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Sample records for early protein expression

  1. Matrix Gla Protein expression pattern in the early avian embryo.

    PubMed

    Correia, Elizabeth; Conceição, Natércia; Cancela, M Leonor; Belo, José A

    2016-01-01

    MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

  2. Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos.

    PubMed

    Bang, Jae-Il; Lee, Hyo-Sang; Deb, Gautam Kumar; Ha, A-Na; Kwon, Young-Sang; Cho, Seong-Keun; Kim, Byeong-Woo; Cho, Kyu-Woan; Kong, Il-Keun

    2013-01-15

    It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term.

  3. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  4. Expression and imaging of fluorescent proteins in the C. elegans gonad and early embryo.

    PubMed

    Green, Rebecca A; Audhya, Anjon; Pozniakovsky, Andrei; Dammermann, Alexander; Pemble, Hayley; Monen, Joost; Portier, Nathan; Hyman, Anthony; Desai, Arshad; Oegema, Karen

    2008-01-01

    The Caenorhabditis elegans gonad and early embryo have recently emerged as an attractive metazoan model system for studying cell and developmental biology. The success of this system is attributable to the stereotypical architecture and reproducible cell divisions of the gonad/early embryo, coupled with penetrant RNAi-mediated protein depletion. These features have facilitated the development of visual assays with high spatiotemporal resolution to monitor specific subcellular processes. Assay development has relied heavily on the emergence of methods to circumvent germline silencing to allow the expression of transgenes encoding fluorescent fusion proteins. In this chapter, we discuss methods for the expression and imaging of fluorescent proteins in the C. elegans germline, including the design of transgenes for optimal expression, the generation of transgenic worm lines by ballistic bombardment, the construction of multimarker lines by mating, and methods for live imaging of the gonad and early embryo.

  5. Redox Protein Expression Predicts Radiotherapeutic Response in Early-Stage Invasive Breast Cancer Patients

    SciTech Connect

    Woolston, Caroline M.; Al-Attar, Ahmad; Storr, Sarah J.; Ellis, Ian O.; Morgan, David A.L.; Martin, Stewart G.

    2011-04-01

    Purpose: Early-stage invasive breast cancer patients have commonly undergone breast-conserving surgery and radiotherapy. In a large majority of these patients, the treatment is effective; however, a proportion will develop local recurrence. Deregulated redox systems provide cancer cells protection from increased oxidative stress, such as that induced by ionizing radiation. Therefore, the expression of redox proteins was examined in tumor specimens from this defined cohort to determine whether such expression could predict response. Methods and Materials: The nuclear and cytoplasmic expression of nine redox proteins (glutathione, glutathione reductase, glutaredoxin, glutathione peroxidase 1, 3, and 4, and glutathione S-transferase-{theta}, -{pi}, and -{alpha}) was assessed using conventional immunohistochemistry on a tissue microarray of 224 tumors. Results: A high cytoplasmic expression of glutathione S-transferase-{theta} significantly correlated with a greater risk of local recurrence (p = .008) and, when combined with a low nuclear expression (p = .009), became an independent predictive factor (p = .002) for local recurrence. High cytoplasmic expression of glutathione S-transferase-{theta} also correlated with a worse overall survival (p = .009). Low nuclear and cytoplasmic expression of glutathione peroxidase 3 (p = .002) correlated with a greater risk of local recurrence and was an independent predictive factor (p = .005). These proteins did not correlate with tumor grade, suggesting their function might be specific to the regulation of oxidative stress rather than alterations of tumor phenotype. Only nuclear (p = .005) and cytoplasmic (p = .001) expression of glutathione peroxidase 4 correlated with the tumor grade. Conclusions: Our results support the use of redox protein expression, namely glutathione S-transferase-{theta} and glutathione peroxidase 3, to predict the response to radiotherapy in early-stage breast cancer patients. If incorporated into

  6. The Etl-1 gene encodes a nuclear protein differentially expressed during early mouse development.

    PubMed

    Schoor, M; Schuster-Gossler, K; Gossler, A

    1993-07-01

    Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae. Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development. ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis. Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm. In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development. After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1. During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1. Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription. In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage.

  7. Early changes in costameric and mitochondrial protein expression with unloading are muscle specific.

    PubMed

    Flück, Martin; Li, Ruowei; Valdivieso, Paola; Linnehan, Richard M; Castells, Josiane; Tesch, Per; Gustafsson, Thomas

    2014-01-01

    We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

  8. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

    ERIC Educational Resources Information Center

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  9. Expression of Mismatch Repair Proteins in Early and Advanced Gastric Cancer in Poland

    PubMed Central

    Karpińska-Kaczmarczyk, Katarzyna; Lewandowska, Magdalena; Ławniczak, Małgorzata; Białek, Andrzej; Urasińska, Elżbieta

    2016-01-01

    Background Mutations in DNA of mismatch repair (MMR) genes result in failure to repair errors that occur during DNA replication in microsatellites, resulting in accumulation of frameshift mutations in these genes and leading to DNA mismatch replication errors and microsatellite instability. Gastric cancers (GCs) with high MSI (MSI-H) are a well-defined subset of carcinomas showing distinctive clinicopathological features. In this study we investigated the rate of MSI and the correlation between MSI status and clinicopathological features of GC. Material/Methods The study included 107 patients with GCs: 61 with advanced gastric cancers (AGC) and 46 with early gastric cancer (EGC). MSI deficiency in GCs was assessed by the immunohistochemical analysis of expression of MMR proteins – MLH1, MSH2, MSH6, and PMS2 – using formalin-fixed and paraffin-embedded tissue. Results A total of 6 (5.6%) MSI-H were observed. The loss of MMR proteins expression was associated with the intestinal type of GC in Lauren classification, and tubular and papillary architecture in WHO classification. There was no statistically significant association between negative MMR expression and other selected clinical parameters: age, sex, tumor location, depth of invasion (EGC and AGC), lymph nodes status, presence of the ulceration, and lymphocytic infiltrate. Conclusions In the present era of personalized medicine, the histological type of GC and MMR proteins status in cancer cells are very important for the proper surveillance of patients with familial GC and sporadic GCs, as well as for selecting the proper follow-up and treatment. Larger collaborative studies are needed to verify the features of MSI-H GCs in Poland. PMID:27527654

  10. NUCLEAR EGFR PROTEIN EXPRESSION PREDICTS POOR SURVIVAL IN EARLY STAGE NON-SMALL CELL LUNG CANCER

    PubMed Central

    Traynor, Anne M.; Weigel, Tracey L.; Oettel, Kurt R.; Yang, David T.; Zhang, Chong; Kim, KyungMann; Salgia, Ravi; Iida, Mari; Brand, Toni M.; Hoang, Tien; Campbell, Toby C.; Hernan, Hilary R.; Wheeler, Deric L.

    2013-01-01

    Introduction Nuclear EGFR (nEGFR) has been identified in various human tumor tissues, including cancers of the breast, ovary, oropharynx, and esophagus, and has predicted poor patient outcomes. We sought to determine if protein expression of nEGFR is prognostic in early stage non-small cell lung cancer (NSCLC). Methods Resected stage I and II NSCLC specimens were evaluated for nEGFR protein expression using immunohistochemistry (IHC). Cases with at least one replicate core containing ≥5% of tumor cells demonstrating strong dot-like nucleolar EGFR expression were scored as nEGFR positive. Results Twenty-three (26.1% of the population) of 88 resected specimens stained positively for nEGFR. Nuclear EGFR protein expression was associated with higher disease stage (45.5% of stage II vs. 14.5% of stage I; p=0.023), histology (41.7% in squamous cell carcinoma vs. 17.1% in adenocarcinoma; p=0.028), shorter progression-free survival (PFS) (median PFS 8.7 months [95% CI 5.1–10.7 mo] for nEGFR positive vs. 14.5 months [95% CI 9.5–17.4 mo] for nEGFR negative; hazard ratio (HR) of 1.89 [95% CI 1.15–3.10]; p=0.011), and shorter overall survival (OS) (median OS 14.1 months [95% CI 10.3–22.7 mo] for nEGFR positive vs. 23.4 months [95% CI 20.1–29.4 mo] for nEGFR negative; HR of 1.83 [95% CI 1.12–2.99]; p=0.014). Conclusions Expression of nEGFR protein was associated with higher stage and squamous cell histology, and predicted shorter PFS and OS, in this patient cohort. Nuclear EGFR serves as a useful independent prognostic variable and as a potential therapeutic target in NSCLC. PMID:23628526

  11. Bone morphogenetic protein 1 is expressed in porcine ovarian follicles and promotes oocyte maturation and early embryonic development

    PubMed Central

    LEI, Xiaocan; CUI, Kuiqing; CAI, Xiaoyan; REN, Yanping; LIU, Qingyou; SHI, Deshun

    2016-01-01

    In the present study, we tried to determine whether bone morphogenetic protein 1 (BMP1) plays a role in ovarian follicular development and early embryo development. We systematically investigated the expression and influence of BMP1 during porcine follicle and early embryonic development. Immunohistochemistry demonstrated that the BMP1 protein is expressed in granular cells and oocytes during follicular development, from primary to pre-ovulatory follicles, including atretic follicles and the corpus luteum. The mRNA expression of BMP1 significantly increased as the porcine follicles grew. Immunofluorescence analysis indicated that BMP1 was expressed in cumulus-oocyte complexes (COCs), oocytes and porcine embryos during early in vitro culture. qPCR and western blot analysis showed that the expression of BMP1 was significantly up-regulated in mature porcine oocytes and COCs compared to immature oocytes and COCs. BMP1 is expressed in early porcine embryos, and its expression reaches a peak at the 8-cell stage. To determine the effect of BMP1 on the maturation of oocytes and the development of early embryos, various concentrations of BMP1 recombinant protein or antibody were added to the in vitro culture media, respectively. BMP1 significantly affected the porcine oocyte maturation rate, the cleavage rate and the blastocyst development rate of embryos cultured in vitro in a positive way, as well as the blastocyst cell number. In conclusion, BMP1 is expressed throughout porcine ovarian follicle development and early embryogenesis, and it promotes oocyte maturation and the developmental ability of embryos during early in vitro culture. PMID:27890905

  12. Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

    PubMed Central

    Jurata, L W; Kenny, D A; Gill, G N

    1996-01-01

    LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876198

  13. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  14. Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene.

    PubMed Central

    Hayek, T; Masucci-Magoulas, L; Jiang, X; Walsh, A; Rubin, E; Breslow, J L; Tall, A R

    1995-01-01

    The human cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins. The activity of CETP results in a reduction in HDL cholesterol levels, but CETP may also promote reverse cholesterol transport. Thus, the net impact of CETP expression on atherogenesis is uncertain. The influence of hypertriglyceridemia and CETP on the development of atherosclerotic lesions in the proximal aorta was assessed by feeding transgenic mice a high cholesterol diet for 16 wk. 13 out of 14 (93%) hypertriglyceridemic human apo CIII (HuCIII) transgenic (Tg) mice developed atherosclerotic lesions, compared to 18 out of 29 (62%) controls. In HuCIII/CETPTg, human apo AI/CIIITg and HuAI/CIII/CETPTg mice, 7 of 13 (54%), 5 of 10 (50%), and 5 of 13 (38%), respectively, developed lesions in the proximal aorta (P < .05 compared to HuCIIITg). The average number of aortic lesions per mouse in HuCIIITg and controls was 3.4 +/- 0.8 and 2.7 +/- 0.6, respectively in HuCIII/CETPTg, HuAI/CIIIg, and HuAI/CIII/CETPTg mice the number of lesions was significantly lower than in HuCIIITg and control mice: 0.9 +/- 0.4, 1.5 +/- 0.5, and 0.9 +/- 0.4, respectively. There were parallel reductions in mean lesion area. In a separate study, we found an increased susceptibility to dietary atherosclerosis in nonhypertriglyceridemic CETP transgenic mice compared to controls. We conclude that CETP expression inhibits the development of early atherosclerotic lesions but only in hypertriglyceridemic mice. PMID:7560101

  15. Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood.

    PubMed

    Clinton, Sarah M; Glover, Matthew E; Maltare, Astha; Laszczyk, Ann M; Mehi, Stephen J; Simmons, Rebecca K; King, Gwendalyn D

    2013-08-21

    Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development.

  16. Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy.

    PubMed

    Yang, Yong-Jun; Liu, Wei-Min; Zhou, Jia-Xi; Cao, Yu-Jing; Li, Jing; Peng, Sha; Wang, Li; Yuan, Jiang-Gang; Duan, En-Kui

    2006-01-11

    Calcyclin-binding protein (Siah-1-Interacting Protein, CacyBP/SIP), is a calcium signaling protein involved in the degradation of beta-catenin, however, little is known about its role in reproductive biology. The present study was to character its temporospatial expression pattern and regulation in mouse uterus and to investigate whether it plays a role in the regulation of normal endometrial events. While prominently expressed in both luminal and glandular epithelia, CacyBP underwent dynamic changes during early pregnancy. CacyBP expression was observed weakly from days 1-4. An intense accumulation in luminal and glandular epithelia as well as decidua surrounding the embryo at later stages (days 5-7) was observed. Most notably, CacyBP accumulation in trophoblast was pronounced at day 7. Using ovariectomized and pseudopregnant mice, we found that progesterone (P(4)) and 17beta-estradiol (E(2)) led to increased expression of CacyBP gene and this could be abolished by Ru486 and tamoxifen, respectively. Antisense oligonucleotides (ODNs) against CacyBP significantly inhibited cultured endometrial stromal cells' (ESCs) apoptosis induced by UV irradiation. Injection of antisense ODNs into mouse uterine horn severely impaired the number of implanted blastocysts. Taken together, our results suggested that CacyBP expression was positively regulated by P(4) and E(2). CacyBP may be involved in the regulation of endometrial cell apoptosis during early pregnancy and play an important role in mouse endometrial events such as pregrancy establishment.

  17. Expression of regulatory proteins in choroid plexus changes in early stages of Alzheimer disease.

    PubMed

    Krzyzanowska, Agnieszka; García-Consuegra, Inés; Pascual, Consuelo; Antequera, Desiree; Ferrer, Isidro; Carro, Eva

    2015-04-01

    Recent studies indicate that the choroid plexus has important physiologic and pathologic roles in Alzheimer disease (AD). To obtain additional insight on choroid plexus function, we performed a proteomic analysis of choroid plexus samples from patients with AD stages I to II (n = 16), III to IV (n = 16), and V to VI (n = 11) and 7 age-matched control subjects. We used 2-dimensional differential gel electrophoresis coupled with mass spectrometry to generate a complete picture of changes in choroid plexus protein expression occurring in AD patients. We identified 6 proteins: 14-3-3 β/α, 14-3-3 ε, moesin, proteasome activator complex subunit 1, annexin V, and aldehyde dehydrogenase, which were significantly regulated in AD patient samples (p < 0.05, >1.5-fold variation in expression vs control samples). These proteins are implicated in major physiologic functions including mitochondrial dysfunction and apoptosis regulation. These findings contribute additional significance to the emerging importance of molecular and functional changes of choroid plexus function in the pathophysiology of AD.

  18. Early changes in protein expression of barley following inoculation with erysiphe graminis f. sp. hordei

    SciTech Connect

    Simons, S.P.; Somerville, S.C. )

    1989-04-01

    Erysiphe graminis f. sp. hordei is an obligate pathogen of barley causing the powdery mildew disease. Resistance to this disease is the product of a highly specific interaction between barley lines with specific resistance alleles and pathogen races carrying complementary avirulence alleles. Using congenic barley lines which differ at the M1-a disease reaction locus, we hope to define the early molecular events of this interaction. Accordingly, resistant and susceptible barley seedlings were labelled with {sup 35}S-methionine and examined by two-dimensional electrophoresis at two hour intervals following inoculation. Infection related changes were observed with both isolines during the four to twelve hour time period. Additional differences existed constitutively between the barley lines. These differences have been quantified. Further characterization of these proteins will yield useful markers for events preceding or coinciding with cytological responses any may lead to identification and cloning of the M1-a gene.

  19. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery.

    PubMed

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-07-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood-brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.

  20. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery

    PubMed Central

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-01-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood–brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke. PMID:23571276

  1. Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

    PubMed

    Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-04-01

    Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

  2. Enhanced expression of LINE-1-encoded ORF2 protein in early stages of colon and prostate transformation

    PubMed Central

    De Luca, Chiara; Guadagni, Fiorella; Sinibaldi-Vallebona, Paola; Sentinelli, Steno; Gallucci, Michele; Hoffmann, Andreas; Schumann, Gerald G.; Spadafora, Corrado; Sciamanna, Ilaria

    2016-01-01

    LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer. We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and tissues. We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively. Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker. PMID:26716650

  3. Growth pattern switch of renal cells and expression of cell cycle related proteins at the early stage of diabetic nephropathy

    SciTech Connect

    Zhang Yanling; Shi Yonghong; Liu Yaling; Dong Hui; Liu, Maodong; Li Ying; Duan Huijun

    2007-11-09

    Renal hypertrophy, partly due to cell proliferation and hypertrophy, has been found correlated to renal function deterioration in diabetes mellitus. We screened the up-regulated cell cycle related genes to investigate cell growth and the expression of cell cycle regulating proteins at the early stage of diabetic nephropathy using STZ-induced diabetic rats. Cyclin E, CDK{sub 2} and P{sup 27} were found significantly up-regulated in diabetic kidney. Increased cell proliferation in the kidney was seen at day 3, peaked at day 5, and returned to normal level at day 30. Cyclin E and CDK{sub 2} expression also peeked at day 5 and P{sup 27} activity peaked at day 14. These findings indicate that a hyperplastic growth period of renal cells is followed by a hypertrophic growth period at the early stage of diabetes. The growth pattern switch may be regulated by cell cycle regulating proteins, Cyclin E, CDK{sub 2}, and P{sup 27}.

  4. Phylogenetic analysis and seasonal cold acclimation-associated expression of early light-induced protein genes of Rhododendron catawbiense.

    PubMed

    Peng, Yanhui; Lin, Wuling; Wei, Hui; Krebs, Stephen L; Arora, Rajeev

    2008-01-01

    The early light-induced proteins (ELIPs) are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting Chl a/b-binding proteins. Recent evidence from physiological and genetic (mutant) studies supports a photoprotective function for ELIPs, particularly when green tissues are exposed to high light intensities at suboptimal temperatures. Broad-leaved evergreens belonging to genus Rhododendron are often exposed to a combination of low temperatures and high light in their natural habitat as the understory plants in deciduous forests and, therefore, are expected to employ photoprotective strategies during overwintering phase. Here we report analysis and characterization of previously identified ELIP expressed sequence tags (ESTs) from winter-collected Rhododendron catawbiense leaves. 5' or 3' rapid amplification of complementary DNA ends (RACEs) coupled with bioinformatic analyses were used to identify seven unique ELIPs from the 40 ESTs and were designated as RcELIP1-RcELIP7. Phylogenetic analysis revealed separate clustering of ELIP homologs from lower plants, monocots and eudicots (including RcELIPs) and further indicated an evolutionary divergence of ELIPs among angiosperms and gymnosperms. To gain insights into the cold acclimation (CA) physiology of rhododendrons, relative and absolute quantitative expression of RcELIPs was examined during seasonal CA of R. catawbiense leaves using real time reverse transcriptase-polymerase chain reaction. All seven RcELIPs were distinctly upregulated during the CA. It is postulated that RcELIPs expression constitutes an adaptive response to cold and high light in winter-adapted rhododendron leaves and perhaps plays a key role in the protection of photosynthetic apparatus from these stresses.

  5. Nuclear Expression of Hepatitis B Virus X Protein Is Associated with Recurrence of Early-Stage Hepatocellular Carcinomas: Role of Viral Protein in Tumor Recurrence

    PubMed Central

    Jin, Jing; Jung, Hae Yoen; Lee, Kyu Ho; Yi, Nam-Joon; Suh, Kyung-Suk; Jang, Ja-June; Lee, Kyoung-Bun

    2016-01-01

    Background: Hepatitis B virus (HBV) plays well-known roles in tumorigenesis of hepatocellular carcinoma (HCC) in infected patients. However, HBV-associated protein status in tumor tissues and the relevance to tumor behavior has not been reported. Our study aimed to examine the expression of HBV-associated proteins in HCC and adjacent nontumorous tissue and their clinicopathologic implication in HCC patients. Methods: HBV surface antigen (HBsAg), HBV core antigen (HBcAg), and HBV X protein (HBx) were assessed in 328 HBV-associated HCCs and in 155 matched nontumorous tissues by immunohistochemistry staining. Results: The positive rates of HBsAg and cytoplasmic HBx staining in tumor tissue were lower than those in nontumorous tissue (7.3% vs. 57.4%, p < .001; 43.4% vs. 81.3%, p < .001). Conversely, nuclear HBx was detected more frequently in tumors than in nontumorous tissue (52.1% vs. 30.3%, p < .001). HCCs expressing HBsAg, HBcAg, or cytoplasmic HBx had smaller size; lower Edmondson-Steiner (ES) nuclear grade, pT stage, and serum alpha-fetoprotein, and less angioinvasion than HCCs not expressing HBV-associated proteins. Exceptionally, nuclear HBx-positive HCCs showed higher ES nuclear grade and more frequent large-vessel invasion than did nuclear HBx-negative HCCs. In survival analysis, only nuclear HBx-positive HCCs had shorter disease-free survival than nuclear HBx-negative HCCs in pT1 and ES nuclear grade 1–2 HCC subgroup (median, 126 months vs. 35 months; p = .015). Conclusions: Our data confirmed that expression of normal HBV-associated proteins generally decreases in tumor cells in comparison to nontumorous hepatocytes, with the exception of nuclear HBx, which suggests that nuclear HBx plays a role in recurrence of well-differentiated and early-stage HCCs. PMID:27086597

  6. Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

    PubMed

    Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

    2016-12-15

    A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans.

  7. Early weaning increases intestinal permeability, alters expression of cytokine and tight junction proteins, and activates mitogen-activated protein kinases in pigs.

    PubMed

    Hu, C H; Xiao, K; Luan, Z S; Song, J

    2013-03-01

    Although weaning stress has been reported to impair intestinal barrier function, the mechanisms have not yet been elucidated. In the present study, the intestinal morphology and permeability and mRNA expressions of tight junction proteins and cytokines in the intestine of piglets during the 2 wk after weaning were assessed. The phosphorylated (activated) ratios of p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular regulated kinases (ERK1/2) were determined to investigate whether mitogen-activated protein kinase (MAPK) signaling pathways are involved in the early weaning process. A shorter villus and deeper crypt were observed on d 3 and 7 postweaning. Although damaged intestinal morphology recovered to preweaning values on d 14 postweaning, the intestinal mucosal barrier, which was reflected by transepithelial electrical resistance (TER) and paracellular flux of dextran (4 kDa) in the Ussing chamber and tight junction protein expression, was not recovered. Compared with the preweaning stage (d 0), jejunal TER and mRNA expressions of occludin and claudin-1 on d 3, 7, and 14 postweaning and Zonula occludens-1 (ZO-1) mRNA on d 3 and 7 postweaning were reduced, and paracellular flux of dextran on d 3, 7, and 14 postweaning was increased. An increase (P < 0.05) of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA on d 3 and d 7 postweaning and an increase (P < 0.05) of interferon-γ (IFN-γ) mRNA on d 3 postweaning were observed compared with d 0. No significant increase of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) mRNA after weaning was observed. The phosphorylated (activated) ratios of JNK and p38 on d 3 and 7 postweaning and the phosphorylated ratio of ERK1/2 on d 3 postweaning were increased (P < 0.05) compared with d 0. The results indicated that early weaning induced sustained impairment in the intestinal barrier, decreased mRNA expression of tight junction proteins, and upregulated the expression of proinflammatory

  8. Differences in heat shock protein 70 expression during larval and early spat development in the Eastern oyster, Crassostrea virginica (Gmelin, 1791).

    PubMed

    Ueda, Nobuo; Boettcher, Anne

    2009-07-01

    For a variety of species, changes in the expression of heat shock proteins (HSP) have been linked to key developmental changes, i.e., gametogenesis, embryogenesis, and metamorphosis. Many marine invertebrates are known to have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis as they transition to the benthic life stage. A series of experiments were run to examine the expression of heat shock protein 70 (HSP 70) during larval and early spat (initial benthic phase) development in the Eastern oyster, Crassostrea virginica. In addition, the impact of thermal stress on HSP 70 expression during these early stages was studied. C. virginica larvae and spat expressed three HSP 70 isoforms, two constitutive, HSC 77 and HSC 72, and one inducible, HSP 69. We found differences in the expression of both the constitutive and inducible forms of HSP 70 among larval and early juvenile stages and in response to thermal stress. Low expression of HSP 69 during early larval and spat development may be associated with the susceptibility of these stages to environmental stress. Although developmental regulation of HSP 70 expression has been widely recognized, changes in its expression during settlement and metamorphosis of marine invertebrates are still unknown. The results of the current study demonstrated a reduction of HSP 70 expression during settlement and metamorphosis in the Eastern oyster, C. virginica.

  9. The autoimmunity-associated BLK haplotype exhibits cis-regulatory effects on mRNA and protein expression that are prominently observed in B cells early in development

    PubMed Central

    Simpfendorfer, Kim R.; Olsson, Lina M.; Manjarrez Orduño, Nataly; Khalili, Houman; Simeone, Alyssa M.; Katz, Matthew S.; Lee, Annette T.; Diamond, Betty; Gregersen, Peter K.

    2012-01-01

    The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4+ and CD8+ T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development. PMID:22678060

  10. Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

    PubMed Central

    Voronina, Ekaterina; Lopez, Manuel; Juliano, Celina E.; Gustafson, Eric; Song, Jia L.; Extavour, Cassandra; George, Sophie; Oliveri, Paola; McClay, David; Wessel, Gary

    2009-01-01

    Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16 cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa-protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed, respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa-expression in this embryo is restricted early by translational regulation to the small micromere lineage. PMID:18191830

  11. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  12. Expressive Education in Early Childhood.

    ERIC Educational Resources Information Center

    Mori, Kimie

    1996-01-01

    Presents a concise overview of early childhood music education in Japan. Japanese early childhood education stresses the natural development of childhood, as well as cultivation of expressive activities. Discusses teaching methods, creative activities, and educational guidelines (MJP)

  13. Effects of an Early Experience of Reward through Maternal Contact or its Denial on Laterality of Protein Expression in the Developing Rat Hippocampus

    PubMed Central

    Raftogianni, Androniki; Stamatakis, Antonios; Papadopoulou, Angeliki; Vougas, Konstantinos; Anagnostopoulos, Athanasios K.; Stylianopoulou, Fotini; Tsangaris, George Th.

    2012-01-01

    Laterality is a basic characteristic of the brain which is detectable early in life. Although early experiences affect laterality of the mature brain, there are no reports on their immediate neurochemical effects during neonatal life, which could provide evidence as to the mechanisms leading to the lateralized brain. In order to address this issue, we determined the differential protein expression profile of the left and right hippocampus of 13-day-old rat control (CTR) pups, as well as following exposure to an early experience involving either receipt (RER) or denial (DER) of the expected reward of maternal contact. Proteomic analysis was performed by 2-dimensional polyacrylamide gel electrophoresis (PAGE) followed by mass spectroscopy. The majority of proteins found to be differentially expressed either between the three experimental groups (DER, RER, CTR) or between the left and right hemisphere were cytoskeletal (34%), enzymes of energy metabolism (32%), and heat shock proteins (17%). In all three groups more proteins were up-regulated in the left compared to the right hippocampus. Tubulins were found to be most often up-regulated, always in the left hippocampus. The differential expression of β-tubulin, β-actin, dihydropyrimidinase like protein 1, glial fibrillary acidic protein (GFAP) and Heat Shock protein 70 revealed by the proteomic analysis was in general confirmed by Western blots. Exposure to the early experience affected brain asymmetry: In the RER pups the ratio of proteins up-regulated in the left hippocampus to those in the right was 1.8, while the respective ratio was 3.6 in the CTR and 3.4 in the DER. Our results could contribute to the elucidation of the cellular mechanisms mediating the effects of early experiences on the vulnerability for psychopathology, since proteins shown in our study to be differentially expressed (e.g. tubulins, dihydropyrimidinase like proteins, 14-3-3 protein, GFAP, ATP synthase, α-internexin) have also been

  14. Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

    PubMed

    Tsuji, Frederick I

    2010-08-01

    The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment.

  15. Time course of immediate early gene protein expression in the spinal cord following conditioning stimulation of the sciatic nerve in rats.

    PubMed

    Bojovic, Ognjen; Panja, Debabrata; Bittins, Margarethe; Bramham, Clive R; Tjølsen, Arne

    2015-01-01

    Long-term potentiation induced by conditioning electrical stimulation of afferent fibers is a widely studied form of synaptic plasticity in the brain and the spinal cord. In the spinal cord dorsal horn, long-term potentiation is induced by a series of high-frequency trains applied to primary afferent fibers. Conditioning stimulation (CS) of sciatic nerve primary afferent fibers also induces expression of immediate early gene proteins in the lumbar spinal cord. However, the time course of immediate early gene expression and the rostral-caudal distribution of expression in the spinal cord have not been systematically studied. Here, we examined the effects of sciatic nerve conditioning stimulation (10 stimulus trains, 0.5 ms stimuli, 7.2 mA, 100 Hz, train duration 2 s, 8 s intervals between trains) on cellular expression of immediate early genes, Arc, c-Fos and Zif268, in anesthetized rats. Immunohistochemical analysis was performed on sagittal sections obtained from Th13- L5 segments of the spinal cord at 1, 2, 3, 6 and 12 h post-CS. Strikingly, all immediate early genes exhibited a monophasic increase in expression with peak increases detected in dorsal horn neurons at 2 hours post-CS. Regional analysis showed peak increases at the location between the L3 and L4 spinal segments. Both Arc, c-Fos and Zif268 remained significantly elevated at 2 hours, followed by a sharp decrease in immediate early gene expression between 2 and 3 hours post-CS. Colocalization analysis performed at 2 hours post-CS showed that all c-Fos and Zif268 neurons were positive for Arc, while 30% and 43% of Arc positive neurons were positive for c-Fos and Zif268, respectively. The present study identifies the spinal cord level and time course of immediate early gene (IEGP) expression of relevance for analysis of IEGPs function in neuronal plasticity and nociception.

  16. Proteomic Analysis on the Alteration of Protein Expression in the Early-Stage Placental Villous Tissue of Electromagnetic Fields Associated With Cell Phone Exposure

    PubMed Central

    Luo, Qiong; Jiang, Ying; Jin, Min

    2013-01-01

    Background: To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Methods: Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Results: Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified— procollagen–proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Conclusion: Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects. PMID:23420827

  17. Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection.

    PubMed

    Gryspeerdt, Annick C; Vandekerckhove, Annelies P; Baghi, Hossein Bannazadeh; Van de Walle, Gerlinde R; Nauwynck, Hans J

    2012-08-01

    Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.

  18. Differential expression of proteins involved in energy production along the crypt-villus axis in early-weaning pig small intestine.

    PubMed

    Xiong, Xia; Yang, Huansheng; Tan, Bie; Yang, Chengbo; Wu, Miaomiao; Liu, Gang; Kim, Sung Woo; Li, Tiejun; Li, Lili; Wang, Junjun; Wu, Guoyao; Yin, Yulong

    2015-08-15

    Weaning of piglets reflects intestinal dysfunction and atrophy and affected the physiological state of enterocytes. However, few studies have defined physiological state of enterocytes along the crypt-villus axis in early-weaning piglets. A total of 16 piglets from 8 litters were used in the experiment. One group of piglets was nursed by sows until age 21 days, and another group was weaned at age 14 days and then fed creep feed instead of breast milk for 7 days. Piglets were killed at 21 days, and the jejunum segments were dissected. After sequential isolation of jejunum epithelial cells along the crypt-villus axis, their proteins were analyzed through the isobaric tags for relative and absolute quantification, and proteins involved in the mammalian target of rapamycin signaling pathway and proliferating cell nuclear antigen abundances in jejunal epithelial cells of weaning or suckling group were determined by Western blotting. The differential proteins in three cell fractions were identified and analyzed. The results showed that proteins involved in the tricarboxylic acid cycle, β-oxidation, and the glycolysis pathway were significantly downregulated in the upper and middle villus of the early-weaned group. However, proteins involved in glycolysis were significantly upregulated in crypt cells. In addition, Western blot analysis showed that the expression of mammalian target of rapamycin pathway-related proteins was decreased (P < 0.05) in the early-weaned group. The present results showed that early-weaning differentially affect the expression of proteins involved in energy production of enterocytes along the jejunal crypt-villus axis.

  19. Increased gene expression of growth associated protein-43 in skin of patients with early-stage peripheral neuropathies.

    PubMed

    Scheytt, Sarah; Riediger, Nadja; Braunsdorf, Silvia; Sommer, Claudia; Üçeyler, Nurcan

    2015-08-15

    Growth associated protein-43 (GAP-43) is one of the neural proteins associated with nerve injury that is upregulated after nerve injury. To investigate whether GAP-43 quantification in skin biopsies would differentiate subtypes of peripheral neuropathies, we analyzed GAP-43 expression in skin from the lateral thigh and the distal leg. We prospectively enrolled 130 patients with peripheral neuropathies and compared data with healthy controls. Intraepidermal nerve fiber density (IENFD) was determined using antibodies against protein gene product 9.5 (PGP 9.5); anti-GAP-43 antibodies were applied to visualize regenerating nerve fibers. PGP 9.5 and GAP-43 gene expression was analyzed using qRT-PCR. Patients with neuropathies had a generalized reduction of IENFD and GAP-43 immunoreactive fibers compared to controls (p<0.01). In contrast, cutaneous GAP-43 gene expression was increased in proximal skin in patients (p<0.05), particularly when disease duration was short (<3 years; p<0.01). While fiber density for both markers decreased with age in healthy skin (p<0.01), age-dependent reduction of skin innervation was absent in neuropathies. Diagnostic subgroups and neuropathic pain had no influence on skin innervation. We conclude that peripheral neuropathies lead to an initial increase in GAP-43 gene expression as a potential mechanism of regeneration, which is not sustained in neuropathies of long duration.

  20. Epigenetic regulations of immediate early genes expression involved in memory formation by the amyloid precursor protein of Alzheimer disease.

    PubMed

    Hendrickx, Aurélie; Pierrot, Nathalie; Tasiaux, Bernadette; Schakman, Olivier; Kienlen-Campard, Pascal; De Smet, Charles; Octave, Jean-Noël

    2014-01-01

    We previously demonstrated that APP epigenetically regulates Egr1 expression both in cultured neurons and in vivo. Since Egr1 is an immediate early gene involved in memory formation, we wondered whether other early genes involved in memory were regulated by APP and we studied molecular mechanisms involved. By comparing prefrontal (PF) cortex from wild type (APP+/+) and APP knockout mice (APP-/-), we observed that APP down regulates expression of four immediate early genes, Egr1, c-Fos, Bdnf and Arc. Down regulation of Egr1, c-Fos and Bdnf transcription resulted from a decreased enrichment of acetylated histone H4 on the corresponding gene promoter. Further characterization of H4 acetylation at Egr1 and c-Fos promoters revealed increased acetylation of H4K5 and H4K12 residues in APP-/- mice. Whereas APP affected Egr1 promoter activity by reducing access of the CREB transcription factor, its effect on c-Fos appeared to depend on increased recruitment of HDAC2 histone deacetylase to the gene promoter. The physiological relevance of the epigenetic regulation of Egr1 and c-Fos gene transcription by APP was further analyzed following exposure of mice to novelty. Although transcription of Egr1 and c-Fos was increased following exposure of APP+/+ mice to novelty, such an induction was not possible in APP-/- mice with a high basal level of expression of these immediate early genes. Altogether, these results demonstrate that APP-mediated regulation of c-Fos and Egr1 by different epigenetic mechanisms is needed for their induction during exposure to novelty.

  1. Early-Life Exposure to Lead (Pb) Alters the Expression of microRNA that Target Proteins Associated with Alzheimer's Disease.

    PubMed

    Masoud, Anwar M; Bihaqi, Syed W; Machan, Jason T; Zawia, Nasser H; Renehan, William E

    2016-01-01

    There is a growing recognition of the impact of environmental toxins on the epigenetic regulation of gene expression, including the genes that play a critical role in neural development, neural function, and neurodegeneration. We have shown previously that exposure to the heavy metal lead (Pb) in early life results in a latent over-expression of AD-related proteins in rodents and primates. The present study provides evidence that early postnatal exposure to Pb also alters the expression of select miRNA. Mice were exposed to 0.2% Pb acetate from Postnatal Day 1 (PND 1, first 24 h after birth) to PND 20 via their mother's milk. Brain tissue was harvested at PND 20, 180, or 700, and miRNA were isolated and quantified by qPCR. This exposure produced a transient increase (relative to control) in the expression of miR-106b (binds to AβPP mRNA), miR-29b (targets the mRNA for the transcription factor SP1) and two miRNAs (miR-29b and miR-132) that have the ability to inhibit translation of proteins involved in promoter methylation. The expression of miR-106b decreased over time in the Pb-exposed animals and was significantly less than the levels exhibited by the control animals at PND700. The level of miR-124, which binds to SP1 mRNA, was also reduced (relative to controls) at PND700. In summary, we show that exposure to the heavy metal Pb in early life has a significant impact on the short- and long-term expression of miRNA that target epigenetic mediators and neurotoxic proteins.

  2. Regulation of desmocollin gene expression in the epidermis: CCAAT/enhancer-binding proteins modulate early and late events in keratinocyte differentiation.

    PubMed Central

    Smith, Conrad; Zhu, Kuichun; Merritt, Anita; Picton, Rhian; Youngs, Denise; Garrod, David; Chidgey, Martyn

    2004-01-01

    Desmocollins (Dscs) are desmosomal cadherins that exhibit differentiation-specific patterns of expression in the epidermis. Dsc3 expression is strongest in basal cell layers, whereas Dsc1 is largely confined to upper, terminally differentiating strata. To understand better the processes by which Dsc expression is regulated in the epidermis, we have isolated Dsc3 and Dsc1 5'-flanking DNAs and analysed their activity in primary keratinocytes. In the present study, we found that transcription factors of the CCAAT/enhancer-binding protein family play a role in the regulation of expression of both Dscs and, in so doing, implicate this class of transcription factors in both early and late events in keratinocyte differentiation. We show that Dscs are differentially regulated by C/EBP (CCAAT/enhancer-binding protein) family members, with Dsc3 expression being activated by C/EBPbeta but not C/EBPalpha, and the reverse being the case for Dsc1. Expression of both Dscs is activated by another family member, C/EBPdelta. These results show for the first time how desmosomal cadherin gene expression is regulated and provide a mechanism for the control of other differentiation-specific genes in the epidermis. PMID:15030314

  3. Maternal Low-Protein Diet Modulates Glucose Metabolism and Hepatic MicroRNAs Expression in the Early Life of Offspring †

    PubMed Central

    Zheng, Jia; Xiao, Xinhua; Zhang, Qian; Wang, Tong; Yu, Miao; Xu, Jianping

    2017-01-01

    Emerging studies revealed that maternal protein restriction was associated with increased risk of type 2 diabetes mellitus in adulthood. However, the mechanisms of its effects on offspring, especially during early life of offspring, are poorly understood. Here, it is hypothesized that impaired metabolic health in offspring from maternal low-protein diet (LPD) is associated with perturbed miRNAs expression in offspring as early as the weaning age. We examined the metabolic effects on the C57BL/6J mice male offspring at weaning from dams fed with LPD or normal chow diet (NCD) throughout pregnancy and lactation. Maternal LPD feeding impaired metabolic health in offspring. Microarray profiling indicated that mmu-miR-615, mmu-miR-124, mmu-miR-376b, and mmu-let-7e were significantly downregulated, while, mmu-miR-708 and mmu-miR-879 were upregulated in LPD offspring. Bioinformatic analysis showed target genes were mapped to inflammatory-related pathways. Serum tumor necrosis factor-α (TNF-α) levels were higher and interleukin 6 (IL-6) had a tendency to be elevated in the LPD group. Finally, both mRNA and protein levels of IL-6 and TNF-α were significantly increased in the LPD group. Our findings provide novel evidence that maternal LPD can regulate miRNAs expression, which may be associated with chronic inflammation status and metabolic health in offspring as early as the weaning age. PMID:28264458

  4. Immediate early gene expression in cat visual cortex during and after the critical period: differences between EGR-1 and Fos proteins.

    PubMed

    Kaplan, I V; Guo, Y; Mower, G D

    1996-02-01

    Immediate early gene (IEG) expression in the cat visual cortex is highly responsive to visual input and may initiate genetic mechanisms responsible for neuronal plasticity. The present study used immunohistochemical methods to address two issues regarding IEG expression in response to visual input. One was to define the differential response of distinct IEG families by comparing EGR-1 (also termed zif-268, NGFI-A, and Krox-24) and Fos proteins. The second was to determine whether IEG expression, in addition to reflecting neural activity, is related to the state of plasticity by comparing young and adult visual cortex. Immunoreactivity of the two IEG proteins was compared between 5-week-old and adult cats under three conditions of visual input: ambient light to assess basal levels of expression, 1 week of darkness to assess the effect of reduced activity, and exposure to light after 1 week of darkness to determine rapid changes in expression as a result of visual input. At both ages, there were marked differences in the expression of the two IEG proteins. EGR-1 responded to visual input with sustained changes in its level of expression. It showed high basal levels, reduced expression in darkness, and a rapid return to high constitutive levels with the introduction of light. Fos showed a markedly different profile. It had very low basal expression which was not demonstrably affected by darkness and its principal response was a marked transient induction upon exposure to light after darkness. These unique changes in expression highlight the complex response across IEGs to environmental input and suggest a genetic "on/off' signaling mechanism. There were marked differences in the laminar distribution of EGR-1 and Fos proteins between young and adult cats. In young animals, cells in all visual cortical layers showed high levels of EGR-1 and Fos proteins. In adults, immunostaining was largely specific to cells located above and below layer IV and only very faint labeling

  5. Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

    PubMed

    Lippolis, Rosa; Siciliano, Rosa Anna; Pacelli, Consiglia; Ferretta, Anna; Mazzeo, Maria Fiorella; Scacco, Salvatore; Papa, Francesco; Gaballo, Antonio; Dell'Aquila, Claudia; De Mari, Michele; Papa, Sergio; Cocco, Tiziana

    2015-09-01

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

  6. Complement regulatory proteins in early human fetal life: CD59, membrane co-factor protein (MCP) and decay-accelerating factor (DAF) are differentially expressed in the developing liver.

    PubMed Central

    Simpson, K L; Houlihan, J M; Holmes, C H

    1993-01-01

    The human fetus appears to be capable of protecting itself from maternal complement (C) from an early stage in development by expressing the C regulatory proteins decay-accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 on fetally derived trophoblast at the feto-maternal interface. In this study we have examined the ontogeny of these proteins within the fetus itself and have focused on the liver which represents a major site of haemopoiesis during development. Immunostaining revealed that DAF, MCP and CD59 are all expressed from at least 6 weeks of gestation in the liver but that these proteins display distinct distribution patterns. CD59 was broadly distributed both within the epithelial and haemopoietic compartments, but expression of C3 convertase regulators was more restricted. DAF expression was limited to isolated cells within haemopoietic nests and the epithelium was DAF-negative. Although MCP expression on haemopoietic cells was also limited, by contrast with DAF the developing hepatic epithelium was strongly MCP-positive. Typical CD59 and MCP components were observed in fetal liver extracts by immunoblotting, although liver MCP components consistently migrated 4000-5000 MW ahead of those observed on placental trophoblast. Differences in the distribution of these proteins were also observed between the fetal and adult liver. In particular, by comparison with fetal hepatic epithelium, there was an apparent loss of MCP expression from adult hepatocytes. Thus, MCP appears to be developmentally regulated in the human liver and is expressed in the absence of DAF on the early hepatic epithelium. Overall, this study suggests that C regulatory proteins, and in particular CD59 and MCP, are required from the very early stages of gestation within the fetus itself. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7505254

  7. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    PubMed

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

  8. Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication.

    PubMed

    Sirikanchana, Kwanrawee; Shisler, Joanna L; Mariñas, Benito J

    2008-06-01

    The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20 degrees C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.

  9. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    PubMed

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  10. Isolation rearing attenuates social interaction-induced expression of immediate early gene protein products in the medial prefrontal cortex of male and female rats.

    PubMed

    Wall, Vanessa L; Fischer, Eva K; Bland, Sondra T

    2012-10-10

    Early life adversity and stress in humans have been related to a number of psychological disorders including anxiety, depression, and addiction. The present study used isolation rearing, a well-characterized animal model of early life adversity, to examine its effects on social behavior and immediate early gene (IEG) expression produced by exposure to a novel social experience. Male and female rats were housed in same-sex groups or in isolation for 4 weeks beginning at weaning and were tested during late adolescence. The protein products of the IEGs c-fos and Arc, as well as the neurotrophic factor BDNF were assessed in medial prefrontal cortex (mPFC) subregions (anterior cingulate, prelimbic and infralimbic) using immunohistochemistry. Aggressive and non-aggressive behaviors during novel social exposure were also assessed. Exposure to a novel conspecific produced increases in Arc and c-fos activation in the mPFC of group reared animals in a sex- and subregion-dependent fashion compared to no social exposure controls, but this increase was blunted or absent in isolated animals. Isolates engaged in more social interactions and more aggressive behavior than group reared rats. Sex differences in some behaviors as well as in Arc and BDNF expression were observed. These results indicate that isolation rearing alters IEG activation in the mPFC produced by exposure to a novel conspecific, in addition to changing social behavior, and that these effects depend in part on sex.

  11. Elfin is expressed during early heart development.

    PubMed

    Kotaka, M; Lau, Y M; Cheung, K K; Lee, S M; Li, H Y; Chan, W Y; Fung, K P; Lee, C Y; Waye, M M; Tsui, S K

    Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.

  12. Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein.

    PubMed

    Azimzadeh, Agnes M; Kelishadi, Sean S; Ezzelarab, Mohamed B; Singh, Avneesh K; Stoddard, Tiffany; Iwase, Hayato; Zhang, Tianshu; Burdorf, Lars; Sievert, Evelyn; Avon, Chris; Cheng, Xiangfei; Ayares, David; Horvath, Keith A; Corcoran, Philip C; Mohiuddin, Muhammad M; Barth, Rolf N; Cooper, David K C; Pierson, Richard N

    2015-01-01

    We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

  13. Differential effect of early antibiotic intervention on bacterial fermentation patterns and mucosal gene expression in the colon of pigs under diets with different protein levels.

    PubMed

    Zhang, Chuanjian; Yu, Miao; Yang, Yuxiang; Mu, Chunlong; Su, Yong; Zhu, Weiyun

    2017-03-01

    The study aimed to evaluate the effects of early antibiotic intervention (EAI) on bacterial fermentation patterns and mucosal immune markers in the colon of pigs with different protein level diets. Eighteen litters of piglets at day (d) 7 were fed creep feed without or with growth promoting antibiotics until d 42. At d 42, pigs within each group were further randomly assigned to a normal- or low-crude protein (CP) diet. At d 77 and d 120, five pigs per group were slaughtered for analyzing colonic bacteria, metabolites, and mucosal gene expressions. Results showed that low-CP diet increased propionate and butyrate concentrations at d 77 but reduced ammonia and phenol concentrations (P < 0.05). EAI increased p-cresol and indole concentrations under normal-CP diet at d 77 (P < 0.05). Low-CP diet significantly affected (P < 0.05) some bacteria groups (Firmicutes, Clostridium cluster IV, Clostridium cluster XIVa, Escherichia coli, and Lactobacillus), but EAI showed limited effects. Low-CP diet down-regulated gene expressions of pro-inflammatory cytokines, toll-like receptor (TLR4), myeloid differentiating factor 88 (MyD88), and nuclear factor-κB p65 (NF-κB p65) (P < 0.05). EAI up-regulated mRNA expressions of interleukin-8 (IL-8) and interferon-γ (IFN-γ) under normal-CP diet at d 77 (P < 0.05). Furthermore, reductions of E. coli and ammonia under low-CP diet were positively correlated with down-regulated gene expressions of pro-inflammatory cytokines, which were positively correlated with the down-regulated TLR4-MyD88-NF-κB signaling pathway. In conclusion, EAI had short-term effects under normal-CP diet with increased aromatic amino acid fermentation and gene expressions of pro-inflammatory cytokines. Low-CP diet markedly reduced protein fermentation, modified microbial communities, and down-regulated gene expressions of pro-inflammatory cytokines possibly via down-regulating TLR4-MyD88-NF-κB signaling pathway.

  14. Low Ki67/high ATM protein expression in malignant tumors predicts favorable prognosis in a retrospective study of early stage hormone receptor positive breast cancer

    PubMed Central

    Feng, Xiaolan; Li, Haocheng; Kornaga, Elizabeth N.; Dean, Michelle; Lees-Miller, Susan P.; Riabowol, Karl; Magliocco, Anthony M.; Morris, Don; Watson, Peter H.; Enwere, Emeka K.; Bebb, Gwyn; Paterson, Alexander

    2016-01-01

    Introduction This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (< 4 cm) and fewer than four positive lymph nodes. Methods 532 formalin-fixed paraffin-embedded specimens of resected primary breast tumors were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Results Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). Conclusions These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC. PMID:27741524

  15. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway.

    PubMed

    Szlachcic, Wojciech J; Switonski, Pawel M; Krzyzosiak, Wlodzimierz J; Figlerowicz, Marek; Figiel, Maciej

    2015-09-01

    Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life.

  16. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway

    PubMed Central

    Szlachcic, Wojciech J.; Switonski, Pawel M.; Krzyzosiak, Wlodzimierz J.; Figlerowicz, Marek; Figiel, Maciej

    2015-01-01

    ABSTRACT Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life. PMID:26092128

  17. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.

    PubMed

    Chishti, M A; Yang, D S; Janus, C; Phinney, A L; Horne, P; Pearson, J; Strome, R; Zuker, N; Loukides, J; French, J; Turner, S; Lozza, G; Grilli, M; Kunicki, S; Morissette, C; Paquette, J; Gervais, F; Bergeron, C; Fraser, P E; Carlson, G A; George-Hyslop, P S; Westaway, D

    2001-06-15

    We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.

  18. Morphological analysis of the early development of telencephalic and diencephalic gonadotropin-releasing hormone neuronal systems in enhanced green fluorescent protein-expressing transgenic medaka lines.

    PubMed

    Takahashi, Akiko; Islam, M Sadiqul; Abe, Hideki; Okubo, Kataaki; Akazome, Yasuhisa; Kaneko, Takeshi; Hioki, Hiroyuki; Oka, Yoshitaka

    2016-03-01

    Teleosts possess two or three paralogs of gonadotropin-releasing hormone (GnRH) genes: gnrh1, gnrh2, and gnrh3. Some species have lost the gnrh1 and/or gnrh3 genes, whereas gnrh2 has been completely conserved in the teleost species analyzed to date. In most teleosts that possess gnrh1, GnRH1 peptide is the authentic GnRH that stimulates gonadotropin release, whereas GnRH2 and GnRH3, if present, are neuromodulatory. Progenitors of GnRH1 and GnRH3 neurons originate from olfactory placodes and migrate to their destination during early development. However, because of the relatively low affinity/specificity of generally available antibodies that recognize GnRH1 or GnRH3, labeling of these neurons has only been possible using genetic manipulation. We used a model teleost, medaka, which possesses all three paralogous gnrh genes, to analyze development of forebrain GnRH neurons composed of GnRH1 and GnRH3 neurons. Here, we newly generated transgenic medaka lines that express enhanced green fluorescent protein under the control of promoters for gnrh1 or gnrh3, to detect GnRH neurons and facilitate immunohistochemical analysis of the neuronal morphology. We used a combination of immunohistochemistry and three-dimensional confocal microscopy image reconstructions to improve identification of neurites from GnRH1 or GnRH3 neuronal populations with greater precision. This led us to clearly identify the hypophysiotropic innervation of GnRH1 neurons residing in the ventral preoptic area (vPOA) from as early as 10 days post hatching. Furthermore, these analyses also revealed retinopetal projections of nonhypophysiotropic GnRH1 neurons in vPOA, prominent during early developmental stages, and multiple populations of GnRH3 neurons with different origins and migratory pathways.

  19. Differential expression of serum glycodelin and insulin-like growth factor binding protein 1 in early pregnancy.

    PubMed

    Douglas, Nataki C; Thornton, Melvin H; Nurudeen, Sahadat K; Bucur, Maria; Lobo, Rogerio A; Sauer, Mark V

    2013-11-01

    This prospective study evaluated whether serum glycodelin and insulin-like growth factor binding protein 1 (IGFBP-1) predict the likelihood of embryo implantation in recipients undergoing donor egg in vitro fertilization. We measured glycodelin and IGFBP-1 at 6 points from lining check to lutenizing hormone (LH) + 31. β-Human chorionic gonadotropin levels were first measured at LH + 17. The recipients were divided into those without embryo implantation (group 1, n = 6) and those with successful implantation (group 2, n = 30). Although this is a negative study in that neither glycodelin nor IGFBP-1 alone reflected endometrial (EM) receptivity, the glycodelin/IGFBP-1 ratio on the day of blastocyst transfer was higher in recipients who achieved pregnancy (P = .05). At LH + 17, glycodelin was higher (P = .04), and IGFBP-1 was lower (P = .004) in recipients who achieved pregnancy when compared to those who did not. These observations are likely due to EM changes induced by successful embryo implantation.

  20. Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

    SciTech Connect

    Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica; Bagnasco, Luca; Orecchia, Paola; Carnemolla, Barbara; Patrone, Eligio; Balbi, Cecilia

    2009-01-15

    Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.

  1. Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

    PubMed

    Salem, K A; Adrian, T E; Qureshi, M A; Parekh, K; Oz, M; Howarth, F C

    2012-12-01

    There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0

  2. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  3. Differential Expression of Serum Glycodelin and Insulin-Like Growth Factor Binding Protein 1 in Early Pregnancy

    PubMed Central

    Thornton, Melvin H.; Nurudeen, Sahadat K.; Bucur, Maria; Lobo, Rogerio A.; Sauer, Mark V.

    2013-01-01

    This prospective study evaluated whether serum glycodelin and insulin-like growth factor binding protein 1 (IGFBP-1) predict the likelihood of embryo implantation in recipients undergoing donor egg in vitro fertilization. We measured glycodelin and IGFBP-1 at 6 points from lining check to lutenizing hormone (LH) + 31. β-Human chorionic gonadotropin levels were first measured at LH + 17. The recipients were divided into those without embryo implantation (group 1, n = 6) and those with successful implantation (group 2, n = 30). Although this is a negative study in that neither glycodelin nor IGFBP-1 alone reflected endometrial (EM) receptivity, the glycodelin/IGFBP-1 ratio on the day of blastocyst transfer was higher in recipients who achieved pregnancy (P = .05). At LH + 17, glycodelin was higher (P = .04), and IGFBP-1 was lower (P = .004) in recipients who achieved pregnancy when compared to those who did not. These observations are likely due to EM changes induced by successful embryo implantation. PMID:23585335

  4. Reduced expression of the immediate-early protein IE0 enables efficient replication of Autographa californica multiple nucleopolyhedrovirus in poorly permissive Spodoptera littoralis cells.

    PubMed

    Lu, Liqun; Du, Quansheng; Chejanovsky, Nor

    2003-01-01

    Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.

  5. Obestatin induction of early-response gene expression in gastrointestinal and adipose tissues and the mediatory role of G protein-coupled receptor, GPR39.

    PubMed

    Zhang, Jian V; Jahr, Holger; Luo, Chin-Wei; Klein, Cynthia; Van Kolen, Kristof; Ver Donck, Luc; De, Ananya; Baart, Esther; Li, Jing; Moechars, Dieder; Hsueh, Aaron J W

    2008-06-01

    Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.

  6. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    PubMed

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  7. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  8. Early Detection of Baculovirus Expression and Infection in Lepidopteran Larvae Fed Occlusion Bodies of an AcMNPV Recombinant Carrying a Red Fluorescent Protein Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method has been devised utilizing a baculovirus recombinant (AcMNPV hsp70Red) carrying a red fluorescent protein (RFP) gene under the early heat shock promoter (hsp70) to assess potential infectivity of larvae fed occlusion bodies. A time study was employed whereby first and third instars of Trich...

  9. A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

    PubMed Central

    Tyndall, C; La Mantia, G; Thacker, C M; Favaloro, J; Kamen, R

    1981-01-01

    Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed. Images PMID:6275353

  10. The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection.

    PubMed Central

    Tollefson, A E; Scaria, A; Saha, S K; Wold, W S

    1992-01-01

    We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit. Images PMID:1316473

  11. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  12. Pre- and early-postnatal nutrition modify gene and protein expressions of muscle energy metabolism markers and phospholipid Fatty Acid composition in a muscle type specific manner in sheep.

    PubMed

    Hou, Lei; Kongsted, Anna H; Ghoreishi, Seyed M; Takhtsabzy, Tasnim K; Friedrichsen, Martin; Hellgren, Lars I; Kadarmideen, Haja N; Vaag, Allan; Nielsen, Mette O

    2013-01-01

    We previously reported that undernutrition in late fetal life reduced whole-body insulin sensitivity in adult sheep, irrespective of dietary exposure in early postnatal life. Skeletal muscle may play an important role in control of insulin action. We therefore studied a range of putative key muscle determinants of insulin signalling in two types of skeletal muscles (longissimus dorsi (LD) and biceps femoris (BF)) and in the cardiac muscle (ventriculus sinister cordis (VSC)) of sheep from the same experiment. Twin-bearing ewes were fed either 100% (NORM) or 50% (LOW) of their energy and protein requirements during the last trimester of gestation. From day-3 postpartum to 6-months of age (around puberty), twin offspring received a high-carbohydrate-high-fat (HCHF) or a moderate-conventional (CONV) diet, whereafter all males were slaughtered. Females were subsequently raised on a moderate diet and slaughtered at 2-years of age (young adults). The only long-term consequences of fetal undernutrition observed in adult offspring were lower expressions of the insulin responsive glucose transporter 4 (GLUT4) protein and peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC1α) mRNA in BF, but increased PGC1α expression in VSC. Interestingly, the HCHF diet in early postnatal life was associated with somewhat paradoxically increased expressions in LD of a range of genes (but not proteins) related to glucose uptake, insulin signalling and fatty acid oxidation. Except for fatty acid oxidation genes, these changes persisted into adulthood. No persistent expression changes were observed in BF and VSC. The HCHF diet increased phospholipid ratios of n-6/n-3 polyunsaturated fatty acids in all muscles, even in adults fed identical diets for 1½ years. In conclusion, early postnatal, but not late gestation, nutrition had long-term consequences for a number of determinants of insulin action and metabolism in LD. Tissues other than muscle may account for reduced whole

  13. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection.

    PubMed Central

    McMahan, L; Schaffer, P A

    1990-01-01

    mutant, which specifies an ICP27 peptide lacking the repressing region between residues 327 and 407, is able to (i) repress early gene expression, consistent with the repressing ability of the d3 mutation in transient expression assays, (ii) induce the synthesis of significant but reduced levels of delayed-early (gamma 1) proteins and no gamma 2 proteins (thus vd3 exhibits a late protein phenotype intermediate between that of the wild-type virus and 5dl 1.2), and (iii) confer altered electrophoretic mobility on ICP4, demonstrating a role for ICP27 in the posttranslational modification of this essential regulatory protein.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2161950

  14. Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi.

    PubMed

    Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo; Chávez-Mardones, Jacqueline; Maldonado-Aguayo, Waleska

    2014-02-15

    The couch potato (CPO) protein is a key biomolecule involved in regulating diapause through the RNA-binding process of the peripheral and central nervous systems in insects and also recently discovered in a few crustacean species. As such, ectoparasitic copepods are interesting model species that have no evidence of developmental arrest. The present study is the first to report on the cloning of a putative CPO gene from the salmon louse Caligus rogercresseyi (CrCPO), as identified by high-throughput transcriptome sequencing. In addition, the transcription expression in larvae and adults was evaluated using quantitative real-time PCR. The CrCPO cDNA sequence showed 3261 base pairs (bp), consisting of 713bp of 5' UTR, 1741bp of 3' UTR, and an open reading frame of 807bp encoding for 268 amino acids. The highly conserved RNA binding regions RNP2 (LFVSGL) and RNP1 (SPVGFVTF), as well the dimerization site (LEF), were also found. Furthermore, eight single nucleotide polymorphisms located in the untranslated regions and one located in the coding region were detected. Gene transcription analysis revealed that CrCPO has ubiquitous expression across larval stages and in adult individuals, with the highest expression from nauplius to copepodid stages. The present study suggests a putative biological function of CrCPO associated with the development of the nervous system in salmon lice and contributes molecular evidence for candidate genes related to host-parasite interactions.

  15. Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

    PubMed Central

    Pawlik, Timothy M; Hawke, David H; Liu, Yanna; Krishnamurthy, Savitri; Fritsche, Herbert; Hunt, Kelly K; Kuerer, Henry M

    2006-01-01

    Background Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. Methods Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. Results 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. Conclusion ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer. PMID:16542425

  16. Krüppel-like factor 4 is widely expressed in the mouse male and female reproductive tract and responds as an immediate early gene to activation of the protein kinase A in TM4 Sertoli cells.

    PubMed

    Godmann, M; Kosan, C; Behr, R

    2010-04-01

    Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor critically involved in cell proliferation, differentiation, and carcinogenesis. Recently, KLF4 has also been used for the generation of induced pluripotent stem cells. In this study, we analyzed Klf4 expression in different mouse tissues using northern blot analysis and immunohistochemistry. Focusing on the male and female reproductive tract, we showed for the first time that KLF4 is expressed in the epithelia of the murine uterus and the vagina. In the male reproductive tract, we detected KLF4 in the epithelia of the epididymis, ductus deferens, coagulating gland, and the penis. As KLF4 is strongly inducible by FSH signaling in Sertoli cells and as this transcription factor is also involved in Sertoli cell development, we employed the mouse Sertoli cell line TM4 as a model system to investigate i) the induction kinetics of Klf4 upon activation of the cAMP/protein kinase A pathway by forskolin and ii) the effects of Klf4 induction on TM4 cell cycle progression. Interestingly, Klf4 mRNA and protein were rapidly but transiently induced, reaching peak levels after 90-120 min and declining to basal levels within 4 h. Compared with the inducible cAMP early repressor, an immediate early response gene, the induction kinetics of Klf4 is much faster. In conclusion, Klf4 is an immediate early gene in TM4 cells and its expression in several epithelia of the male and female reproductive tract suggests an important role of Klf4 in mouse reproductive functions.

  17. ES2, a gene deleted in DiGeorge syndrome, encodes a nuclear protein and is expressed during early mouse development, where it shares an expression domain with a Goosecoid-like gene.

    PubMed

    Lindsay, E A; Harvey, E L; Scambler, P J; Baldini, A

    1998-04-01

    ES2 is a gene deleted in DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) which has homologs in species as distant as Caenorhabditis elegans and Drosophila . The function of ES2 is unknown, and the predicted protein sequence does not contain motifs which suggest a particular role in the developmental defects present in DGS and VCFS. Here we show that the mouse homolog, Es2 , is transcribed in two forms resulting from the use of alternative polyadenylation signals. Structural analysis programs predict that the Es2 -encoded peptide has a coiled-coil domain, and transfection experiments with an Es2 -green fluorescent protein (GFP) fusion construct show that the peptide is recruited into the nucleus. Es2 is highly expressed during mouse embryogenesis from E7 onwards. In situ hybridization with an RNA probe revealed that the gene is widely expressed; however, relatively higher expression was detected in the nervous system, with a particularly high area of expression in a sub-region of the pons. The Es2 expression domain in the pons is shared with a Goosecoid-like gene ( Gscl) which is located upstream of Es2 , and raises the possibility that the two genes share regulatory elements and/or interact in this region of the developing brain. This finding suggests that different genes in the deleted region may be functionally related and might explain the occurrence of the characteristic phenotype in patients with non-overlapping genetic lesions.

  18. Adenovirus Early Proteins and Host Sumoylation

    PubMed Central

    Sohn, Sook-Young

    2016-01-01

    ABSTRACT The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system. PMID:27651358

  19. Expression of potato RNA-binding proteins StUBA2a/b and StUBA2c induces hypersensitive-like cell death and early leaf senescence in Arabidopsis

    PubMed Central

    Na, Jong-Kuk; Kim, Jae-Kwang; Kim, Dool-Yi; Assmann, Sarah M.

    2015-01-01

    The Arabidopsis thaliana genome encodes three RNA-binding proteins (RBPs), UBP1-associated protein 2a (UBA2a), UBA2b, and UBA2c, that contain two RNA-recognition motif (RRM) domains. They play important roles in wounding response and leaf senescence, and are homologs of Vicia faba abscisic-acid-activated protein kinase-interacting protein 1 (VfAKIP1). The potato (Solanum tuberosum) genome encodes at least seven AKIP1-like RBPs. Here, two potato RBPs have been characterized, StUBA2a/b and StUBA2c, that are homologous to VfAKIP1 and Arabidopsis UBA2s. Transient expression of StUBA2s induced a hypersensitive-like cell death phenotype in tobacco leaves, and an RRM-domain deletion assay of StUBA2s revealed that the first RRM domain is crucial for the phenotype. Unlike overexpression of Arabidopsis UBA2s, constitutive expression of StUBA2a/b in Arabidopsis did not cause growth arrest and lethality at the young seedling stage, but induced early leaf senescence. This phenotype was associated with increased expression of defence- and senescence-associated genes, including pathogen-related genes (PR) and a senescence-associated gene (SAG13), and it was aggravated upon flowering and ultimately resulted in a shortened life cycle. Leaf senescence of StUBA2a/b Arabidopsis plants was enhanced under darkness and was accompanied by H2O2 accumulation and altered expression of autophagy-associated genes, which likely cause cellular damage and are proximate causes of the early leaf senescence. Expression of salicylic acid signalling and biosynthetic genes was also upregulated in StUBA2a/b plants. Consistent with the localization of UBA2s-GFPs and VfAKIP1-GFP, soluble-modified GFP-StUBA2s localized in the nucleus within nuclear speckles. StUBA2s potentially can be considered for transgenic approaches to induce potato shoot senescence, which is desirable at harvest. PMID:25944928

  20. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  1. Functional analysis of chloroplast early light inducible proteins (ELIPs)

    SciTech Connect

    Wetzel, Carolyn M

    2005-02-22

    The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identified and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.

  2. Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

    PubMed Central

    Heston, Lee; El-Guindy, Ayman; Countryman, Jill; Dela Cruz, Charles; Delecluse, Henri-Jacques; Miller, George

    2006-01-01

    The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding

  3. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  4. In situ expression of heat-shock proteins and 3-nitrotyrosine in brains of young rats exposed to a WiFi signal in utero and in early life.

    PubMed

    Aït-Aïssa, Saliha; de Gannes, Florence Poulletier; Taxile, Murielle; Billaudel, Bernard; Hurtier, Annabelle; Haro, Emmanuelle; Ruffié, Gilles; Athané, Axel; Veyret, Bernard; Lagroye, Isabelle

    2013-06-01

    The bioeffects of exposure to Wireless High-Fidelity (WiFi) signals on the developing nervous systems of young rodents was investigated by assessing the in vivo and in situ expression levels of three stress markers: 3-Nitrotyrosine (3-NT), an oxidative stress marker and two heat-shock proteins (Hsp25 and Hsp70). These biomarkers were measured in the brains of young rats exposed to a 2450 MHz WiFi signal by immunohistochemistry. Pregnant rats were first exposed or sham exposed to WiFi from day 6 to day 21 of gestation. In addition three newborns per litter were further exposed up to 5 weeks old. Daily 2-h exposures were performed blind in a reverberation chamber and whole-body specific absorption rate levels were 0, 0.08, 0.4 and 4 W/kg. 3-NT and stress protein expression was assayed in different areas of the hippocampus and cortex. No significant difference was observed among exposed and sham-exposed groups. These results suggest that repeated exposure to WiFi during gestation and early life has no deleterious effects on the brains of young rats.

  5. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  6. Evolution, diversification, and expression of KNOX proteins in plants

    PubMed Central

    Gao, Jie; Yang, Xue; Zhao, Wei; Lang, Tiange; Samuelsson, Tore

    2015-01-01

    The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification. PMID:26557129

  7. Protein Expression Dynamics During Postnatal Mouse Brain Development

    PubMed Central

    Laeremans, Annelies; Van de Plas, Babs; Clerens, Stefan; Van den Bergh, Gert; Arckens, Lutgarde; Hu, Tjing-Tjing

    2013-01-01

    We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation. PMID:25157209

  8. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  9. A-TWinnipeg: Pathogenesis of rare ATM missense mutation c.6200C>A with decreased protein expression and downstream signaling, early-onset dystonia, cancer, and life-threatening radiotoxicity.

    PubMed

    Nakamura, Kotoka; Fike, Francesca; Haghayegh, Sara; Saunders-Pullman, Rachel; Dawson, Angelika J; Dörk, Thilo; Gatti, Richard A

    2014-07-01

    We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important.

  10. Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins

    PubMed Central

    Lisak, Robert P; Benjamins, Joyce A; Bealmear, Beverly; Nedelkoska, Liljana; Yao, Bin; Land, Susan; Studzinski, Diane

    2007-01-01

    Background In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells. Methods To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M) on CNS glia after 6 hours of treatment. Results In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells. Conclusion Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system. PMID:18088439

  11. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  12. Cytomegalovirus immediate early proteins promote stemness properties in glioblastoma

    PubMed Central

    Soroceanu, Liliana; Matlaf, Lisa; Khan, Sabeena; Akhavan, Armin; Singer, Eric; Bezrookove, Vladimir; Decker, Stacy; Ghanny, Saleena; Hadaczek, Piotr; Bengtsson, Henrik; Ohlfest, John; Luciani-Torres, Maria-Gloria; Harkins, Lualhati; Perry, Arie; Guo, Hong; Soteropoulos, Patricia; Cobbs, Charles S

    2015-01-01

    Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with onconcogenic properties. Here, we show how HCMV IE are preferentially expressed in glioma stem-like cells (GSC), where they co-localize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSC that are endogenously infected with HCMV, attenuating IE expression by an RNA-i-based strategy, was sufficient to inhibit tumorsphere formation, Sox2 expression, cell cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSC elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSC, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and pro-inflammatory cytokines, resembling the therapeutically-resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miRNA-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSC infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared to mock-infected human GSC. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in glioblastoma cells. PMID:26239477

  13. Contingency Table Browser − prediction of early stage protein structure

    PubMed Central

    Kalinowska, Barbara; Krzykalski, Artur; Roterman, Irena

    2015-01-01

    The Early Stage (ES) intermediate represents the starting structure in protein folding simulations based on the Fuzzy Oil Drop (FOD) model. The accuracy of FOD predictions is greatly dependent on the accuracy of the chosen intermediate. A suitable intermediate can be constructed using the sequence-structure relationship information contained in the so-called contingency table − this table expresses the likelihood of encountering various structural motifs for each tetrapeptide fragment in the amino acid sequence. The limited accuracy with which such structures could previously be predicted provided the motivation for a more indepth study of the contingency table itself. The Contingency Table Browser is a tool which can visualize, search and analyze the table. Our work presents possible applications of Contingency Table Browser, among them − analysis of specific protein sequences from the point of view of their structural ambiguity. PMID:26664034

  14. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

    Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

  15. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  16. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  17. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  18. Heart Failure Protein May Signal Early Brain Damage

    MedlinePlus

    ... 162447.html Heart Failure Protein May Signal Early Brain Damage Higher levels indicated potential trouble, study showed ... a specific heart disease protein are associated with brain damage, a new study suggests. N-terminal Pro- ...

  19. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

    PubMed Central

    Stenberg, R M; Fortney, J; Barlow, S W; Magrane, B P; Nelson, J A; Ghazal, P

    1990-01-01

    trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression. Images PMID:2157043

  20. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  1. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  2. Streamlined expressed protein ligation using split inteins.

    PubMed

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  3. Gene Expression Changes and Early Events in Cotton Fibre Development

    PubMed Central

    Lee, Jinsuk J.; Woodward, Andrew W.; Chen, Z. Jeffrey

    2007-01-01

    Background Cotton is the dominant source of natural textile fibre and a significant oil crop. Cotton fibres, produced by certain species in the genus Gossypium, are seed trichomes derived from individual cells of the epidermal layer of the seed coat. Cotton fibre development is delineated into four distinct and overlapping developmental stages: fibre initiation, elongation, secondary wall biosynthesis and maturation. Scope Recent advances in gene expression studies are beginning to provide new insights into a better understanding of early events in cotton fibre development. Fibre cell development is a complex process involving many pathways, including various signal transduction and transcriptional regulation components. Several analyses using expressed sequence tags and microarray have identified transcripts that preferentially accumulate during fibre development. These studies, as well as complementation and overexpression experiments using cotton genes in arabidopsis and tobacco, indicate some similar molecular events between trichome development from the leaf epidermis and fibre development from the ovule epidermis. Specifically, MYB transcription factors regulate leaf trichome development in arabidopsis and may regulate seed trichome development in cotton. In addition, transcript profiling and ovule culture experiments both indicate that several phytohormones and other signalling pathways mediate cotton fibre development. Auxin and gibberellins promote early stages of fibre initiation; ethylene- and brassinosteroid-related genes are up-regulated during the fibre elongation phase; and genes associated with calmodulin and calmodulin-binding proteins are up-regulated in fibre initials. Additional genomic data, mutant and functional analyses, and genome mapping studies promise to reveal the critical factors mediating cotton fibre cell development. PMID:17905721

  4. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  5. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  6. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  7. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    PubMed

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our

  8. PARP-1 protein expression in glioblastoma multiforme

    PubMed Central

    Galia, A.; Calogero, A.E.; Condorelli, R.A.; Fraggetta, F.; La Corte, C.; Ridolfo, F.; Bosco, P.; Castiglione, R.; Salemi, M.

    2012-01-01

    One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

  9. Soluble expression and complex formation of proteins required for HCMV DNA replication using the SFV expression system.

    PubMed

    McCue, L A; Anders, D G

    1998-08-01

    Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms-a precursor and a product-indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.

  10. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    PubMed

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  11. Sphedgehog is expressed by pigment cell precursors during early gastrulation in Strongylocentrotus purpuratus.

    PubMed

    Egaña, Ana L; Ernst, Susan G

    2004-10-01

    We have sequenced the Sphedgehog (Sphh) gene from the sea urchin Strongylocentrotus purpuratus. Sphh transcripts are detected first at the mesenchyme blastula stage, and they accumulate throughout early embryogenesis. The Sphh protein is produced by precursor pigment cells during early and midgastrulation. NiCl2 inhibits pigment cell differentiation in sea urchins. Here, we show that, in S. purpuratus, nickel affects a process(es) between 17 and 24 hr of development, corresponding to the time period when Sphh mRNA is first detected. However, nickel treatment does not alter the early expression of Sphh.

  12. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  13. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    PubMed

    Miranda, Alberto; Pericuesta, Eva; Ramírez, Miguel Ángel; Gutierrez-Adan, Alfonso

    2011-04-04

    Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  14. Analysis on Gene Expression Profile in Oncospheres and Early Stage Metacestodes from Echinococcus multilocularis

    PubMed Central

    Dang, Zhisheng; Suzuki, Yutaka; Horiuchi, Terumi; Yagi, Kinpei; Kouguchi, Hirokazu; Irie, Takao; Kim, Kyeongsoon; Oku, Yuzaburo

    2016-01-01

    Alveolar echinococcosis is a worldwide zoonosis of great public health concern. Analysis of genome data for Echinococcus multilocularis has identified antigen families that can be used in diagnostic assays and vaccine development. However, little gene expression data is available for antigens of the egg and early larval stages. To address this information gap, we used a Next-Generation Sequencing approach to investigate three different stages (non-activated and activated oncospheres, and early stage metacestodes) of E. multilocularis (Nemuro strain). Transcriptome data analysis revealed that some diagnostic antigen gp50 isoforms and the antigen Eg95 family dominated in activated oncospheres, and the antigen B family dominated in early stage metacestodes. Furthermore, heat shock proteins and antigen II/3 are constantly expressed in the three stages. The expression pattern of various known antigens in E. multilocularis may give fundamental information for choosing candidate genes used in diagnosis and vaccine development. PMID:27092774

  15. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  16. Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

    2010-02-15

    Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing.

  17. Expression and regulation of androgen receptor in the mouse uterus during early pregnancy and decidualization.

    PubMed

    Xu, Jingjie; Li, Mo; Zhang, Lu; Xiong, Hao; Lai, Lidan; Guo, Meijun; Zong, Teng; Zhang, Dalei; Yang, Bei; Wu, Lei; Tang, Min; Kuang, Haibin

    2015-11-01

    The androgen receptor (AR) is a ligand-activated transcription factor that is important for both the male and female reproductive systems. The expression and regulation of AR in the uterine endometrium during early pregnancy and decidualization remain relatively under-investigated, so we sought to immunohistochemically examine the spatiotemporal expression of AR in mouse uteri during the peri-implantation period as well as in response to specific steroid hormones. AR protein was found in the nuclei of uterine stromal cells starting on pregnancy Days 1 and 2, with its abundance increasing on Days 3 and 4. From pregnancy Days 5 to 9, however, the expression of AR markedly declined in stromal zones of uteri. No signal was detected in the decidualized cells surrounding the site of embryo implantation; moreover, no AR immunostaining was observed in decidualized uterine cells in an artificial oil-induced model of decidualization. Progesterone significantly inhibited AR protein expression, whereas estrogen dramatically elevated AR abundance in the stroma of ovariectomized mouse uteri. Taken together, our results are the first to demonstrate that decidualization and progesterone significantly inhibited the AR protein expression in vivo, whereas estrogen increased AR protein levels in the stromal cells of mouse uteri. These responses might be advantageous for the proliferation and differentiation of uterine stroma and for embryo implantation during early pregnancy.

  18. AGL15, a MADS domain protein expressed in developing embryos.

    PubMed Central

    Heck, G R; Perry, S E; Nichols, K W; Fernandez, D E

    1995-01-01

    To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo. PMID:7549483

  19. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    PubMed

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

  20. Wheat germ systems for cell-free protein expression.

    PubMed

    Harbers, Matthias

    2014-08-25

    Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

  1. Trypanosoma cruzi expresses diverse repetitive protein antigens.

    PubMed Central

    Hoft, D F; Kim, K S; Otsu, K; Moser, D R; Yost, W J; Blumin, J H; Donelson, J E; Kirchhoff, L V

    1989-01-01

    We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship. Images PMID:2659529

  2. Gene Expression Profiling Reveals Early Cellular Responses to Intracellular Magnetic Labeling with Superparamagnetic Iron Oxide Nanoparticles

    PubMed Central

    Kedziorek, Dorota A.; Muja, Naser; Walczak, Piotr; Ruiz-Cabello, Jesus; Gilad, Assaf A.; Jie, Chunfa C.; Bulte, Jeff W. M.

    2010-01-01

    With MRI (stem) cell tracking having entered the clinic, studies on the cellular genomic response toward labeling are warranted. Gene expression profiling was applied to C17.2 neural stem cells following superparamagnetic iron oxide/PLL (poly-L-lysine) labeling over the course of 1 week. Relative to unlabeled cells, less than 1% of genes (49 total) exhibited greater than 2-fold difference in expression in response to superparamagnetic iron oxide/PLL labeling. In particular, transferrin receptor 1 (Tfrc) and heme oxygenase 1 (Hmox1) expression was downregulated early, whereas genes involved in lysosomal function (Sulf1) and detoxification (Clu, Cp, Gstm2, Mgst1) were upregulated at later time points. Relative to cells treated with PLL only, cells labeled with superparamagnetic iron oxide/PLL complexes exhibited differential expression of 1399 genes. Though these differentially expressed genes exhibited altered expression over time, the overall extent was limited. Gene ontology analysis of differentially expressed genes showed that genes encoding zinc-binding proteins are enriched after superparamagnetic iron oxide/PLL labeling relative to PLL only treatment, whereas members of the apoptosis/ programmed cell death pathway did not display increased expression. Overexpression of the differentially expressed genes Rnf138 and Abcc4 were confirmed by quantitative real-time polymerase chain reaction. These results demonstrate that, although early reactions responsible for iron homeostasis are induced, overall neural stem cell gene expression remains largely unaltered following superparamagnetic iron oxide/PLL labeling. PMID:20373404

  3. Early Gene Expression Changes in the Retinal Ganglion Cell Layer of a Rat Glaucoma Model

    PubMed Central

    Guo, Ying; Johnson, Elaine C.; Cepurna, William O.; Dyck, Jennifer A.; Doser, Tom

    2011-01-01

    Purpose. To identify patterns of early gene expression changes in the retinal ganglion cell layer (GCL) of a rodent model of chronic glaucoma. Methods. Prolonged elevation of intraocular pressure (IOP) was produced in rats by episcleral vein injection of hypertonic saline (N = 30). GCLs isolated by laser capture microdissection were grouped by grading of the nerve injury (<25% axon degeneration for early injury; >25% for advanced injury). Gene expression was determined by cDNA microarray of independent GCL RNA samples. Quantitative PCR (qPCR) was used to further examine the expression of selected genes. Results. By array analysis, 533 GCL genes (225 up, 308 down) were significantly regulated in early injury. Compared to only one major upregulated gene class of metabolism regulation, more were downregulated, including mitochondria, ribosome, proteasome, energy pathways, protein synthesis, protein folding, and synaptic transmission. qPCR confirmed an early upregulation of Atf3. With advanced injury, 1790 GCL genes were significantly regulated (997 up, 793 down). Altered gene categories included upregulated protein synthesis, immune response, and cell apoptosis and downregulated dendrite morphogenesis and axon extension. Of all the early changed genes, 50% were not present in advanced injury. These uniquely affected genes were mainly associated with upregulated transcription regulation and downregulated protein synthesis. Conclusions. Early GCL gene responses to pressure-induced injury are characterized by an upregulation of Atf3 and extensive downregulation in genes associated with cellular metabolism and neuronal functions. Most likely, these changes represent those specific to RGCs and are thus potentially important for enhancing RGC survival in glaucoma. PMID:21051717

  4. Silencing myotubularin related protein 7 enhances proliferation and early differentiation of C2C12 myoblast.

    PubMed

    Yuan, Zhuning; Chen, Yaosheng; Zhang, Xumeng; Zhou, Xingyu; Li, Mingsen; Chen, Hu; Wu, Ming; Zhang, Ying; Mo, Delin

    2017-03-11

    Myotubularin related protein 7 (MTMR7) is a key member of the highly conserved myotubularin related proteins (MTMRs) family, which has phosphatase activity. MTMR7 was increased during myoblast differentiation and exhibited high expression level at primary fibers formation stages in pigs. This suggests that MTMR7 may be involved in myogenesis. In our study, we investigated the roles of MTMR7 on proliferation and differentiation of C2C12 myoblasts. Knocking down MTMR7 not only enhanced myoblast early differentiation via altering the expression of Myf5, but also promoted myoblast proliferation through increasing cyclinA2 expression. The improved proliferation capacity was related to the increased phosphorylation of AKT. Taken together, our research demonstrates that MTMR7 plays an important role in proliferation and early differentiation of C2C12 myoblast.

  5. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  6. Expression of Glucocorticoid Receptor and Early Growth Response Gene 1 during Postnatal Development of Two Inbred Strains of Mice Exposed to Early Life Stress

    PubMed Central

    Navailles, Sylvia; Zimnisky, Ross; Schmauss, Claudia

    2010-01-01

    Early life stress can elicit profound changes in adult gene expression and behavior. One consequence of early life stress is a decreased expression of glucocorticoid receptors (GRs) in the frontal cortex and hippocampus. However, neither the time of onset nor the mechanism(s) leading to decreased GR expression during postnatal development are known. The present study used two inbred strains of mice that differ in their behavioral responsiveness to stress (Balb/c and C57Bl/6), exposed them to an established paradigm of early life stress (infant maternal separation), and measured their expression of frontal cortical and hippocampal GRs and the putative transcriptional activator of the GR gene, early growth response gene (egr)-1, at defined stages of postnatal development. In both strains, real-time RT-PCR experiments revealed that decreased expression of GR in adolescence and adulthood is, in fact, preceded by increased GR expression during early life stress exposure. Thus, the early life stress-induced disruption of the normal stress-hyporesponsive period during infancy is accompanied by increased GR expression. Moreover, chronic treatment with the antidepressant drug fluoxetine during adolescence or adulthood reversed the effect of early life stress on adult GR mRNA expression. In contrast to the strain-independent effect of early life stress on GR expression, however, changes in egr-1 expression occurred only in Balb/c mice, and unlike the biphasic developmental changes in GR mRNA expression, egr-1 mRNA was decreased throughout postnatal development. Moreover, there was no consistent overlap of anatomic regions affected by decreased GR and egr-1 protein expression. Thus, in Balb/c mice, changes in GR and egr-1 expression can independently contribute to the phenotypes resulting from early life stress exposure. These findings illustrate that the impact of early life stress on gene expression changes is modulated by the genetic background and that the persistent

  7. Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditions

    PubMed Central

    Da Silva, Carla Alexandra; Kassel, Olivier; Lebouquin, Renaud; Lacroix, Emmanuel Jean; Frossard, Nelly

    2003-01-01

    Stem cell factor (SCF) is a major growth factor for mast cells, promoting their differentiation and chemotaxis. Its expression is regulated by glucocorticoids in inflammatory conditions, showing an early increased protein expression, before the expected anti-inflammatory decrease (Da Silva et al., Br. J. Pharmacol. 2002:135,1634). We here evaluated the early kinetic of SCF expression regulated by interleukin (IL)-1β, budesonide and the combination of both in human lung fibroblasts in culture. Budesonide potentiated the IL-1β-enhanced expression of SCF mRNA (+103%) and protein (+98%) very shortly after treatment (at 30 min and 1 h, respectively). A gentle downregulation followed. This potentiating effect of budesonide was related to increased SCF mRNA stability and SCF gene transcription. Deletion of a κB-like site that we identified in the first intron of the SCF gene, in a luciferase reporter system, abolished the potentiation by budesonide, as well as the effect of IL-1β alone, as compared to the wild-type construction activity. All budesonide-induced effects were glucocorticoid-receptor dependent, since they were reproduced by dexamethasone and blocked by RU486. IL-1β+budesonide did not affect the relative expression of the soluble and membrane-bound forms of SCF. In conclusion, our results clearly show that glucocorticoids act very early to adversely increase the expression of SCF mRNA and protein in the inflammatory conditions created by IL-1β, and that this effect involves increased mRNA stability and increased gene expression through activation of the NF-κB-like responsive element. PMID:14662725

  8. Increased expression of neutrophil-related genes in patients with early sepsis-induced ARDS.

    PubMed

    Kangelaris, Kirsten Neudoerffer; Prakash, Arun; Liu, Kathleen D; Aouizerat, Bradley; Woodruff, Prescott G; Erle, David J; Rogers, Angela; Seeley, Eric J; Chu, Jeffrey; Liu, Tom; Osterberg-Deiss, Thomas; Zhuo, Hanjing; Matthay, Michael A; Calfee, Carolyn S

    2015-06-01

    The early sequence of events leading to the development of the acute respiratory distress syndrome (ARDS) in patients with sepsis remains inadequately understood. The purpose of this study was to identify changes in gene expression early in the course of illness, when mechanisms of injury may provide the most relevant treatment and prognostic targets. We collected whole blood RNA in critically ill patients admitted from the Emergency Department to the intensive care unit within 24 h of admission at a tertiary care center. Whole genome expression was compared in patients with sepsis and ARDS to patients with sepsis alone. We selected genes with >1 log2 fold change and false discovery rate <0.25, determined their significance in the literature, and performed pathway analysis. Several genes were upregulated in 29 patients with sepsis with ARDS compared with 28 patients with sepsis alone. The most differentially expressed genes included key mediators of the initial neutrophil response to infection: olfactomedin 4, lipocalin 2, CD24, and bactericidal/permeability-increasing protein. These gene expression differences withstood adjustment for age, sex, study batch, white blood cell count, and presence of pneumonia or aspiration. Pathway analysis demonstrated overrepresentation of genes involved in known respiratory and infection pathways. These data indicate that several neutrophil-related pathways may be involved in the early pathogenesis of sepsis-related ARDS. In addition, identifiable gene expression differences occurring early in the course of sepsis-related ARDS may further elucidate understanding of the neutrophil-related mechanisms in progression to ARDS.

  9. Expression profiling of the mouse early embryo: Reflections and Perspectives

    PubMed Central

    Ko, Minoru S. H.

    2008-01-01

    Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

  10. Gene Expression Analysis of Sporadic Early-Onset Rectal Adenocarcinoma

    PubMed Central

    Nfonsam, V; Xu, W; Koblinski, J; Jandova, J

    2016-01-01

    Background Overall declines in incidence of rectal cancer (RC) in patients older than 50 years have been mostly attributed to improvement in treatment modalities and introduction of age-based screening. Recent studies, however, have shown a rise in the incidence of RC in patients younger than 50 years. The etiology of early-onset (EO) RC is not well understood. The aim of this study is to elucidate the molecular features of (EO) RC and show its uniqueness compared to late-onset (LO) disease. Methods Two cohorts of patients with sporadic RC were identified. Tumors and matching non-involved tissues from six (EO) RC patients (< 50 years) and six (LO) RC patients (>65 years) were obtained from Pathology archives. Deparaffinized tissues were macro-dissected from FFPE sections, RNA isolated and used for expression profiling of 770 cancer related genes representing 13 canonical pathways. Statistical analysis was performed using the Gene Expression R-script module within the nCounter software v2.6. A gene was considered to be above background if the average count for the target gene was greater than the average counts for the eight negative control genes and if the P value of the t-test was less than 0.05. Results When we compared rectal tumors to non-involved rectal tissues, changes in expression levels of 171 genes were statistically significant in early-onset group and 151 genes in late-onset group. Further comparative gene expression analysis between early- and late-onset rectal tumors normalized to their matching non-involved tissues revealed that changes in expression of 65 genes were unique to early-onset rectal tumors with 16 genes being up- and 49 genes down-regulated using the cutoff criteria of expression levels difference >2 fold and p-value <0.01. At the pathway level, MAPK signaling was the most deregulated pathway in early-onset rectal tumors compared to PI3K-AKT signaling pathway being the most deregulated in late-onset rectal tumors. Conclusions Results of

  11. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  12. Protein-guided RNA dynamics during early ribosome assembly.

    PubMed

    Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2014-02-20

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  13. Protein-guided RNA dynamics during early ribosome assembly

    PubMed Central

    Kim, Hajin; Abeysirigunawardena, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-01-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. PMID:24522531

  14. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  15. Dynamic changes in stanniocalcin gene expression in the mouse uterus during early implantation.

    PubMed

    Stasko, S E; DiMattia, G E; Wagner, G F

    2001-03-28

    Blastocyst implantation is accompanied by dramatic changes in gene expression to facilitate decidualization and remodelling of uterine architecture. Stanniocalcin (STC) is a new mammalian polypeptide hormone with roles in ion transport, reproduction and development. Here we report dynamic changes in STC mRNA and protein distributions in the early post-implantation mouse uterus. In the non-pregnant state, STC gene expression was confined to the uterine lumenal epithelium. Following implantation STC gene expression shifted to mesometrial stromal cells bordering the uterine lumen. Between E6.5-E8.5 expression shifted once more to cells of the mesometrial lateral sinusoids, and then declined thereafter. Intriguingly immunoreactive STC did not entirely co-localize with areas of high STC gene activity and instead appeared to accumulate in presumptive targets of the hormone (uterine epithelium, stromal and decidual cells, trophoblastic giant cells). STC is only the fourth gene identified as being expressed mesometrially in the uterus following implantation.

  16. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

    PubMed

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

  17. Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

    PubMed Central

    Sauvegarde, Caroline; Paul, Delphine; Bridoux, Laure; Jouneau, Alice; Degrelle, Séverine; Hue, Isabelle; Rezsohazy, René; Donnay, Isabelle

    2016-01-01

    Background We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. Results The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. Conclusions Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals. PMID:27798681

  18. Comparative Transcriptome Analysis Reveals Early Pregnancy-Specific Genes Expressed in Peripheral Blood of Pregnant Sows

    PubMed Central

    Zhu, Shien; Shi, Wenqing; Hu, Maishun; Fu, Xiangwei; Wang, Chuduan; Wang, Yachun; Zhang, Qin; Yu, Ying

    2014-01-01

    Early and accurate diagnosis of pregnancy is important for effective management of an economical pig farm. Besides the currently available methods used in early diagnosis of sows, circulating nucleic acids in peripheral blood may contain some early pregnancy-specific molecular markers. For the first time, microarray analysis of peripheral blood from pregnant sows versus non-pregnant sows identified 127 up-regulated and 56 down-regulated genes at day 14 post-insemination. Gene Ontology annotation grouped the total differently expressed genes into 3 significantly enriched terms, cell surface receptor linked signal transduction, G-protein coupled receptor protein signaling pathway and regulation of vesicle-mediated transport. Signaling pathway analysis revealed the only one significantly changed pathway was arachidonic acid metabolism. Of the differently expressed genes, nine (including LPAR3, RXFP4, GALP, CBR1, CBR2, GPX6, USP18, LHB and NR5A1) were found to exert function related to early pregnancy processes. This study provides a clue that differentially abundant RNAs in maternal peripheral blood can help to identify the molecular markers of early pregnancy in pigs. PMID:25479131

  19. Effects of immunosuppressive treatment on protein expression in rat kidney

    PubMed Central

    Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

    2014-01-01

    The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

  20. Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation.

    PubMed

    Tada, H; Lashgari, M; Rappaport, J; Khalili, K

    1989-01-01

    We analyzed control sequences of the human papovavirus JC virus (JCV) to define the cis-acting elements that regulate specific expression of the viral early region genes in glial cells. Nuclear run-on transcription, S1 analysis, and chloramphenicol acetyltransferase enzyme activity in a transient transfection assay established that the cell type-specific expression of JCV early genes is determined at the transcriptional level. Using DNase footprinting analysis of nuclear proteins prepared from glial and nonglial cells, we located four regions within the JCV control sequences that specifically interacted with the proteins. In glial cells, all four domains contributed to the specific expression of a heterologous promoter, whereas in nonglial cells, two protein-binding regions showed no effect on basal transcriptional activity and the other two domains significantly downregulated transcription of the promoter. We conclude that cell type-specific transcription of the JCV early promoter is under both positive and negative regulation in eucaryotic cells.

  1. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  2. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  3. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  4. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  5. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins.

  6. Expression of the orexin system in the porcine uterus, conceptus and trophoblast during early pregnancy.

    PubMed

    Smolinska, N; Kiezun, M; Dobrzyn, K; Szeszko, K; Maleszka, A; Kaminski, T

    2015-11-01

    Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.

  7. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

    2005-01-01

    Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

  8. SSAO/VAP-1 protein expression during mouse embryonic development.

    PubMed

    Valente, Tony; Solé, Montse; Unzeta, Mercedes

    2008-09-01

    SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation.

  9. Early Impact of Fontan Operation on Enteric Protein Loss

    PubMed Central

    Patel, Jyoti K.; Loomes, Kathleen M.; Goldberg, David J.; Mercer-Rosa, Laura; Dodds, Kathryn; Rychik, Jack

    2016-01-01

    Background Protein losing enteropathy (PLE) is a challenging complication after Fontan operation. Subclinical enteric protein loss may precede development of overt PLE. We evaluated the acute effects of Fontan circulation on enteric protein loss and mesenteric vascular resistance. Methods A prospective cohort study was performed evaluating enteric protein loss in children undergoing Fontan operation. Stool alpha-1-antitrypsin (A1AT) concentration was measured in the pre-operative, early post-operative, and intermediate post-operative (3–9 months) periods. The intestinal circulation was characterized by Doppler-derived resistance indices of the superior mesenteric artery, and serum albumin and protein levels were obtained. Results We enrolled 33 subjects at a median age at operation of 3.0 (2.5–3.3) years. No clinical PLE was observed. Six of the 93 stool A1AT samples obtained were elevated (>54 mg/dl), with two abnormal samples at each of the three time points. Two of the five subjects with elevated stool A1AT values had significant hemodynamic disturbances requiring intervention (junctional bradycardia or tricuspid stenosis). There was no difference in superior mesenteric artery resistance in the pre-operative versus early post-operative period (p=0.9). Serum albumin levels were lower in the early post-operative period compared to the pre-operative period (3.2 mg/dl [IQR 2.9–3.5] vs. 4.1 mg/dl [IQR 3.4–4.5], p=0.01) but did not correlate with abnormal stool A1AT concentration or superior mesenteric artery resistance indices. Conclusions The Fontan operation does not commonly result in acute development of increased enteric protein loss. However, increased enteric protein loss may occur in children before or after Fontan operation, particularly when hemodynamic disturbances are present. PMID:26652137

  10. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.

    PubMed

    Hayashi, Kokoro; Kojima, Chojiro

    2010-11-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  11. Differential expression of prostaglandin E receptor subtype EP2 in rat uterus during early pregnancy.

    PubMed

    Shi, J J; Ma, X H; Diao, H L; Ni, H; Xu, L B; Zhu, H; Yang, Z M

    2005-10-01

    PGE2 is essential for mammalian female reproduction. This study was to examine the expression of EP2 gene in the rat uterus during early pregnancy, delayed implantation and artificial decidualization by in situ hybridization and immunohistochemistry. There was no detectable EP2 mRNA expression in the uterus from days 1 to 4 of pregnancy (day 1 = day of vaginal sperm). A low level of EP2 immunostaining was observed in the luminal and glandular epithelium from days 1 to 4 of pregnancy. Both EP2 mRNA and protein expression were highly detected in the luminal epithelium at implantation sites on day 6 of pregnancy. EP2 expression decreased from day 7 of pregnancy and was undetectable on days 8 and 9 of pregnancy. After delayed implantation was terminated by estrogen treatment and the embryo implanted, both EP2 mRNA and protein expression were strongly observed in the luminal epithelium at the implantation site. There was no detectable EP2 expression in both control and decidualized uteri. In conclusion, these data suggest that EP2 expression at implantation site may play an important role during embryo implantation in rats.

  12. Altered representation of facial expressions after early visual deprivation

    PubMed Central

    Gao, Xiaoqing; Maurer, Daphne; Nishimura, Mayu

    2013-01-01

    We investigated the effects of early visual deprivation on the underlying representation of the six basic emotions. Using multi-dimensional scaling (MDS), we compared the similarity judgments of adults who had missed early visual input because of bilateral congenital cataracts to control adults with normal vision. Participants made similarity judgments of the six basic emotional expressions, plus neutral, at three different intensities. Consistent with previous studies, the similarity judgments of typical adults could be modeled with four underlying dimensions, which can be interpreted as representing pleasure, arousal, potency and intensity of expressions. As a group, cataract-reversal patients showed a systematic structure with dimensions representing pleasure, potency, and intensity. However, an arousal dimension was not obvious in the patient group's judgments. Hierarchical clustering analysis revealed a pattern in patients seen in typical 7-year-olds but not typical 14-year-olds or adults. There was also more variability among the patients than among the controls, as evidenced by higher stress values for the MDS fit to the patients' data and more dispersed weightings on the four dimensions. The findings suggest an important role for early visual experience in shaping the later development of the representations of emotions. Since the normal underlying structure for emotion emerges postnatally and continues to be refined until late childhood, the altered representation of emotion in adult patients suggests a sleeper effect. PMID:24312071

  13. Store-Operated Ca2+ Channels in Mesangial Cells Inhibit Matrix Protein Expression.

    PubMed

    Wu, Peiwen; Wang, Yanxia; Davis, Mark E; Zuckerman, Jonathan E; Chaudhari, Sarika; Begg, Malcolm; Ma, Rong

    2015-11-01

    Accumulation of extracellular matrix derived from glomerular mesangial cells is an early feature of diabetic nephropathy. Ca(2+) signals mediated by store-operated Ca(2+) channels regulate protein production in a variety of cell types. The aim of this study was to determine the effect of store-operated Ca(2+) channels in mesangial cells on extracellular matrix protein expression. In cultured human mesangial cells, activation of store-operated Ca(2+) channels by thapsigargin significantly decreased fibronectin protein expression and collagen IV mRNA expression in a dose-dependent manner. Conversely, inhibition of the channels by 2-aminoethyl diphenylborinate significantly increased the expression of fibronectin and collagen IV. Similarly, overexpression of stromal interacting molecule 1 reduced, but knockdown of calcium release-activated calcium channel protein 1 (Orai1) increased fibronectin protein expression. Furthermore, 2-aminoethyl diphenylborinate significantly augmented angiotensin II-induced fibronectin protein expression, whereas thapsigargin abrogated high glucose- and TGF-β1-stimulated matrix protein expression. In vivo knockdown of Orai1 in mesangial cells of mice using a targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV, and mice showed significant mesangial expansion compared with controls. Similarly, in vivo knockdown of stromal interacting molecule 1 in mesangial cells by recombinant adeno-associated virus-encoded shRNA markedly increased collagen IV protein expression in renal cortex and caused mesangial expansion in rats. These results suggest that store-operated Ca(2+) channels in mesangial cells negatively regulate extracellular matrix protein expression in the kidney, which may serve as an endogenous renoprotective mechanism in diabetes.

  14. Nfix Expression Critically Modulates Early B Lymphopoiesis and Myelopoiesis

    PubMed Central

    O’Connor, Caitríona; Campos, Joana; Osinski, Jason M.; Gronostajski, Richard M.; Michie, Alison M.; Keeshan, Karen

    2015-01-01

    The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification. PMID:25780920

  15. Early changes in corticospinal excitability when seeing fearful body expressions

    PubMed Central

    Borgomaneri, Sara; Vitale, Francesca; Avenanti, Alessio

    2015-01-01

    Quick inhibition of approach tendencies in response to signals of potential threats is thought to promote survival. However, little is known about the effect of viewing fearful expressions on the early dynamics of the human motor system. We used the high temporal resolution of single-pulse and paired-pulse transcranial magnetic stimulation (TMS) over the motor cortex to assess corticospinal excitability (CSE) and intracortical facilitation (ICF) during observation of happy, fearful and neutral body postures. To test motor circuits involved in approach tendencies, CSE and ICF were recorded from the first dorsal interosseous (FDI), a muscle involved in grasping, and the abductor pollicis brevis (APB), which served as a control. To test early motor dynamics, CSE and ICF were measured 70–90 ms after stimulus onset. We found a selective reduction in CSE in the FDI when participants observed fearful body expressions. No changes in ICF or in the excitability of APB were detected. Our study establishes an extremely rapid motor system reaction to observed fearful body expressions. This motor modulation involves corticospinal downstream projections but not cortical excitatory mechanisms, and appears to reflect an inhibition of hand grasping. Our results suggest a fast visuo-motor route that may rapidly inhibit inappropriate approaching actions. PMID:26388400

  16. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  17. Elevated nuclear S100P expression is associated with poor survival in early breast cancer patients.

    PubMed

    Maciejczyk, Adam; Łacko, Aleksandra; Ekiert, Marcin; Jagoda, Ewa; Wysocka, Teresa; Matkowski, Rafał; Hałoń, Agnieszka; Györffy, Balázs; Lage, Hermann; Surowiak, Paweł

    2013-04-01

    S100P - low molecular weight acidic protein has been shown to be involved in processes of proliferation, survival, angiogenesis, multidrug resistance and metastasis in various human malignancies. In breast cancer, S100P expression is associated with immortalization of neoplastic cells and aggressive tumour behaviour, indicating that this protein may have adverse prognostic value. We analyzed nuclear and cytoplasmic expression of S100P in 85 stage II breast cancer patients with a median follow up of 17 years. Immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against S100P. We also studied prognostic value of S100P mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. Moreover, the relationship was examined between expression of S100P in cells of four breast cancer cell lines and their sensitivity to the 11 most frequently applied cytotoxic drugs. Univariate and multivariate analyses showed that higher expression of nuclear S100P (S100Pn) was typical for cases of a shorter overall survival and disease-free time. KM plotter analysis showed that elevated S100P expression was specific for cases of a relapse-free survival and distant metastases-free survival. No relationship could be documented between expression of S100P and sensitivity of breast cancer cells to cytostatic drugs. We demonstrated that a high S100Pn expression level was associated with poor survival in early stage breast cancer patients. Since preliminary data indicated that expression of S100P was up-regulated by activation of glucocorticoid receptor and several agents manifested potential to activate or inhibit S100P promoter activity, this protein might become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value.

  18. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  19. Cell-free protein synthesis as a promising expression system for recombinant proteins.

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

    Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

  20. Xenopus homeobox-containing cDNAs expressed in early development.

    PubMed Central

    Fritz, A; De Robertis, E M

    1988-01-01

    We report the isolation of six different homeobox-containing genes in Xenopus laevis which are expressed during early embryogenesis. cDNA clones of all of them were partially sequenced including two from previously isolated Xenopus genomic loci and found to contain homeodomains which share a high degree of homology with the Antennapedia protein (50 to 59 out of 60 amino acids). We find a short region of homology (consensus Ile Tyr Pro Trp Met) in four of the cDNAs, which is also present in Antennapedia and that may correspond to a bend region preceding the homeodomain. Northern blots have been performed to show the transcriptional pattern through early frog embryogenesis. Three of the genes are expressed only during a very narrow period of embryogenesis, reinforcing the view that homeobox genes are developmentally regulated. Images PMID:2894634

  1. Translational Control of Protein Synthesis: The Early Years

    PubMed Central

    Lodish, Harvey F.

    2012-01-01

    For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of α- and β-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments. PMID:22988251

  2. Signatures of unfolding in the early stages of protein denaturation

    NASA Astrophysics Data System (ADS)

    Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

    2012-04-01

    A comparative study of the early stages of unfolding of five proteins: cyt c, c-b 562, cyt c‧, azurin, and lysozyme is reported. From crystallographic data, helical regions and intervening non-helical (or 'turning') regions are identified in each. Exploiting a previously introduced geometrical model, the paper describes quantitatively the stepwise extension of a polypeptide chain subject to the geometrical constraint that the spatial relationship among the residues of each triplet is fixed by native-state crystallographic data. Despite differences among the above-cited proteins, remarkable universality of behavior is found in the early stages of unfolding. At the very earliest stages, internal residues in each helical region have a common unfolding history; the terminal residues, however, are extraordinarily sensitive to structural perturbations. Residues in non-helical sections of the polypeptide unfold after residues in the internal helical regions, but with increasing steric perturbation playing a dominant role in advancing denaturation.

  3. Translational control of protein synthesis: the early years.

    PubMed

    Lodish, Harvey F

    2012-10-19

    For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of α- and β-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments.

  4. Computational Simulations of the Early Steps of Protein Aggregation

    PubMed Central

    Wei, Guanghong; Mousseau, Normand

    2007-01-01

    There is strong evidence that the oligomers of key proteins, formed during the early steps of aggregation, could be the primary toxic species associated with human neurodegenerative diseases, such as Alzheimer's and prion diseases. Here, we review recent progress in the development of computational approaches in order to understand the structures, dynamics and free energy surfaces of oligomers. We also discuss possible research directions for the coming years. PMID:19164927

  5. Temporal and spatial expression of AIF in the mouse uterus during early pregnancy.

    PubMed

    Zhang, Li-Ying; Hua, Yu-Ping; An, Tie-Zhu; Zuo, Ru-Juan; Nie, Wei-Tian; Wang, Lian-Bang; Shan, Chun-Hua

    2012-01-01

    Apoptosis-inducing factor (AIF) is a phylogenetically old, bifunctional protein with a pro-apoptotic function and redox activity. AIF regulates apoptosis and also plays a role in the defense against stress depending on its subcellular localization. Embryo implantation is a complicated process, in which an activated blastocyst interacts with a receptive uterus. The expression and regulation of AIF were investigated in this study in the mouse uterus during early pregnancy, pseudopregnancy, delayed implantation, artificial decidualization and under hormonal treatment using in situ hybridization, immunohistochemistry and real-time PCR. During early pregnancy, temporally and spatially regulated patterns of AIF expression were found in the mouse uterus; AIF expression in the luminal epithelium and glandular epithelium is regulated by steroid hormones; AIF mRNA expression in the stroma is influenced by the active blastocyst; and AIF protein was found to be located in the cytoplasm rather than the nucleus through confocal microscope. Our data suggest that AIF might play an important role during mouse embryo implantation and that the role of AIF might be implemented through its physiological activity rather than through its pro-apoptotic function in the mouse uterus during this period.

  6. Characterization of sea urchin unconventional myosins and analysis of their patterns of expression during early embryogenesis.

    PubMed

    Sirotkin, V; Seipel, S; Krendel, M; Bonder, E M

    2000-10-01

    Early sea urchin development requires a dynamic reorganization of both the actin cytoskeleton and cytoskeletal interactions with cellular membranes. These events may involve the activities of multiple members of the superfamily of myosin motor proteins. Using RT-PCR with degenerate myosin primers, we identified 11 myosin mRNAs expressed in unfertilized eggs and coelomocytes of the sea urchin Strongylocentrotus purpuratus. Seven of these sea urchin myosins belonged to myosin classes Igamma, II, V, VI, VII, IX, and amoeboid-type I, and the remaining four may be from novel classes. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I were either completely or partially cloned and their molecular structures characterized. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I shared a high degree of sequence identity with their respective family members from vertebrates and they retained their class-specific structure and domain organization. Analysis of expression of myosin-V, -VI, -VII, and amoeboid-type-I mRNAs during development revealed that each myosin mRNA displayed a distinct temporal pattern of expression, suggesting that myosins might be involved in specific events of early embryogenesis. Interestingly, the onset of gastrulation appeared to be a pivotal point in modulation of myosin mRNA expression. The presence of multiple myosin mRNAs in eggs and embryos provides insight into the potential involvement of multiple specific motor proteins in the actin-dependent events of embryo development.

  7. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  8. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  9. Social isolation stress down-regulates cortical early growth response 1 (Egr-1) expression in mice.

    PubMed

    Matsumoto, Kinzo; Ono, Kazuya; Ouchi, Hirofumi; Tsushima, Ryohei; Murakami, Yukihisa

    2012-07-01

    Social isolation stress induces behavioral disturbances such as aggression, cognitive impairments, and deficits in prepulse inhibition in mice. Social isolation mice have, therefore, been studied as an animal model of neuropsychiatric disorders such as schizophrenia. Recently, the decrease in early growth response (Egr) gene expression levels were reported in the post-mortem brains of schizophrenia patients. In this study, we investigate the effects of social isolation stress on the expression levels of Egr mRNA and protein in the frontal cortex. Social isolation stress exposure significantly down-regulated the expression of Egr-1 protein and Egr-1 gene transcript in nucleus of cortical neurons in a manner dependent on a social isolation period. This stress had no effect on the expression level of Egr-1 in the striatum or the expression levels of other Egr family members (Egr-2, -3, and -4) in the frontal cortex. These results suggest that the decrease in Egr-1 expression in the frontal cortex may be involved in social isolation stress-induced behavioral abnormalities.

  10. Regulation of chick early B-cell factor-1 gene expression in feather development.

    PubMed

    El-Magd, Mohammed Abu; Sayed-Ahmed, Ahmed; Awad, Ashraf; Shukry, Mustafa

    2014-05-01

    The chick Ebf1 (early B-cell factor-1) gene is a member of a novel family of helix loop helix transcription factors. The expression profile, regulation and significance of this gene have been extensively studied in lymphatic, nervous, adipose and muscular tissues. However, cEbf1 expression, regulation and function in the feather of chick embryo have not yet been investigated. cEbf1 expression was first detected throughout the mesenchymal core of some few feather placodes (D7-D7.5). After feathers became mature and grew distally (D9 and D10), the mesenchymal expression of cEbf1 became confined to the caudal margin of the proximal half of all formed feather buds. Because this dynamic pattern of expression resembles that of Sonic Hedgehog (Shh) protein and bone morphogenetic protein (Bmp4) plus the crucial role of these two major signals in feather development, we hypothesized that cEbf1 expression in the feather may be regulated by Shh and Bmp4. In a feather explant culture system, Shh signals are necessary to initiate and maintain cEbf1 expression in the posterior half of the feather bud, while Bmp4 is crucial for the initial cEbf1 expression in the anterior half of the feather bud. Inhibition of Shh, not only down-regulates cEbf1, but also changes the morphology of feather buds, which become irregular and fused. This is the first study to demonstrate that cEbf1 expression in the feather bud is under the control of Shh and Bmp4 signals and that expression may play a role in the normal development of feathers.

  11. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  12. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  13. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  14. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  15. Expression of heat shock protein genes in insect stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  16. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  17. A gene expression atlas of early craniofacial development.

    PubMed

    Brunskill, Eric W; Potter, Andrew S; Distasio, Andrew; Dexheimer, Phillip; Plassard, Andrew; Aronow, Bruce J; Potter, S Steven

    2014-07-15

    We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical microregions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the cranial mesenchyme, composed of mixed neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. At E9.5 cells from the cranial mesenchyme, overlying olfactory placode/epidermal ectoderm, and underlying neuroepithelium, as well as the emerging mandibular and maxillary arches were sampled. At E10.5, as the facial prominences form, cells from the medial and lateral prominences, the olfactory pit, multiple discrete regions of underlying neuroepithelium, the mandibular and maxillary arches, including both their mesenchymal and ectodermal components, as well as Rathke's pouch, were similarly sampled and profiled using both microarray and RNA-seq technologies. Further, we performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm. Taken together, and analyzable by a variety of biological network approaches, these data provide a complementing and cross validating resource capable of fueling discovery of novel compartment specific markers and signatures whose combinatorial interactions of transcription factors and growth factors/receptors are responsible for providing the master genetic blueprint for craniofacial development.

  18. Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

    PubMed

    Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

    2010-12-01

    Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

  19. Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis

    PubMed Central

    1987-01-01

    A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpole parvalbumin is highly similar to previously characterized beta- parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos. PMID:3558484

  20. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  1. Plasma protein insudation as an index of early coronary atherogenesis.

    PubMed Central

    Zhang, Y.; Cliff, W. J.; Schoefl, G. I.; Higgins, G.

    1993-01-01

    Two hundred ninety-nine paraffin-embedded coronary artery blocks from 68 autopsy cases were serially sectioned. The blocks were selected to provide a range from normal through various stages of atherosclerosis, and sections were examined with the indirect immunofluorescence technique for intramural distribution of plasma albumin, fibrinogen, and immunoglobulin gamma (IgG). Cryostat-sections of 44 blocks from 22 of the same cases were examined with the same technique for distribution of apolipoprotein B. Alteration of protein insudation in the artery wall was a sensitive index of coronary atherogenesis. The sequence in which these proteins were involved in the initiation and development of early atherosclerotic lesions was analyzed by determining the average relative intimal thickness and relative lumen size that was associated with the first occurrence of altered insudation of each of these proteins. Results indicate that changed plasma albumin insudation is the earliest sign of a focal intimal lesion, and increasing albumin insudation shows the strongest association with intimal plaque growth. The other proteins tested showed altered insudation, in the order IgG, fibrinogen, apolipoprotein B. The results indicate that a progressive increase in permeability of the coronary artery endothelium occurs in the early stages of atherogenesis. Patterns of IgG localization provide evidence of both early systemic and subsequent local immune reactions being involved in atherogenesis. Altered albumin and apolipoprotein B insudation levels have stronger correlation coefficients with relative intimal thickness and relative lumen size than do those IgG and fibrinogen. The extremely high correlation coefficients shown by albumin emphasizes the importance of edema in determining plaque size and lumen stenosis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8342598

  2. Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

    PubMed

    Fernández-Bussy, Rodrigo; Mouguelar, Valeria; Banchio, Claudia; Coux, Gabriela

    2015-04-01

    In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.

  3. Transient protein expression in three Pisum sativum (green pea) varieties.

    PubMed

    Green, Brian J; Fujiki, Masaaki; Mett, Valentina; Kaczmarczyk, Jon; Shamloul, Moneim; Musiychuk, Konstantin; Underkoffler, Susan; Yusibov, Vidadi; Mett, Vadim

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

  4. Fungal and algal gene expression in early developmental stages of lichen-symbiosis.

    PubMed

    Joneson, Suzanne; Armaleo, Daniele; Lutzoni, François

    2011-01-01

    How plants and microbes recognize each other and interact to form long-lasting relationships remains one of the central questions in cellular communication. The symbiosis between the filamentous fungus Cladonia grayi and the single-celled green alga Asterochloris sp. was used to determine fungal and algal genes upregulated in vitro in early lichen development. cDNA libraries of upregulated genes were created with suppression subtractive hybridization in the first two stages of lichen development. Quantitative PCR subsequently was used to verify the expression level of 41 and 33 candidate fungal and algal genes respectively. Induced fungal genes showed significant matches to genes putatively encoding proteins involved in self and non-self recognition, lipid metabolism, and negative regulation of glucose repressible genes, as well as to a putative d-arabitol reductase and two dioxygenases. Upregulated algal genes included a chitinase-like protein, an amino acid metabolism protein, a dynein-related protein and a protein arginine methyltransferase. These results also provided the first evidence that extracellular communication without cellular contact can occur between lichen symbionts. Many genes showing slight variation in expression appear to direct the development of the lichen symbiosis. The results of this study highlight future avenues of investigation into the molecular biology of lichen symbiosis.

  5. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  6. Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses

    PubMed Central

    Chudotvorova, Ilona; Ivanov, Anton; Rama, Sylvain; Hübner, Christian A; Pellegrino, Christophe; Ben-Ari, Yehezkel; Medina, Igor

    2005-01-01

    The development of GABAergic synapses is associated with an excitatory to inhibitory shift of the actions of GABA because of a reduction of [Cl−]i. This is due to a delayed postnatal expression of the K+–Cl− cotransporter KCC2, which has low levels at birth and peaks during the first few postnatal weeks. Whether the expression of the cotransporter and the excitatory to inhibitory shift have other consequences on the operation of GABAA receptors and synapses is not yet known. We have now expressed KCC2 in immature neurones at an early developmental stage and determined the consequences on the formation of GABA and glutamate synapses. We report that early expression of the cotransporter selectively enhances GABAergic synapses: there is a significant increase of the density of GABAA receptors and synapses and an increase of the frequency of GABAergic miniature postsynaptic currents. The density of glutamate synapses and frequency of AMPA miniature postsynaptic currents are not affected. We conclude that the expression of KCC2 and the reduction of [Cl−]i play a critical role in the construction of GABAergic networks that extends beyond the excitatory to inhibitory shift of the actions of GABA. PMID:15961425

  7. Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses.

    PubMed

    Chudotvorova, Ilona; Ivanov, Anton; Rama, Sylvain; Hübner, Christian A; Pellegrino, Christophe; Ben-Ari, Yehezkel; Medina, Igor

    2005-08-01

    The development of GABAergic synapses is associated with an excitatory to inhibitory shift of the actions of GABA because of a reduction of [Cl-]i. This is due to a delayed postnatal expression of the K+ -Cl- cotransporter KCC2, which has low levels at birth and peaks during the first few postnatal weeks. Whether the expression of the cotransporter and the excitatory to inhibitory shift have other consequences on the operation of GABA(A) receptors and synapses is not yet known. We have now expressed KCC2 in immature neurones at an early developmental stage and determined the consequences on the formation of GABA and glutamate synapses. We report that early expression of the cotransporter selectively enhances GABAergic synapses: there is a significant increase of the density of GABA(A) receptors and synapses and an increase of the frequency of GABAergic miniature postsynaptic currents. The density of glutamate synapses and frequency of AMPA miniature postsynaptic currents are not affected. We conclude that the expression of KCC2 and the reduction of [Cl-]i play a critical role in the construction of GABAergic networks that extends beyond the excitatory to inhibitory shift of the actions of GABA.

  8. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

    PubMed Central

    da Silva, Haroldo Cid; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-01-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  9. Identification of genes differentially expressed in dorsal and ventral chick midbrain during early Development

    PubMed Central

    Chittka, A; Volff, JN; Wizenmann, A

    2009-01-01

    Background During the development of the central nervous system (CNS), patterning processes along the dorsoventral (DV) axis of the neural tube generate different neuronal subtypes. As development progresses these neurons are arranged into functional units with varying cytoarchitecture, such as laminae or nuclei for efficient relaying of information. Early in development ventral and dorsal regions are similar in size and structure. Different proliferation rates and cell migration patterns are likely to result in the formation of laminae or nuclei, eventually. However, the underlying molecular mechanisms that establish these different structural arrangements are not well understood. We undertook a differential display polymerase chain reaction (DD-PCR) screen to identify genes with distinct expression patterns between dorsal and ventral regions of the chick midbrain in order to identify genes which regulate the sculpturing of such divergent neuronal organisation. We focused on the DV axis of the early chick midbrain since mesencephalic alar plate and basal plate develop into laminae and nuclei, respectively. Results We identified 53 differentially expressed bands in our initial screen. Twenty-six of these could be assigned to specific genes and we could unambiguously show the differential expression of five of the isolated cDNAs in vivo by in situ mRNA expression analysis. Additionally, we verified differential levels of expression of a selected number of genes by using reverse transcriptase (RT) PCR method with gene-specific primers. One of these genes, QR1, has been previously cloned and we present here a detailed study of its early developmental time course and pattern of expression providing some insights into its possible function. Our phylogenetic analysis of QR1 shows that it is the chick orthologue of Sparc-like 1/Hevin/Mast9 gene in mice, rats, dogs and humans, a protein involved in cell adhesion. Conclusion This study reveals some possible networks, which

  10. Early expressed genes showing a dichotomous developing pattern in the lancelet embryo.

    PubMed

    Yasui, K; Saiga, H; Wang, Y; Zhang, P J; Semba, I

    2001-04-01

    Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3beta class genes. The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region, respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling. The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.

  11. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  12. Neuroendocrine secretory protein 7B2: structure, expression and functions.

    PubMed Central

    Mbikay, M; Seidah, N G; Chrétien, M

    2001-01-01

    7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation. PMID:11439082

  13. Expression Analysis of Cathepsin F during Embryogenesis and Early Developmental Stage in Olive Flounder (Paralichthys olivaceus)

    PubMed Central

    Lee, Jang-Wook; Lee, Young Mee; Yang, Hyun; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Hwang, In Joon; Kim, Sung Yeon; Lee, Jeong-Ho

    2013-01-01

    Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87-98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder. PMID:25949137

  14. Analysis of the Expression of Tachykinins and Tachykinin Receptors in the Rat Uterus During Early Pregnancy.

    PubMed

    Pinto, Francisco M; Bello, Aixa R; Gallardo-Castro, Manuel; Valladares, Francisco; Almeida, Teresa A; Tena-Sempere, Manuel; Candenas, Luz

    2015-08-01

    The peptides of the tachykinin family participate in the regulation of reproductive function acting at both central and peripheral levels. Our previous data showed that treatment of rats with a tachykinin NK3R antagonist caused a reduction of litter size. In the present study, we analyzed the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy. Uterine samples were obtained from early pregnant rats (Days 1-9 of pregnancy) and from nonpregnant rats during the proestrus stage of the ovarian cycle, and real-time quantitative RT-PCR, immunohistochemistry, and Western blot studies were used to investigate the pattern of expression of tachykinins and tachykinin receptors. We found that all tachykinins and tachykinin receptors were locally synthesized in the uterus of early pregnant rats. The expression of substance P, neurokinin B, and the tachykinin receptors NK1R and NK3R mRNAs and proteins underwent major changes during the days around implantation and they were widely distributed in implantation sites, being particularly abundant in decidual cells. These findings support the involvement of the tachykinin system in the series of uterine events that occur around embryo implantation in the rat.

  15. Protein dynamics modulated electron transfer kinetics in early stage photosynthesis

    NASA Astrophysics Data System (ADS)

    Kundu, Prasanta; Dua, Arti

    2013-01-01

    A recent experiment has probed the electron transfer kinetics in the early stage of photosynthesis in Rhodobacter sphaeroides for the reaction center of wild type and different mutants [Science 316, 747 (2007)]. By monitoring the changes in the transient absorption of the donor-acceptor pair at 280 and 930 nm, both of which show non-exponential temporal decay, the experiment has provided a strong evidence that the initial electron transfer kinetics is modulated by the dynamics of protein backbone. In this work, we present a model where the electron transfer kinetics of the donor-acceptor pair is described along the reaction coordinate associated with the distance fluctuations in a protein backbone. The stochastic evolution of the reaction coordinate is described in terms of a non-Markovian generalized Langevin equation with a memory kernel and Gaussian colored noise, both of which are completely described in terms of the microscopics of the protein normal modes. This model provides excellent fits to the transient absorption signals at 280 and 930 nm associated with protein distance fluctuations and protein dynamics modulated electron transfer reaction, respectively. In contrast to previous models, the present work explains the microscopic origins of the non-exponential decay of the transient absorption curve at 280 nm in terms of multiple time scales of relaxation of the protein normal modes. Dynamic disorder in the reaction pathway due to protein conformational fluctuations which occur on time scales slower than or comparable to the electron transfer kinetics explains the microscopic origin of the non-exponential nature of the transient absorption decay at 930 nm. The theoretical estimates for the relative driving force for five different mutants are in close agreement with the experimental estimates obtained using electrochemical measurements.

  16. Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early

    DTIC Science & Technology

    2012-07-01

    10-1-0422 TITLE: Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early PRINCIPAL...molecular imaging 7 cdrescher@fhcrc.org Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early Page 3...Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early Charles W Drescher, MD, Principle Investigator

  17. Postnatal developmental expression of regulator of G protein signaling 14 (RGS14) in the mouse brain.

    PubMed

    Evans, Paul R; Lee, Sarah E; Smith, Yoland; Hepler, John R

    2014-01-01

    Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and mitogen-activated protein kinase (MAPK) signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal-dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti-RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0), with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7, with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain.

  18. Protein Production for Structural Genomics Using E. coli Expression

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Li, Hui; Zhou, Min; Joachimiak, Grazyna; Babnigg, Gyorgy; Joachimiak, Andrzej

    2014-01-01

    The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail. PMID:24590711

  19. Two novel transcripts encoding two Ankyrin repeat containing proteins have preponderant expression during the mouse spermatogenesis.

    PubMed

    Wang, Fei; Hu, Jiarui; Song, Ping; Gong, Wuming

    2007-12-01

    The clone 4921537P18 expressed preponderantly in mouse testis was identified by screening the Riken cDNA database, and two new full-length isoforms of this clone, which were named gsarp1 (Gonad Specific Ankyrin Repeat (ANK) Protein 1) and gsarp2, were found and isolated from mouse testis in the course of the research. Both of the GSARP1 and GSARP2 contain an ANK region circular composed by seven ANKs, and their structural feature is very similar to that of the IkappaB family proteins, while IkappaB proteins associate with the transcription factor NF-kappaB via their ANKs in the NF-kappaB pathway. We investigated the expression pattern at the mRNA level by Reverse transcription PCR. The gsarp1 has high expression level in mouse testis, while has low expression level in the ovary, and the gsarp2 is only expressed in mouse testis. The gsarp1 and gsarp2 begin to be detected at the early and later pachytene stage of meiosis separately, while both have high-expression level at the stage of MI and MII. The result of in situ hybridization reveals that the gsarp1 is primarily expressed in spermatocytes, while gsarp2 is expressed in spermatocytes and spermatids. In view of the structural feature and expression pattern of the GSARP1 and GSARP2, we speculate that they may play a certain role in a signal pathway of meiosis.

  20. Protein microarrays and quantum dot probes for early cancer detection.

    PubMed

    Zajac, Aleksandra; Song, Dansheng; Qian, Wei; Zhukov, Tatyana

    2007-08-01

    We describe here a novel approach for detection of cancer markers using quantum dot protein microarrays. Both relatively new technologies; quantum dots and protein microarrays, offer very unique features that together allow detection of cancer markers in biological specimens (serum, plasma, body fluids) at pg/ml concentration. Quantum dots offer remarkable photostability and brightness. They do not exhibit photobleaching common to organic fluorophores. Moreover, the high emission amplitude for QDs results in a marked improvement in the signal to noise ratio of the final image. Protein microarrays allow highly parallel quantitation of specific proteins in a rapid, low-cost and low sample volume format. Furthermore the multiplexed assay enables detection of many proteins at once in one sample, making it a powerful tool for biomarker analysis and early cancer diagnostics. In a series of multiplexing experiments we investigated ability of the platform to detect six different cytokines in protein solution. We were able to detect TNF-alpha, IL-8, IL-6, MIP-1beta, IL-13 and IL-1beta down to picomolar concentration, demonstrating high sensitivity of the investigated detection system. We have also constructed and investigated two different models of quantum dot probes. One by conjugation of nanocrystals to antibody specific to the selected marker--IL-10, and the second by use of streptavidin coated quantum dots and biotinylated detector antibody. Comparison of those two models showed better performance of streptavidin QD-biotinylated detector antibody model. Data quantitated using custom designed computer program (CDAS) show that proposed methodology allows monitoring of changes in biomarker concentration in physiological range.

  1. Proteins and an Inflammatory Network Expressed in Colon Tumors

    PubMed Central

    Zhu, Wenhong; Fang, Changming; Gramatikoff, Kosi; Niemeyer, Christina C.; Smith, Jeffrey W.

    2011-01-01

    The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of Multidimensional Protein Identification Technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from ApcMin/+ mice. Twenty-seven proteins were found to be up-regulated in colon tumors and twenty-five down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as “seeds” to search for co-expressed genes. This approach revealed a co-expression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The co-expression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer. PMID:21366352

  2. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression.

  3. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  4. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

    PubMed

    Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

  5. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  6. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  7. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  8. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  9. A Statistical Study on Oscillatory Protein Expression

    NASA Astrophysics Data System (ADS)

    Yan, Shiwei

    Motivated by the experiments on the dynamics of a common network motif, p53 and Mdm2 feedback loop, by Lahav et al. [Nat. Genet 36, 147(2004)] in individual cells and Lev Bar-or et al. [Proc. Natl. Acad. Sci. USA 97, 11250(2000)] at the population of cells, we propose a statistical signal-response model with aiming to describe the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and in population of cells in a unified way. At the cellular level, the activities of p53 and Mdm2 proteins are described by a group of nonlinear dynamical equations where the damage-derived signal is assumed to have the form with abrupt transition (”on” leftrightarrow ”off”) as soon as signal strength passes forth and back across a threshold. Each cell responses to the damage with different time duration within which the oscillations persist. For the case of population of cells, the activities of p53 and Mdm2 proteins will be the population average of the individual cells, which results damped oscillations, due to the averaging over the cell population with the different response time.

  10. Protein Z: A putative novel biomarker for early detection of ovarian cancer.

    PubMed

    Russell, Matthew R; Walker, Michael J; Williamson, Andrew J K; Gentry-Maharaj, Aleksandra; Ryan, Andy; Kalsi, Jatinderpal; Skates, Steven; D'Amato, Alfonsina; Dive, Caroline; Pernemalm, Maria; Humphryes, Phillip C; Fourkala, Evangelia-Ourania; Whetton, Anthony D; Menon, Usha; Jacobs, Ian; Graham, Robert L J

    2016-06-15

    Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded-evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down-regulated up to 2 years pre-diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up-regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA-125 and CA-125 combined with Protein Z showed a statistically significant (p = 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA-125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes.

  11. TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion.

    PubMed

    Rostasy, Kevin; Augood, Sarah J; Hewett, Jeffrey W; Leung, Joanne Chung-on; Sasaki, Hikaru; Ozelius, Laurie J; Ramesh, Vijaya; Standaert, David G; Breakefield, Xandra O; Hedreen, John C

    2003-02-01

    Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. Most cases are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which is widely expressed in human brain and encodes the protein torsinA. This study compares neuropathology and torsinA expression in the normal human brain with that in dystonia cases with and without the GAG deletion. TorsinA-like protein was expressed in neuronal cytoplasm throughout the human brain, including cerebellum, substantia nigra, hippocampus, and neostriatum, with higher levels in specific neurons. This immunostaining pattern was not discernibly different in dystonia and normal brains in midbrain and neostriatal regions. However, nigral dopaminergic neurons appeared to be larger in both GAG-deletion and non-GAG-deletion dystonia brains compared to normal, and may be more closely spaced in GAG-deletion brains. Beyond these apparent changes in neuronal size and spacing in dystonia brains, there was no indication of neuron loss, inflammation, DNA strand breaks, or altered distribution of torsin-like immunoreactivity, supporting a functional rather than degenerative etiology of early onset torsion dystonia.

  12. Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

    PubMed

    Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

    2017-04-01

    The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.

  13. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  14. Expression of Escherichia coli cspA during early exponential growth at 37 °C.

    PubMed

    Brandi, Anna; Pon, Cynthia L

    2012-01-25

    CspA is a small (7.4 kDa) nucleic acid binding protein of Escherichia coli whose expression is stimulated after cold-stress but whose level is also extraordinarily high during the early phase of growth of non-stressed cells. In this study the relationship existing between cspA transcription/translation on the one hand and the acquisition of critical mass for cell division and chromosome replication, on the other, in stationary phase cells subjected to a nutritional up-shift at 37 °C has been analyzed. Measurements of optical density and viable counts, pulse-chase, real-time PCR and immunodetection experiments, as well as cytofluorimetric and DNA duplication analyses show that synthesis of new CspA molecules at 37 °C is not only restricted to the lag phase ensuing the nutritional up-shift, but continues also during the first stages of logarithmic growth, when cells have already started dividing; although the early synthesized molecules are diluted by the following cell divisions and new synthesis occurs at an extremely low level, cspA mRNA and CspA continue to be present. A possible explanation for the apparent paradox that cspA is activated not only following cold stress, but also under non-stress and other stress conditions which entail a down-regulation of bulk gene expression and protein synthesis is presented.

  15. Expression of growth hormone gene during early development of Siberian sturgeon (Acipenser baerii)

    PubMed Central

    Abdolahnejad, Zeinab; Pourkazemi, Mohammad; Khoshkholgh, Majid Reza; Yarmohammadi, Mahtab

    2015-01-01

    The mRNA expression of growth hormone (GH) gene in early development stages of Siberian sturgeon was investigated using RT-PCR method. Samples were collected from unfertilized eggs up to 50 days post hatched (dph) larvae in 11 different times. Ribosomal protein L6 (RPL6) transcripts were used as the internal standard during quantification of GH mRNA expression. The results showed that the GH mRNA could be observed in the eyed eggs and even at unfertilized eggs of Siberian sturgeon. The highest amounts of GH mRNA were found at 25 and 50 dph larvae, while the lowest levels were detected at 1 and 3 dph larvae stage. These findings suggest that, the GH mRNA play a key role during developmental stages of Siberian sturgeon. PMID:27844010

  16. Expression of growth hormone gene during early development of Siberian sturgeon (Acipenserbaerii).

    PubMed

    Abdolahnejad, Zeinab; Pourkazemi, Mohammad; Khoshkholgh, Majid Reza; Yarmohammadi, Mahtab

    2015-12-01

    The mRNA expression of growth hormone (GH) gene in early development stages of Siberian sturgeon was investigated using RT-PCR method. Samples were collected from unfertilized eggs up to 50 days post hatched (dph) larvae in 11 different times. Ribosomal protein L6 (RPL6) transcripts were used as the internal standard during quantification of GH mRNA expression. The results showed that the GH mRNA could be observed in the eyed eggs and even at unfertilized eggs of Siberian sturgeon. The highest amounts of GH mRNA were found at 25 and 50 dph larvae, while the lowest levels were detected at 1 and 3 dph larvae stage. These findings suggest that, the GH mRNA play a key role during developmental stages of Siberian sturgeon.

  17. Comprehensive analysis of TCP transcription factors and their expression during cotton (Gossypium arboreum) fiber early development.

    PubMed

    Ma, Jun; Liu, Fang; Wang, Qinglian; Wang, Kunbo; Jones, Don C; Zhang, Baohong

    2016-02-09

    TCP proteins are plant-specific transcription factors implicated to perform a variety of physiological functions during plant growth and development. In the current study, we performed for the first time the comprehensive analysis of TCP gene family in a diploid cotton species, Gossypium arboreum, including phylogenetic analysis, chromosome location, gene duplication status, gene structure and conserved motif analysis, as well as expression profiles in fiber at different developmental stages. Our results showed that G. arboreum contains 36 TCP genes, distributing across all of the thirteen chromosomes. GaTCPs within the same subclade of the phylogenetic tree shared similar exon/intron organization and motif composition. In addition, both segmental duplication and whole-genome duplication contributed significantly to the expansion of GaTCPs. Many these TCP transcription factor genes are specifically expressed in cotton fiber during different developmental stages, including cotton fiber initiation and early development. This suggests that TCP genes may play important roles in cotton fiber development.

  18. Serum Protein Markers for the Early Detection of Lung Cancer: A Focus on Autoantibodies.

    PubMed

    Broodman, Ingrid; Lindemans, Jan; van Sten, Jenny; Bischoff, Rainer; Luider, Theo

    2017-01-06

    Lung cancer has the highest mortality rate among cancer patients in the world, in particular because most patients are only diagnosed at an advanced and noncurable stage. Computed tomography (CT) screening on high-risk individuals has shown that early detection could reduce the mortality rate. However, the still high false-positive rate of CT screening may harm healthy individuals because of unnecessary follow-up scans and invasive follow-up procedures. Alternatively, false-negative and indeterminate results may harm patients due to the delayed diagnosis and treatment of lung cancer. Noninvasive biomarkers, complementary to CT screening, could lower the false-positive and false-negative rate of CT screening at baseline and thereby reduce the number of patients that need follow-up and diagnose patients at an earlier stage of lung cancer. Lung cancer tissue generates lung cancer-associated proteins to which the immune system might produce high-affinity autoantibodies. This autoantibody response to tumor-associated antigens starts during early stage lung cancer and may endure over years. Identification of tumor-associated antigens or the corresponding autoantibodies in body fluids as potential noninvasive biomarkers could thus be an effective approach for early detection and monitoring of lung cancer. We provide an overview of differentially expressed protein, antigen, and autoantibody biomarkers that combined with CT imaging might be of clinical use for early detection of lung cancer.

  19. Early Fluid and Protein Shifts in Men During Water Immersion

    NASA Technical Reports Server (NTRS)

    Hinghofer-Szalkay, H.; Harrison, M. H.; Greenleaf, J. E.

    1987-01-01

    High precision blood and plasma densitometry was used to measure transvascular fluid shifts during water immersion to the neck. Six men (28-49 years) undertook 30 min of standing immersion in water at 35.0 +/- 0.2 C; immersion was preceded by 30 min control standing in air at 28 +/- 1 C. Blood was sampled from an antecubital catheter for determination of Blood Density (BD), Plasma Density (PD), Haematocrit (Ht), total Plasma Protein Concentration (PPC), and Plasma Albumin Concentration (PAC). Compared to control, significant decreases (p less than 0.01) in all these measures were observed after 20 min immersion. At 30 min, plasma volume had increased by 11.0 +/- 2.8%; the average density of the fluid shifted from extravascular fluid into the vascular compartment was 1006.3 g/l; albumin moved with the fluid and its albumin concentration was about one-third of the plasma protein concentration during early immersion. These calculations are based on the assumption that the F-cell ratio remained unchanged. No changes in erythrocyte water content during immersion were found. Thus, immersion-induced haemodilution is probably accompanied by protein (mainly albumin) augmentation which accompanies the intra-vascular fluid shift.

  20. Early Days of Pressure Denaturation Studies of Proteins.

    PubMed

    Suzuki, Keizo

    2015-01-01

    The denaturation of protein by pressure has been generally well known since the findings of the perfect coagulation of egg white by a pressure of 7,000 atm within 30 min by Bridgman (J Biol Chem 19:511-512, 1914), and Kiyama and Yanagimoto (Rev Phys Chem Jpn 21:41-43, 1951) confirmed that the coagulation occurs above 3,880 kg cm(-2). Grant et al. (Science 94:616, 1941) and Suzuki and Kitamura (Abstracts of 30th annual meeting of Japanese Biochemical Society, 1957) found that SH groups are detected at the compressed sample of ovalbumin. On the other hand, Johnson and Campbell (J Cell Comp Physiol 26:43-49, 1945), Tongur (Kolloid Zhur 11:274-279, 1949; Biokhimiya 17:495-503, 1952) and Suzuki et al. (Mem Res Inst Sci Eng Ritsumeikan Univ 3:1-4, 1958) reported that the thermal denaturation of proteins is retarded in a few examples by the low pressure of about 1,000 atm. Before 1960, the studies of denaturation under high pressure were, however, rare and almost qualitative compared with those by heat, acid, urea and so on, so that there was no theory for the influence of hydrostatic pressure on the mechanism of denaturation. Here I review how I started experiments and analysis on pressure denaturation of proteins in early days of 1950s and 1960s in my laboratory and others.

  1. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  2. Vectors for the expression of tagged proteins in Drosophila.

    PubMed

    Parker, L; Gross, S; Alphey, L

    2001-12-01

    Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

  3. Extracellular Stiffness Modulates the Expression of Functional Proteins and Growth Factors in Endothelial Cells.

    PubMed

    Santos, Lívia; Fuhrmann, Gregor; Juenet, Maya; Amdursky, Nadav; Horejs, Christine-Maria; Campagnolo, Paola; Stevens, Molly M

    2015-08-13

    Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.

  4. Expression of an early Epstein-Barr virus antigen (EA-D) in E. coli. Brief report.

    PubMed

    Roeckel, D; Boos, H; Mueller-Lantzsch, N

    1987-01-01

    The 1.34 kb BcII-BgIII-fragment of the BamHI-M region of Epstein-Barr virus genome, comprising the complete BMRF1 open reading frame, was cloned into the tryptophan regulated E. coli expression vector pATH1. The resulting fusion protein, having a molecular weight of 80 kd, is recognized not only by anti-early antigen (EA)-positive human sera but also by the monoclonal antibody R3 directed against the diffuse component of EA (EA-D). A possible use for this fusion protein as an indicator protein in diagnosis of IgA antibodies against EA-D is presented.

  5. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  6. Gene Expression Profiles Deciphering Leaf Senescence Variation between Early- and Late-Senescence Cotton Lines

    PubMed Central

    Kong, Xiangqiang; Luo, Zhen; Dong, Hezhong; Eneji, A. Egrinya; Li, Weijiang; Lu, Hequan

    2013-01-01

    Leaf senescence varies greatly among genotypes of cotton (Gossypium hirsutium L), possibly due to the different expression of senescence-related genes. To determine genes involved in leaf senescence, we performed genome-wide transcriptional profiling of the main-stem leaves of an early- (K1) and a late-senescence (K2) cotton line at 110 day after planting (DAP) using the Solexa technology. The profiling analysis indicated that 1132 genes were up-regulated and 455 genes down-regulated in K1 compared with K2 at 110 DAP. The Solexa data were highly consistent with, and thus were validated by those from real-time quantitative PCR (RT-PCR). Most of the genes related to photosynthesis, anabolism of carbohydrates and other biomolecules were down-regulated, but those for catabolism of proteins, nucleic acids, lipids and nutrient recycling were mostly up-regulated in K1 compared with K2. Fifty-one differently expressed hormone-related genes were identified, of which 5 ethylene, 3 brassinosteroid (BR), 5 JA, 18 auxin, 8 GA and 1 ABA related genes were up-regulated in K1 compared with K2, indicating that these hormone-related genes might play crucial roles in early senescence of K1 leaves. Many differently expressed transcription factor (TF) genes were identified and 11 NAC and 8 WRKY TF genes were up-regulated in K1 compared with K2, suggesting that TF genes, especially NAC and WRKY genes were involved in early senescence of K1 leaves. Genotypic variation in leaf senescence was attributed to differently expressed genes, particularly hormone-related and TF genes. PMID:23922821

  7. Expression analysis of a novel p75(NTR) signaling protein, which regulates cell cycle progression and apoptosis.

    PubMed

    Kendall, Stephen E; Goldhawk, Donna E; Kubu, Chris; Barker, Philip A; Verdi, Joseph M

    2002-09-01

    Neurotrophin receptor-interacting MAGE (NRAGE) is the most recently identified p75 neurotrophin receptor (p75(NTR)) intracellular binding protein. Previously, NRAGE over-expression was shown to mediate cell cycle arrest and facilitate nerve growth factor (NGF) dependent apoptosis of sympathetic neuroblasts in a p75(NTR) specific manner. Here we have examined the temporal and spatial expression patterns of NRAGE over the course of murine embryogenesis to determine whether NRAGE's expression is consistent with its proposed functions. We demonstrate that NRAGE mRNA and protein are expressed throughout embryonic and adult tissues. The mRNA is constitutively expressed within each tissue across development. However, expression of NRAGE protein displays a tight temporal tissue specific regulation. During early CNS development, NRAGE protein is expressed throughout the neural tube, but by later stages of neurogenesis, NRAGE protein is restricted within the ventricular zone, subplate and cortical plate. Moreover, NRAGE protein expression is limited to proliferative neural subpopulations as we fail to detect NRAGE expression co-localized with mature/differentiation associated neuronal markers. Interestingly, NRAGE's expression is not restricted solely to areas of p75(NTR) expression suggesting that NRAGE may mediate proliferation and/or apoptosis from other environmental signals in addition to NGF within the CNS. Our data support previously characterized roles for NRAGE as a mediator of precursor apoptosis and a repressor of cell cycle progression in neural development.

  8. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  9. Prolonged morphine administration alters protein expression in the rat myocardium

    PubMed Central

    2011-01-01

    Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart. PMID:22129148

  10. Aquaporin expression in the cerebral cortex is increased at early stages of Alzheimer disease.

    PubMed

    Pérez, Esther; Barrachina, Marta; Rodríguez, Agustín; Torrejón-Escribano, Benjamín; Boada, Mercé; Hernández, Isabel; Sánchez, Marisa; Ferrer, Isidre

    2007-01-12

    Abnormalities in the cerebral microvasculature are common in Alzheimer disease (AD). Expression levels of the water channels aquaporin 1 and aquaporin 4 (AQP1, AQP4) were examined in AD cases by gel electrophoresis and Western blotting, and densitometric values normalized with beta-actin were compared with corresponding values in age-matched controls processed in parallel. In addition, samples of cases with Pick disease (PiD) were examined for comparative purposes. A significant increase in the expression levels of AQP1 was observed in AD stage II (following Braak and Braak classification). Individual variations were seen in advanced stages which resulted in non-significant differences between AD stages V-VI and age-matched controls. No differences in AQP1 levels were observed between familial AD cases (FAD, all of them at advanced stages) and corresponding age-matched controls. Immunohistochemistry showed increased AQP1 in astrocytes at early stages of AD. Double-labelling immunofluorescence and confocal microscopy disclosed AQP1 immunoreactivity at the cell surface of astrocytes which were recognized with anti-glial fibrillary acidic protein antibodies. No differences in the levels of AQP4 were observed in AD, FAD and PiD when compared with corresponding controls. These results indicate abnormal expression of AQP1 in astrocytes in AD, and they add support to the idea that abnormal regulation of mechanisms involved in the control of water fluxes occurs at early stages in AD.

  11. Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions.

    PubMed

    McReynolds, Jayme R; Donowho, Kyle; Abdi, Amin; McGaugh, James L; Roozendaal, Benno; McIntyre, Christa K

    2010-03-01

    Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a beta-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of beta-adrenoceptors with propranolol (0.5 microg/0.2 microL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA beta-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.

  12. Analysis of protein expression profile of oral squamous cell carcinoma by MALDI-TOF-MS.

    PubMed

    Roman, Eric; Lunde, Mai Lill Suhr; Miron, Talia; Warnakulasauriya, Saman; Johannessen, Anne Christine; Vasstrand, Endre Normann; Ibrahim, Salah Osman

    2013-03-01

    In this study, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF-MS) technology was used to examine differentially expressed proteins in oral squamous cell carcinoma (OSCC) tissues from Norway (n=15) and the UK (n=45). Twenty-nine proteins were found to be significantly overexpressed in the OSCCs examined compared to the normal controls. Identified proteins included, family of annexin proteins that play important roles in signal transduction pathways and regulation of cellular growth, keratin-1, heat-shock proteins (HSP), squamous cell carcinoma antigen (SCC-Ag), cytoskeleton proteins, and proteins involved in mitochondrial and intracellular signalling pathways. The expression of four selected proteins (annexin II and V, HSP-27, and SCC-Ag) was verified using western blot analysis of 76 fresh tissue biopsy specimens in total, from Norway (n=53) and the UK (n=23). Proteomic analysis of OSCCs examined here demonstrated involvement of several proteins that might function as potential biomarkers and molecular targets for early cancer diagnostics, and may contribute to a novel approach to therapeutics and for predicting prognosis of OSCC.

  13. Dynamic methylation and expression of Oct4 in early neural stem cells

    PubMed Central

    Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

    2010-01-01

    Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form ‘induced pluripotent stem cells’ (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages. PMID:20646110

  14. Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.

    PubMed

    Adler, M J; Coronel, C; Shelton, E; Seegmiller, J E; Dewji, N N

    1991-01-01

    The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4- to 5-kDa amyloid beta-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of beta-amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyclonal antibodies to a synthetic peptide of the beta-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. Regulated expression of beta-amyloid precursor protein may be an important determinant of growth and metabolic responses to serum and growth factors under physiological as well as pathological conditions.

  15. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    PubMed Central

    Chakrabarti, Sanjukta; Barrow, Colin J.; Kanwar, Rupinder K.; Ramana, Venkata; Kanwar, Jagat R.

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

  16. Expression profiling identifies genes expressed early during lint fibre initiation in cotton.

    PubMed

    Wu, Yingru; Machado, Adriane C; White, Rosemary G; Llewellyn, Danny J; Dennis, Elizabeth S

    2006-01-01

    Cotton fibres are a subset of single epidermal cells that elongate from the seed coat to produce the long cellulose strands or lint used for spinning into yarn. To identify genes that might regulate lint fibre initiation, expression profiles of 0 days post-anthesis (dpa) whole ovules from six reduced fibre or fibreless mutants were compared with wild-type linted cotton using cDNA microarrays. Numerous clones were differentially expressed, but when only those genes that are normally expressed in the ovule outer integument (where fibres develop) were considered, just 13 different cDNA clones were down-regulated in some or all of the mutants. These included: a Myb transcription factor (GhMyb25) similar to the Antirrhinum Myb AmMIXTA, a putative homeodomain protein (related to Arabidopsis ATML1), a cyclin D gene, some previously identified fibre-expressed structural and metabolic genes, such as lipid transfer protein, alpha-expansin and sucrose synthase, as well as some unknown genes. Laser capture microdissection and reverse transcription-PCR were used to show that both the GhMyb25 and the homeodomain gene were predominantly ovule specific and were up-regulated on the day of anthesis in fibre initials relative to adjacent non-fibre ovule epidermal cells. Their spatial and temporal expression pattern therefore coincided with the time and location of fibre initiation. Constitutive overexpression of GhMyb25 in transgenic tobacco resulted in an increase in branched long-stalked leaf trichomes. The involvement of cell cycle genes prompted DNA content measurements that indicated that fibre initials, like leaf trichomes, undergo DNA endoreduplication. Cotton fibre initiation therefore has some parallels with leaf trichome development, although the detailed molecular mechanisms are clearly different.

  17. Recent patents on alphavirus protein expression and vector production.

    PubMed

    Aranda, Alejandro; Ruiz-Guillen, Marta; Quetglas, Jose I; Bezunartea, Jaione; Casales, Erkuden; Smerdou, Cristian

    2011-12-01

    Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

  18. Identification of c-Yes expression in the nuclei of hepatocellular carcinoma cells: involvement in the early stages of hepatocarcinogenesis.

    PubMed

    Nonomura, Takako; Masaki, Tsutomu; Morishita, Asahiro; Jian, Gong; Uchida, Naohito; Himoto, Takashi; Izuishi, Kunihiko; Iwama, Hisakazu; Yoshiji, Hitoshi; Watanabe, Seishiro; Kurokohchi, Kazutaka; Kuriyama, Shigeki

    2007-01-01

    It is thought that the subcellular distribution of Src-family tyrosine kinases, including c-Yes binding to the cellular membrane, is membranous and/or cytoplasmic. c-Yes protein tyrosine kinase is known to be related to malignant transformation. However, the expression patterns of c-Yes in hepatocellular carcinoma (HCC) remains unknown. In the present study, we report that c-Yes is expressed not only in the membrane and cytoplasm, but also in the nuclei of cancer cells in some human HCC tissues and in a human HCC cell line. We examined the expression and localization of c-Yes in human HCC cell lines (HLE, HLF, PLC/PRF/5 and Hep 3B) by Western blotting and immunohistochemical analyses; we also examined the expression of c-Yes by immunohistochemistry and Western blotting in the tissues of various liver diseases, including 39 samples from HCC patients. We used an antibody array to detect proteins that bind to nuclear c-Yes in PLC/PRF/5 cell line. c-Yes was found to be expressed in the membranes and cytoplasm of HLE, HLF and Hep 3B HCC cells; it was also detected in the nuclei in addition to the membranes and cytoplasm of PLC/PRF/5 HCC cells. HCC with nuclear c-Yes was detected in 5 of 39 cases (13.0%), and nuclear c-Yes expression was not detected in normal, chronic hepatitis or cirrhotic livers. All HCCs with nuclear c-Yes expression were well-differentiated, small tumors at the early stages. In the PLC/PRF/5 cell line, the nuclear localization of c-Yes with cyclin-dependent kinase 1 was confirmed by a protein antibody array. In conclusion, nuclear c-Yes expression was found in cancer cells at the early stages of hepatocarcinogenesis, suggesting that nucleus-located c-Yes may be a useful marker to detect early-stage HCC.

  19. Temporal expression and immunogold localization of Plodia interpunctella granulosis virus structural proteins

    NASA Technical Reports Server (NTRS)

    Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Monospecific antisera were produced against four structural proteins (VP12, VP17, VP31, and granulin) of the Plodia interpunctella granulosis virus using polypeptides derived by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or acid extraction. The antisera were shown to be specific on immunoblots of SDS-PAGE separated granulosis virus and were further used to detect structural proteins in infected fat body lysates. Immunoblots of fat body lysates from early stages of infection indicated that VP12, VP17, VP31, and granulin were expressed by 2.5 days post-infection. Immunogold labeling of the virus using the monospecific antisera and electron microscopy confirmed earlier reports that granulin is located in the protein matrix, V17 is an envelope protein, and VP31 is a capsid protein.

  20. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  1. Oog1, an oocyte-specific protein, interacts with Ras and Ras-signaling proteins during early embryogenesis

    SciTech Connect

    Tsukamoto, Satoshi; Ihara, Ryo; Aizawa, Akira; Kishida, Shosei; Kikuchi, Akira; Imai, Hiroshi; Minami, Naojiro . E-mail: naojiro@kais.kyoto-u.ac.jp

    2006-05-19

    We previously identified an oocyte-specific gene, Oogenesin 1 (Oog1), that encodes 326 amino acids containing a leucine zipper structure and a leucine-rich repeat. In the present study, to identify the interacting proteins of Oog1, we performed a yeast two-hybrid screening using a GV-oocyte cDNA library and found that Ral guanine nucleotide dissociation stimulator (RalGDS) is the binding partner of Oog1. Coimmunoprecipitation assay confirmed the interaction between Oog1 and RalGDS proteins. Colocalization experiments provide the evidence that the nuclear localization of RalGDS depends on the expression of Oog1. Interestingly, RalGDS protein localized in the nucleus rather than the cytoplasm between late 1-cell and early 2-cell stages, the time when Oog1 localizes in the nucleus. We also examined the interaction between Oog1 and Ras by GST pull-down assay and revealed that Oog1 interacts with Ras in a GTP-dependent manner. These findings suggest a role of Oog1 as a Ras-binding protein.

  2. Visualizing changes in circuit activity resulting from denervation and reinnervation using immediate early gene expression.

    PubMed

    Temple, Meredith D; Worley, Paul F; Steward, Oswald

    2003-04-01

    We describe a novel strategy to evaluate circuit function after brain injury that takes advantage of experience-dependent immediate early gene (IEG) expression. When normal rats undergo training or are exposed to a novel environment, there is a strong induction of IEG expression in forebrain regions, including the hippocampus. This gene induction identifies the neurons that are engaged during the experience. Here, we demonstrate that experience-dependent IEG induction is diminished after brain injury in young adult rats (120-200 gm), specifically after unilateral lesions of the entorhinal cortex (EC), and then recovers with a time course consistent with reinnervation. In situ hybridization techniques were used to assess the expression of the activity-regulated cytoskeleton-associated protein Arc at various times after the lesion (4, 8, 12, 16, or 30 d). One group of rats was allowed to explore a complex novel environment for 1 hr; control operated animals remained in their home cage. In unoperated animals, exposure to the novel environment induced Arc mRNA levels in most pyramidal neurons in CA1, in many pyramidal neurons in CA3, and in a small number of dentate granule cells. This characteristic pattern of induction was absent at early time points after unilateral EC lesions (4 and 8 d) but recovered progressively at later time points. The recovery of Arc expression occurred with approximately the same time course as the reinnervation of the dentate gyrus as a result of postlesion sprouting. These results document a novel approach for quantitatively assessing activity-regulated gene expression in polysynaptic circuits after trauma.

  3. Expression and biochemical characterization of recombinant human epididymis protein 4.

    PubMed

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

  4. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

    Gibbs, David L.; Baratt, Arie; Baric, Ralph; Kawaoka, Yoshihiro; Smith, Richard D.; Orwoll, Eric S.; Katze, Michael G.; Mcweeney, Shannon K.

    2013-06-01

    Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology. We have evaluated the construction of network models using methods derived from weighted gene co-expression network analysis (WGCNA). We show that approximately scale-free peptide networks, composed of statistically significant modules, are feasible and biologically meaningful using two mouse lung experiments and one human plasma experiment. Within each network, peptides derived from the same protein are shown to have a statistically higher topological overlap and concordance in abundance, which is potentially important for inferring protein abundance. The module representatives, called eigenpeptides, correlate significantly with biological phenotypes. Furthermore, within modules, we find significant enrichment for biological function and known interactions (gene ontology and protein-protein interactions). Biological networks are important tools in the analysis of complex systems. In this paper we evaluate the application of weighted co-expression network analysis to quantitative proteomics data. Protein co-expression networks allow novel approaches for biological interpretation, quality control, inference of protein abundance, a framework for potentially resolving degenerate peptide-protein mappings, and a biomarker signature discovery.

  5. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

    PubMed Central

    Smits, Arne H.; Lindeboom, Rik G.H.; Perino, Matteo; van Heeringen, Simon J.; Veenstra, Gert Jan C.; Vermeulen, Michiel

    2014-01-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs. PMID:25056316

  6. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

    PubMed

    Smits, Arne H; Lindeboom, Rik G H; Perino, Matteo; van Heeringen, Simon J; Veenstra, Gert Jan C; Vermeulen, Michiel

    2014-09-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

  7. Decreased Expression of the Early Mitotic Gene, CHFR, Contributes to the Acquisition of Breast Cancer Phenotypes

    DTIC Science & Technology

    2008-03-01

    AD_________________ Award Number: W81XWH-06-1-0332 TITLE: Decreased Expression of the Early... Decreased Expression of the Early Mitotic Gene, CHFR, Contributes to the Acquisition of Breast Cancer Phenotypes 5b. GRANT NUMBER W81XWH-06-1-0332 5c... decreased CHFR expression compared to normal cells and tissues and the loss of CHFR expression by RNAi in cell culture models leads to the acquisition of

  8. Thyroid-Related Protein Expression in the Human Thymus

    PubMed Central

    Park, Do Joon; Jung, Kyeong Cheon

    2017-01-01

    Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS. PMID:28386277

  9. NR2F6 Expression Correlates with Pelvic Lymph Node Metastasis and Poor Prognosis in Early-Stage Cervical Cancer

    PubMed Central

    Niu, Chunhao; Sun, Xiaoying; Zhang, Weijing; Li, Han; Xu, Liqun; Li, Jun; Xu, Benke; Zhang, Yanna

    2016-01-01

    Background: There is an abnormal expression of nuclear receptor subfamily 2 group F member 6 (NR2F6) in human cancers such as breast cancer, colon cancer, and acute myelogenous leukemia. However, its clinical significance in cervical cancer has not been established. We explored NR2F6 expression and its clinicopathological significance in early-stage cervical cancer. Methods: NR2F6 expression in cervical cancer cell lines and cervical cancer tissues was determined by Western blotting, real-time PCR, and immunochemistry (IHC). NR2F6 expression in 189 human early-stage cervical cancer tissue samples was evaluated using IHC. The relevance between NR2F6 expression and early-stage cervical cancer prognosis and clinicopathological features was determined. Results: There was marked NR2F6 mRNA and protein overexpression in the cervical cancer cells and clinical tissues compared with an immortalized squamous cell line and adjacent noncancerous cervical tissues, respectively. In the 189 cervical cancer samples, NR2F6 expression was positively related to International Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.006), squamous cell carcinoma antigen (p = 0.006), vital status (p < 0.001), tumor recurrence (p = 0.001), chemotherapy (p = 0.039), and lymph node metastasis (p < 0.001). Overall and disease-free survival was shorter in patients with early-stage cervical cancer and higher NR2F6 levels than in patients with lower levels of NR2F6. Univariate and multivariate analysis determined that NR2F6 was an independent prognostic factor of survival in early-stage cervical cancer. Conclusions: Taken together, our findings suggest that high NR2F6 expression predicts pelvic lymph node metastasis, tumor recurrence and poor prognosis in early-stage cervical cancer. NR2F6 might be a novel prognostic biomarker and potential therapeutic target of cervical cancer. PMID:27775588

  10. MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos.

    PubMed Central

    Hopwood, N D; Pluck, A; Gurdon, J B

    1989-01-01

    We describe the cloning, cDNA sequence and embryonic expression of a Xenopus homologue of MyoD, a mouse gene encoding a DNA-binding protein that can activate muscle gene expression in cultured cells. The predicted Xenopus MyoD protein sequence is remarkably similar to mouse MyoD. Zygotic expression of MyoD begins in early gastrulae, but there is a low level of unlocalized maternal message. Northern blot analysis of dissected embryos and in situ hybridization show that MyoD RNA is restricted to the gastrula mesoderm and to the somites of neurulae and tailbud embryos. The time and place of MyoD expression are consistent with a role for MyoD in the activation of other muscle genes in the somites of the frog embryo. However, MyoD is skeletal muscle-specific and is not expressed even in the early embryonic heart, which co-expresses cardiac and skeletal actin isoforms. Striated muscle genes can therefore be activated in some embryonic tissues in the absence of MyoD. The concentration of MyoD in the somites falls once they have formed, suggesting that MyoD may act there transiently to establish muscle gene expression. MyoD transcription is activated following mesoderm induction, and is the earliest muscle-specific response to mesoderm-inducing factors so far described. Images PMID:2555164

  11. Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

    PubMed

    van Opstal, Angélique; Bijvelt, José; van Donselaar, Elly; Humbel, Bruno M; Boonstra, Johannes

    2012-04-01

    Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.

  12. Expression of protein kinase C genes during ontogenic development of the central nervous system

    SciTech Connect

    Sposi, N.M.; Bottero, L.; Testa, U.; Peschle, C.; Russo, G.

    1989-05-01

    The authors have analyzed the RNA expression of three protein kinase C (PKC) genes in (/alpha/, /beta/, and /gamma/) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)/sup +/ RNA indicates that expression of PKC /alpha/ and /beta/ genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage,and that the PKC /gamma/ gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)/sup +/ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenic patterns were similar to those observed for human brain. Furthermore, they observed that the expression of PKC /gamma/ is induced in the peri- and postnatal phases. These results suggest that expression of PKC /alpha/, /beta/, and /gamma/ genes possibly mediates the development of central neuronal functions, and expression of PKC /gamma/ in particular may be involved in the development of peri- and postnatal functions.

  13. Iridovirus Bcl-2 protein inhibits apoptosis in the early stage of viral infection.

    PubMed

    Lin, Pei-Wen; Huang, Yi-Jen; John, Joseph Abraham Christopher; Chang, Ya-Nan; Yuan, Chung-Hsiang; Chen, Wen-Ya; Yeh, Chiao-Hwa; Shen, San-Tai; Lin, Fu-Pang; Tsui, Wen-Huei; Chang, Chi-Yao

    2008-01-01

    The grouper iridovirus (GIV) belongs to the family Iridoviridae, whose genome contains an antiapoptotic B-cell lymphoma (Bcl)-2-like gene. This study was carried-out to understand whether GIV blocks apoptosis in its host. UV-irradiated grouper kidney (GK) cells underwent apoptosis. However, a DNA fragmentation assay of UV-exposed GK cells after GIV infection revealed an inhibition of apoptosis. The UV- or heat-inactivated GIV failed to inhibit apoptosis, implying that a gene or protein of the viral particle might contribute to an apoptosis inhibitory function. The DNA ladder assay for GIV-infected GK cells after UV irradiation confirmed that apoptosis inhibition was an early process which occurred as early as 5 min post-infection. A GIV-Bcl sequence comparison showed distant sequence similarities to that of human and four viruses; however, all possessed the putative Bcl-2 homology (BH) domains of BH1, BH2, BH3, and BH4, as well as a transmembrane domain. Northern blot hybridization showed that GIV-Bcl transcription began at 2 h post-infection, and the mRNA level significantly increased in the presence of cycloheximide or aphidicolin, indicating that this GIV-Bcl is an immediate-early gene. This was consistent with the Western blot results, which also revealed that the virion carries the Bcl protein. We observed the localization of GIV-Bcl on the mitochondrial membrane and other defined intracellular areas. By immunostaining, it was proven that GIV-Bcl-expressing cells effectively inhibited apoptosis. Taken together, these results demonstrate that GIV inhibits the promotion of apoptosis by GK cells, which is mediated by the immediate early expressed viral Bcl gene.

  14. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    PubMed Central

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2013-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease. PMID:23012103

  15. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  16. Using ion exchange chromatography to purify a recombinantly expressed protein.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  17. Atrial expression of the CCN1 and CCN2 proteins in chronic heart failure.

    PubMed

    Bonda, Tomasz A; Kamiński, Karol A; Dziemidowicz, Magdalena; Litvinovich, Sergey; Kożuch, Marcin; Hirnle, Tomasz; Dmitruk, Iwona; Chyczewski, Lech; Winnicka, Maria M

    2012-04-24

    Previous studies have reported the upregulation of CCN proteins early after acute heart injury. The aim of the present work was to evaluate the expression of the CCN1 and CCN2 proteins and their regulation by angiotensin II in the atrial myocardium of a chronically failing heart. Male adult mice were subjected to ligation of the left coronary artery to produce myocardial infarction (the MI group), and 16 of them were treated for 12 weeks with the AT1 receptor antagonist telmisartan (the MI-Tel group). Sham-operated mice served as controls. The expression of proteins was evaluated by immunohistochemistry 12 weeks after the operation. In shamoperated mice, stainings for CCN1 and CCN2 proteins were positive within atrial cardiomyocytes. CCN1-positive reaction revealed diffused cytoplasmic localization, while CCN2 was present mainly within the perinuclear cytoplasm. CCN1 was upregulated in the MI group, while CCN2 remained at basal level. Telmisartan prevented the upregulation of CCN1 and decreased CCN2 level. We compared the experimental data with the expression of CCN1 and CCN2 proteins in human right atrial appendages. We found an inverse, but not significant, relation between the level of either protein and the left ventricular ejection fraction. This suggests a similar atrial regulation of CCN1 and CCN2 expression also in humans. We conclude that in the murine atria, CCN1 and CCN2 proteins are expressed constitutively. In chronic heart failure, CCN proteins tend to be upregulated, which may be related to the action of angiotensin II.

  18. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  19. Expression profile of ribosomal protein L10a throughout gonadal development in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Makkapan, Walaiporn; Yoshizaki, Goro; Tashiro, Masami; Chotigeat, Wilaiwan

    2014-08-01

    Ribosomal protein L10a (RpL10A) has been previously established as a stimulator during the early stages of ovarian development in both the banana prawn and the fruit fly. In order to develop a greater understanding of the role of this protein in vertebrates, the present study aimed to characterize the expression profile of rpl10a during gonadal development in fish. It was determined that the expression of rpl10a within genital ridges increased during embryonic development. Although rpl10a expression was observed in both gonadal somatic cells and primordial germ cells, higher levels of both transcript and protein expression were detected in somatic cells. rpl10a transcripts were observed in all of the adult tissues examined. Cellular level expression of rpl10a was subsequently characterized across various maturational stages using in situ hybridization and immunohistochemistry of both testes and ovaries. Analysis of tissue derived from the testis showed high levels of rpl10a expression within spermatogonia and the Sertoli cells attached to them. In ovarian tissue, rpl10a was strongly expressed in chromatin-nucleolus-stage and peri-nucleolus-stage oocytes. The relationship between rpl10a expression and regulation of gonadal development was confirmed using real-time PCR, which was performed in order to analyze rpl10a expression in testicular and ovarian tissues subsequent to incubation with salmon pituitary extract and various sex steroids for 24 h. Among them, 11-ketotestosterone at 100 ng/mL effectively up-regulated expression of rpl10a in testicular tissues, while 17β-estradiol down-regulated rpl10a expression in ovarian tissues. These results suggested that rpl10a played a role in the regulation of gonadal development in fish.

  20. SPINK 1 Protein Expression and Prostate Cancer Progression

    PubMed Central

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  1. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  2. Enhancement of G Protein-Coupled Receptor Surface Expression

    PubMed Central

    Dunham, Jill H.; Hall, Randy A.

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

  3. Spatio-Temporal Gene Expression Profiling during In Vivo Early Ovarian Folliculogenesis: Integrated Transcriptomic Study and Molecular Signature of Early Follicular Growth

    PubMed Central

    Bonnet, Agnes; Servin, Bertrand; Mulsant, Philippe; Mandon-Pepin, Beatrice

    2015-01-01

    Background The successful achievement of early ovarian folliculogenesis is important for fertility and reproductive life span. This complex biological process requires the appropriate expression of numerous genes at each developmental stage, in each follicular compartment. Relatively little is known at present about the molecular mechanisms that drive this process, and most gene expression studies have been performed in rodents and without considering the different follicular compartments. Results We used RNA-seq technology to explore the sheep transcriptome during early ovarian follicular development in the two main compartments: oocytes and granulosa cells. We documented the differential expression of 3,015 genes during this phase and described the gene expression dynamic specific to these compartments. We showed that important steps occurred during primary/secondary transition in sheep. We also described the in vivo molecular course of a number of pathways. In oocytes, these pathways documented the chronology of the acquisition of meiotic competence, migration and cellular organization, while in granulosa cells they concerned adhesion, the formation of cytoplasmic projections and steroid synthesis. This study proposes the involvement in this process of several members of the integrin and BMP families. The expression of genes such as Kruppel-like factor 9 (KLF9) and BMP binding endothelial regulator (BMPER) was highlighted for the first time during early follicular development, and their proteins were also predicted to be involved in gene regulation. Finally, we selected a data set of 24 biomarkers that enabled the discrimination of early follicular stages and thus offer a molecular signature of early follicular growth. This set of biomarkers includes known genes such as SPO11 meiotic protein covalently bound to DSB (SPO11), bone morphogenetic protein 15 (BMP15) and WEE1 homolog 2 (S. pombe)(WEE2) which play critical roles in follicular development but other

  4. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  5. Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages.

    PubMed

    Miyazaki, A; Sakashita, N; Lee, O; Takahashi, K; Horiuchi, S; Hakamata, H; Morganelli, P M; Chang, C C; Chang, T Y

    1998-10-01

    The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.

  6. Expression and in vitro functional analyses of recombinant Gam1 protein.

    PubMed

    Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

    2015-01-01

    Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication.

  7. Temporal and spatial expression of caudal-type homeobox proteins in the midgut of human embryos

    PubMed Central

    Tang, Xiao-Bing; Zhang, Jin; Wang, Wei-Lin; Yuan, Zheng-Wei; Bai, Yu-Zuo

    2015-01-01

    Background: This study aimed to determine the spatiotemporal expression of caudal-type homeobox genes (CDX1, CDX2 and CDX4) during development of the midgut in human embryos and to explore the possible roles of CDX genes during the morphogenesis of human midgut. Human embryos (n=28) were sectioned serially and sagittally and CDX1, CDX2 and CDX4 proteins were detected on the midline from the 5th to 9th weeks of gestation by immunohistochemical staining. Results: CDX1, CDX2 and CDX4 proteins were weakly expressed in epithelium and mesenchyme of the midgut in the 6th and 7th weeks of gestation and reached estimated optimal level on the 8th and 9th weeks of gestation. In the 9th week of gestation, immunoreactivities specific to CDX1, CDX2 and CDX4 were restricted in epithelium of the midgut. Conclusions: CDX1, CDX2 and CDX4 proteins began to express in human midgut in the 6th week of gestation. From the 6th to 9th week of gastation, the expression of CDX1, CDX2 and CDX4 proteins gradually increase and exhibited overlapping expression patterns, suggesting that CDX genes may be involved in early development of the epithelium of human midgut. Cross-regulatory interactions may exist among CDX genes with respect to human midgut development. PMID:26884902

  8. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  9. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  10. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  11. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  12. AB223. Expression of tight junction proteins in rat vagina

    PubMed Central

    Oh, Kyung Jin; Lee, Hyun-Suk; Chung, Ho Suck; Ahn, Kyu Youn; Park, Kwangsung

    2014-01-01

    Aim Tight junction plays a role in apical cell-to-cell adhesion and epithelial polarity. In this study, we investigated the expression of tight junction proteins, such as Claudin-1, zonula occludens (ZO)-1, junction adhesion molecule (JAM)-A, and occludin in rat vagina. Methods Female Sprague-dawley rats (230-240 g, n=20) were divided into two groups: control (n=10) and bilateral ovariectomy (n=10). The expression and cellular localization of claudin-1, ZO-1, JAM-A, and occludin were determined in each group by immunohistochemistry and Western blot. Results Immunolabeling of ZO-1 was mainly expressed in the capillaries and venules of the vagina. Claudin-1, JAM-A, and occludin were expressed in the epithelium of the vagina. The immunoreactivity and protein expression of claudin-1 was significantly decreased in the ovariectomy group compared with the control group. Conclusions Our results suggest that tight junction proteins may have an important role in the vagina. Further studies are needed to clarify the role of each tight junction protein on vaginal lubrication.

  13. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

    PubMed

    Carmona-Rodríguez, Bruno; Alvarez-Pérez, Marco Antonio; Narayanan, A Sampath; Zeichner-David, Margarita; Reyes-Gasga, José; Molina-Guarneros, Juan; García-Hernández, Ana Lilia; Suárez-Franco, José Luis; Chavarría, Ivet Gil; Villarreal-Ramírez, Eduardo; Arzate, Higinio

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  14. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  15. Expression and clinical significance of Kelch-like epichlorohydrin-associated protein 1 in breast cancer.

    PubMed

    Zhang, L; Yang, W P; Wu, L Y; Zhu, X; Wei, C Y

    2016-05-12

    Our objective was to explore the expression and clinical significance of Kelch-like epichlorohydrin-associated protein 1 (Keap1) in breast cancer tissue. Eighty-one breast cancer patients having undergone surgical treatment in our hospital between March 2002 and December 2008 were enrolled in this study. Normal tissue adjacent to tumors was used for the control samples. Diagnoses for all patients were confirmed by postoperative pathological examination. Immunohistochemical assays were used to measure the expression of Keap1 protein in breast cancer tissue and adjacent normal tissue, and its clinical significance was explored. We observed that 24.6% breast cancer tissue samples were positive for Keap1, a significantly lower proportion than that seen with adjacent normal tissue specimens (80.2%; P < 0.05). The presence of Keap1 expression did not correlate with age, tumor size, pathological classification, or degree of differentiation. However, it was found to be significantly associated with tumor-node-metastasis stage and the presence of lymphatic metastasis. Kaplan-Meier survival analysis showed a remarkably higher five-year survival rate among patients with positive Keap1 expression than in those lacking detectable levels of the protein (P = 0.032). Keap1 expression is significantly decreased in breast cancer tissue; therefore, the early detection of its expression might have great significance in determining prognosis for breast cancer patients.

  16. Fe-S Proteins that Regulate Gene Expression

    PubMed Central

    Mettert, Erin L.; Kiley, Patricia J.

    2014-01-01

    Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

  17. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  18. Expression of Aequorea green fluorescent protein in plant cells.

    PubMed

    Hu, W; Cheng, C L

    1995-08-07

    The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

  19. Early Evolution of Vertebrate Mybs: An Integrative Perspective Combining Synteny, Phylogenetic, and Gene Expression Analyses.

    PubMed

    Campanini, Emeline B; Vandewege, Michael W; Pillai, Nisha E; Tay, Boon-Hui; Jones, Justin L; Venkatesh, Byrappa; Hoffmann, Federico G

    2015-10-15

    The genes in the Myb superfamily encode for three related transcription factors in most vertebrates, A-, B-, and c-Myb, with functionally distinct roles, whereas most invertebrates have a single Myb. B-Myb plays an essential role in cell division and cell cycle progression, c-Myb is involved in hematopoiesis, and A-Myb is involved in spermatogenesis and regulating expression of pachytene PIWI interacting RNAs, a class of small RNAs involved in posttranscriptional gene regulation and the maintenance of reproductive tissues. Comparisons between teleost fish and tetrapods suggest that the emergence and functional divergence of the Myb genes were linked to the two rounds of whole-genome duplication early in vertebrate evolution. We combined phylogenetic, synteny, structural, and gene expression analyses of the Myb paralogs from elephant shark and lampreys with data from 12 bony vertebrates to reconstruct the early evolution of vertebrate Mybs. Phylogenetic and synteny analyses suggest that the elephant shark and Japanese lamprey have copies of the A-, B-, and c-Myb genes, implying their origin could be traced back to the common ancestor of lampreys and gnathostomes. However, structural and gene expression analyses suggest that their functional roles diverged between gnathostomes and cyclostomes. In particular, we did not detect A-Myb expression in testis suggesting that the involvement of A-Myb in the pachytene PIWI interacting RNA pathway is probably a gnathostome-specific innovation. We speculate that the secondary loss of a central domain in lamprey A-Myb underlies the functional differences between the cyclostome and gnathostome A-Myb proteins.

  20. Expression of wilms' tumor gene and protein localization during ovarian formation and follicular development in sheep.

    PubMed

    Logan, Kathleen A; McNatty, Kenneth P; Juengel, Jennifer L

    2003-02-01

    Wilms' tumor protein (WT1) is a transcriptional repressor essential for the development of mammalian kidneys and gonads. To gain insight into possible roles of WT1 in ovarian formation and follicular function, we studied patterns of mRNA and protein localization throughout fetal gonadal development and in ovaries of 4-wk-old and adult sheep. At Day 24 after conception, strong expression of WT1 mRNA and protein was observed in the coelomic epithelial region of the mesonephros where the gonad was forming. By Day 30, expression was observed in the surface epithelium and in many mesenchymal and endothelial cells of the gonad. Epithelial cells continued to express WT1 throughout gonadal development, as did pregranulosa cells during the process of follicular formation. However, WT1 expression was not observed in germ cells. During follicular growth, granulosa cells expressed WT1 from the type 1 (primordial) to the type 4 stages, but thereafter expression was reduced in type 5 (antral) follicles, consistent with the differentiation of granulosa cells into steroid-producing cells. The possible progenitor cells for the theca interna (i.e., the cell streams in the ovarian interstitium) expressed WT1 heterogeneously. However, differentiated theca cells in antral follicles did not express WT1. Strong expression of WT1 was observed during gonadal development, which is consistent with a role for WT1 in ovarian and follicular formation in the ewe. WT1 was identified in many cells of the neonatal and adult ovaries, including granulosa cells, suggesting that this factor is important for preantral follicular growth. However, the decline in WT1 expression in antral follicles suggests that WT1 may prevent premature differentiation of somatic cells of the follicle during early follicular growth.

  1. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  2. Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.

    PubMed

    Schild, Christof; Beyeler, Michael; Lang, Niklaus P; Trueb, Beat

    2009-02-01

    Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP.

  3. Selection of soluble protein expression constructs: the experimental determination of protein domain boundaries.

    PubMed

    Dyson, Michael R

    2010-08-01

    Proteins can contain multiple domains each of which is capable of possessing a separate independent function and three-dimensional structure. It is often useful to clone and express individual protein domains to study their biochemical properties and for structure determination. However, the annotated domain boundaries in databases such as Pfam or SMART are not always accurate. The present review summarizes various strategies for the experimental determination of protein domain boundaries.

  4. Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

    PubMed

    Nguyen, Tien Dung; Moon, Sunok; Nguyen, Van Ngoc Tuyet; Gho, Yunsil; Chandran, Anil Kumar Nalini; Soh, Moon-Soo; Song, Jong Tae; An, Gynheung; Oh, Sung Aeong; Park, Soon Ki; Jung, Ki-Hong

    2016-09-01

    Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

  5. Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

    PubMed Central

    Burns, Tara A.; Dours-Zimmermann, Maria T.; Zimmermann, Dieter R.; Krug, Edward L.; Comte-Walters, Susana; Reyes, Leticia; Davis, Monica A.; Schey, Kevin L.; Schwacke, John H.; Kern, Christine B.; Mjaatvedt, Corey H.

    2014-01-01

    The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles. PMID:24586547

  6. Properties of a novel gene isolated from a Hodgkin's disease cell line that is expressed early during lymphoid cell activation

    SciTech Connect

    Bennett, J.S.; Tredway, T.L.; Dizikes, G.J.; Nawrocki, J.F. Hines Veterans Administration Hospital, IL )

    1994-03-01

    The authors have isolated a novel 667-bp cDNA clone, designated epag, from a Hodgkin's-disease cell line-derived library that is expressed in association with T cell activation and which is not related to any known gene family. By using reverse transcription/PCR, the authors have demonstrated that epag mRNA is expressed as early as 1 h after stimulation of normal PBMCs with anti-CD3. The levels of mRNA peaked by 4 h, and no expression was detectable by 12 h postactivation or in resting cells incubated in culture without activation. Expression of epag was also detected in PMA- and PHA-stimulated, but not in nonstimulated Jurkat cells, and overall its expression in transformed cell lines of hemopoietic origin is highly restricted. Sequence analysis of multiple independent cDNA clones showed that epag expressed in the Hodgkin's-disease cell line L428 is identical to the gene expressed in normal activated PBMC. Epag expression was detected by reverse transcription/PCR in RNA preparations made from various normal nonlymphoid tissues. Computer analysis of the sequence identified an open reading frame encoding a putative protein of 13.2 kDa initiating at a CUG translational codon. In vitro translation and Western blot analysis with anti-peptide serum supported this analysis. The authors hypothesize that epag functions as an early signal that helps mediate the activation of T cells. 63 refs., 11 figs.

  7. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  8. Computational codon optimization of synthetic gene for protein expression

    PubMed Central

    2012-01-01

    Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. PMID:23083100

  9. Expression and detection of LINE-1 ORF-encoded proteins.

    PubMed

    Dai, Lixin; LaCava, John; Taylor, Martin S; Boeke, Jef D

    2014-01-01

    LINE-1 (L1) elements are endogenous retrotransposons active in mammalian genomes. The L1 RNA is bicistronic, encoding two non-overlapping open reading frames, ORF1 and ORF2, whose protein products (ORF1p and ORF2p) bind the L1 RNA to form a ribonucleoprotein (RNP) complex that is presumed to be a critical retrotransposition intermediate. However, ORF2p is expressed at a significantly lower level than ORF1p; these differences are thought to be controlled at the level of translation, due to a low frequency ribosome reinitiation mechanism controlling ORF2 expression. As a result, while ORF1p is readily detectable, ORF2p has previously been very challenging to detect in vitro and in vivo. To address this, we recently tested several epitope tags fused to the N- or C-termini of the ORF proteins in an effort to enable robust detection and affinity purification from native (L1RP) and synthetic (ORFeus-Hs) L1 constructs. An analysis of tagged RNPs from both L1RP and ORFeus-Hs showed similar host-cell-derived protein interactors. Our observations also revealed that the tag sequences affected the retrotransposition competency of native and synthetic L1s differently although they encode identical ORF proteins. Unexpectedly, we observed apparently stochastic expression of ORF2p within seemingly homogenous L1-expressing cell populations.

  10. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  11. Effects of maternal separation and methamphetamine exposure on protein expression in the nucleus accumbens shell and core.

    PubMed

    Dimatelis, J J; Russell, V A; Stein, D J; Daniels, W M

    2012-09-01

    Early life adversity has been suggested to predispose an individual to later drug abuse. The core and shell sub-regions of the nucleus accumbens are differentially affected by both stressors and methamphetamine. This study aimed to characterize and quantify methamphetamine-induced protein expression in the shell and core of the nucleus accumbens in animals exposed to maternal separation during early development. Isobaric tagging (iTRAQ) which enables simultaneous identification and quantification of peptides with tandem mass spectrometry (MS/MS) was used. We found that maternal separation altered more proteins involved in structure and redox regulation in the shell than in the core of the nucleus accumbens, and that maternal separation and methamphetamine had differential effects on signaling proteins in the shell and core. Compared to maternal separation or methamphetamine alone, the maternal separation/methamphetamine combination altered more proteins involved in energy metabolism, redox regulatory processes and neurotrophic proteins. Methamphetamine treatment of rats subjected to maternal separation caused a reduction of cytoskeletal proteins in the shell and altered cytoskeletal, signaling, energy metabolism and redox proteins in the core. Comparison of maternal separation/methamphetamine to methamphetamine alone resulted in decreased cytoskeletal proteins in both the shell and core and increased neurotrophic proteins in the core. This study confirms that both early life stress and methamphetamine differentially affect the shell and core of the nucleus accumbens and demonstrates that the combination of early life adversity and later methamphetamine use results in more proteins being affected in the nucleus accumbens than either treatment alone.

  12. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  13. Optimization of Translation Profiles Enhances Protein Expression and Solubility

    PubMed Central

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. PMID:25965266

  14. Optimization of translation profiles enhances protein expression and solubility.

    PubMed

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  15. A characterization of structural proteins expressed by Bombyx mori bidensovirus.

    PubMed

    Lü, Peng; Xing, Yali; Hu, Zhaoyang; Yang, Yanhua; Pan, Ye; Chen, Kangmin; Zhu, Feifei; Zhou, Yajing; Chen, Keping; Yao, Qin

    2017-03-01

    Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5kb (viral DNA 1, VD1) and 6.0kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22-24nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism.

  16. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  17. Differentiating disease subtypes by using pathway patterns constructed from gene expressions and protein networks.

    PubMed

    Hung, Fei-Hung; Chiu, Hung-Wen

    2015-01-01

    Gene expression profiles differ in different diseases. Even if diseases are at the same stage, such diseases exhibit different gene expressions, not to mention the different subtypes at a single lesion site. Distinguishing different disease subtypes at a single lesion site is difficult. In early cases, subtypes were initially distinguished by doctors. Subsequently, further differences were found through pathological experiments. For example, a brain tumor can be classified according to its origin, its cell-type origin, or the tumor site. Because of the advancements in bioinformatics and the techniques for accumulating gene expressions, researchers can use gene expression data to classify disease subtypes. Because the operation of a biopathway is closely related to the disease mechanism, the application of gene expression profiles for clustering disease subtypes is insufficient. In this study, we collected gene expression data of healthy and four myelodysplastic syndrome subtypes and applied a method that integrated protein-protein interaction and gene expression data to identify different patterns of disease subtypes. We hope it is efficient for the classification of disease subtypes in adventure.

  18. Expression of glutamine metabolism-related proteins in thyroid cancer

    PubMed Central

    Kim, Hye Min; Lee, Yu Kyung; Koo, Ja Seung

    2016-01-01

    Purpose This study aimed to investigate the expression of glutamine metabolism-related protein in tumor and stromal compartments among the histologic subtypes of thyroid cancer. Results GLS1 and GDH expression in tumor and stromal compartments were the highest in AC than in other subtypes. Tumoral ASCT2 expression was higher in MC but lower in FC (p < 0.001). In PTC, tumoral GLS1 and tumoral GDH expression was higher in the conventional type than in the follicular variant (p = 0.043 and 0.001, respectively), and in PTC with BRAF V600E mutation than in PTC without BRAF V600E mutation (p<0.001). Stromal GDH positivity was the independent factor associated with short overall survival (hazard ratio: 21.48, 95% confidence interval: 2.178-211.8, p = 0.009). Methods We performed tissue microarrays with 557 thyroid cancer cases (papillary thyroid carcinoma [PTC]: 344, follicular carcinoma [FC]: 112, medullary carcinoma [MC]: 70, poorly differentiated carcinoma [PDC]: 23, and anaplastic carcinoma [AC]: 8) and 152 follicular adenoma (FA) cases. We performed immunohistochemical staining of glutaminolysis-related proteins (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter-2 [ASCT-2]). Conclusion Glutamine metabolism-related protein expression differed among the histologic subtypes of thyroid cancer. PMID:27447554

  19. Methods and constructs for expression of foreign proteins in photosynthetic organisms

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2002-01-01

    A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

  20. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  1. Dark proteins: effect of inclusion body formation on quantification of protein expression.

    PubMed

    Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

    2008-09-01

    Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

  2. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  3. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  4. Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body

    PubMed Central

    Seita, Yasunari; Tsukiyama, Tomoyuki; Iwatani, Chizuru; Tsuchiya, Hideaki; Matsushita, Jun; Azami, Takuya; Okahara, Junko; Nakamura, Shinichiro; Hayashi, Yoshitaka; Hitoshi, Seiji; Itoh, Yasushi; Imamura, Takeshi; Nishimura, Masaki; Tooyama, Ikuo; Miyoshi, Hiroyuki; Saitou, Mitinori; Ogasawara, Kazumasa; Sasaki, Erika; Ema, Masatsugu

    2016-01-01

    Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson’s disease and Alzheimer’s disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research. PMID:27109065

  5. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

  6. Correlation of gene expression and protein production rate - a system wide study

    PubMed Central

    2011-01-01

    Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR). PMID:22185473

  7. Grape seed extract inhibits VEGF expression via reducing HIF-1α protein expression

    PubMed Central

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-01-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1α. The inhibitory effect of GSE on HIF-1α expression was mainly through inhibiting HIF-1α protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1α protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1α and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1α, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1α protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE. PMID:19131542

  8. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  9. Morphine Withdrawal Modifies Prion Protein Expression in Rat Hippocampus

    PubMed Central

    Mattei, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Garofalo, Tina; Candelise, Niccolò; Caruso, Alessandra; Sorice, Maurizio; Scaccianoce, Sergio

    2017-01-01

    The hippocampus is a vulnerable brain structure susceptible to damage during aging and chronic stress. Repeated exposure to opioids may alter the brain so that it functions normally when the drugs are present, thus, a prolonged withdrawal might lead to homeostatic changes headed for the restoration of the physiological state. Abuse of morphine may lead to Reacting Oxygen Species-induced neurodegeneration and apoptosis. It has been proposed that during morphine withdrawal, stress responses might be responsible, at least in part, for long-term changes of hippocampal plasticity. Since prion protein is involved in both, Reacting Oxygen Species mediated stress responses and synaptic plasticity, in this work we investigate the effect of opiate withdrawal in rats after morphine treatment. We hypothesize that stressful stimuli induced by opiate withdrawal, and the subsequent long-term homeostatic changes in hippocampal plasticity, might modulate the Prion protein expression. Our results indicate that abstinence from the opiate induced a time-dependent and region-specific modification in Prion protein content, indeed during morphine withdrawal a selective unbalance of hippocampal Prion Protein is observable. Moreover, Prion protein overexpression in hippocampal tissue seems to generate a dimeric structure of Prion protein and α-cleavage at the hydrophobic domain. Stress factors or toxic insults can induce cytosolic dimerization of Prion Protein through the hydrophobic domain, which in turn, it stimulates the α-cleavage and the production of neuroprotective Prion protein fragments. We speculate that this might be the mechanism by which stressful stimuli induced by opiate withdrawal and the subsequent long-term homeostatic changes in hippocampal plasticity, modulate the expression and the dynamics of Prion protein. PMID:28081197

  10. Expression and Localization of Plant Protein Disulfide Isomerase.

    PubMed Central

    Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

    1993-01-01

    A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

  11. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo

    PubMed Central

    Hostettler, Lola; Grundy, Laura; Käser-Pébernard, Stéphanie; Wicky, Chantal; Schafer, William R.; Glauser, Dominique A.

    2017-01-01

    The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro. The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans—a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3–5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression. PMID:28108553

  12. Gene expression profiling during spermatogenesis in early maturing male Atlantic salmon parr testes.

    PubMed

    Maugars, Gersende; Schmitz, Monika

    2008-01-01

    The initiation of sexual maturation and spermatogenesis are complex processes that require the highly coordinated regulation of a number of key genes. The endocrine system plays crucial roles in these processes, but the precise mechanisms involved in sexual maturation of fish are poorly understood. We investigated the expression of genes encoding proteins involved in sex steroid biosynthesis (Ff1b (FTZ-F1 homolog), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17), cytochrome P450 11beta-hydroxylase (P45011beta) and 11beta-hydroxysteroid dehydrogenase (11beta-HSD)) and the anti-Müllerian hormone (AMH) homolog during early sexual maturation of one-summer-old male Atlantic salmon parr by RT-PCR. Genes encoding Ff1b, StAR, 3beta-HSD, P450c17 and 11beta-HSD were upregulated during spermatogonial proliferation. During the course of spermatogenesis expression profiles of Ff1b, StAR, 3beta-HSD, P450scc, P450c17, P45011beta, and 11beta-HSD were similar; transcript levels being low during early stages, then strongly increasing during spermiogenesis. These results indicate that coordinated de novo transcription of genes encoding StAR as well as 3beta-HSD, P450c17 and 11beta-HSD might be required for sex steroids production during the initiation of spermatogenesis in salmon. In contrast, transcription levels of AMH were comparatively high in immature testes, decreased when spermatogenesis was initiated, and were lowest during spermiogenesis, suggesting that AMH suppression plays a crucial role in the process of spermatogenesis in salmonids. Correlation analyses show that FSH and LH might be differentially involved in the regulation of several of these genes studied.

  13. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  14. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis.

    PubMed

    Khan, Junaid A; Wang, Qi; Sjölund, Richard D; Schulz, Alexander; Thompson, Gary A

    2007-04-01

    Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae) tissue cultures, recognizes an antigen in the Arabidopsis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve elements, but not companion cells, and accumulates at the earliest stages of sieve element differentiation. The identity of the RS6 antigen was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins that have a similar overall domain structure of an amino-terminal signal peptide, plastocyanin-like copper-binding domain, proline/serine-rich domain, and carboxy-terminal hydrophobic domain. The amino- and carboxy-terminal domains of the 21.5-kD sieve element-specific ENOD are posttranslationally cleaved from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both floral and vegetative tissues, the sieve element-specific ENOD is expressed only within the phloem. Members of the ENOD subfamily of the cupredoxin superfamily do not appear to bind copper and have unknown functions. Phenotypic analysis of homozygous T-DNA insertion mutants for the gene At3g20570 shows minimal alteration in vegetative growth but a significant reduction in the overall reproductive potential.

  15. Beta-amyloid oligomers induce early loss of presynaptic proteins in primary neurons by caspase-dependent and proteasome-dependent mechanisms.

    PubMed

    Jang, Bong Geum; In, Sua; Choi, Boyoung; Kim, Min-Ju

    2014-11-12

    Beta-amyloid is a major pathogenic molecule for Alzheimer's disease (AD) and can be aggregated into a soluble oligomer, which is a toxic intermediate, before amyloid fibril formation. Beta-amyloid oligomers are associated closely with early synaptic loss in AD. However, it is still unknown which synaptic proteins are involved in the synaptotoxicity, and a direct comparison among the synaptic proteins should also be addressed. Here, we investigated changes in the expression of several presynaptic and postsynaptic proteins in primary neurons after treatment with a low-molecular weight and a high-molecular weight beta-amyloid oligomer. Both oligomers induced early neuronal dysfunction after 4 h and significantly reduced presynaptic protein (synaptophysin, syntaxin, synapsin, and synaptotagmin) expression. However, the expression of postsynaptic proteins (PSD95, NMDAR2A/B, and GluR2/3), except NMDAR1 was not reduced, and some protein expression levels were increased. Glutamate treatment, which is correlated with postsynaptic activation, showed more postsynaptic-specific protein loss compared with beta-amyloid oligomer treatment. Finally, the caspase inhibitor zVAD and the proteasomal inhibitor MG132 attenuated presynaptic protein loss. Thus, our data showed changes in synaptic proteins by beta-amyloid oligomers, which provides an understanding of early synaptotoxicity and suggests new approaches for AD treatment.

  16. Heat shock protein gene expression and function in amphibian model systems.

    PubMed

    Heikkila, John J

    2010-05-01

    Heat shock proteins (HSPs) are molecular chaperones that are involved in protein folding and translocation. During heat shock, both constitutive and stress-inducible HSPs bind to and inhibit irreversible aggregation of denatured protein and facilitate their refolding once normal cellular conditions are re-established. Recent interest in HSPs has been propelled by their association with various human diseases. Amphibian model systems, as shown in this review, have had a significant impact on our understanding of hsp gene expression and function. Some amphibian hsp genes are expressed constitutively during oogenesis and embryogenesis, while others are developmentally regulated and enriched in selected tissues in a stress-inducible fashion. For example, while hsp70 genes are heat-inducible after the midblastula stage, hsp30 genes are not inducible until late neurula/early tailbud. This particular phenomenon is likely controlled by chromatin structure. Also, hsp genes are expressed during regeneration, primarily in response to wounding-associated trauma. The availability of amphibian cultured cells has enabled the analysis of hsp gene expression induced by different stresses (e.g. cadmium, arsenite, proteasome inhibitors etc.), HSP intracellular localization, and their involvement in stress resistance. Furthermore, hyperthermia treatment of adult amphibians reveals that certain tissues were more sensitive than others in terms of hsp gene expression. Finally, this review details the evidence available for the role of amphibian small HSPs as molecular chaperones.

  17. Low Density Lipoprotein Receptor-Related Protein and Apolipoprotein E Expression is Altered in Schizophrenia

    PubMed Central

    Gibbons, Andrew Stuart; Thomas, Elizabeth A.; Scarr, Elizabeth; Dean, Brian

    2010-01-01

    Our recent microarray study reported altered mRNA expression of several low density lipoprotein receptor-related proteins (LRP) associated with the first 4 years following diagnosis with schizophrenia. Whilst this finding is novel, apolipoprotein E (APOE), which mediates its activity through LRPs, has been reported by several studies to be altered in brains of subjects with schizophrenia. We used qPCR to measure the expression of LRP2, LRP4, LRP6, LRP8, LRP10 and LRP12 mRNA in Brodmann's area (BA) 46 of the dorsolateral prefrontal cortex in 15 subjects with short duration of illness schizophrenia (SDS) and 15 pair matched controls. We also used Western blotting to measure APOE protein expression in BA46 from these subjects. Amongst the LRPs examined, LRP10 expression was significantly increased (P = 0.03) and LRP12 was significantly decreased (P < 0.01) in SDS. APOE protein expression was also increased in SDS (P = 0.01). No other marker examined in this study was altered with diagnosis. Our data supports a role for distinct members of the LRP family in the pathology of schizophrenia and adds weight to the hypothesis that aberrant apolipoprotein signaling is involved in the early stages of schizophrenia. PMID:21423430

  18. Tools to cope with difficult-to-express proteins.

    PubMed

    Saccardo, Paolo; Corchero, José Luís; Ferrer-Miralles, Neus

    2016-05-01

    The identification of DNA coding sequences contained in the genome of many organisms coupled to the use of high throughput approaches has fueled the field of recombinant protein production. Apart from basic research interests, the growing relevance of this field is highlighted by the global sales of the top ten biopharmaceuticals on the market, which exceeds the trillion USD in a steady increasing tendency. Therefore, the demand of biological compounds seems to have a long run on the market. One of the most popular expression systems is based on Escherichia coli cells which apart from being cost-effective counts with a large selection of resources. However, a significant percentage of the genes of interest are not efficiently expressed in this system, or the expressed proteins are accumulated within aggregates, degraded or lacking the desired biological activity, being finally discarded. In some instances, expressing the gene in a homologous expression system might alleviate those drawbacks but then the process usually increases in complexity and is not as cost-effective as the prokaryotic systems. An increasing toolbox is available to approach the production and purification of those difficult-to-express proteins, including different expression systems, promoters with different strengths, cultivation media and conditions, solubilization tags and chaperone coexpression, among others. However, in most cases, the process follows a non-integrative trial and error strategy with discrete success. This review is focused on the design of the whole process by using an integrative approach, taken into account the accumulated knowledge of the pivotal factors that affect any of the key processes, in an attempt to rationalize the efforts made in this appealing field.

  19. Digital Gene Expression Tag Profiling Analysis of the Gene Expression Patterns Regulating the Early Stage of Mouse Spermatogenesis

    PubMed Central

    Meng, Lijun; Liu, Meiling; Zhao, Lina; Hu, Fen; Ding, Cunbao; Wang, Yang; He, Baoling; Pan, Yuxin; Fang, Wei; Chen, Jing; Hu, Songnian; Jia, Mengchun

    2013-01-01

    Detailed characterization of the gene expression patterns in spermatogonia and primary spermatocytes is critical to understand the processes which occur prior to meiosis during normal spermatogenesis. The genome-wide expression profiles of mouse type B spermatogonia and primary spermatocytes were investigated using the Solexa/Illumina digital gene expression (DGE) system, a tag based high-throughput transcriptome sequencing method, and the developmental processes which occur during early spermatogenesis were systematically analyzed. Gene expression patterns vary significantly between mouse type B spermatogonia and primary spermatocytes. The functional analysis revealed that genes related to junction assembly, regulation of the actin cytoskeleton and pluripotency were most significantly differently expressed. Pathway analysis indicated that the Wnt non-canonical signaling pathway played a central role and interacted with the actin filament organization pathway during the development of spermatogonia. This study provides a foundation for further analysis of the gene expression patterns and signaling pathways which regulate the molecular mechanisms of early spermatogenesis. PMID:23554914

  20. EARLY SENESCENCE1 Encodes a SCAR-LIKE PROTEIN2 That Affects Water Loss in Rice1[OPEN

    PubMed Central

    Rao, Yuchun; Yang, Yaolong; Xu, Jie; Li, Xiaojing; Leng, Yujia; Dai, Liping; Huang, Lichao; Shao, Guosheng; Ren, Deyong; Hu, Jiang; Guo, Longbiao; Pan, Jianwei; Zeng, Dali

    2015-01-01

    The global problem of drought threatens agricultural production and constrains the development of sustainable agricultural practices. In plants, excessive water loss causes drought stress and induces early senescence. In this study, we isolated a rice (Oryza sativa) mutant, designated as early senescence1 (es1), which exhibits early leaf senescence. The es1-1 leaves undergo water loss at the seedling stage (as reflected by whitening of the leaf margin and wilting) and display early senescence at the three-leaf stage. We used map-based cloning to identify ES1, which encodes a SCAR-LIKE PROTEIN2, a component of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous complex involved in actin polymerization and function. The es1-1 mutants exhibited significantly higher stomatal density. This resulted in excessive water loss and accelerated water flow in es1-1, also enhancing the water absorption capacity of the roots and the water transport capacity of the stems as well as promoting the in vivo enrichment of metal ions cotransported with water. The expression of ES1 is higher in the leaves and leaf sheaths than in other tissues, consistent with its role in controlling water loss from leaves. GREEN FLUORESCENT PROTEIN-ES1 fusion proteins were ubiquitously distributed in the cytoplasm of plant cells. Collectively, our data suggest that ES1 is important for regulating water loss in rice. PMID:26243619

  1. Expression of low molecular weight proteins in patients with leukaemia.

    PubMed

    Sheikh, N; Abid, R; Qureshi, A W; Basheer, T

    2012-06-01

    The current study is conducted to observe the differences in the level of low molecular weight proteins in the sera of patients with leukaemia in comparison to healthy subjects (control group). The sera of patients with leukaemia showed 15 peaks in the densitometric curve in comparison to the seven peaks of the controls. The peaks in the experimental samples that coincide with those in the control were of 134.14, 113.15, 76.06, 63.25, 48.07, 22.85 and 16.47 kDa molecular weights, respectively. Most of the new peaks appeared between the proteins of molecular weight 36-29 kDa in the experimental groups. Mean density of the 134.14 kDa protein band showed an increase in the protein in experimental groups I and II only whereas 113.15 and 22.85 kDa protein were increased in all experimental groups of patients with leukaemia. The expression of 76.06 and 63.25 kDa protein fraction was downregulated in the patients with leukaemia. A decline in the level of the protein of 48.07 kDa was observed in patients with leukaemia except in group I. Unlike the other protein fractions, the level of the protein of 16.47 kDa was significantly (p < 0.05) increased with a maximum density in group II. Intergroup experimental) comparison revealed an increasing pattern of 95.44 and 89.21 kDa with maximum level in group III sera. However the protein fractions of 38.07 and 34.94 kDa varied in the serum with maximum density in Group IV Protein fractions of 32.92 and 31.24 kDa were expressed in all age groups of patients with leukaemia with a maximum density in group III whereas the percentage densities of 14.42 and 13.56 kDa protein were quite different. This preliminary study will provide a basis to study the role of different proteins in patients with leukaemia.

  2. FIAT is co-expressed with its dimerization target ATF4 in early osteoblasts, but not in osteocytes.

    PubMed

    Yu, Vionnie W C; Akhouayri, Omar; St-Arnaud, René

    2009-06-01

    FIAT represses osteocalcin gene transcription by heterodimerizing with ATF4 and preventing it from binding to DNA. We report here the expression profiles of FIAT and ATF4 during osteoblastogenesis. Messenger RNA levels for the osteoblast transcriptional regulators Satb2, Runx2, Fiat, and Atf4 were quantified using real-time reverse-transcription PCR (RT-qPCR) and respective protein levels monitored by immunodetection in differentiating primary osteoblast cultures. Satb2, Fiat, and Atf4 mRNA levels remained constant throughout the differentiation sequence, whereas Runx2 transcript levels were significantly increased by 12 days post-confluency. Using immunofluorescence, the SATB2, RUNX2, and ATF4 signals appeared to increase as a function of time in culture. FIAT protein expression was readily detected in early cultures, but signal intensity decreased thereafter. When immunoblotting was used to quantify the relative amounts of FIAT and ATF4 proteins, the expression levels of the two proteins were found to be inversely correlated. The decrease in FIAT protein levels coincided with increased binding of ATF4 to the osteocalcin gene promoter, and with increased osteocalcin expression measured by RT-qPCR or immunoblotting. Immunohistochemistry of long bones from mice at E16.5 and 2 days post-natal revealed that both proteins are initially expressed in osteoblasts. In adult bone, FIAT was detected in osteocytes, while ATF4 expression was observed in active osteoblasts and lining cells, but not in osteocytes. Taken together, these data support the idea that a stoichiometric excess of ATF4 over FIAT in mature osteoblasts releases ATF4 from sequestration by FIAT, thereby allowing ATF4 homodimerization and subsequent transactivation of the osteocalcin gene.

  3. Early postnatal proteolipid promoter-expressing progenitors produce multilineage cells in vivo.

    PubMed

    Guo, Fuzheng; Ma, Joyce; McCauley, Erica; Bannerman, Peter; Pleasure, David

    2009-06-03

    Proteolipid promoter (plp promoter) activity in the newborn mouse CNS is restricted to NG2-expressing oligodendroglial progenitor cells and oligodendrocytes. There are two populations of NG2 progenitors based on their plp promoter expression. Whereas the general population of NG2 progenitors has been shown to be multipotent in vitro and after transplantation, it is not known whether the subpopulation of plp promoter-expressing NG2 progenitors [i.e., plp promoter-expressing NG2 progenitors (PPEPs)] has the potential to generate multilineage cells during normal development in vivo. We addressed this issue by fate mapping Plp-Cre-ER(T2)/Rosa26-EYFP (PCE/R) double-transgenic mice, which carried an inducible Cre gene under the control of the plp promoter. Expression of the enhanced yellow fluorescent protein (EYFP) reporter gene in PPEPs was elicited by administering tamoxifen to postnatal day 7 PCE/R mice. We have demonstrated that early postnatal PPEPs, which had been thought to be restricted to the oligodendroglial lineage, also unexpectedly gave rise to a subset of immature, postmitotic, protoplasmic astrocytes in the gray matter of the spinal cord and ventral forebrain, but not in white matter. Furthermore, these PPEPs also gave rise to small numbers of immature, DCX (doublecortin)-negative neurons in the ventral forebrain, dorsal cerebral cortex, and hippocampus. EYFP-labeled representatives of each of these lineages survived to adulthood. These findings indicate that there are regional differences in the fates of neonatal PPEPs, which are multipotent in vivo, giving rise to oligodendrocytes, astrocytes, and neurons.

  4. Expression and serological reactivity of hemorrhagic enteritis virus hexon protein.

    PubMed

    Lobová, Dana; Celer, Vladimír

    2016-05-01

    The aim of this work was to express the recombinant hexon protein of the hemorrhagic enteritis virus, to establish the diagnostic value of this protein for serological detection of antibodies in turkey serum samples and to assess seroprevalence of the infection in the Czech Republic. The N' terminal part of the hexon protein was expressed in a bacterial expression system and used as an antigen in an ELISA test for the detection of hemorrhagic enteritis virus specific antibodies in turkey sera. Validation of the test was performed by comparison with a commercially available ELISA test. Serological reactivity was assessed on a panel of 126 turkey sera by a newly developed ELISA test. Serum samples were taken from turkey farms with the history of hemorrhagic enteritis virus infection, from farms with animals free of infection, and from turkey farms following vaccination. Both ELISA kits gave identical results (100 %) with the tested sera. ELISA based on the recombinant hexon protein thus proved useful and cheaper for detection of antibodies in turkey flocks infected with the hemorrhagic enteritis virus.

  5. Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

    SciTech Connect

    Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.; Bilimoria, Shaen L.

    2008-01-20

    Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV{sub XS}; 400 {mu}g/ml), UV-irradiated virus (CIV{sub UV}; 10 {mu}g/ml) and CVPE (CIV protein extract; 10 {mu}g/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 {mu}g/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV{sub UV} or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV{sub UV} particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV{sub UV}, CIV{sub XS} or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae

  6. Prion protein expression in bovine podocytes and extraglomerular mesangial cells.

    PubMed

    Amselgruber, W M; Steffl, M; Didier, A; Märtlbauer, E; Pfaff, E; Büttner, M

    2006-06-01

    The cellular form of the prion protein (PrP(c)) is thought to be a substrate for an abnormal isoform of the prion protein (PrP(sc)). One emerging hypothesis is that the proposed conversion phenomenon takes place at the site at which the infectious agent meets PrP(c). PrP(c) is abundant in the central nervous system, but little is known about the cell-type-specific distribution of PrP(c) in non-neuronal tissues of cattle. We have studied whether PrP(c), a protein found predominantly in neurons, also exists in bovine podocytes, since neurons and podocytes share a large number of similarities. We have therefore examined the expression of PrP(c) by immunohistochemistry, reverse transcription/polymerase chain reaction and enzyme-linked immunosorbent analysis. Immunostained serial sections and specific antibodies against PrP(c) have revealed that PrP(c) is selectively localized in podocytes and is particularly strongly expressed in extraglomerular mesangial cells but not in endothelial or intraglomerular mesangial cells. The selective expression of PrP(c) in podocytes is of special importance, as it suggests that these cells represent possible targets for peripheral infection with prions and demonstrates that PrP(c) can be added to the list of neuronal factors expressed in mammalian podocytes.

  7. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  8. Protein Phosphatase-1 Regulates Expression of Neuregulin-1

    PubMed Central

    Ammosova, Tatiana; Washington, Kareem; Rotimi, Jamie; Kumari, Namita; Smith, Kahli A.; Niu, Xiaomei; Jerebtsova, Marina; Nekhai, Sergei

    2016-01-01

    Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner. PMID:27918433

  9. Progesterone regulates secretin expression in mouse uterus during early pregnancy.

    PubMed

    Huang, Zhu; Wang, Tong-Song; Qi, Qian-Rong; Zuo, Ru-Juan; Liang, Xiao-Huan; Zhao, Xu-Yu; Yang, Zeng-Ming

    2014-06-01

    Secretin, a classical gastrointestinal and neuroendocrine peptide, plays an important role in maintaining the body fluid balance. However, the expression and regulation of secretin in the reproductive system are still unknown. In our study, secretin is specifically expressed in the decidua on days 5 to 8 of pregnancy. Secretin expression is not detected under delayed implantation but is stimulated after estrogen activation and under artificial decidualization. Progesterone induces secretin expression in ovariectomized mice and cultured stromal cells, which is abrogated by specific LY294002. Because secretin is mainly localized in the decidua and also strongly expressed during in vitro decidualization, secretin may play a role during mouse decidualization through regulating cyclic adenosine monophosphate level.

  10. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    PubMed

    Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2011-01-01

    It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  11. Tissue-Specific Protein Expression in Plant Mitochondria.

    PubMed Central

    Conley, C. A.; Hanson, M. R.

    1994-01-01

    Although the physiological role of plant mitochondria is thought to vary in different tissues at progressive stages of development, there has been little documentation that the complement of mitochondrial proteins is altered in different plant organs. Because the phenomenon of cytoplasmic male sterility suggests an unusual function for mitochondria in floral buds, we examined the tissue-specific expression of mitochondrial proteins in petunia buds at several stages of development, using both fertile and cytoplasmic male sterile plants. On tissue prints of cryostat-sectioned buds, antibodies recognizing subunit A of the mitochondrial ATPase (ATPA) localized very differently from antibodies recognizing subunit II of the cytochrome oxidase (COXII), which indicated that mitochondria in the same tissue could differentially express mitochondrially encoded proteins. The petunia cytoplasmic male sterility-associated fused (pcf) gene encodes a protein that colocalized with ATPA and the nuclear-encoded mitochondrial alternative oxidase (AOA) in sporogenous tissues, where little COXII protein was found. These overlapping and differential localization patterns may provide clues to the molecular mechanism of cytoplasmic male sterility. PMID:12244222

  12. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  13. Eosinophil granule proteins expressed in ocular cicatricial pemphigoid

    PubMed Central

    Heiligenhaus, A.; Schaller, J.; Mauss, S.; Engelbrecht, S.; Dutt, J.; Foster, C; Steuhl, K.

    1998-01-01

    BACKGROUND—Blister formation and tissue damage in bullous pemphigoid have been attributed to the release of eosinophil granule proteins—namely, to eosinophil derived cationic protein (ECP) and major basic protein (MBP). In the present investigation these eosinophil granule proteins were studied in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP).
METHODS—Conjunctival biopsy specimens obtained from patients with subacute (n=8) or chronic conjunctival disease (n=13) were analysed histologically and immunohistochemically using antibodies directed against EG1 (stored and secreted ECP), EG2 (secreted ECP), MBP, CD45 (common leucocyte antigen), CD3 (pan T cell marker), and HLA-DR (class II antigen).
RESULTS—Subepithelial mononuclear cells, mast cells, and neutrophils were detected in all specimens. The number of mononuclear cells, neutrophils, CD45+ cells, CD3+ cells, and the HLA-DR expression were significantly higher in the subacute than in the chronic disease group. Some eosinophils were found in specimens from five of eight patients with subacute OCP, but in none of the patients with chronic disease. The eosinophil granule proteins (ECP and MBP) were found in the epithelium and substantia propria in patients with subacute conjunctivitis.
CONCLUSIONS—Subepithelial cell infiltration in the conjunctiva greatly differs between subacute and chronic ocular cicatricial pemphigoid specimens. The findings suggest that eosinophil granule proteins may participate in tissue damage in acute phase of inflammation in OCP.

 Keywords: ocular cicatricial pemphigoid; conjunctivitis; eosinophil derived cationic protein; major basic protein PMID:9602632

  14. Somatostatin regulates tight junction proteins expression in colitis mice.

    PubMed

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P<0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P<0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P<0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P<0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression.

  15. Somatostatin regulates tight junction proteins expression in colitis mice

    PubMed Central

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P < 0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P < 0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression. PMID:24966923

  16. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  17. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

  18. Epithelial membrane protein 1 expression in ovarian serous tumors.

    PubMed

    Demirag, Guzin Gonullu; Kefeli, Mehmet; Kemal, Yasemin; Yucel, Idris

    2016-03-01

    The present study aimed to analyze the clinical significance of epithelial membrane protein 1 (EMP1) expression in ovarian serous tumors. A total of 84 cases of ovarian serous tumor (50 patients with malignant ovarian serous tumors and 34 patients with borderline and benign serous tumors) were retrospectively analyzed. Differences in the expression levels of EMP1 between the malignant and non-malignant tumor groups were evaluated by immunohistochemical staining. In addition, the association between EMP1 expression and prognostic factors in malignant ovarian serous tumors was investigated. The expression levels of EMP1 were significantly reduced in all the 50 malignant ovarian serous tumors, compared with the 34 non-malignant ovarian serous tumors (P<0.000). Reduced expression of EMP1 was correlated with high grade (P=0.009) and stage (P<0.000) of malignant tumors. EMP1 expression was not observed to be correlated with any other investigated parameters, including surgery, type of operation and chemotherapy response (P>0.005). These results indicated that EMP1 may have a significant role as a negative regulator in ovarian serous tumors, and reduced EMP1 expression in serous tumors may be associated with increased disease severity.

  19. [Expression of transmitter receptor genes in early development of sea urchin Paracentrotus lividus].

    PubMed

    Nikishin, D A; Semenova, M N; Shmukler, Iu B

    2012-01-01

    Neurotransmitters (including serotonin and acetylcholine) perform a number of prenervous functions in early sea urchin development. To detect the particular receptor components involved in these processes, we carried out a database search and nucleotide sequences homologous to serotonin receptor type 4, and the alpha6- and alpha10-subunits of nicotinic acetylcholine receptor were found among EST-clones from early Paracentrotus lividus embryos. Expression of these transcripts during early development was demonstrated using RT-PCR. These results are the first molecular biology evidence ofserotonin and acetylcholine receptor expression in sea urchin early embryogenesis.

  20. Label-Free Proteome Analysis of Plasma from Patients with Breast Cancer: Stage-Specific Protein Expression

    PubMed Central

    Lobo, Marina Duarte Pinto; Moreno, Frederico Bruno Mendes Batista; Souza, Gustavo Henrique Martins Ferreira; Verde, Sara Maria Moreira Lima; Moreira, Renato de Azevedo; Monteiro-Moreira, Ana Cristina de Oliveira

    2017-01-01

    Breast cancer is one of the most commonly diagnosed types of cancer among women. Breast cancer mortality rates remain high probably because its diagnosis is hampered by inaccurate detection methods. Since changes in protein expression as well as modifications in protein glycosylation have been frequently reported in cancer development, the aim of this work was to study the differential expression as well as modifications of glycosylation of proteins from plasma of women with breast cancer at different stages of disease (n = 30) compared to healthy women (n = 10). A proteomics approach was used that depleted albumin and IgG from plasma followed by glycoprotein enrichment using immobilized Moraceae lectin (frutalin)-affinity chromatography and data-independent label-free mass spectrometric analysis. Data are available via ProteomeXchange with identifier PXD003106. As result, 57,016 peptides and 4,175 proteins among all samples were identified. From this, 40 proteins present in unbound (PI—proteins that did not interact with lectin) and bound (PII—proteins that interacted with lectin) fractions were differentially expressed. High levels of apolipoprotein A-II were detected here that were elevated significantly in the early and advanced stages of the disease. Apolipoprotein C-III was detected in both fractions, and its level was increased slightly in the PI fraction of patients with early-stage breast cancer and expressed at higher levels in the PII fraction of patients with early and intermediate stages. Clusterin was present at higher levels in both fractions of patients with early and intermediate stages of breast cancer. Our findings reveal a correlation between alterations in protein glycosylation, lipid metabolism, and the progression of breast cancer. PMID:28210565

  1. Generation of transgenic dogs that conditionally express green fluorescent protein.

    PubMed

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  2. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  3. Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells

    PubMed Central

    Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

    2016-01-01

    The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS. PMID:26934632

  4. Autophagy and lysosomal related protein expression patterns in human glioblastoma.

    PubMed

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Mitrakas, Achileas; Kalamida, Dimitra; Zois, Christos E; Haider, Syed; Piperidou, Charitomeni; Pappa, Aglaia; Gatter, Kevin C; Harris, Adrian L; Koukourakis, Michael I

    2014-01-01

    Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

  5. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

  6. Disposable bioreactors for inoculum production and protein expression.

    PubMed

    Eibl, Regine; Löffelholz, Christian; Eibl, Dieter

    2014-01-01

    Disposable bioreactors have been increasingly implemented over the past ten years. This relates to both R & D and commercial manufacture, in particular, in animal cell-based processes. Among the numerous disposable bioreactors which are available today, wave-mixed bag bioreactors and stirred bioreactors are predominant. Whereas wave-mixed bag bioreactors represent the system of choice for inoculum production, stirred systems are often preferred for protein expression. For this reason, the authors present protocols instructing the reader how to use the wave-mixed BIOSTAT CultiBag RM 20 L for inoculum production and the stirred UniVessel SU 2 L for recombinant protein production at benchtop scale. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing the human placental secreted alkaline phosphatase (SEAP).

  7. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  8. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  9. Downregulation of hepatic stimulator substance during the early phase of liver regeneration inhibits E-cadherin expression in mice.

    PubMed

    Zhang, Haifeng; Dong, Ling-Yue; Sun, Guangyong; An, Wei

    2014-02-01

    Hepatic stimulatory substance (HSS), which encodes a sulfhydryl oxidase enzyme, promotes liver regeneration (LR) and maintains the viability of hepatocytes. Surprisingly, we found that the levels of the HSS mRNA and expressed protein were both strongly repressed at 12h after a 70% partial hepatectomy (PH) in mice. Understanding the mechanism and effect of this extraordinary suppression can provide a novel path for exploring the molecular function of HSS during LR. We observed that the EGF levels in the serum were negatively correlated with HSS expression in regenerating livers. Treating primary mouse hepatocytes or Hepa1-6 cells with EGF suppressed HSS mRNA expression. This suppression was transcriptional and was mediated by the effect of EGF on the phosphorylation of CCAAT/enhancer-binding protein β (C/EBPβ), which regulates HSS expression. We further showed that the enhanced phosphorylation of C/EBPβ after PH promoted its interaction with the HSS promoter and repressed HSS expression at early time-points after PH. Interestingly, the knockdown of HSS caused a dramatic decrease in E-cadherin expression in hepatocytes. E-cadherin expression was also significantly suppressed at 12h after PH. Moreover, the pre-injection of HSS-expressing adenovirus vectors prevented E-cadherin suppression after PH. Treatment with C/EBPβ siRNA reversed the EGF-mediated inhibition of HSS expression and led to enhanced E-cadherin expression and reduced cell migration. Our findings suggest that C/EBPβ directly inhibits the HSS promoter after PH and that this inhibition can downregulate E-cadherin expression. These data provide novel insight into the potential role of HSS in hepatic structural reconstruction during LR.

  10. Hippocampal expression of the calcium sensor protein visinin-like protein-1 in schizophrenia.

    PubMed

    Bernstein, Hans-Gert; Braunewell, Karl-Heinz; Spilker, Christina; Danos, Peter; Baumann, Bruno; Funke, Sieglinde; Diekmann, Silvia; Gundelfinger, Eckart D; Bogerts, Bernhard

    2002-03-25

    Hippocampal cytoarchitectural abnormalities may be part of the cerebral substrate of schizophrenia. Amongst the chemical components being abnormal in brains of schizophrenics are altered calcium concentrations and reduced expression of the neurotrophin receptor, trkB. We studied by immunohistochemical methods the distribution of visinin-like protein-1 (VILIP-1), which is a calcium sensor protein and at the same time a trkB mRNA binding protein, in hippocampi of nine schizophrenic patients and nine matched control subjects. In normal hippocampi VILIP-1 immunoreactivity was found in multiple pyramidal cells and interneurons. A portion of VILIP-1 immunoreactive interneurons co-express calretinin (60%) and parvalbumin (<10%). In schizophrenics fewer pyramidal cells but more interneurons were immunostained. Our data point to an involvement of the protein in the altered hippocampal circuitry in schizophrenia.

  11. Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.

    PubMed

    Hannula, Mirva J; Männistö, Pekka T; Myöhänen, Timo T

    2011-01-01

    Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage.

  12. Shared Understanding and Idiosyncratic Expression in Early Vocabularies

    ERIC Educational Resources Information Center

    Mayor, Julien; Plunkett, Kim

    2014-01-01

    To what extent do toddlers have shared vocabularies? We examined CDI data collected from 14,607 infants and toddlers in five countries and measured the amount of variability between individual lexicons during development for both comprehension and production. Early lexicons are highly overlapping. However, beyond 100 words, toddlers share more…

  13. Transcriptome Sequencing, De Novo Assembly and Differential Gene Expression Analysis of the Early Development of Acipenser baeri

    PubMed Central

    Song, Wei; Jiang, Keji; Zhang, Fengying; Lin, Yu; Ma, Lingbo

    2015-01-01

    The molecular mechanisms that drive the development of the endangered fossil fish species Acipenser baeri are difficult to study due to the lack of genomic data. Recent advances in sequencing technologies and the reducing cost of sequencing offer exclusive opportunities for exploring important molecular mechanisms underlying specific biological processes. This manuscript describes the large scale sequencing and analyses of mRNA from Acipenser baeri collected at five development time points using the Illumina Hiseq2000 platform. The sequencing reads were de novo assembled and clustered into 278167 unigenes, of which 57346 (20.62%) had 45837 known homologues proteins in Uniprot protein databases while 11509 proteins matched with at least one sequence of assembled unigenes. The remaining 79.38% of unigenes could stand for non-coding unigenes or unigenes specific to A. baeri. A number of 43062 unigenes were annotated into functional categories via Gene Ontology (GO) annotation whereas 29526 unigenes were associated with 329 pathways by mapping to KEGG database. Subsequently, 3479 differentially expressed genes were scanned within developmental stages and clustered into 50 gene expression profiles. Genes preferentially expressed at each stage were also identified. Through GO and KEGG pathway enrichment analysis, relevant physiological variations during the early development of A. baeri could be better cognized. Accordingly, the present study gives insights into the transcriptome profile of the early development of A. baeri, and the information contained in this large scale transcriptome will provide substantial references for A. baeri developmental biology and promote its aquaculture research. PMID:26359664

  14. High-level protein expression following single and dual gene cloning of infectious bronchitis virus N and S genes using baculovirus systems.

    PubMed

    Abdel-Moneim, Ahmed S; Giesow, Katrin; Keil, Günther M

    2014-03-01

    Baculovirus is an efficient system for the gene expression that can be used for gene transfer to both insect and different vertebrate hosts. The nucleocapsid gene (N) of the infectious bronchitis virus was cloned in a baculovirus expression system for insect cell expression. Dual expression vectors containing IBV N and spike (S) proteins of the avian infectious bronchitis virus were engineered under the control of human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in both vertebrate and insect cells. Transduction of the N gene in the insect Sf9 cells revealed a high level of protein expression. The expressed protein, used in ELISA, effectively detected chicken anti-IBV antibodies with high specificity. Transduction of mammalian and avian cells with BacMam viruses revealed that dual expression cassettes yielded high levels of protein from both transcription units.

  15. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

    PubMed

    Allen, Robert S; Tilbrook, Kimberley; Warden, Andrew C; Campbell, Peter C; Rolland, Vivien; Singh, Surinder P; Wood, Craig C

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

  16. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

    PubMed Central

    Allen, Robert S.; Tilbrook, Kimberley; Warden, Andrew C.; Campbell, Peter C.; Rolland, Vivien; Singh, Surinder P.; Wood, Craig C.

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. PMID:28316608

  17. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    PubMed

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy.

  18. Differential expression of ribosome-inactivating protein genes during somatic embryogenesis in spinach (Spinacia oleracea).

    PubMed

    Kawade, Kensuke; Ishizaki, Takuma; Masuda, Kiyoshi

    2008-10-01

    Root segments from spinach (Spinacia oleracea L. cv. Jiromaru) seedlings form embryogenic callus (EC) that responded to exogenous GA(3) by accumulating a 31-kDa glycoprotein [BP31 or S. oleracea ribosome-inactivating protein (EC 3.2.2.22) (SoRIP1)] in association with the expression of embryogenic potential. Microsequencing of this protein revealed significant similarity with type 1 RIPs. We identified cDNAs for SoRIP1 and S. oleracea RIP2 (SoRIP2), a novel RIP having a consensus shiga/ricin toxic domain and performed a comparative analysis of the expression of SoRIPs during somatic embryogenesis. Western blotting and quantitative polymerase chain reaction analyses revealed that the expression of SoRIP1 in calli increased remarkably in association with the acquisition of embryogenic potential, although the expression in somatic embryos decreased moderately with their development. However, the expression of SoRIP2 in calli remained low and constant but increased markedly with the development of somatic embryos. Treatment of callus with GA(3) and/or ABA for 24 h, or with ABA for a longer period, failed to stimulate the expression of either gene. Immunohistochemistry showed that SoRIP1 preferentially accumulated in the proembryos and peripheral meristem of somatic embryos early in development. Appreciable expression of SoRIP2 was not detected in the callus, but intense expression was found in the epidermis of somatic embryos. These results suggest that the expression of spinach RIP genes is differentially regulated in a development-dependent fashion during somatic embryogenesis in spinach.

  19. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

    PubMed Central

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  20. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

    PubMed

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-05-24

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.

  1. The E4 protein; structure, function and patterns of expression.

    PubMed

    Doorbar, John

    2013-10-01

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1^E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein's flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1^E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1^E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1^E4, these kinases regulate one of

  2. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  3. Phylogeny and expression of carbonic anhydrase-related proteins

    PubMed Central

    2010-01-01

    Background Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain

  4. Early and Late Postnatal Myocardial and Vascular Changes in a Protein Restriction Rat Model of Intrauterine Growth Restriction

    PubMed Central

    Menendez-Castro, Carlos; Fahlbusch, Fabian; Cordasic, Nada; Amann, Kerstin; Münzel, Kathrin; Plank, Christian; Wachtveitl, Rainer; Rascher, Wolfgang; Hilgers, Karl F.; Hartner, Andrea

    2011-01-01

    Intrauterine growth restriction (IUGR) is a risk factor for cardiovascular disease in later life. Early structural and functional changes in the cardiovascular system after IUGR may contribute to its pathogenesis. We tested the hypothesis that IUGR leads to primary myocardial and vascular alterations before the onset of hypertension. A rat IUGR model of maternal protein restriction during gestation was used. Dams were fed low protein (LP; casein 8.4%) or isocaloric normal protein diet (NP; casein 17.2%). The offspring was reduced to six males per litter. Immunohistochemical and real-time PCR analyses were performed in myocardial and vascular tissue of neonates and animals at day 70 of life. In the aortas of newborn IUGR rats expression of connective tissue growth factor (CTGF) was induced 3.2-fold. At day 70 of life, the expression of collagen I was increased 5.6-fold in aortas of IUGR rats. In the hearts of neonate IUGR rats, cell proliferation was more prominent compared to controls. At day 70 the expression of osteopontin was induced 7.2-fold. A 3- to 7-fold increase in the expression of the profibrotic cytokines TGF-β and CTGF as well as of microfibrillar matrix molecules was observed. The myocardial expression and deposition of collagens was more prominent in IUGR animals compared to controls at day 70. In the low-protein diet model, IUGR leads to changes in the expression patterns of profibrotic genes and discrete structural abnormalities of vessels and hearts in adolescence, but, with the exception of CTGF, not as early as at the time of birth. Invasive and non-invasive blood pressure measurements confirmed that IUGR rats were normotensive at the time point investigated and that the changes observed occurred independently of an increased blood pressure. Hence, altered matrix composition of the vascular wall and the myocardium may predispose IUGR animals to cardiovascular disease later in life. PMID:21655297

  5. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    PubMed Central

    Nalbant, Demet; Youn, Hyewon; Nalbant, S Isil; Sharma, Savitha; Cobos, Everardo; Beale, Elmus G; Du, Yang; Williams, Simon C

    2005-01-01

    Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of

  6. Label-Free Proteomic Analysis of Protein Changes in the Striatum during Chronic Ethanol Use and Early Withdrawal

    PubMed Central

    Ayers-Ringler, Jennifer R.; Oliveros, Alfredo; Qiu, Yanyan; Lindberg, Daniel M.; Hinton, David J.; Moore, Raymond M.; Dasari, Surendra; Choi, Doo-Sup

    2016-01-01

    The molecular mechanisms underlying the neuronal signaling changes in alcohol addiction and withdrawal are complex and multifaceted. The cortico-striatal circuit is highly implicated in these processes, and the striatum plays a significant role not only in the early stages of addiction, but in the developed-addictive state as well, including withdrawal symptoms. Transcriptional analysis is a useful method for determining changes in gene expression, however, the results do not always accurately correlate with protein levels. In this study, we employ label-free proteomic analysis to determine changes in protein expression within the striatum during chronic ethanol use and early withdrawal. The striatum, composed primarily of medium spiny GABAergic neurons, glutamatergic and dopaminergic nerve terminals and astrocytes, is relatively homogeneous for proteomic analysis. We were able to analyze more than 5000 proteins from both the dorsal (caudate and putamen) and ventral (nucleus accumbens) striatum and identified significant changes following chronic intermittent ethanol exposure and acute (8 h) withdrawal compared to ethanol naïve and ethanol exposure groups respectively. Our results showed significant changes in proteins involved in glutamate and opioid peptide signaling, and also uncovered novel pathways including mitochondrial function and lipid/cholesterol metabolism, as revealed by changes in electron transport chain proteins and RXR activation pathways. These results will be useful in the development of novel treatments for alcohol withdrawal and thereby aid in recovery from alcohol use disorder. PMID:27014007

  7. Piwi proteins and piRNAs in mammalian oocytes and early embryos: From sample to sequence

    PubMed Central

    Rosenkranz, David; Han, Chung-Ting; Roovers, Elke F.; Zischler, Hans; Ketting, René F.

    2015-01-01

    The role of the Piwi/piRNA pathway during mammalian oogenesis has remained enigmatic thus far, especially since experiments with Piwi knockout mice did not reveal any phenotypic defects in female individuals. This is in striking contrast with results obtained from other species including flies and zebrafish. In mouse oocytes, however, only low levels of piRNAs are found and they are not required for their function. We recently demonstrated dynamic expression of PIWIL1, PIWIL2, and PIWIL3 during mammalian oogenesis and early embryogenesis. In addition, small RNA analysis of human, crab-eating macaque and cattle revealed that piRNAs are also expressed in the female germline and closely resemble piRNAs from testis. Here, we thoroughly describe the experimental and computational methods that we applied for the generation, processing and analyses of next generation sequencing (NGS) data associated with our study on Piwi proteins and piRNAs in mammalian oocytes and embryos (Roovers et al., 2015). The complete sequence data is available at NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession GSE64942. PMID:26484274

  8. cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells

    PubMed Central

    1992-01-01

    A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase. Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level. By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase. Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm. During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes. The nuclear locale reappears during telophase. In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T. Hunter. 1991. J. Cell Biol. 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity. PMID:1500423

  9. Early gene expression in Pseudomonas fluorescens exposed to a polymetallic solution.

    PubMed

    Gómez-Sagasti, María T; Becerril, José M; Epelde, Lur; Alkorta, Itziar; Garbisu, Carlos

    2015-02-01

    The molecular response of Pseudomonas fluorescens cells exposed to a mixture of heavy metals remains largely unknown. Here, we studied the temporal changes in the early gene expression of P. fluorescens cells exposed to three doses of a polymetallic solution over two exposure times, through the application of a customized cDNA microarray. At the lowest metal dose (MD/4), we observed a repression of the Hsp70 chaperone system, MATE and MFS transporters, TonB membrane transporter and histidine kinases, together with an overexpression of metal transport (ChaC, CopC), chemotaxis and glutamine synthetase genes. At the intermediate metal dose (MD), several amino acid transporters, a response regulator (CheY), a TonB-dependent receptor and the mutT DNA repair gene were repressed; by contrast, an overexpression of genes associated with the antioxidative stress system and the transport of chelates and sulfur was observed. Finally, at the highest metal dose (4MD), a repression of genes encoding metal ion transporters, drug resistance and alginate biosynthesis was found, together with an overexpression of genes encoding antioxidative proteins, membrane transporters, ribosomal proteins, chaperones and proteases. It was concluded that P. fluorescens cells showed, over exposure time, a highly complex molecular response when exposed to a polymetallic solution, involving mechanisms related with chemotaxis, signal transmission, membrane transport, cellular redox state, and the regulation of transcription and ribosomal activity.

  10. Assessment of cathepsin mRNA expression and enzymatic activity during early embryonic development in the yellowtail kingfish Seriola lalandi.

    PubMed

    Palomino, Jaime; Herrera, Giannina; Torres-Fuentes, Jorge; Dettleff, Phillip; Patel, Alok; Martínez, Víctor

    2017-02-21

    In pelagic species such as Seriola lalandi, survival of both the eggs and embryos depends on yolk processing during oocyte maturation and embryo development. The main enzymes involved in these processes are the cathepsins, which are essential for the hydration process, acquiring buoyancy and nutrition of the embryo before hatching. This study aimed to investigate the mRNA expression profiles of cathepsins B, D and L (catb, catd and catl) and the activity of these enzymes during early development in S. lalandi. We included previtellogenic oocytes (PO). All three enzymes were highly expressed in PO, but the expression was reduced throughout development. Between PO and recently spawned eggs (E1) the transcript to catb and catd decreased, unlike catl. Cathepsin B activity, showed stable levels between PO until blastula stage (E4). High activities levels of cathepsins D and L were observed in E1 in comparison with later developmental stages. Cathepsin L activity remained constant until E1, consistent with observations in other pelagic spawners, where its participation in a second protolithic cleavage of the yolk proteins, has been proposed for this enzyme. Their profiles of both mRNA expression and enzymatic activity indicate the importance of these enzymes during early development and suggest different roles in egg yolk processing for the hydration process and nutrition in early embryos in this species.

  11. Insights into the early evolution of SOX genes from expression analyses in a ctenophore.

    PubMed

    Jager, Muriel; Quéinnec, Eric; Chiori, Roxane; Le Guyader, Hervé; Manuel, Michaël

    2008-12-15

    SOX genes encode transcription factors acting in various developmental processes in bilaterian animals, such as stem cell maintenance and the control of specification and differentiation of cell types in a variety of contexts, notably in the developing nervous system. To gain insights into the early evolution of this important family of developmental regulators, we investigated the expression of one subgroup B, two subgroup E, one subgroup F and two divergent SOX genes in the cydippid larva and in the adult of the ctenophore Pleurobrachia pileus. Transcripts of the two unclassified SOX (PpiSOX2/12) were detected in the female germ line and in various populations of putative somatic stem cells/undifferentiated progenitors. The remaining genes had spatially restricted expression patterns in ciliated epithelial cells, notably within neuro-sensory territories. These data are compatible with an ancient involvement of SOX proteins in controlling aspects of stem cell maintenance, cellular differentiation and specification, notably within neuro-sensory epithelia. In addition, the results highlight the complexity of the ctenophore anatomy and suggest that the SOX played an important role in the elaboration of the unique ctenophore body plan during evolution, through multiple gene co-option.

  12. Retinoic acid increases zif268 early gene expression in rat preosteoblastic cells.

    PubMed Central

    Suva, L J; Ernst, M; Rodan, G A

    1991-01-01

    In this study we demonstrate that retinoic acid (RA) increases the expression of transcription factor zif268 mRNA in primary cultures of fetal rat calvarial cells and in simian virus 40-immortalized clonal rat calvarial preosteoblastic cells (RCT-1), which differentiate in response to RA, but not in the more differentiated RCT-3 and ROS 17/2.8 cells. The increased expression of zif268 mRNA is rapid (maximal within 1 h), transient (returns to basal levels by 3 h), detectable at RA doses of 10(-12)M, and independent of protein synthesis. The relative stimulation of zif268 mRNA by RA was much larger than that of other early genes, including c-fos, c-jun, and junB. The rate of transcription of RA-stimulated RCT-1 cells, estimated by nuclear run-on assays, was elevated, suggesting that RA regulation of zif268 gene transcription was at least in part transcriptional. Moreover, RA stimulated the transcriptional activity of a Zif268CAT (chloramphenicol acetyltransferase) plasmid containing 632 bp of zif268 5' regulatory sequences in RCT-1 cells but not in the more differentiated RCT-3 cells. These in vitro data support the in vivo observations which localize zif268 and RA receptor-gamma transcripts to bone and cartilage during development, suggesting that both RA and zif268 may play a role in osteoblast differentiation. Images PMID:1708092

  13. miR-29 regulates Tet1 expression and contributes to early differentiation of mouse ESCs

    PubMed Central

    Yang, Dehua; Li, Song; Le, Weidong

    2016-01-01

    The ten-eleven translocation-1 (Tet1), which converts 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC), plays important roles in many important biological processes, such as mouse embryonic stem cells (ESCs) maintenance. However, the mechanisms for Tet-1 regulation remain largely unknown. Here we showed that miR-29 family (miR-29a, miR-29b and miR-29c) can directly repress Tet1 expression. We found that Tet1 was highly expressed and 5hmC was presented at relatively high levels in mouse ESCs, but the levels of both Tet1 and 5hmC were reduced during the early differentiation of ESCs. On the contrary, miR-29 level was increased in this process. ESCs stably transfecting with miR-29 precursors showed lower levels of Tet1 protein and 5hmC. Furthermore, we demonstrated that miR-29 overexpression selectively affected cell lineage markers and skewed ESC differentiation, which was similar in Tet1 knockdown ESCs. Our results indicate that miR-29 is a direct regulator of Tet1 in mouse ESCs. PMID:27449105

  14. HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation.

    PubMed

    Johansson, Cecilia; Somberg, Monika; Li, Xiaoze; Backström Winquist, Ellenor; Fay, Joanna; Ryan, Fergus; Pim, David; Banks, Lawrence; Schwartz, Stefan

    2012-05-22

    We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.

  15. HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation

    PubMed Central

    Johansson, Cecilia; Somberg, Monika; Li, Xiaoze; Backström Winquist, Ellenor; Fay, Joanna; Ryan, Fergus; Pim, David; Banks, Lawrence; Schwartz, Stefan

    2012-01-01

    We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2. PMID:22617423

  16. Circulating promyelocytes and low levels of CD16 expression on polymorphonuclear leukocytes accompany early-onset periodontitis.

    PubMed Central

    Nemoto, E; Nakamura, M; Shoji, S; Horiuchi, H

    1997-01-01

    Early-onset periodontitis (EOP) is characterized by rapidly progressive alveolar bone loss, chemotactic defects of neutrophils, and significant familial aggregation. We found immature myeloid lineage cells, defined as promyelocytes, in the peripheral blood in patients with EOP. A hematological examination of peripheral blood cells showed normal reference values regarding cell proportions. Flow cytometry revealed significantly lower expression of CD16, a glycosylphosphatidylinositol (GPI)-anchored protein, on peripheral neutrophils in patients compared with those in age- and sex-matched healthy controls, whereas the levels of CD11a and CD11b expression were similar. The chemotactic response of neutrophils was lower toward not only formyl-methionyl-leucyl-phenylalanine but also complement fragment C5a than that of healthy controls. The expression of another GPI-anchored protein, CD14, was equally expressed by controls and patients. Therefore, the low level of CD16 expression was not due to the incomplete synthesis of the GPI anchor. GPI anchors of CD16 on neutrophils from controls and patients were both partially resistant to phosphatidylinositol-specific phospholipase C. The presence of promyelocytes in peripheral blood, low expression of CD16, and low chemotactic response of neutrophils suggest that patients with EOP have an abnormal maturation system in myeloid lineage cells in the bone marrow, which may be associated with the onset and course of EOP. PMID:9284170

  17. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments.

  18. Structure and Expression of a Novel Compact Myelin Protein - Small VCP-Interacting Protein (SVIP)

    PubMed Central

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Li, Jun

    2013-01-01

    SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes. PMID:24055875

  19. Differential expression of murine adult hemoglobins in early ontogeny

    SciTech Connect

    Wawrzyniak, C.J.; Lewis, S.E.; Popp, R.A.

    1985-01-01

    A hemoglobin mutation is described that permits study of the expression of the two adult ..beta..-globin genes throughout fetal and postnatal development. Mice with a mutation at the Hbb/sup s/, ..beta..-globin locus, were used to study the relative levels of ..beta..-s2major and ..beta..-sminor globins specified by the mutant Hbb/sup s2/ haplotype during development. At 11.5 days of gestation ..beta..-sminor comprised over 80% and ..beta..-s2major under 20% of the adult beta-globin. The relative level of ..beta..-sminor decreased through fetal development; at birth ..beta..-sminor represented 33.7% of the ..beta..-globin. The adult values of 71.0% ..beta..-s2major and 29.0% ..beta..-sminor globin are expressed in mice six days after birth. Because the two ..beta..-globin genes are expressed in mice of the Hbb/sup 2s/ haplotype, both the ..beta..-smajor and ..beta..-sminor genes must be expressed in mice of the Hbb/sup s/ haplotype. Expression of the ..beta..-sminor gene is elevated to 35.6% in Hbb/sup s2/ mice that have been bled repeatedly. Thus, the 5' ..beta..-s2major and 3' ..beta..-sminor genes of the Hbb/sup s2/ haplotype and, presumably the 5' ..beta..-smajor and 3' ..beta..-sminor genes of the Hbb/sup s/ haplotype, are regulated independently and are homologous to the 5' ..beta..-dmajor and 3' ..beta..-dminor genes of the Hbb/sup d/ haplotype. Mice of the Hbb/sup s2/ haplotype are better than mice of the Hbb/sup d/ haplotytpe for studying the mechanisms of hemoglobin switching because the Hbb/sup s2/ each of the three embryonic and two adult hemoglobins can be separated by electrophoresis. 17 refs., 3 figs.

  20. Embryonic Expression and Evolution of Duplicated E-Protein Genes in Xenopus Laevis: Parallels with Ancestral E-Protein Genes

    PubMed Central

    Shain, D. H.; Neuman, T.; Zuber, M. X.

    1997-01-01

    E-proteins comprise a subfamily of helix-loop-helix transcription factors that have been identified in arthropods and several chordate taxa. In mammals, there are three classes of E-protein genes (E2A, E2-2, and HEB) that encode related, and often interchangeable, gene products. We have determined that the clawed frog Xenopus laevis contains twice the number of transcriptionally active E-protein genes when compared with other vertebrate species. Based upon genomic Southern blots and nucleotide sequence comparisons, it is likely that the additional X. laevis genes arose from tetraploidization. During embryogenesis, XE2A (homologue of mammalian E2A) transcripts were broadly expressed in anterior and posterior regions of the embryo while homologues of E2-2 (XE2.2) and HEB (XE1.2) appeared in vertebrate-specific structures including the pineal gland, olfactory bulb, and brachial arches. A phylogenetic analysis of these genes and other known metazoan E-proteins suggests that there were two periods of marked E-protein gene expansion; one that predated the radiation of vertebrates, and the other that coincided with Xenopus tetraploidization. Both of these periods were characterized by the rapid evolution of E2-2 and HEB-class genes, but not of E2A. We propose that the former genes acquired new or specialized roles during early chordate evolution and also more recently in Xenopus, as reflected by the stereotypic expression patterns of these genes during X. laevis development. PMID:9136023

  1. Effect of hyperoxic exposure during early development on neurotrophin expression in the carotid body and nucleus tractus solitarii

    PubMed Central

    Chavez-Valdez, Raul; Mason, Ariel; Nunes, Ana R.; Northington, Frances J.; Tankersley, Clarke; Ahlawat, Rajni; Johnson, Sheree M.

    2012-01-01

    Synaptic activity can modify expression of neurotrophins, which influence the development of neuronal circuits. In the newborn rat, early hyperoxia silences the synaptic activity and input from the carotid body, impairing the development and function of chemoreceptors. The purpose of this study was to determine whether early hyperoxic exposure, sufficient to induce hypoplasia of the carotid body and decrease the number of chemoafferents, would also modify neurotrophin expression within the nucleus tractus solitarii (nTS). Rat pups were exposed to hyperoxia (fraction of inspired oxygen 0.60) or normoxia until 7 or 14 days of postnatal development (PND). In the carotid body, hyperoxia decreased brain-derived neurotrophic factor (BDNF) protein expression by 93% (P = 0.04) after a 7-day exposure, followed by a decrease in retrogradely labeled chemoafferents by 55% (P = 0.004) within the petrosal ganglion at 14 days. Return to normoxia for 1 wk after a 14-day hyperoxic exposure did not reverse this effect. In the nTS, hyperoxia for 7 days: 1) decreased BDNF gene expression by 67% and protein expression by 18%; 2) attenuated upregulation of BDNF mRNA levels in response to acute hypoxia; and 3) upregulated p75 neurotrophic receptor, truncated tropomyosin kinase B (inactive receptor), and cleaved caspase-3. These effects were not observed in the locus coeruleus (LC). Hyperoxia for 14 days also decreased tyrosine hydroxylase levels by 18% (P = 0.04) in nTS but not in the LC. In conclusion, hyperoxic exposure during early PND reduces neurotrophin levels in the carotid body and the nTS and shifts the balance of neurotrophic support from prosurvival to proapoptotic in the nTS, the primary brain stem site for central integration of sensory and autonomic inputs. PMID:22422797

  2. Medial amygdala lesions modify aggressive behavior and immediate early gene expression in oxytocin and vasopressin neurons during intermale exposure.

    PubMed

    Wang, Yu; He, Zhiyi; Zhao, Chuansheng; Li, Lei

    2013-05-15

    The medial amygdala and neuropeptides oxytocin (OXT) and vasopressin (VSP) have been associated aggressive behavior regulation. However, the specific mechanism involved in OXT and VSP modulation in distinct brain regions during hostile intermale aggressive behavior is undetermined. A retrograde tracer mouse model was employed using male C57BL/6 mice injected with rhodamine-conjugated latex microsphere suspensions in the right hypothalamic paraventricular nucleus. Adult male C57BL/6 mice (aged 14-16 weeks) were subjected to resident-intruder testing using juvenile intruder mice (aged 3 weeks) or adult intruder mice (aged 8 weeks). Following exposure, Fos protein expression was increased in the medial amygdala neurons of resident mice receiving the retrograde tracer. Thus, medial amygdala neurons projecting to or localized in the vicinity of the hypothalamic paraventricular nucleus showed immediate early gene (IEG) expression following resident-intruder testing that was considered an indirect marker of activation. Additionally, intermale aggression-related behaviors were inhibited or modified by exposure to juvenile or adult intruders, respectively, in mice that underwent medial amygdala lesioning. Furthermore, Fos protein expression in OXT-positive neurons was attenuated. Thus, ablation of medial amygdala neurons prevented immediate early gene expression in OXT- and VSP-positive neurons in the hypothalamus, bed nucleus of stria terminalis, and medial preoptic area during intermale exposure. These findings indicate that the medial amygdala likely modulates hostile aggressive behavior associated with immediate early gene expression in OXT and VSP neurons in specific brain areas, which may actually be instrumental in beneficial social interaction-related aggressive responses associated with mating, territorial defense, and offspring protection.

  3. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins

    PubMed Central

    2013-01-01

    Background Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. Results The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. Conclusion The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags

  4. Expression of P53 protein after exposure to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

    2001-10-01

    One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

  5. Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos.

    PubMed

    Hirasawa, Ryutaro; Sasaki, Hiroyuki

    2009-01-01

    The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.

  6. Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4.

    PubMed Central

    DeLuca, N A; Schaffer, P A

    1985-01-01

    To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal

  7. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    PubMed

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  8. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    SciTech Connect

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory . E-mail: vchinchar@microbio.umsmed.edu

    2007-02-20

    Frog virus 3 (FV3) is a large DNA virus that encodes {approx} 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-II{alpha}). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-II{alpha} triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

  9. Dynamic expression of the Slit-Robo GTPase activating protein genes during development of the murine nervous system.

    PubMed

    Bacon, Claire; Endris, Volker; Rappold, Gudrun

    2009-03-10

    We investigated the expression of the three known Slit-Robo GTPase activating protein (srGAP) genes in the developing murine nervous system using in situ hybridization. The three genes are expressed during embryonic and early postnatal development in the murine nervous system, showing a distinct pattern of expression in the olfactory system, the eye, forebrain and midbrain structures, the cerebellum, the spinal cord, and dorsal root ganglia, which we discuss in relation to Slit-Robo expression patterns and signaling pathways. We also report srGAP2 expression in zones of neuronal differentiation and srGAP3 in ventricular zones of neurogenesis in many different tissues of the central nervous system (CNS). Compared to srGAP2 and srGAP3, the onset of srGAP1 expression is later in most CNS tissues. We propose that these differences in expression point to functional differences between these three genes in the development of neural tissues.

  10. Protein expression in salivary glands of rats with streptozotocin diabetes

    PubMed Central

    Mednieks, Maija I; Szczepanski, Andrew; Clark, Brett; Hand, Arthur R

    2009-01-01

    Diabetes mellitus (DM) is a widespread disease with high morbidity and health care costs. An experimental animal model was employed, using morphological and biochemical methods, to investigate the effects of DM on the expression and compartmentation of salivary gland proteins. The distribution of proline-rich proteins (PRP), submandibular mucin (Muc10) and the regulatory (RI and RII) subunits of cyclic AMP-dependent protein kinase type I and type II was determined in the parotid and submandibular (SMG) glands of rats treated with streptozotocin. Quantitative immunocytochemistry of secretory granules in diabetic glands revealed decreases of 30% for PRP in both the parotid and SMG, and a 40% decrease in Muc10 in the SMG. Immunogold labelling showed that RII decreased in nuclei and the cytoplasm in diabetic acinar cells while labelling of secretory granules was similar in control and diabetic parotid. Electrophoresis and Western blotting of tissue extracts of two secretory proteins showed that the response to DM and insulin treatment was gland specific: PRP showed little change in the SMG, but decreased in the parotid in DM and was partially restored after insulin treatment. Photoaffinity labelling showed only RI present in the SMG and mainly RII in the parotid. The results of this and previous studies demonstrating highly specific changes in salivary protein expression indicate that the oral environment is significantly altered by DM, and that oral tissues and their function can be compromised. These findings may provide a basis for future studies to develop tests using saliva for diabetic status or progression in humans. PMID:19659899

  11. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

    PubMed

    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01

    The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

  12. Expression of FGFs during early mouse tongue development

    PubMed Central

    Du, Wen; Prochazka, Jan; Prochazkova, Michaela; Klein, Ophir D.

    2016-01-01

    The fibroblast growth factors (FGFs) constitute one of the largest growth factor families, and several ligands and receptors in this family are known to play critical roles during tongue development. In order to provide a comprehensive foundation for research into the role of FGFs during the process of tongue formation, we measured the transcript levels by quantitative PCR and mapped the expression patterns by in situ hybridization of all 22 Fgfs during mouse tongue development between embryonic days (E) 11.5 and E14.5. During this period, Fgf5, Fgf6, Fgf7, Fgf9, Fgf10, Fgf13, Fgf15, Fgf16 and Fgf18 could all be detected with various intensities in the mesenchyme, whereas Fgf1 and Fgf2 were expressed in both the epithelium and the mesenchyme. Our results indicate that FGF signaling regulates tongue development at multiple stages. PMID:26748348

  13. Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

    PubMed

    Boiocchi, C; Monti, M C; Osera, C; Mallucci, G; Pistono, C; Ferraro, O E; Nosari, G; Romani, A; Cuccia, M; Govoni, S; Pascale, A; Montomoli, C; Bergamaschi, R

    2016-09-15

    Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity.

  14. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

    PubMed Central

    Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

    2016-01-01

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

  15. Recombinant protein expression in Lactococcus lactis using the P170 expression system.

    PubMed

    Jørgensen, Casper M; Vrang, Astrid; Madsen, Søren M

    2014-02-01

    The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L(-1).

  16. Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection.

    PubMed

    Enjapoori, Ashwantha Kumar; Grant, Tom R; Nicol, Stewart C; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

    2014-09-22

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female's abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples.

  17. Monotreme Lactation Protein Is Highly Expressed in Monotreme Milk and Provides Antimicrobial Protection

    PubMed Central

    Enjapoori, Ashwantha Kumar; Grant, Tom R.; Nicol, Stewart C.; Lefèvre, Christophe M.; Nicholas, Kevin R.; Sharp, Julie A.

    2014-01-01

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female’s abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples. PMID:25245409

  18. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  19. Dynamic Alterations of miR-34c Expression in the Hypothalamus of Male Rats after Early Adolescent Traumatic Stress

    PubMed Central

    Li, Chuting; Liu, Yuan; Liu, Dexiang; Jiang, Hong; Pan, Fang

    2016-01-01

    Several types of microRNA (miRNA) overexpression in the brain are associated with stress. One of the targets of miR-34c is the stress-related corticotrophin releasing factor receptor 1 mRNA (CRFR1 mRNA). Here we will probe into the short-term effect and long-term effect of early adolescent traumatic stress on the expression of miR-34c and CRFR1 mRNA. Traumatic stress was established by electric foot shock for six consecutive days using 28-day rats. The anxiety-like behaviors, memory damage, CRFR1 protein, CRFR1 mRNA, and miR-34c expression were detected in our study. The results of our study proved that exposure to acute traumatic stress in early adolescent can cause permanent changes in neural network, resulting in dysregulation of CRFR1 expression and CRFR1 mRNA and miR-34c expression in hypothalamus, anxiety-like behavior, and memory impairment, suggesting that the miR-34c expression in hypothalamus may be an important factor involved in susceptibility to PTSD. PMID:26925271

  20. Paraxial left-sided nodal expression and the start of left-right patterning in the early chick embryo.

    PubMed

    Tsikolia, Nikoloz; Schröder, Silke; Schwartz, Peter; Viebahn, Christoph

    2012-12-01

    A common element during early left-right patterning of the vertebrate body is left-sided nodal expression in the early-somite stage lateral plate mesoderm. Leftward cell movements near the node of the gastrulating chick embryo recently offered a plausible mechanism for breaking the presomite-stage molecular symmetry in those vertebrates which lack rotating cilia on the notochord or equivalent tissues. However, the temporal and functional relationships between generation of the known morphological node asymmetry, onset of leftward cell movements and establishment of stable molecular asymmetry in the chick remain unresolved. This study uses high-resolution light microscopy and in situ gene expression analysis to show that intranodal cell rearrangement during the phase of counter-clockwise node torsion at stage 4+ is immediately followed by symmetry loss and rearrangement of shh and fgf8 expression in node epiblast between stages 5- and 5+. Surprisingly, left-sided nodal expression starts at stage 5-, too, but lies in the paraxial mesoderm next to the forming notochordal plate, and can be rendered symmetrical by minimal mechanical disturbance of distant tissue integrity at stage 4. The "premature" paraxial nodal expression together with morphological and molecular asymmetries in, and near, midline compartments occurring at defined substages of early gastrulation help to identify a new narrow time window for early steps in left-right patterning in the chick and support the concept of a causal relationship between a-still enigmatic-chiral (motor) protein, cell movements and incipient left-right asymmetry in the amniote embryo.

  1. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    PubMed

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  2. Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses.

    PubMed

    González, Enrique; de Leon, Maria del Carmen García; Meza, Isaura; Ocadiz-Delgado, Rodolfo; Gariglio, Patricio; Silva-Olivares, Angelica; Galindo-Gómez, Silvia; Shibayama, Mineko; Morán, Patricia; Valadez, Alicia; Limón, Angelica; Rojas, Liliana; Hernández, Eric G; Cerritos, René; Ximenez, Cecilia

    2011-02-01

    Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.

  3. FGFR1, 2 and 3 protein overexpression and molecular aberrations of FGFR3 in early stage non-small cell lung cancer.

    PubMed

    Theelen, Willemijn Sme; Mittempergher, Lorenza; Willems, Stefan M; Bosma, Astrid J; Peters, Dennis Dgc; van der Noort, Vincent; Japenga, Eva J; Peeters, Ton; Koole, Koos; Šuštić, Tonći; Blaauwgeers, J L; van Noesel, Carel J; Bernards, René; van den Heuvel, Michel M

    2016-10-01

    This study aimed to determine protein expression levels of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in early stage non-small cell lung cancer (NSCLC). Additionally, a screen to define the frequency of FGFR3-TACC3 translocation and FGFR3 amplification was performed. Archived tissues from 653 NSCLC samples (adenocarcinoma (AC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC)) were analysed with immunohistochemistry (IHC) for expression of FGFR1, 2 and 3. Expression levels of FGFR1, 2 and 3 were correlated with clinicopathological features. The presence of FGFR3-TACC3 translocation was detected by RT-PCR and FGFR3 amplification was detected by fluorescence in situ hybridization. FGFR1, 2 and 3 proteins were highly expressed in 64 (10.6%), 76 (12.9%) and 20 (3.3%) NSCLC tumour samples, respectively. Protein expression of FGFR1 was significantly related to worse overall survival in NSCLC. Furthermore, FGFR1 protein expression was associated with light smoking and histological subtype (AC), FGFR2 protein expression with female gender, younger age, histological subtype (AC) and lower tumour stage, and FGFR3 protein was significantly overexpressed in tumours of older patients and SCC histology. The FGFR3-TACC3 fusion was detected in 3.0% (6/200) of NSCLC samples and the FGFR3 gene was amplified in 4.7% of IHC positive NSCLC samples (2/43). FGFR1, 2 and 3 proteins are expressed in a high number of early stage NSCLC and FGFR1 protein expression may serve as a prognostic biomarker. Recurrent translocations and amplifications in FGFR3 can be found in NSCLC. This study shows that FGFR family members are frequently aberrant in NSCLC and could be interesting therapeutic targets for the treatment of NSCLC.

  4. FGFR1, 2 and 3 protein overexpression and molecular aberrations of FGFR3 in early stage non‐small cell lung cancer

    PubMed Central

    Mittempergher, Lorenza; Willems, Stefan M; Bosma, Astrid J; Peters, Dennis DGC; van der Noort, Vincent; Japenga, Eva J; Peeters, Ton; Koole, Koos; Šuštić, Tonći; Blaauwgeers, JL; van Noesel, Carel J; Bernards, René

    2016-01-01

    Abstract This study aimed to determine protein expression levels of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in early stage non‐small cell lung cancer (NSCLC). Additionally, a screen to define the frequency of FGFR3‐TACC3 translocation and FGFR3 amplification was performed. Archived tissues from 653 NSCLC samples (adenocarcinoma (AC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC)) were analysed with immunohistochemistry (IHC) for expression of FGFR1, 2 and 3. Expression levels of FGFR1, 2 and 3 were correlated with clinicopathological features. The presence of FGFR3‐TACC3 translocation was detected by RT‐PCR and FGFR3 amplification was detected by fluorescence in situ hybridization. FGFR1, 2 and 3 proteins were highly expressed in 64 (10.6%), 76 (12.9%) and 20 (3.3%) NSCLC tumour samples, respectively. Protein expression of FGFR1 was significantly related to worse overall survival in NSCLC. Furthermore, FGFR1 protein expression was associated with light smoking and histological subtype (AC), FGFR2 protein expression with female gender, younger age, histological subtype (AC) and lower tumour stage, and FGFR3 protein was significantly overexpressed in tumours of older patients and SCC histology. The FGFR3‐TACC3 fusion was detected in 3.0% (6/200) of NSCLC samples and the FGFR3 gene was amplified in 4.7% of IHC positive NSCLC samples (2/43). FGFR1, 2 and 3 proteins are expressed in a high number of early stage NSCLC and FGFR1 protein expression may serve as a prognostic biomarker. Recurrent translocations and amplifications in FGFR3 can be found in NSCLC. This study shows that FGFR family members are frequently aberrant in NSCLC and could be interesting therapeutic targets for the treatment of NSCLC. PMID:27785367

  5. A Simple Method for Immunohistochemical Staining of Zebrafish Brain Sections for c-fos Protein Expression.

    PubMed

    Chatterjee, Diptendu; Tran, Steven; Shams, Soaleha; Gerlai, Robert

    2015-12-01

    Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapidly, for example, in the brain in response to environmental stimuli. c-fos is an IEG often used as a marker of neuronal activation. c-fos mRNA expression has started to be quantified and localized in the zebrafish brain following environmental manipulations but analysis of the expression of c-fos protein in the zebrafish brain has rarely been attempted. Here, we describe an immunofluorescence staining method for quantifying c-fos protein expression in different regions of the zebrafish brain. In addition, we expose zebrafish to caffeine, a positive control for c-fos activation in the brain. To confirm cell nucleus specific binding of the c-fos antibody, we counterstained brain sections with the nuclear fluorescent stain DAPI. Furthermore, we describe a method for reducing background autofluorescence often observed in zebrafish brain tissue. Our analysis showed that exposure to caffeine increased the number of c-fos protein-positive cells in specific zebrafish brain regions detected by the immunofluorescence method. Our results demonstrate the feasibility of immunofluorescence-based methods in the analysis of neuronal activation in the zebrafish brain, and reinforce the utility of the zebrafish in behavioral neuroscience research.

  6. Redox modulation of the expression of bacterial genes encoding cysteine-rich proteins in plant protoplasts.

    PubMed Central

    Piñeiro, M; García-Olmedo, F; Diaz, I

    1994-01-01

    Activity of neomycin phosphotransferase II (NPTII; gene, neo; five cysteines) in tobacco protoplasts transfected with fusions of the octopine TR2' or cauliflower mosaic virus 35S promoter and the neo gene, with or without a signal peptide, increased up to 8-fold in response to externally added dithiothreitol at concentrations that did not affect protoplast viability (up to 2.5 mM). Activity of phosphinothricin acetyltransferase (PAT; gene, bar; one cysteine) expressed under control of the TR1' or 35S promoter was not similarly affected, thus excluding a redox modulation of transcription as the mechanism of NPTII activation by dithiothreitol. Western-blot analyses showed an increase in the amount of protein in response to dithiothreitol, whereas neither the steady-state level of NPTII mRNA nor the specific activity of the purified enzyme was affected. The same type of modulation was observed for transiently expressed beta-glucuronidase (nine cysteines) produced from a fusion with the 35S promoter, with or without a signal peptide. Limitation of cotranslational and/or early posttranslational steps by excessively oxidizing sulfhydryl/disulfide redox potentials is postulated to explain the low net accumulation of cysteine-rich proteins of bacterial origin (i.e., NPTII and beta-glucuronidase) when expressed in plant protoplasts, and the marked increase in such proteins in response to externally added dithiothreitol. Images PMID:8171004

  7. Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development.

    PubMed

    Marcorelles, Pascale; Gillet, Danièle; Friocourt, Gaëlle; Ledé, Françoise; Samaison, Laura; Huguen, Geneviève; Ferec, Claude

    2012-03-01

    Sterility due to bilateral destruction in utero or in early infancy resulting in congenital absence of the vas deferens is the rule in male patients with cystic fibrosis. To understand the developmental pattern of this anomaly, the microscopic morphology of the male excretory system was analyzed during development and the expression of the cystic fibrosis transmembrane conductance regulator protein was explored by immunohistochemistry. We observed that cystic fibrosis fetuses had no excretory ducts agenesis or obstruction until 22 weeks of gestation. However, a focal inflammatory pattern and mucinous plugs in the oldest cystic fibrosis case suggested a disruptive mechanism. Immunolabeling of cytoplasmic epithelial cystic fibrosis transmembrane conductance regulator protein was demonstrated in all cystic fibrosis and control cases with a similar pattern of expression of the protein between age-matched controls and cystic fibrosis cases. At midgestation, an apical intensification appeared in both cystic fibrosis and control cases and was stable during the remainder of fetal life. No gradient of intensity could be detected between the different segments of the excretory tract. These findings are different from those reported in adults. The absence of any morphologic anomaly until 22 weeks of gestation, the focal destruction of the epithelial structures during the second trimester, and the chronological pattern of expression of cystic fibrosis transmembrane conductance regulator are of interest for a better understanding of the pathophysiology of this disease.

  8. Detection of differentially expressed genes in the early developmental stage of the mouse mandible.

    PubMed

    Yamaza, H; Matsuo, K; Kiyoshima, T; Shigemura, N; Kobayashi, I; Wada, H; Akamime, A; Sakai, H

    2001-06-01

    We previously examined the development of the mouse mandible, and demonstrated that odontogenesis occurs between embryonic day 10.5 (E10.5) and E12. Based on the histological findings, we performed cDNA subtraction between the E10.5 and E12 mandibles to detect any differentially expressed genes which might be involved in the initiation of odontogenesis. By sequencing, homology search and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), we thus found Pgk-1, Ccte, Hsp86, Nucleolin, Hsc73, Frg1, N-ras, Set alpha and Hsj2 from the E10.5 mandible, and E25, ATPase6, Mum2, Thymosin beta4 and L21 from the E12 mandible to be differentially expressed genes. These genes are functionally related to protein transport, signal transduction, transcription, translation and molecular chaperon activity. In situ hybridization analyses of Set alpha and E25 showed that Set alpha was detected in the tooth germ at E12 and E14.5, thus indicating a close relationship of this gene to odontogenesis. Meanwhile, the in situ signal of E25 was found in the muscular layer of the tongue, thus suggesting E25 to be related to the differentiation of muscular tissue. In conclusion, we found 15 differentially expressed genes in the course of the early developmental stage of the mouse mandible using a combination of the cDNA subtraction and semi-quantitative RT-PCR methods, while in addition, two genes were demonstrated to be related to the initiation and the development of both tooth germ and the tongue according to the in situ hybridization technique.

  9. Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

    PubMed Central

    Chen, Fangyu; Jiang, Liangrong; Zheng, Jingsheng; Huang, Rongyu; Wang, Houcong; Hong, Zonglie; Huang, Yumin

    2014-01-01

    Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development. PMID:24699514

  10. Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)

    SciTech Connect

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Li, Jun

    2013-10-11

    Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

  11. Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

    PubMed

    Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

    2013-04-01

    Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

  12. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

    PubMed

    Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

    2013-05-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

  13. Brevibacillus expression system: host-vector system for efficient production of secretory proteins.

    PubMed

    Mizukami, Makoto; Hanagata, Hiroshi; Miyauchi, Akira

    2010-04-01

    Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (approximately 30 g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various heterologous proteins, including cytokines, enzymes, antigens, and adjuvants.

  14. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    PubMed

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  15. Transgenic expression of therapeutic proteins in Arabidopsis thaliana seed.

    PubMed

    Nykiforuk, Cory L; Boothe, Joseph G

    2012-01-01

    The production of therapeutic proteins in plant seed augments alternative production platforms such as microbial fermentation, cell-based systems, transgenic animals, and other recombinant plant production systems to meet increasing demands for the existing biologics, drugs under evaluation, and undiscovered therapeutics in the future. We have developed upstream purification technologies for oilseeds which are designed to cost-effectively purify therapeutic proteins amenable to conventional downstream manufacture. A very useful tool in these endeavors is the plant model system Arabidopsis thaliana. The current chapter describes the rationale and methods used to over-express potential therapeutic products in A. thaliana seed for evaluation and definitive insight into whether our production platform, Safflower, can be utilized for large-scale manufacture.

  16. Expression and putative role of mitochondrial transport proteins in cancer.

    PubMed

    Lytovchenko, Oleksandr; Kunji, Edmund R S

    2017-03-22

    Cancer cells undergo major changes in energy and biosynthetic metabolism. One of them is the Warburg effect, in which pyruvate is used for fermentation rather for oxidative phosphorylation. Another major one is their increased reliance on glutamine, which helps to replenish the pool of Krebs cycle metabolites used for other purposes, such as amino acid or lipid biosynthesis. Mitochondria are central to these alterations, as the biochemical pathways linking these processes run through these organelles. Two membranes, an outer and inner membrane, surround mitochondria, the latter being impermeable to most organic compounds. Therefore, a large number of transport proteins are needed to link the biochemical pathways of the cytosol and mitochondrial matrix. Since the transport steps are relatively slow, it is expected that many of these transport steps are altered when cells become cancerous. In this review, changes in expression and regulation of these transport proteins are discussed as well as the role of the transported substrates.

  17. Development of Conformation Independent Computational Models for the Early Recognition of Breast Cancer Resistance Protein Substrates

    PubMed Central

    Gantner, Melisa Edith; Di Ianni, Mauricio Emiliano; Ruiz, María Esperanza; Bruno-Blanch, Luis E.

    2013-01-01

    ABC efflux transporters are polyspecific members of the ABC superfamily that, acting as drug and metabolite carriers, provide a biochemical barrier against drug penetration and contribute to detoxification. Their overexpression is linked to multidrug resistance issues in a diversity of diseases. Breast cancer resistance protein (BCRP) is the most expressed ABC efflux transporter throughout the intestine and the blood-brain barrier, limiting oral absorption and brain bioavailability of its substrates. Early recognition of BCRP substrates is thus essential to optimize oral drug absorption, design of novel therapeutics for central nervous system conditions, and overcome BCRP-mediated cross-resistance issues. We present the development of an ensemble of ligand-based machine learning algorithms for the early recognition of BCRP substrates, from a database of 262 substrates and nonsubstrates compiled from the literature. Such dataset was rationally partitioned into training and test sets by application of a 2-step clustering procedure. The models were developed through application of linear discriminant analysis to random subsamples of Dragon molecular descriptors. Simple data fusion and statistical comparison of partial areas under the curve of ROC curves were applied to obtain the best 2-model combination, which presented 82% and 74.5% of overall accuracy in the training and test set, respectively. PMID:23984415

  18. The p66(Shc) adaptor protein controls oxidative stress response in early bovine embryos.

    PubMed

    Betts, Dean H; Bain, Nathan T; Madan, Pavneesh

    2014-01-01

    The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

  19. Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

    PubMed

    Masseguin, C; Corcoran, M; Carcenac, C; Daunton, N G; Güell, A; Verkman, A S; Gabrion, J

    2000-03-01

    Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.

  20. Expression of S-100 protein in renal cell neoplasms.

    PubMed

    Lin, Fan; Yang, Wannian; Betten, Mark; Teh, Bin Tean; Yang, Ximing J

    2006-04-01

    Polyclonal antibody to S-100 protein has been routinely applied for initial screening of various types of tumors, including, melanocytic tumors and neurogenic tumors. S-100 protein has been shown to have a broad distribution in human tissues, including renal tubules. The potential utility of S-100 protein in renal cell neoplasms has not been extensively investigated. Using an EnVision-Horseradish Peroxidase (HRP; Dako, Carpinteria, Calif) kit, we evaluated the diagnostic value of S-100 protein on tissue microarray sections from 175 cases of renal epithelial neoplasm (145 primary renal neoplasms and 30 metastatic renal cell carcinomas) and 24 non-neoplastic renal tissues. Immunohistochemical stains for pancytokeratin, HMB-45, and Mart-1 were also performed. Western blot using the same antibody (anti-S-100 protein) was performed on 10 cases of renal cell neoplasm. The results demonstrated that nuclear and cytoplasmic staining pattern for S-100 protein was observed in 56 (69%) of 81 conventional (clear cell) renal cell carcinomas (RCCs), 10 (30%) of 33 papillary RCCs, 1 (6%) of 16 ChRCCs, and 13 (87%) of 15 oncocytomas. Among the 81 cases of CRCC, positivity for S-100 protein was seen in 41 (71%) of 58 and 15 (65%) of 23 cases with Furhman nuclear grade I/II and III/IV, respectively. Focal immunostaining was present in 22 (92%) of 24 normal renal tubules. Similar staining pattern was observed in 21 (70%) of 30 metastatic RCCs. Western blotting demonstrated the S-100 protein expression in both renal cell neoplasm and normal renal tissue. Overexpression of S-100 in oncocytomas compared with ChRCCs was confirmed by the data of Western blot and cDNA microarray analysis. Importantly, 14.8% (12/81) of clear cell RCC and 13.3% (4/30) of metastatic RCC revealed an immunostaining profile of pancytokeratin (-)/S-100 protein (+). These data indicate that caution should be taken in interpreting an unknown primary with S-100 positivity and cytokeratin negativity. In addition, it

  1. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  2. Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

    PubMed

    Rezvani, Hamid Reza; Mahfouf, Walid; Ali, Nsrein; Chemin, Cecile; Ged, Cecile; Kim, Arianna L; de Verneuil, Hubert; Taïeb, Alain; Bickers, David R; Mazurier, Frédéric

    2010-01-01

    The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

  3. Plasmodium vivax merozoite surface protein 8 cloning, expression, and characterisation.

    PubMed

    Perez-Leal, Oscar; Sierra, Adriana Y; Barrero, Carlos A; Moncada, Camilo; Martinez, Pilar; Cortes, Jimena; Lopez, Yolanda; Torres, Elizabeth; Salazar, Luz M; Patarroyo, Manuel A

    2004-11-26

    Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespread throughout the world, leading to nearly 80 million cases per year, mainly in Latin-America and Asia. An open reading frame encoding the Plasmodium falciparum merozoite surface protein 8 P. vivax homologue has been identified in the present study by screening the current data obtained from this parasite's partially sequenced genome. This new protein contains 487 amino-acids, two epidermal growth factor like domains, hydrophobic regions at the N- and C-termini compatible with a signal peptide, and a glycosylphosphatidylinositol anchor site, respectively. This gene's transcription and its encoded protein expression have been assessed, as well as its recognition by P. vivax-infected patients' sera. Based on this recognition, and a previous study showing that mice immunised with the Plasmodium yoelii homologous protein were protected, we consider the PvMSP8 a good candidate to be included in a multi-stage multi-antigen P. vivax vaccine.

  4. Expression and purification of recombinant human EGFL7 protein.

    PubMed

    Picuric, Srdjan; Friedrich, Matthias; Oess, Stefanie

    2009-11-01

    The secreted epidermal growth factor-like protein 7 (EGFL7) plays an important role in angiogenesis, especially in the recruitment of endothelial and smooth muscle cells to the site of the nascent vessel and their ordered assembly into functional vasculature. However, progress in the understanding of the underlying mechanisms is to date greatly hindered by the lack of recombinant EGFL7 protein in a stable, soluble, native state, thus preventing e.g. the characterization of the proposed functional receptor as well as investigation of additional biological effects of EGFL7. So far all attempts to produce sufficient amounts of recombinant EGFL7 protein by various groups have failed. In this study we describe a procedure for the expression and purification of human EGFL7 from Sf9 cells and for the first time provide means to isolate biologically functional EGFL7 protein in sufficient quantities for its further biological characterization. We believe that the availability of EGFL7 will greatly accelerate our understanding of the precise role of EGFL7 and the underlying molecular mechanisms of EGFL7 action in the fundamentally important process of angiogenesis.

  5. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction.

    PubMed

    Markholt, S; Grøndahl, M L; Ernst, E H; Andersen, C Yding; Ernst, E; Lykke-Hartmann, K

    2012-02-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

  6. AP-1(c-Jun/FosB) mediates xFoxD5b expression in Xenopus early developmental neurogenesis.

    PubMed

    Yoon, Jaeho; Kim, Jung-Ho; Lee, Ok-Joo; Lee, Sung-Young; Lee, Seung-Hwan; Park, Jae-Bong; Lee, Jae-Yong; Kim, Sung-Chan; Kim, Jaebong

    2013-01-01

    AP-1 (activator protein-1) is composed of Jun and Fos proteins and functions in cell proliferation, apoptosis and differentiation. Previous studies have demonstrated that different AP-1 complexes participate in the determination of various cell fates. However, the role of different AP-1 complexes during early vertebrate development is not yet fully understood. In the present study, we demonstrate that AP-1(c-Jun/FosB) regulates neurogenesis via FoxD5b expression. We show that FoxD5 was induced by the inhibition of BMP and that FoxD5b plays roles in neurogenesis. Additionally, we show that the FoxD5b promoter region within -1336 and -1316 contains an AP-1 binding site, which is required for the transcriptional regulation of FoxD5b and is induced by the inhibition of BMP signaling in animal cap explants. Moreover, c-Jun, a component of AP-1, directly binds to the AP-1 binding site of the FoxD5b promoter and induces FoxD5b expression cooperatively with FosB, but not with c-Fos or Fra-1. Altogether, these results suggest that AP-1(c-Jun/FosB) may play a role in neurogenesis via the induction of FoxD5b expression during early vertebrate development.

  7. Chronic morphine treatment enhances sciatic nerve stimulation-induced immediate early gene expression in the rat dorsal horn.

    PubMed

    Bojovic, Ognjen; Bramham, Clive R; Tjølsen, Arne

    2015-01-01

    Synaptic plasticity is a property of neurons that can be induced by conditioning electrical stimulation (CS) of afferent fibers in the spinal cord. This is a widely studied property of spinal cord and hippocampal neurons. CS has been shown to trigger enhanced expression of immediate early gene proteins (IEGPs), with peak increases observed 2 hour post stimulation. Chronic morphine treatment has been shown to promoteinduce opioid-induced hyperalgesia, and also to increase CS-induced central sensitization in the dorsal horn. As IEGP expression may contribute to development of chronic pain states, we aimed to determine whether chronic morphine treatment affects the expression of IEGPs following sciatic nerve CS. Changes in expression of the IEGPs Arc, c-Fos or Zif268 were determined in cells of the lumbar dorsal horn of the spinal cord. Chronic Morphine pretreatment over 7 days led to a significant increase in the number of IEGP positive cells observed at both 2 h and 6 h after CS. The same pattern of immunoreactivity was obtained for all IEGPs, with peak increases occurring at 2 h post CS. In contrast, morphine treatment alone in sham operated animals had no effect on IEGP expression. We conclude that chronic morphine treatment enhances stimulus-induced expression of IEGPs in the lumbar dorsal horn. These data support the notion that morphine alters gene expression responses linked to nociceptive stimulation and plasticity.

  8. Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system.

    PubMed

    Breier, G; Breviario, F; Caveda, L; Berthier, R; Schnürch, H; Gotsch, U; Vestweber, D; Risau, W; Dejana, E

    1996-01-15

    An early step in the formation of the extraembryonic and intraembryonic vasculature is endothelial cell differentiation and organization in blood islands and vascular structures. This involves the expression and function of specific adhesive molecules at cell-to-cell junctions. Previous work showed that endothelial cells express a cell-specific cadherin (vascular endothelial [VE]-cadherin, or 7B4/cadherin-5) that is organized at cell-to-cell contacts in cultured cells and is able to promote intercellular adhesion. In this study, we investigated whether VE-cadherin could be involved in early cardiovascular development in the mouse embryo. We first cloned and sequenced the mouse VE-cadherin cDNA. At the protein level, murine VE-cadherin presented 75% identity (90%, considering conservative amino acid substitutions) with the human homologue. Transfection of murine VE-cadherin cDNA in L cells induced Ca(++)-dependent cell-to-cell aggregation and reduced cell detachment from monolayers. In situ hybridization of adult tissues showed that the murine molecule is specifically expressed by endothelial cells. In mouse embryos, VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5) in mesodermal cells of the yolk sac mesenchyme. At E9.5, expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cell