statistically significant increases were found for the Rett population compared to the controls. The reason for this…

Effects of periodontal therapy on C-reactive protein and HDL in serum of subjects with periodontitis.

PubMed

Leite, Anne Carolina Eleutério; Carneiro, Valéria Martins de Araújo; Guimarães, Maria do Carmo Machado

2014-01-01

To investigate the effects of nonsurgical periodontal therapy on levels of high-sensitivity C-reactive protein in the sera and its association with body mass index and high density lipoprotein in subjects with severe periodontitis. Sera from 28 subjects (mean age: 34.36±6.24; 32% men) with severe periodontitis and 27 healthy controls (mean age: 33.18±6.42; 33% men) were collected prior to periodontal therapy. Blood samples were obtained from 23 subjects who completed therapy (9-12 months). Oral and systemic parameters such as the number of blood cells, glucose examination, lipid profile, and high-sensitivity C-reactive protein levels accessed by high-sensitivity immunonephelometry assay, were included. Before therapy, in the periodontitis group, the ratio of subjects with high-sensitivity C-reactive protein <0.3 mg/dL was statistically lower than in the control group (P<0.0216). After therapy, the ratio of subjects with high-sensitivity C-reactive protein <0.3 mg/dL was significantly higher (65.22%) (P<0.0339). The mean value for body mass index was statistically lower in subjects with high-sensitivity C-reactive protein <0.3 mg/dL (24.63±4.19), compared with those with high-sensitivity C-reactive protein >0.3 mg/dL (28.91±6.03) (P<0.0411). High density lipoprotein presented a mean value statistically higher after therapy (P<0.0027). In systemically healthy subjects with periodontitis, periodontal therapy was associated with decreased levels of circulating high-sensitivity C-reactive protein and increase of high density lipoprotein in serum. The clinical trial was registered at http://www.clinicaltrials.gov.br/, No. RBR-24T799.

Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) weremore » dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. - Highlights: • Ovarian gap junction proteins are affected by ovarian aging and obesity. • DMBA exposure negatively impacts gap junction proteins. • Altered gap junction proteins may contribute to infertility.« less

Prepuberal intranasal dopamine treatment in an animal model of ADHD ameliorates deficient spatial attention, working memory, amino acid transmitters and synaptic markers in prefrontal cortex, ventral and dorsal striatum.

PubMed

Ruocco, L A; Treno, C; Gironi Carnevale, U A; Arra, C; Mattern, C; Huston, J P; de Souza Silva, M A; Nikolaus, S; Scorziello, A; Nieddu, M; Boatto, G; Illiano, P; Pagano, C; Tino, A; Sadile, A G

2014-09-01

Intranasal application of dopamine (IN-DA) has been shown to increase motor activity and to release DA in the ventral (VS) and dorsal striatum (DS) of rats. The aim of the present study was to assess the effects of IN-DA treatment on parameters of DA and excitatory amino acid (EAA) function in prepuberal rats of the Naples high-excitability (NHE) line, an animal model for attention-deficit hyperactivity disorder (ADHD) and normal random bred (NRB) controls. NHE and NRB rats were daily administered IN-DA (0.075, 0.15, 0.30 mg/kg) or vehicle for 15 days from postnatal days 28-42 and subsequently tested in the Làt maze and in the Eight-arm radial Olton maze. Soluble and membrane-trapped L-glutamate (L-Glu) and L-aspartate (L-Asp) levels as well as NMDAR1 subunit protein levels were determined after sacrifice in IN-DA- and vehicle-treated NHE and NRB rats in prefrontal cortex (PFc), DS and VS. Moreover, DA transporter (DAT) protein and tyrosine hydroxylase (TH) levels were assessed in PFc, DS, VS and mesencephalon (MES) and in ventral tegmental area (VTA) and substantia nigra, respectively. In NHE rats, IN-DA (0.30 mg/kg) decreased horizontal activity and increased nonselective attention relative to vehicle, whereas the lower dose (0.15 mg/kg) increased selective spatial attention. In NHE rats, basal levels of soluble EAAs were reduced in PFc and DS relative to NRB controls, while membrane-trapped EAAs were elevated in VS. Moreover, basal NMDAR1 subunit protein levels were increased in PFc, DS and VS relative to NRB controls. In addition, DAT protein levels were elevated in PFc and VS relative to NRB controls. IN-DA led to a number of changes of EAA, NMDAR1 subunit protein, TH and DAT protein levels in PFc, DS, VS, MES and VTA, in both NHE and NRB rats with significant differences between lines. Our findings indicate that the NHE rat model of ADHD may be characterized by (1) prefrontal and striatal DAT hyperfunction, indicative of DA hyperactivty, and (2) prefrontal and striatal NMDA receptor hyperfunction indicative of net EAA hyperactivty. IN-DA had ameliorative effects on activity level, attention, and working memory, which are likely to be associated with DA action at inhibitory D2 autoreceptors, leading to a reduction in striatal DA hyperactivity and, possibly, DA action on striatal EAA levels, resulting in a decrease of striatal EAA hyperfunction (with persistence of prefrontal EAA hyperfunction). Previous studies on IN-DA treatment in rodents have indicated antidepressant, anxiolytic and anti-parkinsonian effects in relation to enhanced central DAergic activity. Our present results strengthen the prospects of potential therapeutic applications of intranasal  DA by indicating an enhancement of selective attention and working memory in a deficit model.

The Role of ABC Proteins in Drug-Resistant Breast Cancer Cells

DTIC Science & Technology

2007-04-01

and a biotin acceptor domain) under control of the alcohol oxidase promoter (Figure 2). Upon methanol induction, the yeast expressed high levels of...as native cDNA. Therefore, we backtranslated the protein into a nucleotide sequence codon-optimized for expression in Pichia pastoris yeast. Yeast

Decreased serum protein binding of diazepam and its major metabolite in the neonate during the first postnatal week relate to increased free fatty acid levels.

PubMed Central

Nau, H; Luck, W; Kuhnz, W

1984-01-01

The protein binding of diazepam (D) and its major active metabolite N-desmethyl diazepam (DD) was investigated in vitro in the serum of 14 mothers at birth, 21 foetuses at birth, in 100 neonates between 1 and 11 days of age and in 16 control subjects. The free (unbound) fractions of D and DD in the foetus were similar to those in the controls, but lower than those in the mothers. During the first day of life the free fractions of D and DD doubled in the neonates and subsequently declined slowly to reach near control levels at 1 week of age. The sharp increase and slow decrease of the free fractions of D and DD during the first postnatal week was closely paralleled by sharply increasing and decreasing free fatty acid (FFA) concentrations. Bilirubin and albumin levels were of lower importance in regard to the protein binding of D and DD. These results indicate that the greatly increased FFA levels shortly after birth result in increased free fractions of D and DD. Because of the known immaturity of the neonatal hepatic elimination capacity, these elevated free fractions may result in elevated free concentrations of the two compounds, which may help to explain the adverse effects observed clinically in some D-exposed neonates. PMID:6419763

Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

PubMed

Liu, Mao-Sen; Ko, Miau-Hwa; Li, Hui-Chun; Tsai, Shwu-Jene; Lai, Ying-Mi; Chang, You-Ming; Wu, Min-Tze; Chen, Long-Fang O

2014-08-28

Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, "Green King". Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined.

Compositional and Proteomic Analyses of Genetically Modified Broccoli (Brassica oleracea var. italica) Harboring an Agrobacterial Gene

PubMed Central

Liu, Mao-Sen; Ko, Miau-Hwa; Li, Hui-Chun; Tsai, Shwu-Jene; Lai, Ying-Mi; Chang, You-Ming; Wu, Min-Tze; Chen, Long-Fang O.

2014-01-01

Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, “Green King”. Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined. PMID:25170807

[Effects of low-protein diet plus alpha-keto acid on micro-inflammation and the relationship between micro-inflammation and nutritional status in patients performing continuous ambulatory peritoneal dialysis: a randomized controlled trial].

PubMed

Chen, Wei; Guo, Zhi-Yong; Wu, Hao; Sun, Li-Jing; Cai, Li-Li; Xu, Hai-Yan

2008-05-01

To investigate the effects of the combination of alpha-keto acid and low-protein diet on the levels of serum cytokines in patients performing continuous ambulatory peritoneal dialysis (CAPD) and to explore the relationship between inflammation and malnutrition in CAPD patients. Eighty-nine CAPD patients were randomized into three groups, and 78 cases completed a one-year follow-up and with complete data. There were 31 cases in low-protein diet plus alpha-keto acid group, 26 cases in low-protein diet group and 21 cases in routine-protein diet group. The levels of serum albumin (Alb), prealbumin (PA), retinol-binding protein (RBP), transferrin (TRF), cholesterol (TC), triglycerides (TG), leptin, and triceps skinfold thickness (TSF), mid-arm muscle circumference (MAMC), body mass index (BMI) were measured. The changes of serum interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP) were also detected. Compared with low-protein diet group, serum levels of PA, RBP and TRF were significantly increased both in low-protein diet plus alpha-keto acid and routine-protein diet groups ( P<0.01), however, there was no significant difference in the levels of PA, RBP and TRF between low-protein diet plus alpha-keto acid group and routine-protein diet group. There was an increased tendency in the content of Alb, TC, TG, BMI, TSF and MAMC, but there were no significant differences. The plasma levels of IL-1alpha, IL-6 and TNF-alpha in low-protein diet plus alpha-keto acid group were decreased as compared with the routine-protein diet group, but there were no significant differences. The plasma level of CRP in low-protein diet plus alpha-keto acid group was lower than that in the routine-protein diet group ( P<0.01). The combination of alpha-keto acid and low-protein diet can ameliorate malnutrition and micro-inflammation in CAPD patients.

Protein tyrosine phosphatase 1B as a target for the treatment of impaired glucose tolerance and type II diabetes.

PubMed

Liu, Gang; Trevillyan, James M

2002-11-01

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin signal transduction cascade, initiated when insulin binds to the insulin receptor. PTP1B-deficient mice are more sensitive to insulin, and have improved glycemic control and resistance to diet-induced obesity than wild-type control mice. Diabetic mice treated with PTP1B antisense oligonucleotides intraperitoneally have lower PTP1B protein levels in liver and fat, reduced plasma insulin, blood glucose and hemoglobin A1c (HbA1c) levels. These studies validate PTP1B as a promising drug discovery target for the treatment of insulin resistance, diabetes and obesity. Herein we review the recent advances in the structure-based design of potent and selective small molecule inhibitors of PTP1B, and discuss th e challenge of developing compounds with improved cell permeability and bioavailability.

Oxidant-antioxidant status and pulmonary function in welding workers.

PubMed

Fidan, Fatma; Unlü, Mehmet; Köken, Tülay; Tetik, Levent; Akgün, Sema; Demirel, Reha; Serteser, Mustafa

2005-07-01

Welding is a process during which fumes, gases, electromagnetic radiation and noise are emitted as by-products. Metal oxide particles are particularly hazardous components of welding fumes. Welding has been found to be associated with respiratory symptoms and our objective in the present study was to study the effects of welding on pulmonary function and serum oxidant-antioxidant status. Fifty-one welding workers and 31 control subjects were recruited. Face to face interviews were conducted using the respiratory illness questionnaire adapted from the American Thoracic Society with the addition of demographic characteristics, work history and working conditions. Additionally physical examinations and spirometric measurements were performed at workplaces. Thiobarbituric acid reactive substances (TBARS), protein carbonyls, protein sulfhydryls (SH) and erythrocyte reduced glutathione (GSH) levels were measured to evaluate oxidant-antioxidant status in 34 welding workers and in 20 control subjects. No statistically significant differences were observed in age, height, weight, body mass index (BMI), smoking status and annual working durations between welding workers and controls. Coughing, sputting and wheezing were significantly higher in welding workers (p<0.05). When adjusted for age, BMI and smoking status in logistic regression, welding work showed a significant risk for chronic bronchitis (OR: 4.78, 95%CI: 1.30-17.54). Forced expiratory volume in one second (FEV(1))/forced vital capacity (FVC) and four parameters of forced expiratory flow (FEF: FEF(25), FEF(50), FEF(75), FEF(25-75)) levels measured in the welding workers were significantly lower than those in the control group (p<0.05). Serum TBARS and protein carbonyl levels were higher in welding workers than those in controls (p<0.001, p<0.05, respectively). On the other hand, total protein SH groups and GSH levels were significantly lower in welders than those in controls (p<0.05, p<0.001, respectively). Pulmonary function tests and oxidant-antioxidant status were found to be negatively affected in welding workers chronically exposed to welding fumes and gases. Preventive measures should be taken to improve the health status of these workers.

Tau proteins in the cerebrospinal fluid of patients with subacute sclerosing panencephalitis.

PubMed

Yuksel, Deniz; Yilmaz, Deniz; Uyar, Neval Y; Senbil, Nesrin; Gurer, Yavuz; Anlar, Banu

2010-06-01

Neurodegenerative diseases characterized by cytoskeletal deformation and neurofibrillary tangles are associated with altered levels of tau and related proteins in cerebrospinal fluid (CSF). Neuronal or glial fibrillary tangles have been shown in 20% of subacute sclerosing panencephalitis (SSPE) patients. We therefore investigated CSF samples from 60 newly diagnosed SSPE and 31 neurological control patients for total tau (t-tau), phosphorylated tau (p-tau), and S100-B levels by ELISA. There was no difference between patient and control groups in t-tau and S100-B levels. p-Tau was lower in the SSPE group (p=0.009). Past history of measles infection, measles immunization status, latent period between measles and onset of SSPE, duration of symptoms, frequency of myoclonia, neurological deficit index, stage and progression rate of the disease, CSF glucose levels and cell counts, CSF and serum measles IgG titer, distribution of lesions on brain magnetic resonance imaging were not related to t-tau, p-tau and S100-B levels. Mental status and age were negatively correlated with t-tau, and male gender and EEG abnormalities were associated with higher t-tau levels. The levels of tau proteins in our patients suggest there is no, or only scarce and immature, neurofibrillary tangle formation in SSPE. Autopsy studies showing neurofibrillary tangles might have examined older patients with longer disease and more parenchymal involvement. Copyright (c) 2009 Elsevier B.V. All rights reserved.

MCPIP-1, alias Regnase-1 controls epithelial inflammation by post-transcriptional regulation of IL-8 production

PubMed Central

Dobosz, E.; Wilamowski, M.; Lech, M.; Bugara, B.; Jura, J.; Potempa, J.; Koziel, J.

2016-01-01

Pattern recognition receptors are critical for the detection of invading microorganisms. They activate multiple pathways that lead to the induction of pro-inflammatory responses and pathogen clearance. The intensity and duration of this immune reaction must be tightly controlled spatially and temporally in every tissue by different negative regulators. We hypothesized that monocyte chemoattractant protein-1–induced protein-1 (MCPIP-1) might play a role in maintaining immune homeostasis in the epithelium both under physiological conditions and upon bacterial infection. To this end, we examined the distribution of MCPIP-1 transcript and protein in various tissues. The MCPIP-1 protein level was higher in epithelial cells than in myeloid cells. MCPIP-1 exerted RNase activity towards the IL-8 transcript and the life-span of IL-8 was determined by the presence of the stem-loops/hairpin (SL) structures at the 3′ UTR region of IL-8 mRNA. Moreover, using fully active, purified recombinant MCPIP-1 protein, we elucidated the mechanism by which MCPIP-1 controls the IL-8 mRNA level. In conclusion, we uncovered a novel IL-8–dependent mechanism via which MCPIP-1 maintains epithelial homeostasis. This study reveals for the first time that MCPIP-1 plays a crucial anti-inflammatory role not only in myeloid cells but also in epithelial cells. PMID:27513529

MCPIP-1, Alias Regnase-1, Controls Epithelial Inflammation by Posttranscriptional Regulation of IL-8 Production.

PubMed

Dobosz, Ewelina; Wilamowski, Mateusz; Lech, Maciej; Bugara, Beata; Jura, Jolanta; Potempa, Jan; Koziel, Joanna

2016-01-01

Pattern recognition receptors are critical for the detection of invading microorganisms. They activate multiple pathways that lead to the induction of proinflammatory responses and pathogen clearance. The intensity and duration of this immune reaction must be tightly controlled spatially and temporally in every tissue by different negative regulators. We hypothesized that monocyte chemoattractant protein-1-induced protein-1 (MCPIP-1) might play a role in maintaining immune homeostasis in the epithelium both under physiological conditions and upon bacterial infection. To this end, we examined the distribution of the MCPIP-1 transcript and protein in various tissues. The MCPIP-1 protein level was higher in epithelial cells than in myeloid cells. MCPIP-1 exerted RNase activity towards the interleukin (IL)-8 transcript and the lifespan of IL-8 was determined by the presence of the stem-loops/hairpin structures at the 3'UTR region of IL-8 mRNA. Moreover, using fully active, purified recombinant MCPIP-1 protein, we elucidated the mechanism by which MCPIP-1 controls the IL-8 mRNA level. In conclusion, we uncovered a novel IL-8-dependent mechanism via which MCPIP-1 maintains epithelial homeostasis. This study reveals for the first time that MCPIP-1 plays a crucial anti-inflammatory role not only in myeloid cells but also in epithelial cells. © 2016 S. Karger AG, Basel.

Dietary Japanese millet protein ameliorates plasma levels of adiponectin, glucose, and lipids in type 2 diabetic mice.

PubMed

Nishizawa, Naoyuki; Togawa, Tubasa; Park, Kyung-Ok; Sato, Daiki; Miyakoshi, Yo; Inagaki, Kazuya; Ohmori, Norimasa; Ito, Yoshiaki; Nagasawa, Takashi

2009-02-01

Millet is an important food crop in Asia and Africa, but the health benefits of dietary millet are little known. This study defined the effects of dietary Japanese millet on diabetic mice. Feeding of a high-fat diet containing Japanese millet protein concentrate (JMP, 20% protein) to type 2 diabetic mice for 3 weeks significantly increased plasma levels of adiponectin and high-density lipoprotein cholesterol (HDL cholesterol) and decreased the levels of glucose and triglyceride as compared to control. The starch fraction of Japanese millet had no effect on glucose or adiponectin levels, but the prolamin fraction beneficially modulated plasma glucose and insulin concentrations as well as adiponectin and tumor necrosis factor-alpha gene expression. Considering the physiological significance of adiponectin and HDL cholesterol levels in type 2 diabetes, insulin resistance, and cardiovascular disease, our findings imply that dietary JMP has the potential to ameliorate these diseases.

A global view of Escherichia coli Rsd protein and its interactions.

PubMed

Piper, Sarah E; Mitchell, Jennie E; Lee, David J; Busby, Stephen J W

2009-12-01

The Escherichia coli Rsd protein forms 1 : 1 complexes with sigma(70) protein, which is the major factor in determining promoter recognition by RNA polymerase. Here we describe measurements of the levels of Rsd, RNA polymerase, sigma(70) and the alternative sigma(38) factor. Rsd levels are sufficient to sequester a significant proportion of sigma(70) and immunoaffinity pull-down experiments show that this occurs in stationary phase but not in exponentially growing cells. Rsd expression is controlled by two promoters, P1 and P2. Experiments with lac fusions show that the P2 promoter is stronger than P1, that P2 is active in all phases of growth, and that this accounts for the high levels of Rsd.

Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation

NASA Technical Reports Server (NTRS)

Harter, L. V.; Hruska, K. A.; Duncan, R. L.

1995-01-01

Exposure of osteosarcoma cell lines to chronic intermittent strain increases the activity of mechano-sensitive cation (SA-cat) channels. The impact of mechano-transduction on osteoblast function has not been well studied. We analyzed the expression and production of bone matrix proteins in human osteoblast-like osteosarcoma cells, OHS-4, in response to chronic intermittent mechanical strain. The OHS-4 cells exhibit type I collagen production, 1,25-Dihydroxyvitamin D-inducible osteocalcin, and mineralization of the extracellular matrix. The matrix protein message level was determined from total RNA isolated from cells exposed to 1-4 days of chronic intermittent strain. Northern analysis for type I collagen indicated that strain increased collagen message after 48 h. Immunofluorescent labeling of type I collagen demonstrated that secretion was also enhanced with mechanical strain. Osteopontin message levels were increased several-fold by the application of mechanical load in the absence of vitamin D, and the two stimuli together produced an additive effect. Osteocalcin secretion was also increased with cyclic strain. Osteocalcin levels were not detectable in vitamin D-untreated control cells. However, after 4 days of induced load, significant levels of osteocalcin were observed in the medium. With vitamin D present, osteocalcin levels were 4 times higher in the medium of strained cells compared to nonstrained controls. We conclude that mechanical strain of osteoblast-like cells is sufficient to increase the transcription and secretion of matrix proteins via mechano-transduction without hormonal induction.

Post-mortem biochemistry of NSE and S100B: A supplemental tool for detecting a lethal traumatic brain injury?

PubMed

Sieber, Monique; Dreßler, Jan; Franke, Heike; Pohlers, Dirk; Ondruschka, Benjamin

2018-04-01

Traumatic brain injury (TBI) is a very common entity that leads to numerous fatalities all over the world. Therefore, forensic pathologists are in desperate need of supplemental methodological tools for the diagnosis of TBI in everyday practice besides the standard autopsy. The present study determined post-mortem neuron specific enolase (NSE) and S100 calcium-binding protein B (S100B) levels as biological markers of an underlying TBI in autopsy cases. Paired serum and CSF samples of 92 fatalities were collected throughout routine autopsies. Afterwards, the marker levels were assessed using commercially available immunoassays (ECLIA, Roche Diagnostics). For statistical analysis, we compared the TBI cases to three control groups (sudden natural death by acute myocardial infarction, traumatic death without impact on the head, cerebral hypoxia). Moreover, the TBI cases were subdivided according to their survival time of the trauma. Brain specimens have been collected and stained immunohistochemically against the aforementioned proteins to illustrate their typical cellular staining patterns with an underlying TBI compared to non-TBI fatalities. CSF NSE and S100B levels were elevated after TBI compared to all control groups (p < 0.001). Although this finding can already be investigated among the TBI cases dying immediately subsequent to the trauma, the marker levels in CSF increase with longer survival times until a peak level within the first three days after trauma. There is a strong correlation between both marker levels in CSF (r = 0.67). The presence or absence of cerebral tissue contusion following the initial trauma does not seem to affect the CSF levels of both proteins (p > 0.05). Post-mortem serum levels of both proteins were not elevated in TBI cases compared to controls (p > 0.05). Former elaborated cut-off values in CSF were confirmed and were only exceeded when a TBI survival time of at least 30 min was reached. The present results report that post-mortem NSE and S100B CSF levels are significantly elevated subsequent to a fatal TBI. Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

High-level expression of a synthetic gene encoding a sweet protein, monellin, in Escherichia coli.

PubMed

Chen, Zhongjun; Cai, Heng; Lu, Fuping; Du, Lianxiang

2005-11-01

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.

Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

NASA Technical Reports Server (NTRS)

Evans, G. L.; Morey-Holton, E.; Turner, R. T.

1998-01-01

In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

Association of vitamin D and vitamin D binding protein (DBP) gene polymorphism with susceptibility of type 2 diabetes mellitus in Bangladesh.

PubMed

Rahman, Md Mostafijur; Hosen, Md Bayejid; Faruk, Md Omar; Hasan, Md Mehedi; Kabir, Yearul; Howlader, M Zakir Hossain

2017-12-15

Polymorphism in vitamin D binding protein gene may have an impact on serum vitamin D transport and thus may have relation with type 2 diabetes mellitus. In our study, we investigated the association of serum vitamin D level and vitamin D-binding protein gene polymorphism with the onset of type 2 diabetes mellitus. Blood samples were collected from 104 type 2 diabetic patients and 107 healthy volunteers. Serum vitamin D was measured by high-performance liquid chromatography. Genetic analysis of vitamin D-binging protein gene was carried out by polymerase chain reaction - restriction fragment length polymorphism method. We found significantly (p<0.001) lower level of vitamin D in type 2 diabetic patients compared to control subjects. A significantly negative correlation (r=-0.25, p<0.05) between vitamin D level and fasting blood glucose level was found among type 2 diabetic subjects. The Glu/Glu at codon 416 (rs7041) (p<0.05) and Lys/Lys at codon 420 (rs4588) (p<0.01) variants of vitamin D binding protein gene was significantly higher in type 2 diabetic subjects than controls. The patients with Glu/Glu and Lys/Lys genotypes respectively at codon 416 (odds ratio=2.87; 95% confidence interval=1.19 to 6.95) and 420 (odds ratio=8.9; 95% confidence interval=1.89 to 41.99) were at high risk of developing type 2 diabetes. Our present study strongly suggests that there might have an association of vitamin D, and vitamin D-binding protein gene (codon 416 & 420) polymorphisms with the occurrence of type 2 diabetes mellitus. Copyright © 2017 Elsevier B.V. All rights reserved.

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

PubMed

Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

2012-07-01

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

A winged helix forkhead (FOXD2) tunes sensitivity to cAMP in T lymphocytes through regulation of cAMP-dependent protein kinase RIalpha.

PubMed

Johansson, C Christian; Dahle, Maria K; Blomqvist, Sandra Rodrigo; Grønning, Line M; Aandahl, Einar M; Enerbäck, Sven; Taskén, Kjetil

2003-05-09

Forkhead/winged helix (FOX) transcription factors are essential for control of the cell cycle and metabolism. Here, we show that spleens from Mf2-/- (FOXD2-/-) mice have reduced mRNA (50%) and protein (35%) levels of the RIalpha subunit of the cAMP-dependent protein kinase. In T cells from Mf2-/- mice, reduced levels of RIalpha translates functionally into approximately 2-fold less sensitivity to cAMP-mediated inhibition of proliferation triggered through the T cell receptor-CD3 complex. In Jurkat T cells, FOXD2 overexpression increased the endogenous levels of RIalpha through induction of the RIalpha1b promoter. FOXD2 overexpression also increased the sensitivity of the promoter to cAMP. Finally, co-expression experiments demonstrated that protein kinase Balpha/Akt1 work together with FOXD2 to induce the RIalpha1b promoter (10-fold) and increase endogenous RIalpha protein levels further. Taken together, our data indicate that FOXD2 is a physiological regulator of the RIalpha1b promoter in vivo working synergistically with protein kinase B to induce cAMP-dependent protein kinase RIalpha expression, which increases cAMP sensitivity and sets the threshold for cAMP-mediated negative modulation of T cell activation.

Genomic atlas of the human plasma proteome.

PubMed

Sun, Benjamin B; Maranville, Joseph C; Peters, James E; Stacey, David; Staley, James R; Blackshaw, James; Burgess, Stephen; Jiang, Tao; Paige, Ellie; Surendran, Praveen; Oliver-Williams, Clare; Kamat, Mihir A; Prins, Bram P; Wilcox, Sheri K; Zimmerman, Erik S; Chi, An; Bansal, Narinder; Spain, Sarah L; Wood, Angela M; Morrell, Nicholas W; Bradley, John R; Janjic, Nebojsa; Roberts, David J; Ouwehand, Willem H; Todd, John A; Soranzo, Nicole; Suhre, Karsten; Paul, Dirk S; Fox, Caroline S; Plenge, Robert M; Danesh, John; Runz, Heiko; Butterworth, Adam S

2018-06-01

Although plasma proteins have important roles in biological processes and are the direct targets of many drugs, the genetic factors that control inter-individual variation in plasma protein levels are not well understood. Here we characterize the genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study. We identify 1,927 genetic associations with 1,478 proteins, a fourfold increase on existing knowledge, including trans associations for 1,104 proteins. To understand the consequences of perturbations in plasma protein levels, we apply an integrated approach that links genetic variation with biological pathway, disease, and drug databases. We show that protein quantitative trait loci overlap with gene expression quantitative trait loci, as well as with disease-associated loci, and find evidence that protein biomarkers have causal roles in disease using Mendelian randomization analysis. By linking genetic factors to diseases via specific proteins, our analyses highlight potential therapeutic targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.

Replacement of fish meal protein by surimi by-product protein in the diet of blue gourami Trichogaster trichopterus fingerlings.

PubMed

Mohanta, K N; Subramanian, S; Korikanthimath, V S

2013-02-01

Based on the nutrient requirement of Trichogaster trichopterus, a fish meal-based basal diet with 350 g/kg diet crude protein and 16.7 MJ/kg energy was formulated, in which the fish meal protein was replaced by surimi by-product protein at 0.0 (control), 12.5, 25, 50, 75 and 100% levels. The formulated diets were fed ad libitum to T. trichopterus fingerlings (4.80 ± 0.03 g) in triplicate groups for 45 days in a closed water system. Eighteen fibre-reinforced plastic tanks with 200 l of water were used for rearing the fish. Weight gain, specific growth rate, feed/gain ratio, protein efficiency ratio, nutrient retention and digestibility (protein and energy) of fish were not affected (p > 0.05) up to 50% fish meal protein replacement level by surimi by-product protein. While whole-body protein content of fish was marginally decreased, the lipid content was increased with increase in surumi by-product incorporation level in the diet. The study results suggest that the fish meal protein, which is scarce and costly nowadays, could be replaced up to 50% by surimi by-product protein in the diet of blue gourami without hampering the growth and nutrient utilization of fish. © 2011 Blackwell Verlag GmbH.

Biochemical and nutritional evaluation of patients with visceral leishmaniasis before and after treatment with leishmanicidal drugs.

PubMed

Gatto, Mariana; de Abreu, Mariana Miziara; Tasca, Karen Ingrid; Simão, José Cláudio; Fortaleza, Carlos Magno Castelo Branco; Pereira, Paulo Câmara Marques; Calvi, Sueli Aparecida

2013-01-01

Visceral leishmaniasis (VL) is caused by the intracellular protozoan Leishmania donovani complex. VL may be asymptomatic or progressive and is characterized by fever, anemia, weight loss and the enlargement of the spleen and liver. The nutritional status of the patients with VL is a major determinant of the progression, severity and mortality of the disease, as it affects the clinical progression of the disease. Changes in lipoproteins and plasma proteins may have major impacts in the host during infection. Thus, our goal was evaluate the serum total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, glucose, albumin, globulin and total protein levels, as well as the body composition, of VL patients before and after treatment. Nutritional evaluation was performed using the bioelectrical impedance analysis (BIA) to assess body composition. Biochemical data on the serum total cholesterol, HDL, LDL, triglycerides, glucose, albumin, globulin and total protein were collected from the medical charts of the patients. BIA indicated that both pre-treatment and post-treatment patients exhibited decreased phase angles compared to the controls, which is indicative of disease. Prior to treatment, the patients exhibited lower levels of total body water compared to the controls. Regarding the biochemical evaluation, patients with active VL exhibited lower levels of total cholesterol, HDL, LDL and albumin and higher triglyceride levels compared to patients after treatment and the controls. Treatment increased the levels of albumin and lipoproteins and decreased the triglyceride levels. Our results suggest that patients with active VL present biochemical and nutritional changes that are reversed by treatment.

Homochiral stereochemistry: the missing link of structure to energetics in protein folding.

PubMed

Kumar, Anil; Ramakrishnan, Vibin; Ranbhor, Ranjit; Patel, Kirti; Durani, Susheel

2009-12-24

The notion is tested that homochiral stereochemistry being ubiquitous to protein structure could be critical to protein folding as well, causing it to become frustrated energetically providing the basis for its solvent- and sequence-mediated control. The proof in support of the notion is found in a consensus of experiment and computation according to which suitable oligopeptides are in their folding-unfolding equilibria, at both macrostate and microstate levels, susceptible to dielectric because of the conflict of peptide-chain electrostatics with interpeptide hydrogen bonds when the structure is poly-L but not when it is alternating-L,D. The argument is thus made that homochiral stereochemistry may in protein folding provide the unifying basis for its solvent- and sequence-mediated control based on screening of peptide-chain electrostatics under conflict with folding of the chain due to homochiral stereochemistry. Dielectric is brought into spotlight as the effect comparatively obscure but presumably critical to the folding in protein structure for its control.

Wrecked regulation of intrinsically disordered proteins in diseases: pathogenicity of deregulated regulators

PubMed Central

Uversky, Vladimir N.

2014-01-01

Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs) are typically related to regulation, signaling, and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases. PMID:25988147

Effect of Replacing Animal Protein with Plant Protein on Glycemic Control in Diabetes: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

PubMed Central

Viguiliouk, Effie; Stewart, Sarah E.; Jayalath, Viranda H.; Ng, Alena Praneet; Mirrahimi, Arash; de Souza, Russell J.; Hanley, Anthony J.; Bazinet, Richard P.; Blanco Mejia, Sonia; Leiter, Lawrence A.; Josse, Robert G.; Kendall, Cyril W.C.; Jenkins, David J.A.; Sievenpiper, John L.

2015-01-01

Previous research on the effect of replacing sources of animal protein with plant protein on glycemic control has been inconsistent. We therefore conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) to assess the effect of this replacement on glycemic control in individuals with diabetes. We searched MEDLINE, EMBASE, and Cochrane databases through 26 August 2015. We included RCTs ≥ 3-weeks comparing the effect of replacing animal with plant protein on HbA1c, fasting glucose (FG), and fasting insulin (FI). Two independent reviewers extracted relevant data, assessed study quality and risk of bias. Data were pooled by the generic inverse variance method and expressed as mean differences (MD) with 95% confidence intervals (CIs). Heterogeneity was assessed (Cochran Q-statistic) and quantified (I2-statistic). Thirteen RCTs (n = 280) met the eligibility criteria. Diets emphasizing a replacement of animal with plant protein at a median level of ~35% of total protein per day significantly lowered HbA1c (MD = −0.15%; 95%-CI: −0.26, −0.05%), FG (MD = −0.53 mmol/L; 95%-CI: −0.92, −0.13 mmol/L) and FI (MD = −10.09 pmol/L; 95%-CI: −17.31, −2.86 pmol/L) compared with control arms. Overall, the results indicate that replacing sources of animal with plant protein leads to modest improvements in glycemic control in individuals with diabetes. Owing to uncertainties in our analyses there is a need for larger, longer, higher quality trials. Trial Registration: ClinicalTrials.gov registration number: NCT02037321. PMID:26633472

Light-induced protein degradation in human-derived cells.

PubMed

Sun, Wansheng; Zhang, Wenyao; Zhang, Chao; Mao, Miaowei; Zhao, Yuzheng; Chen, Xianjun; Yang, Yi

2017-05-27

Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

An innovative brioche enriched in protein and energy improves the nutritional status of malnourished nursing home residents compared to oral nutritional supplement and usual breakfast: FARINE+ project.

PubMed

Van Wymelbeke, Virginie; Brondel, Laurent; Bon, Francis; Martin-Pfitzenmeyer, Isabelle; Manckoundia, Patrick

2016-10-01

To compare the effects of a 12-week nutritional intervention, in which an innovative protein-and-energy-enriched brioche, an oral nutritional supplement or a usual breakfast were eaten, on food intake and nutritional status in nursing home residents. Three-armed, multicentre, controlled trial. Eight nursing homes in Burgundy, France. Sixty-eight malnourished participants aged between 70 and 99 years old. Participants were randomly assigned to one of three groups according to the breakfast provided: brioche group, one portion of 65 g brioche enriched in protein and energy (12.8 g and 180 kcal) added to usual breakfast; supplement group, 200-ml of a ready-to-use, energy-dense liquid (14 g protein and 200 kcal) added to usual breakfast or control group, a usual breakfast only. Total energy intakes were assessed for three days at different periods of the study (day 0, day 30 and day 90); blood parameters, nutritional status (mini nutritional assessment, weight) and functional capacities (grip strength and activity level) were measured at the beginning and at the end of the nutritional intervention study (day 0 and day 90). The participants of the brioche group had higher total energy intakes at day 30 (p value 0.004) and at day 90 (p value 0.018) compared with the supplement group and the control group. At the end of the interventional study, 72% of the participants in the brioche group had reached the recommended minimum level of protein of 0.8 g/kg/day, compared with 53% in the supplement group and 36% in the control group (p value 0.036). In addition, between day 0 and day 90 in the brioche group, blood levels of vitamins B 9 , B 2 , D (all p value <0.001), B 6 (p value 0.026) and B 12 (p value 0.036) had increased and plasma homocysteine had decreased (p value 0.024). The protein-and-energy-enriched brioche effectively increased energy and protein intakes and improved the nutritional status of elderly people living in nursing homes. It could be a good alternative to oral liquid nutritional supplements to counteract protein-energy-malnutrition. Copyright © 2016 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.

Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

NASA Astrophysics Data System (ADS)

El-Niely, Hania F. G.

2007-06-01

The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas ( Pisum satinum L), cowpeas ( Vigna unguiculata L.Walp), lentils ( Lens culinaris Med), kidneybeans ( Phaseolus vulgaris L), and chickpeas ( Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly ( p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes.

Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

PubMed

Zhang, Li; Tran, Ngoc-Tung; Su, Hairui; Wang, Rui; Lu, Yuheng; Tang, Haiping; Aoyagi, Sayura; Guo, Ailan; Khodadadi-Jamayran, Alireza; Zhou, Dewang; Qian, Kun; Hricik, Todd; Côté, Jocelyn; Han, Xiaosi; Zhou, Wenping; Laha, Suparna; Abdel-Wahab, Omar; Levine, Ross L; Raffel, Glen; Liu, Yanyan; Chen, Dongquan; Li, Haitao; Townes, Tim; Wang, Hengbin; Deng, Haiteng; Zheng, Y George; Leslie, Christina; Luo, Minkui; Zhao, Xinyang

2015-11-17

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.

Immunoglobulins and dietary protein antibodies in childhood coeliac disease 1

PubMed Central

Kenrick, K. G.; Walker-Smith, J. A.

1970-01-01

Twenty-four children with coeliac disease were compared with a control group, comprising 17 children with a variety of gastroenterological disorders, with respect to serum immunoglobulins and dietary protein antibodies. Elevated levels of IgA and abnormally low levels of IgM were demonstrated in one third of the coeliac patients. Antibodies to at least one of eight dietary proteins were found in 50% of coeliac children. Three children with raised levels of serum IgA and two with deficient IgM were re-examined after varying periods on a gluten-free diet. Antibodies to dietary proteins had waned and immunoglobulin levels returned to normal in all cases. The raised IgA was considered to have resulted from an extensive immunological response to antigens of dietary origin which had entered through the abnormal gut mucosa. It is suggested that IgM deficiency was due to specific inhibition of IgM synthesis by dietary components which had also entered through the mucosa. PMID:4097173