Science.gov

Sample records for age modified proteins

  1. Morphological remodeling of C. elegans neurons during aging is modified by compromised protein homeostasis

    PubMed Central

    Vayndorf, Elena M; Scerbak, Courtney; Hunter, Skyler; Neuswanger, Jason R; Toth, Marton; Parker, J Alex; Neri, Christian; Driscoll, Monica; Taylor, Barbara E

    2016-01-01

    Understanding cellular outcomes, such as neuronal remodeling, that are common to both healthy and diseased aging brains is essential to the development of successful brain aging strategies. Here, we used Caenorhabdits elegans to investigate how the expression of proteotoxic triggers, such as polyglutamine (polyQ)-expanded huntingtin and silencing of proteostasis regulators, such as the ubiquitin–proteasome system (UPS) and protein clearance components, may impact the morphological remodeling of individual neurons as animals age. We examined the effects of disrupted proteostasis on the integrity of neuronal cytoarchitecture by imaging a transgenic C. elegans strain in which touch receptor neurons express the first 57 amino acids of the human huntingtin (Htt) gene with expanded polyQs (128Q) and by using neuron-targeted RNA interference in adult wild-type neurons to knockdown genes encoding proteins involved in proteostasis. We found that proteostatic challenges conferred by polyQ-expanded Htt and knockdown of specific genes involved in protein homeostasis can lead to morphological changes that are restricted to specific domains of specific neurons. The age-associated branching of PLM neurons is suppressed by N-ter polyQ-expanded Htt expression, whereas ALM neurons with polyQ-expanded Htt accumulate extended outgrowths and other soma abnormalities. Furthermore, knockdown of genes important for ubiquitin-mediated degradation, lysosomal function, and autophagy modulated these age-related morphological changes in otherwise normal neurons. Our results show that the expression of misfolded proteins in neurodegenerative disease such as Huntington’s disease modifies the morphological remodeling that is normally associated with neuronal aging. Our results also show that morphological remodeling of healthy neurons during aging can be regulated by the UPS and other proteostasis pathways. Collectively, our data highlight a model in which morphological remodeling during

  2. Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro.

    PubMed

    Jørgensen, Peter; Milkovic, Lidija; Zarkovic, Neven; Waeg, Georg; Rattan, Suresh I S

    2014-02-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

  3. Adhesives from modified soy protein

    DOEpatents

    Sun, Susan; Wang, Donghai; Zhong, Zhikai; Yang, Guang

    2008-08-26

    The, present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

  4. Age and continuous lactose challenge modify lactase protein expression and enzyme activity in gut epithelium in the rat.

    PubMed

    Peuhkuri, K; Hukkanen, M; Beale, R; Polak, J M; Vapaatalo, H; Korpela, R

    1997-12-01

    The activity of lactase enzyme declines after weaning. This study was to investigate changes in the lactase expression in the whole gastrointestinal tract during the development and the possibility that this and activity can be induced by lactose. Expression of lactase protein in the gut of 1-12-weeks old rats was studied by immunocytochemistry. Possible induction was evaluated by immunohistochemical and biochemical techniques in 8-week-old rats after lactose challenge for seven days. Lactase immunoreactivity was detected only in the small intestine and it decreased 20% during the week after weaning. A steady level of 40% lower than in the sucklings was found in the adult rats. In the lactose-challenged rats the optical density of immunoreactivity increased by about 30% in those that consumed the highest concentration of lactose. In the proximal jejunum, elevation of the enzymatic activity was three-fold. In the rat lactase protein expression decreased rapidly after weaning and expression and activity were induced by lactose-rich diet, most notably in the proximal jejunum.

  5. Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.

    PubMed

    Nishimune, Hiroshi; Numata, Tomohiro; Chen, Jie; Aoki, Yudai; Wang, Yonghong; Starr, Miranda P; Mori, Yasuo; Stanford, John A

    2012-01-01

    The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

  6. Protein glycation, diabetes, and aging.

    PubMed

    Ulrich, P; Cerami, A

    2001-01-01

    Biological amines react with reducing sugars to form a complex family of rearranged and dehydrated covalent adducts that are often yellow-brown and/or fluorescent and include many cross-linked structures. Food chemists have long studied this process as a source of flavor, color, and texture changes in cooked, processed, and stored foods. During the 1970s and 1980s, it was realized that this process, called the Maillard reaction or advanced glycation, also occurs slowly in vivo. Advanced glycation endproducts (AGEs) that form are implicated, causing the complications of diabetes and aging, primarily via adventitious and crosslinking of proteins. Long-lived proteins such as structural collagen and lens crystallins particularly are implicated as pathogenic targets of AGE processes. AGE formation in vascular wall collagen appears to be an especially deleterious event, causing crosslinking of collagen molecules to each other and to circulating proteins. This leads to plaque formation, basement membrane thickening, and loss of vascular elasticity. The chemistry of these later-stage, glycation-derived crosslinks is still incompletely understood but, based on the hypothesis that AGE formation involves reactive carbonyl groups, the authors introduced the carbonyl reagent aminoguanidine hydrochloride as an inhibitor of AGE formation in vivo in the mid 1980s. Subsequent studies by many researchers have shown the effectiveness of aminoguanidine in slowing or preventing a wide range of complications of diabetes and aging in animals and, recently, in humans. Since, the authors have developed a new class of agents, exemplified by 4,5-dimethyl-3-phenacylthiazolium chloride (DPTC), which can chemically break already-formed AGE protein-protein crosslinks. These agents are based on a new theory of AGE crosslinking that postulates that alpha-dicarbonyl structures are present in AGE protein-protein crosslinks. In studies in aged animals, DPTC has been shown to be capable of reverting

  7. Protein Requirements during Aging.

    PubMed

    Courtney-Martin, Glenda; Ball, Ronald O; Pencharz, Paul B; Elango, Rajavel

    2016-01-01

    Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals. PMID:27529275

  8. Protein Requirements during Aging

    PubMed Central

    Courtney-Martin, Glenda; Ball, Ronald O.; Pencharz, Paul B.; Elango, Rajavel

    2016-01-01

    Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals. PMID:27529275

  9. Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

    PubMed

    Kapphahn, Rebecca J; Giwa, Babatomiwa M; Berg, Kristin M; Roehrich, Heidi; Feng, Xiao; Olsen, Timothy W; Ferrington, Deborah A

    2006-07-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification. PMID:16530755

  10. Highly Charged Proteins: The Achilles' Heel of Aging Proteomes.

    PubMed

    de Graff, Adam M R; Hazoglou, Michael J; Dill, Ken A

    2016-02-01

    As cells and organisms age, their proteins sustain increasing amounts of oxidative damage. It is estimated that half of all proteins are damaged in old organisms, yet the dominant mechanisms by which damage affects proteins and cellular phenotypes are not known. Here, we show that random modification of side chain charge induced by oxidative damage is likely to be a dominant source of protein stability loss in aging cells. Using an established model of protein electrostatics, we find that short, highly charged proteins are particularly susceptible to large destabilization from even a single side chain oxidation event. This mechanism identifies 20 proteins previously established to be important in aging that are at particularly high risk for oxidative destabilization, including transcription factors, histone and histone-modifying proteins, ribosomal and telomeric proteins, and proteins essential for homeostasis. Cellular processes enriched in high-risk proteins are shown to be particularly abundant in the aggregates of old organisms. PMID:26724998

  11. Scavenger receptor for aldehyde-modified proteins.

    PubMed

    Horiuchi, S; Murakami, M; Takata, K; Morino, Y

    1986-04-15

    This paper describes an unexpectedly broad ligand specificity of a scavenger receptor of sinusoidal liver cells that is responsible for endocytic uptake of formaldehyde-treated bovine serum albumin (f-Alb). Binding of 125I-f-Alb to the isolated cells was effectively inhibited by bovine serum albumin (BSA) modified with aliphatic aldehydes such as glycolaldehye, DL-glyceraldehyde, and propionaldehyde whereas albumin preparations modified by aromatic aldehydes such as pyridoxal, pyridoxal phosphate, salicylaldehyde, and benzaldehyde did not affect this binding process. Binding of 125I-glycolaldehyde-treated BSA to the cells exhibited a saturation kinetics with an apparent Kd = 3.3 micrograms of the ligand/ml. This binding process was inhibited by unlabeled f-Alb as well as by the antibody raised against the f-Alb receptor. Indeed, 125I-glycolaldehyde-treated BSA underwent a rapid plasma clearance (t1/2 approximately 2 min) which was markedly retarded by unlabeled f-Alb. Upon treatment by these aldehydes, other proteins such as ovalbumin, soybean trypsin inhibitor, and hemoglobin were also converted to active ligands for the f-Alb receptor, while no ligand activity was generated with gamma-globulin and RNase A. These results clearly show that the f-Alb receptor, originally described as being specific for f-Alb, exhibits a broad ligand specificity in terms of both aldehydes and proteins and, hence, should be described as a scavenger receptor for aldehyde-modified proteins.

  12. Aged Garlic Extract Modifies Human Immunity.

    PubMed

    Percival, Susan S

    2016-02-01

    Garlic contains numerous compounds that have the potential to influence immunity. Immune cells, especially innate immune cells, are responsible for the inflammation necessary to kill pathogens. Two innate lymphocytes, γδ-T and natural killer (NK) cells, appear to be susceptible to diet modification. The purpose of this review was to summarize the influence of aged garlic extract (AGE) on the immune system. The author's laboratory is interested in AGE's effects on cell proliferation and activation and inflammation and to learn whether those changes might affect the occurrence and severity of colds and flu. Healthy human participants (n = 120), between 21 and 50 y of age, were recruited for a randomized, double-blind, placebo-controlled parallel-intervention study to consume 2.56 g AGE/d or placebo supplements for 90 d during the cold and flu season. Peripheral blood mononuclear cells were isolated before and after consumption, and γδ-T and NK cell function was assessed by flow cytometry. The effect on cold and flu symptoms was determined by using daily diary records of self-reported illnesses. After 45 d of AGE consumption, γδ-T and NK cells proliferated better and were more activated than cells from the placebo group. After 90 d, although the number of illnesses was not significantly different, the AGE group showed reduced cold and flu severity, with a reduction in the number of symptoms, the number of days participants functioned suboptimally, and the number of work/school days missed. These results suggest that AGE supplementation may enhance immune cell function and may be partly responsible for the reduced severity of colds and flu reported. The results also suggest that the immune system functions well with AGE supplementation, perhaps with less accompanying inflammation. This trial was registered at clinicaltrials.gov as NCT01390116.

  13. Aged Garlic Extract Modifies Human Immunity.

    PubMed

    Percival, Susan S

    2016-02-01

    Garlic contains numerous compounds that have the potential to influence immunity. Immune cells, especially innate immune cells, are responsible for the inflammation necessary to kill pathogens. Two innate lymphocytes, γδ-T and natural killer (NK) cells, appear to be susceptible to diet modification. The purpose of this review was to summarize the influence of aged garlic extract (AGE) on the immune system. The author's laboratory is interested in AGE's effects on cell proliferation and activation and inflammation and to learn whether those changes might affect the occurrence and severity of colds and flu. Healthy human participants (n = 120), between 21 and 50 y of age, were recruited for a randomized, double-blind, placebo-controlled parallel-intervention study to consume 2.56 g AGE/d or placebo supplements for 90 d during the cold and flu season. Peripheral blood mononuclear cells were isolated before and after consumption, and γδ-T and NK cell function was assessed by flow cytometry. The effect on cold and flu symptoms was determined by using daily diary records of self-reported illnesses. After 45 d of AGE consumption, γδ-T and NK cells proliferated better and were more activated than cells from the placebo group. After 90 d, although the number of illnesses was not significantly different, the AGE group showed reduced cold and flu severity, with a reduction in the number of symptoms, the number of days participants functioned suboptimally, and the number of work/school days missed. These results suggest that AGE supplementation may enhance immune cell function and may be partly responsible for the reduced severity of colds and flu reported. The results also suggest that the immune system functions well with AGE supplementation, perhaps with less accompanying inflammation. This trial was registered at clinicaltrials.gov as NCT01390116. PMID:26764332

  14. Investigation of modified cottonseed protein adhesives for wood composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several modified cottonseed protein isolates were studied and compared to corresponding soy protein isolates for their adhesive properties when bonded to wood composites. Modifications included treatments with alkali, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and urea. Wood composites...

  15. Measuring Children's Age Stereotyping Using a Modified Piagetian Conservation Task

    ERIC Educational Resources Information Center

    Kwong See, Sheree T.; Rasmussen, Carmen; Pertman, S. Quinn

    2012-01-01

    We examined five-year-old-children's age stereotyping using a modified Piagetian conservation task. Children were asked if two lines of objects were the "same" after one line had been made longer (transformed). A conversational account posits that children's answers reflect assumptions about the asker's motivation for the question (Schwarz, 1996).…

  16. The Protein-Sparing Modified Fast Diet

    PubMed Central

    Bakhach, Marwan; Shah, Vaishal; Harwood, Tara; Lappe, Sara; Bhesania, Natalie; Mansoor, Sana; Alkhouri, Naim

    2016-01-01

    Objectives: The protein-sparing modified fast (PSMF) is a rigorous way of rapidly losing a large amount of weight. Although adult studies have shown the PSMF to be effective, data in adolescents are lacking. The aim of this study was to determine the efficacy and safety of the PSMF in severely obese adolescents. Methods: 12 subjects who were evaluated in the Obesity Management Program at the Cleveland Clinic from 2011 to 2014 were included. The subjects were initiated on the PSMF after failing other conventional methods of weight loss. Once the goal weight was achieved, subjects were transitioned to the refeeding phase for weight maintenance. Results: Follow-up was scheduled at 3-month (11 patients) and 6-month (6 patients) intervals. At the 6-month follow-up visit, the average weight loss was 11.19 kg (95% confidence interval = -5.4, -27.8, P = .028), with average of 9.8% from baseline. Fifty percent of subjects had >5% weight loss and 20% had >10% weight loss. Four patients were lost to the follow-up (40%). An improvement was noted in total cholesterol and high-density lipoprotein. Due to a small sample size these results were not statistically significant. Side effects reported by subjects were mild dehydration due to nausea (2 patients), decreased energy (1 patient), and transient labile mood (1 patient). No life-threatening side effects were reported. Conclusion: Our results show that the PSMF diet can be used as an effective and safe method in the outpatient setting for rapid weight loss in adolescents with severe obesity. PMID:27335996

  17. The ligand activity of AGE-proteins to scavenger receptors is dependent on their rate of modification by AGEs.

    PubMed

    Nagai, Ryoji; Mera, Katsumi; Nakajou, Keisuke; Fujiwara, Yukio; Iwao, Yasunori; Imai, Hiroki; Murata, Toshinori; Otagiri, Masaki

    2007-12-01

    The cellular interaction of proteins modified with advanced glycation end-products (AGEs) is believed to induce several different biological responses, which are involved in the development of diabetic vascular complications. We report here that the ratio of protein glycation is implicated in its ligand activity to scavenger receptors. Although highly-modified AGE-bovine serum albumin (high-AGE-BSA) was significantly recognized by human monocyte-derived macrophages and Chinese hamster ovary cells which overexpress such scavenger receptors as CD36, SR-BI (scavenger receptor class B type-I), and LOX-1 (Lectin-like Ox-LDL receptor-1), the mildly-modified-AGE-BSA (mild-AGE-BSA) did not show any ligand activity to these cells. Furthermore, when (111)In-labeled high- or mild-AGE-BSA were injected into the tail vein of mice, the high-AGE-BSA was rapidly cleared from the circulation whereas the clearance rate of the mild-AGE-BSA was very slow, similar to the native BSA. These results demonstrate the first evidence that the ligand activity of the AGE-proteins to the scavenger receptors and its pharmacokinetic properties depend on their rate of modification by AGEs, and we should carefully prepare the AGE-proteins in vitro to clarify the physiological significance of the interaction between the AGE-receptors and AGE-proteins.

  18. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins.

    PubMed

    Takata, K; Horiuchi, S; Araki, N; Shiga, M; Saitoh, M; Morino, Y

    1988-10-15

    Long term incubation of proteins with glucose, named the Maillard reaction (Maillard, L. C. (1912) C. R. Acad. Sci. (Paris) 154, 66-68), gives rise to advanced glycosylation end product (AGE) with fluorescence, color, as well as cross-linked properties. The receptor-mediated endocytosis of AGE-proteins by macrophages was reported (Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5588-5592). The present study on the binding of AGE-bovine serum albumin (BSA) to rat peritoneal macrophages and sinusoidal liver cells demonstrated the presence of a saturable, high affinity receptor for AGE-BSA with Kd = 2.4 x 10(-7) M (macrophages) and 2.1 x 10(-7) M (sinusoidal cells). The cellular binding of AGE-BSA and its endocytic uptake by these cells were competitively inhibited by BSA preparations modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, ligands known to be specific for a scavenger receptor for aldehyde-modified proteins (Horiuchi, S., Murakami, M., Takata, K., and Morino, Y. (1986). J. Biol. Chem. 261, 4962-4966). These ligands also had a profound in vivo effect on the plasma clearance of 125I-AGE-BSA as well as its hepatic uptake. Thus, endocytic uptake of AGE-proteins by macrophages appeared to be mediated by a scavenger receptor for aldehyde-modified proteins. This provides evidence for the biological importance of the scavenger receptor in eliminating senescent macromolecules from the circulation.

  19. Protein homeostasis and molecular chaperones in aging.

    PubMed

    Arslan, Mehmet Alper; Csermely, Péter; Soti, Csaba

    2006-01-01

    Molecular chaperones are ubiquitous, highly conserved proteins responsible for the maintenance of protein folding homeostasis in cells. Environmental stress causes proteotoxic damage, which triggers chaperone induction and the subsequent reparation of cellular damage by chaperones, including disposing irreparable protein ensembles. We summarize the current view of protein damage, turnover, the stress response and chaperone function in aging, and review novel data showing that accumulation of misfolded proteins outcompete and overload the limited resources of the protein folding, maintenance and turnover system, compromising general protein homeastasis and cellular function. Possible involvement of chaperones and proteolysis in immunosenescence is highlighted. Defects in zinc metabolism are also addressed in relation to aging and changes in chaperone levels. PMID:16964526

  20. Rho-modifying bacterial protein toxins.

    PubMed

    Aktories, Klaus

    2015-12-01

    Rho proteins are targets of numerous bacterial protein toxins, which manipulate the GTP-binding proteins by covalent modifications, including ADP ribosylation, glycosylation, adenylylation, proteolytic cleavage and deamidation. Bacterial toxins are important virulence factors but are also potent and efficient pharmacological tools to study the physiological functions of their eukaryotic targets. Recent studies indicate that amazing variations exist in the molecular mechanisms by which toxins attack Rho proteins, which are discussed here.

  1. Melatonin modifies the rhythm of protein synthesis.

    PubMed

    Brodsky, V Y; Dubovaya, N D; Zvezdina, T K; Fateeva, V I; Mal'chenko, L A

    2010-07-01

    Melatonin (5 nM) added to medium with primary hepatocyte cultures shifted the phase of circahoralian rhythm of protein synthesis and hence, can be a factor synchronizing fluctuations in protein synthesis and rhythm organizer in the hepatocyte population. Blockade of melatonin receptors with luzindole (20 nM) arrested rhythm organization of protein synthesis by melatonin. Prospects of studying biochemical mechanisms of protein synthesis rhythm organization with other drugs (calcium agonists, similarly to melatonin) are discussed.

  2. Genetically modified proteins: functional improvement and chimeragenesis

    PubMed Central

    Balabanova, Larissa; Golotin, Vasily; Podvolotskaya, Anna; Rasskazov, Valery

    2015-01-01

    This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit. PMID:26211369

  3. Preparation and evaluation of tara-modified proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quebracho, a vegetable tannin, can be used to modify gelatin to produce a product that has been applied effectively as a filler in leather processing, as described in our previous report. In this ongoing study, another vegetable tannin tara is examined for its possible application in protein modifi...

  4. Protein carbamylation is a hallmark of aging.

    PubMed

    Gorisse, Laëtitia; Pietrement, Christine; Vuiblet, Vincent; Schmelzer, Christian E H; Köhler, Martin; Duca, Laurent; Debelle, Laurent; Fornès, Paul; Jaisson, Stéphane; Gillery, Philippe

    2016-02-01

    Aging is a progressive process determined by genetic and acquired factors. Among the latter are the chemical reactions referred to as nonenzymatic posttranslational modifications (NEPTMs), such as glycoxidation, which are responsible for protein molecular aging. Carbamylation is a more recently described NEPTM that is caused by the nonenzymatic binding of isocyanate derived from urea dissociation or myeloperoxidase-mediated catabolism of thiocyanate to free amino groups of proteins. This modification is considered an adverse reaction, because it induces alterations of protein and cell properties. It has been shown that carbamylated proteins increase in plasma and tissues during chronic kidney disease and are associated with deleterious clinical outcomes, but nothing is known to date about tissue protein carbamylation during aging. To address this issue, we evaluated homocitrulline rate, the most characteristic carbamylation-derived product (CDP), over time in skin of mammalian species with different life expectancies. Our results show that carbamylation occurs throughout the whole lifespan and leads to tissue accumulation of carbamylated proteins. Because of their remarkably long half-life, matrix proteins, like type I collagen and elastin, are preferential targets. Interestingly, the accumulation rate of CDPs is inversely correlated with longevity, suggesting the occurrence of still unidentified protective mechanisms. In addition, homocitrulline accumulates more intensely than carboxymethyl-lysine, one of the major advanced glycation end products, suggesting the prominent role of carbamylation over glycoxidation reactions in age-related tissue alterations. Thus, protein carbamylation may be considered a hallmark of aging in mammalian species that may significantly contribute in the structural and functional tissue damages encountered during aging.

  5. Protein carbamylation is a hallmark of aging

    PubMed Central

    Gorisse, Laëtitia; Pietrement, Christine; Vuiblet, Vincent; Schmelzer, Christian E. H.; Duca, Laurent; Debelle, Laurent; Fornès, Paul; Jaisson, Stéphane; Gillery, Philippe

    2016-01-01

    Aging is a progressive process determined by genetic and acquired factors. Among the latter are the chemical reactions referred to as nonenzymatic posttranslational modifications (NEPTMs), such as glycoxidation, which are responsible for protein molecular aging. Carbamylation is a more recently described NEPTM that is caused by the nonenzymatic binding of isocyanate derived from urea dissociation or myeloperoxidase-mediated catabolism of thiocyanate to free amino groups of proteins. This modification is considered an adverse reaction, because it induces alterations of protein and cell properties. It has been shown that carbamylated proteins increase in plasma and tissues during chronic kidney disease and are associated with deleterious clinical outcomes, but nothing is known to date about tissue protein carbamylation during aging. To address this issue, we evaluated homocitrulline rate, the most characteristic carbamylation-derived product (CDP), over time in skin of mammalian species with different life expectancies. Our results show that carbamylation occurs throughout the whole lifespan and leads to tissue accumulation of carbamylated proteins. Because of their remarkably long half-life, matrix proteins, like type I collagen and elastin, are preferential targets. Interestingly, the accumulation rate of CDPs is inversely correlated with longevity, suggesting the occurrence of still unidentified protective mechanisms. In addition, homocitrulline accumulates more intensely than carboxymethyl-lysine, one of the major advanced glycation end products, suggesting the prominent role of carbamylation over glycoxidation reactions in age-related tissue alterations. Thus, protein carbamylation may be considered a hallmark of aging in mammalian species that may significantly contribute in the structural and functional tissue damages encountered during aging. PMID:26712018

  6. [Carbonyl stress and oxidatively modified proteins in chronic renal failure].

    PubMed

    Bargnoux, A-S; Morena, M; Badiou, S; Dupuy, A-M; Canaud, B; Cristol, J-P

    2009-01-01

    Oxidative stress is commonly observed in chronic renal failure patients resulting from an unbalance between overproduction of reactive oxygen species and impairement of defense mechanisms. Proteins appear as potential targets of uremia-induced oxidative stress and may undergo qualitative modifications. Proteins could be directly modified by reactive oxygen species which leads to amino acid oxydation and cross-linking. Proteins could be indirectly modified by reactive carbonyl compounds produced by glycoxidation and lipo-peroxidation. The resulting post-traductional modifications are known as carbonyl stress. In addition, thiols could be oxidized or could react with homocystein leading to homocysteinylation. Finally, tyrosin could be oxidized by myeloperoxidase leading to advanced oxidative protein products (AOPP). Oxidatively modified proteins are increased in chronic renal failure patients and may contribute to exacerbate the oxidative stress/inflammation syndrome. They have been involved in long term complications of uremia such as amyloidosis and accelerated atherosclerosis. PMID:19297289

  7. Modifiable diarrhoea risk factors in Egyptian children aged <5 years.

    PubMed

    Mansour, A M; Mohammady, H El; Shabrawi, M El; Shabaan, S Y; Zekri, M Abou; Nassar, M; Salem, M E; Mostafa, M; Riddle, M S; Klena, J D; Messih, I A Abdel; Levin, S; Young, S Y N

    2013-12-01

    By conducting a case-control study in two university hospitals, we explored the association between modifiable risk behaviours and diarrhoea. Children aged <5 years attending outpatient clinics for diarrhoea were matched by age and sex with controls. Data were collected on family demographics, socioeconomic indicators, and risk behaviour practices. Two rectal swabs and a stool specimen were collected from cases and controls. Samples were cultured for bacterial pathogens using standard techniques and tested by ELISA to detect rotavirus and Cryptosporidium spp. Four hundred cases and controls were enrolled between 2007 and 2009. The strongest independent risk factors for diarrhoea were: presence of another household member with diarrhoea [matched odds ratio (mOR) 4.9, 95% CI 2.8-8.4] in the week preceding the survey, introduction to a new kind of food (mOR 3, 95% CI 1.7-5.4), and the child being cared for outside home (mOR 2.6, 95% CI 1.3-5.2). While these risk factors are not identifiable, in some age groups more easily modifiable risk factors were identified including: having no soap for handwashing (mOR 6.3, 95% CI 1.2-33.9) for children aged 7-12 months, and pacifier use (mOR 1.9, 95% CI 1.0-3.5) in children aged 0-6 months. In total, the findings of this study suggest that community-based interventions to improve practices related to sanitation and hygiene, handwashing and food could be utilized to reduce the burden of diarrhoea in Egyptian children aged <5 years. PMID:23433452

  8. Modifiable diarrhoea risk factors in Egyptian children aged <5 years.

    PubMed

    Mansour, A M; Mohammady, H El; Shabrawi, M El; Shabaan, S Y; Zekri, M Abou; Nassar, M; Salem, M E; Mostafa, M; Riddle, M S; Klena, J D; Messih, I A Abdel; Levin, S; Young, S Y N

    2013-12-01

    By conducting a case-control study in two university hospitals, we explored the association between modifiable risk behaviours and diarrhoea. Children aged <5 years attending outpatient clinics for diarrhoea were matched by age and sex with controls. Data were collected on family demographics, socioeconomic indicators, and risk behaviour practices. Two rectal swabs and a stool specimen were collected from cases and controls. Samples were cultured for bacterial pathogens using standard techniques and tested by ELISA to detect rotavirus and Cryptosporidium spp. Four hundred cases and controls were enrolled between 2007 and 2009. The strongest independent risk factors for diarrhoea were: presence of another household member with diarrhoea [matched odds ratio (mOR) 4.9, 95% CI 2.8-8.4] in the week preceding the survey, introduction to a new kind of food (mOR 3, 95% CI 1.7-5.4), and the child being cared for outside home (mOR 2.6, 95% CI 1.3-5.2). While these risk factors are not identifiable, in some age groups more easily modifiable risk factors were identified including: having no soap for handwashing (mOR 6.3, 95% CI 1.2-33.9) for children aged 7-12 months, and pacifier use (mOR 1.9, 95% CI 1.0-3.5) in children aged 0-6 months. In total, the findings of this study suggest that community-based interventions to improve practices related to sanitation and hygiene, handwashing and food could be utilized to reduce the burden of diarrhoea in Egyptian children aged <5 years.

  9. Age and cancer risk: a potentially modifiable relationship.

    PubMed

    White, Mary C; Holman, Dawn M; Boehm, Jennifer E; Peipins, Lucy A; Grossman, Melissa; Henley, S Jane

    2014-03-01

    This article challenges the idea that cancer cannot be prevented among older adults by examining different aspects of the relationship between age and cancer. Although the sequential patterns of aging cannot be changed, several age-related factors that contribute to disease risk can be. For most adults, age is coincidentally associated with preventable chronic conditions, avoidable exposures, and modifiable risk behaviors that are causally associated with cancer. Midlife is a period of life when the prevalence of multiple cancer risk factors is high and incidence rates begin to increase for many types of cancer. However, current evidence suggests that for most adults, cancer does not have to be an inevitable consequence of growing older. Interventions that support healthy environments, help people manage chronic conditions, and promote healthy behaviors may help people make a healthier transition from midlife to older age and reduce the likelihood of developing cancer. Because the number of adults reaching older ages is increasing rapidly, the number of new cancer cases will also increase if current incidence rates remain unchanged. Thus, the need to translate the available research into practice to promote cancer prevention, especially for adults at midlife, has never been greater. PMID:24512933

  10. Self-Organization of Aging in a Modified Penna Model

    NASA Astrophysics Data System (ADS)

    Kim, Gi Ok; Shim, Sugie

    The Penna model for biological aging is modified so that the fertility of each individual is determined by means of the number of activated mutations at that time. A new concept of "good" mutation, which makes an individual to mature enough to reproduce, is introduced. It is assumed that each individual can reproduce only during adulthood, which is determined by the number of activated mutations. The results of Monte Carlo calculations using the modified model show that the ranges of the reproductive age are broadened as time goes by, thus showing self-organization in the biological aging to the direction of the maximum self-conservation. In addition, the population, the survival rate, and the average life span were calculated and analyzed by changing the number of new mutations at birth. It is observed that the higher is the considered number of new mutations at birth, the shorter is the obtained average life span. The mortality functions are also calculated and they showed the exponential increase in adulthood, satisfying the Gompertz law.

  11. Altered Proteins in the Aging Brain

    PubMed Central

    Elobeid, Adila; Libard, Sylwia; Leino, Marina; Popova, Svetlana N.

    2016-01-01

    We assessed the prevalence of common altered brain proteins in 296 cognitively unimpaired subjects ranging from age 50 to 102 years. The incidence and the stage of hyperphosphorylated-τ (HPτ), β-amyloid, α-synuclein (αS), and transactive response DNA (TDP) binding protein 43 (TDP43)-immunoreactivity (-IR) increased with age. HPτ-IR was observed in 98% of the subjects; the locus coeruleus was solely affected in 46%, and 79% of the subjects were in Braak stages a to II. β-Amyloid was seen in 47% of subjects and the Thal phase correlated with the HPτ Braak stage and age. Intermediate Alzheimer disease-related pathology (ADRP) was seen in 12%; 52% of the subjects with HPτ-IR fulfilled criteria for definite primary age-related tauopathy (PART). The incidence of concomitant pathology (αS, TDP43) did not differ between those with PART and those with ADRP but the former were younger. TDP43-IR was observed in 36%; the most frequently affected region was the medulla; αS-IR was observed in 19% of subjects. In 41% of the subjects from 80 to 89 years at death, 3 altered proteins were seen in the brain. Thus, altered proteins are common in the brains of cognitively unimpaired aged subjects; this should be considered while developing diagnostic biomarkers, particularly for identifying subjects at early stages of neurodegenerative diseases. PMID:26979082

  12. Altered Proteins in the Aging Brain.

    PubMed

    Elobeid, Adila; Libard, Sylwia; Leino, Marina; Popova, Svetlana N; Alafuzoff, Irina

    2016-04-01

    We assessed the prevalence of common altered brain proteins in 296 cognitively unimpaired subjects ranging from age 50 to 102 years. The incidence and the stage of hyperphosphorylated-τ (HPτ), β-amyloid, α-synuclein (αS), and transactive response DNA (TDP) binding protein 43 (TDP43)-immunoreactivity (-IR) increased with age. HPτ-IR was observed in 98% of the subjects; the locus coeruleus was solely affected in 46%, and 79% of the subjects were in Braak stages a to II. β-Amyloid was seen in 47% of subjects and the Thal phase correlated with the HPτ Braak stage and age. Intermediate Alzheimer disease-related pathology (ADRP) was seen in 12%; 52% of the subjects with HPτ-IR fulfilled criteria for definite primary age-related tauopathy (PART). The incidence of concomitant pathology (αS, TDP43) did not differ between those with PART and those with ADRP but the former were younger. TDP43-IR was observed in 36%; the most frequently affected region was the medulla; αS-IR was observed in 19% of subjects. In 41% of the subjects from 80 to 89 years at death, 3 altered proteins were seen in the brain. Thus, altered proteins are common in the brains of cognitively unimpaired aged subjects; this should be considered while developing diagnostic biomarkers, particularly for identifying subjects at early stages of neurodegenerative diseases. PMID:26979082

  13. Effects of Chemically Modified Messenger RNA on Protein Expression.

    PubMed

    Li, Bin; Luo, Xiao; Dong, Yizhou

    2016-03-16

    Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Here, we describe the synthesis of two sets of chemically modified mRNAs [encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively], evaluation of protein expression, and correlation analysis of expression level under various conditions. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with N1-methylpseudouridine (me(1)ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Consequently, me(1)ψ, 5moU, and ψ are promising nucleotides for chemical modification of mRNAs. PMID:26906521

  14. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  15. Modifiable risk factors for age-related macular degeneration.

    PubMed

    Guymer, Robyn H; Chong, Elaine Wei-Tinn

    2006-05-01

    Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in Australia and other Western countries. As there is no cure for AMD, and treatments to stop its progression have met with limited success, there is an interest in identifying modifiable risk factors to prevent or slow disease progression. To date, smoking is the only proven modifiable risk factor for AMD. Other factors under study include (i) cardiovascular risk factors such as hypertension, body mass index, and atherosclerosis; and (ii) dietary risk factors including fat and antioxidant intake, but so far these studies have produced conflicting results. Dietary fat in relation to AMD has recently attracted media attention. Despite very limited work supporting an association between vegetable fat and AMD, widespread publicity advocating margarine as a cause of AMD and encouraging use of butter instead has caused confusion and anxiety among sufferers of AMD and the general public, as well as concern among health professionals. The antioxidant carotenoids--lutein and zeaxanthin--found in dark green or yellow vegetables exist in high concentrations in the macula and are hypothesised to play a protective role. Of nine controlled trials of supplementation with carotenoids and other antioxidants, three suggested that various combinations of antioxidants and carotenoids were protective. While a low-fat diet rich in dark green and yellow vegetables is advocated in general, any specific recommendations regarding certain fats or antioxidant supplementation and AMD are not based on consistent findings at this stage. PMID:16646746

  16. Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction.

    PubMed

    Gendron, Joshua M; Webb, Kristofor; Yang, Bing; Rising, Lisa; Zuzow, Nathan; Bennett, Eric J

    2016-08-01

    Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations.

  17. Intakes of PUFAs were inversely associated with plasma C-reactive protein 12 years later in a middle-aged population with vitamin E intake as an effect modifier.

    PubMed

    Julia, Chantal; Touvier, Mathilde; Meunier, Nathalie; Papet, Isabelle; Galan, Pilar; Hercberg, Serge; Kesse-Guyot, Emmanuelle

    2013-11-01

    Although n-3 (ω-3) polyunsaturated fatty acids (PUFAs) are considered anti-inflammatory components, the role of dietary n-6 PUFAs in inflammation remains controversial. Some mechanistic evidence suggests vitamin E as a potential effect modifier in the relationship between PUFAs and inflammation. Our objectives were to evaluate the long-term associations between dietary intakes of PUFAs and elevated plasma C-reactive protein (CRP) and to investigate potential effect modification by vitamin E. Individuals in the placebo group of the SU.VI.MAX trial who had available CRP measurements in 2007-2009 were included in the study (n = 843). Dietary intakes of n-3 PUFAs, n-6 PUFAs, and vitamin E were assessed in 1994-1996 with at least 6 dietary records. The logistic regression OR for elevated CRP (>3 mg/L) and 95% CI were estimated for individual PUFAs and for total n-3 and n-6 PUFA intakes. Models were adjusted for sociodemographical, lifestyle, anthropometric, and dietary variables. Interactions with vitamin E intakes were also assessed. Inverse associations were observed between intakes of total n-3 PUFAs [α-linolenic acid (ALA; 18:3n-3), ALA + eicosapentaenoic acid (EPA; 20:5n-3), EPA + docosapentaenoic acid (DPA; 22:5n-3), DPA + docosahexaenoic acid (DHA; 22:6n-3)] and n-6 PUFA [linoleic acid (18:2n-6) + arachidonic acid (20:4n-6)] and elevated CRP (OR for tertile 3 vs. tertile 1 of intake: 0.41; 95% CI: 0.21, 0.77; P-trend = 0.01; and OR 0.38; 95% CI: 0.21, 0.70; P-trend = 0.002, respectively). Stratification on vitamin E intakes showed that inverse associations between dietary n-3 and n-6 PUFA intakes and elevated CRP were substantial only in individuals with low intakes of vitamin E. Our results supported the contention that intakes of both n-3 and n-6 PUFAs are inversely associated with plasma CRP concentrations. Vitamin E is a potential effect modifier and should therefore be taken into account in such investigations. This trial was registered at clinicaltrials

  18. Numerical solution of the Penna model of biological aging with age-modified mutation rate

    NASA Astrophysics Data System (ADS)

    Magdoń-Maksymowicz, M. S.; Maksymowicz, A. Z.

    2009-06-01

    In this paper we present results of numerical calculation of the Penna bit-string model of biological aging, modified for the case of a -dependent mutation rate m(a) , where a is the parent’s age. The mutation rate m(a) is the probability per bit of an extra bad mutation introduced in offspring inherited genome. We assume that m(a) increases with age a . As compared with the reference case of the standard Penna model based on a constant mutation rate m , the dynamics of the population growth shows distinct changes in age distribution of the population. Here we concentrate on mortality q(a) , a fraction of items eliminated from the population when we go from age (a) to (a+1) in simulated transition from time (t) to next time (t+1) . The experimentally observed q(a) dependence essentially follows the Gompertz exponential law for a above the minimum reproduction age. Deviation from the Gompertz law is however observed for the very old items, close to the maximal age. This effect may also result from an increase in mutation rate m with age a discussed in this paper. The numerical calculations are based on analytical solution of the Penna model, presented in a series of papers by Coe [J. B. Coe, Y. Mao, and M. E. Cates, Phys. Rev. Lett. 89, 288103 (2002)]. Results of the numerical calculations are supported by the data obtained from computer simulation based on the solution by Coe

  19. Serotonin signaling mediates protein valuation and aging

    PubMed Central

    Ro, Jennifer; Pak, Gloria; Malec, Paige A; Lyu, Yang; Allison, David B; Kennedy, Robert T; Pletcher, Scott D

    2016-01-01

    Research into how protein restriction improves organismal health and lengthens lifespan has largely focused on cell-autonomous processes. In certain instances, however, nutrient effects on lifespan are independent of consumption, leading us to test the hypothesis that central, cell non-autonomous processes are important protein restriction regulators. We characterized a transient feeding preference for dietary protein after modest starvation in the fruit fly, Drosophila melanogaster, and identified tryptophan hydroxylase (Trh), serotonin receptor 2a (5HT2a), and the solute carrier 7-family amino acid transporter, JhI-21, as required for this preference through their role in establishing protein value. Disruption of any one of these genes increased lifespan up to 90% independent of food intake suggesting the perceived value of dietary protein is a critical determinant of its effect on lifespan. Evolutionarily conserved neuromodulatory systems that define neural states of nutrient demand and reward are therefore sufficient to control aging and physiology independent of food consumption. DOI: http://dx.doi.org/10.7554/eLife.16843.001 PMID:27572262

  20. Serotonin signaling mediates protein valuation and aging.

    PubMed

    Ro, Jennifer; Pak, Gloria; Malec, Paige A; Lyu, Yang; Allison, David B; Kennedy, Robert T; Pletcher, Scott D

    2016-01-01

    Research into how protein restriction improves organismal health and lengthens lifespan has largely focused on cell-autonomous processes. In certain instances, however, nutrient effects on lifespan are independent of consumption, leading us to test the hypothesis that central, cell non-autonomous processes are important protein restriction regulators. We characterized a transient feeding preference for dietary protein after modest starvation in the fruit fly, Drosophila melanogaster, and identified tryptophan hydroxylase (Trh), serotonin receptor 2a (5HT2a), and the solute carrier 7-family amino acid transporter, JhI-21, as required for this preference through their role in establishing protein value. Disruption of any one of these genes increased lifespan up to 90% independent of food intake suggesting the perceived value of dietary protein is a critical determinant of its effect on lifespan. Evolutionarily conserved neuromodulatory systems that define neural states of nutrient demand and reward are therefore sufficient to control aging and physiology independent of food consumption. PMID:27572262

  1. Effect of ageing on rheological properties of storage-stable SBS/sulfur-modified asphalts.

    PubMed

    Zhang, Feng; Yu, Jianying; Wu, Shaopeng

    2010-10-15

    Oxidative ageing as an inevitable process in practical road paving has a great effect on the properties of polymer-modified asphalts (PMAs). In this article, the effect of short-term and long-term oxidative ageing on the rheological, physical properties and the morphology of the styrene-butadiene-styrene (SBS)- and storage-stable SBS/sulfur-modified asphalts was studied, respectively. The analysis on the rheological and physical properties of the PMAs before and after ageing showed the two major effects of ageing. On one hand, ageing prompted the degradation of polymer and increased the viscous behaviour of the modified binders, on the other, ageing changed the asphalt compositions and improved the elastic behaviour of the modified binders. The final performance of the aged binders depended on the combined effect. After ageing, the storage-stable SBS/sulfur-modified asphalts showed an obvious viscous behaviour compare with the SBS-modified asphalts and this led to an improved low-temperature creep property. The rutting resistance of the SBS-modified asphalts declined by the addition of sulfur due to the structural instability of the SBS/sulfur-modified asphalts. The rheological properties of the modified binders before and after ageing also depended strongly on the structural characteristics of SBS. The observation by using optical microscopy showed the compatibility between asphalt and SBS was improved with further ageing, especially for the storage-stable SBS/sulfur-modified asphalts.

  2. Effect of ageing on rheological properties of storage-stable SBS/sulfur-modified asphalts.

    PubMed

    Zhang, Feng; Yu, Jianying; Wu, Shaopeng

    2010-10-15

    Oxidative ageing as an inevitable process in practical road paving has a great effect on the properties of polymer-modified asphalts (PMAs). In this article, the effect of short-term and long-term oxidative ageing on the rheological, physical properties and the morphology of the styrene-butadiene-styrene (SBS)- and storage-stable SBS/sulfur-modified asphalts was studied, respectively. The analysis on the rheological and physical properties of the PMAs before and after ageing showed the two major effects of ageing. On one hand, ageing prompted the degradation of polymer and increased the viscous behaviour of the modified binders, on the other, ageing changed the asphalt compositions and improved the elastic behaviour of the modified binders. The final performance of the aged binders depended on the combined effect. After ageing, the storage-stable SBS/sulfur-modified asphalts showed an obvious viscous behaviour compare with the SBS-modified asphalts and this led to an improved low-temperature creep property. The rutting resistance of the SBS-modified asphalts declined by the addition of sulfur due to the structural instability of the SBS/sulfur-modified asphalts. The rheological properties of the modified binders before and after ageing also depended strongly on the structural characteristics of SBS. The observation by using optical microscopy showed the compatibility between asphalt and SBS was improved with further ageing, especially for the storage-stable SBS/sulfur-modified asphalts. PMID:20637542

  3. Prion Protein Accumulation in Lipid Rafts of Mouse Aging Brain

    PubMed Central

    Agostini, Federica; Dotti, Carlos G.; Pérez-Cañamás, Azucena; Ledesma, Maria Dolores; Benetti, Federico; Legname, Giuseppe

    2013-01-01

    The cellular form of the prion protein (PrPC) is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrPC. In old mice, this change favors PrPC accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrPC translocation into detergent-resistant membranes (DRMs), we looked at PrPC compartmentalization in hippocampi from acid sphingomyelinase (ASM) knockout (KO) mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrPC in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases. PMID:24040215

  4. Quantification of Poly(ADP-ribose)-Modified Proteins in Cerebrospinal Fluid from Infants and Children after Traumatic Brain Injury

    PubMed Central

    Fink, Ericka L.; Lai, Yichen; Zhang, Xiaopeng; Janesko-Feldman, Keri; Adelson, P. David; Szabó, Csaba; Berger, Rachel P.; Sarnaik, Ajit A.; Kochanek, Patrick M.; Clark, Robert S. B.

    2008-01-01

    Poly-ADP-ribosylation (PAR) of proteins by poly(ADP-ribose) polymerases (PARP) occurs after experimental traumatic brain injury (TBI) and modulates neurological outcome. Several promising pharmacological PARP inhibitors have been developed for use in humans, but there is currently no clinically relevant means of monitoring treatment effects. We therefore utilized an enzyme-linked immunosorbent assay (ELISA) to measure PAR-modified proteins in cerebrospinal fluid (CSF). CSF samples from 17 pediatric TBI and 15 control patients were plated overnight then incubated with polyclonal antibody against PAR. Histone-1, a PARP substrate, was incubated with active PARP, NAD, and nicked DNA, and served as the standard. Both peak and mean CSF PAR-modified protein were increased in TBI patients versus controls. Peak CSF PAR-modified protein levels occurred on day 1 and levels remained increased on day 2 after TBI. Increases in peak CSF PAR-modified protein concentrations were independently associated with age and male sex, but not initial Glasgow coma scale score, Glasgow outcome score, or mechanism of injury. The increase in PAR-modified proteins in CSF after TBI may be due to increased PARP activation, decreased PAR degradation, or both. Since PAR-modified protein concentration correlated with age and male sex, developmental and sex-dependent roles for PARP after TBI are implicated. PMID:18506195

  5. Identification of Proteins that Modify Cataract of the Eye Lens

    PubMed Central

    Hoehenwarter, Wolfgang; Tang, Yajun; Ackermann, Renate; Pleissner, Klaus-Peter; Schmid, Monika; Stein, Robert; Zimny-Arndt, Ursula; Kumar, Nalin M.; Jungblut, Peter R.

    2010-01-01

    The occurrence of a nuclear cataract in the eye lens due to disruption of theα3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and post-translational modifications occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29 and syntaxin binding protein 6 in the eye lens. DNA polymorphisms resulting in non-conservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1 and possibly gamma N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat shock proteins have a major role for influencing cataract formation in humans. PMID:19003866

  6. Preparation of Modified Films with Protein from Grouper Fish

    PubMed Central

    Tecante, A.; Granados-Navarrete, S.; Martínez-García, C.

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials. PMID:27597950

  7. Preparation of Modified Films with Protein from Grouper Fish

    PubMed Central

    Tecante, A.; Granados-Navarrete, S.; Martínez-García, C.

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials.

  8. Preparation of Modified Films with Protein from Grouper Fish.

    PubMed

    Valdivia-López, M A; Tecante, A; Granados-Navarrete, S; Martínez-García, C

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials.

  9. Preparation of Modified Films with Protein from Grouper Fish.

    PubMed

    Valdivia-López, M A; Tecante, A; Granados-Navarrete, S; Martínez-García, C

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials. PMID:27597950

  10. Synthesis, modification and turnover of proteins during aging.

    PubMed

    Rattan, Suresh I S

    2010-01-01

    Iterations in the rate and extent of protein synthesis, accuracy, post-translational modifications and turnover are among the main molecular characteristics of aging. A decline in the cellular capacity through proteasomal and lysosomal pathways to recognize and preferentially degrade damaged proteins leads to the accumulation of abnormal proteins during aging. The consequent increase in molecular heterogeneity and impaired functioning of proteins is the basis of several age-related pathologies, such as cataracts, sarcopenia and neurodegerative diseases. Understanding the proteomic spectrum and its functional implications during aging can facilitate developing effective means of intervention, prevention and therapy of aging and age-related diseases.

  11. Interaction of receptor-activity-modifying protein1 with tubulin.

    PubMed

    Kunz, Thomas H; Mueller-Steiner, Sarah; Schwerdtfeger, Kerstin; Kleinert, Peter; Troxler, Heinz; Kelm, Jens M; Ittner, Lars M; Fischer, Jan A; Born, Walter

    2007-08-01

    Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors. PMID:17493758

  12. SERF protein is a direct modifier of amyloid fiber assembly.

    PubMed

    Falsone, S Fabio; Meyer, N Helge; Schrank, Evelyne; Leitinger, Gerd; Pham, Chi L L; Fodero-Tavoletti, Michelle T; Holmberg, Mats; Dulle, Martin; Scicluna, Benjamin; Gesslbauer, Bernd; Rückert, Hanna-Marie; Wagner, Gabriel E; Merle, David A; Nollen, Ellen A; Kungl, Andreas J; Hill, Andrew F; Cappai, Roberto; Zangger, Klaus

    2012-08-30

    The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway.

  13. SERF Protein Is a Direct Modifier of Amyloid Fiber Assembly

    PubMed Central

    Falsone, S. Fabio; Meyer, N. Helge; Schrank, Evelyne; Leitinger, Gerd; Pham, Chi L.L.; Fodero-Tavoletti, Michelle T.; Holmberg, Mats; Dulle, Martin; Scicluna, Benjamin; Gesslbauer, Bernd; Rückert, Hanna-Marie; Wagner, Gabriel E.; Merle, David A.; Nollen, Ellen A.; Kungl, Andreas J.; Hill, Andrew F.; Cappai, Roberto; Zangger, Klaus

    2012-01-01

    Summary The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway. PMID:22854022

  14. Does the Aging Process Significantly Modify the Mean Heart Rate?

    PubMed Central

    Santos, Marcos Antonio Almeida; Sousa, Antonio Carlos Sobral; Reis, Francisco Prado; Santos, Thayná Ramos; Lima, Sonia Oliveira; Barreto-Filho, José Augusto

    2013-01-01

    Background The Mean Heart Rate (MHR) tends to decrease with age. When adjusted for gender and diseases, the magnitude of this effect is unclear. Objective To analyze the MHR in a stratified sample of active and functionally independent individuals. Methods A total of 1,172 patients aged ≥ 40 years underwent Holter monitoring and were stratified by age group: 1 = 40-49, 2 = 50-59, 3 = 60-69, 4 = 70-79, 5 = ≥ 80 years. The MHR was evaluated according to age and gender, adjusted for Hypertension (SAH), dyslipidemia and non-insulin dependent diabetes mellitus (NIDDM). Several models of ANOVA, correlation and linear regression were employed. A two-tailed p value <0.05 was considered significant (95% CI). Results The MHR tended to decrease with the age range: 1 = 77.20 ± 7.10; 2 = 76.66 ± 7.07; 3 = 74.02 ± 7.46; 4 = 72.93 ± 7.35; 5 = 73.41 ± 7.98 (p < 0.001). Women showed a correlation with higher MHR (p <0.001). In the ANOVA and regression models, age and gender were predictors (p < 0.001). However, R2 and ETA2 < 0.10, as well as discrete standardized beta coefficients indicated reduced effect. Dyslipidemia, hypertension and DM did not influence the findings. Conclusion The MHR decreased with age. Women had higher values of MHR, regardless of the age group. Correlations between MHR and age or gender, albeit significant, showed the effect magnitude had little statistical relevance. The prevalence of SAH, dyslipidemia and diabetes mellitus did not influence the results. PMID:24029962

  15. [Protein assay by the modified Dumas method applied to preparations of plasma proteins].

    PubMed

    Blondel, P; Vian, L

    1993-01-01

    Quantify protein according Pharmacopoeia method, based on Kjeldahl method, needs a long time to do. The development of an automaton which used the modified Dumas method divide the analysis time by 15 (6 minutes versus over 90 minutes). The results show no statistical differences between official method and this one. PMID:8154798

  16. Justification of sexual reproduction by modified Penna model of ageing

    NASA Astrophysics Data System (ADS)

    Sá Martins, J. S.; Stauffer, D.

    2001-05-01

    We generalize the standard Penna bit-string model of biological ageing by assuming that each deleterious mutation diminishes the survival probability in every time interval by a small percentage. This effect is added to the usual lethal but age-dependent effect of the same mutation. We then find strong advantages or disadvantages of sexual reproduction (with males and females) compared to asexual cloning, depending on parameters.

  17. Why and How We Age, and Is That Process Modifiable?

    NASA Astrophysics Data System (ADS)

    Arking, R.

    Aging is an almost-universal biological process that is better understood in terms of an evolutionary explanation than in terms of a medical or adaptationist explanation. The major advances in human longevity which took place in developed countries during the past century arose from decreases in external (e.g., environmental) sources of mortality, and not from any effect on the aging process. Laboratory studies show that the aging process is under genetic control, can be manipulated, and can be expressed in three different phenotypes. The adult lifespan consists of the health span (ages 20-55 yrs) and the senescent span (ages 55+), with a relatively short but variable transition phase between the two. The most socially desirable phenotype would be that where the transition phase is delayed and the health span extended with little effect on the senescent span. The genetic, nutritional, cell-signaling and pharmecutical interventions inducing this phenotype are discussed. The genetic architecture of senescence is discussed and its stochastic nature made clear. The social and ethical consequences of pharmecutical intervention into the aging process are briefly discussed.

  18. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity.

    PubMed

    Brehme, Marc; Voisine, Cindy

    2016-08-01

    Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  19. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    PubMed Central

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  20. Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers1

    PubMed Central

    Kumar, G.N. Mohan; Houtz, Robert L.; Knowles, N. Richard

    1999-01-01

    Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers. PMID:9880350

  1. Age-induced protein modifications and increased proteolysis in potato seed-tubers

    SciTech Connect

    Kumar, G.N.M.; Knowles, N.R.; Houtz, R.L.

    1999-01-01

    Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as in increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed daughter tubers that contained 3-fold more protein than mother tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Their results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; the authors also identify a role for PMC in regulating protein turnover in potato tubers.

  2. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

    2016-01-01

    Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

  3. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

    PubMed Central

    Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

    2016-01-01

    Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis. PMID:26776680

  4. An attempt to age mallards using eye lens proteins

    USGS Publications Warehouse

    Henny, C.J.; Ludke, J.L.

    1974-01-01

    An analysis of insoluble protein content of eye lenses from 59 known-age mallards (Anas platyrhynchos) indicated a slight increase between 8-9 months and 7 years of age. Nearly a complete overlapping of the insoluble protein content of individuals of different ages was apparent showing that the technique cannot be used to separate adult year classes of mallards. These results are contrary to findings reported for selected mammalian species; a possible explanation for the dissimilarity is discussed.

  5. Protein Oxidation in Aging: Does It Play a Role in Aging Progression?

    PubMed Central

    Reeg, Sandra

    2015-01-01

    Abstract Significance: A constant accumulation of oxidized proteins takes place during aging. Oxidation of proteins leads to a partial unfolding and, therefore, to aggregation. Protein aggregates impair the activity of cellular proteolytic systems (proteasomes, lysosomes), resulting in further accumulation of oxidized proteins. In addition, the accumulation of highly crosslinked protein aggregates leads to further oxidant formation, damage to macromolecules, and, finally, to apoptotic cell death. Furthermore, protein oxidation seems to play a role in the development of various age-related diseases, for example, neurodegenerative diseases. Recent Advances: The highly oxidized lipofuscin accumulates during aging. Lipofuscin formation might cause impaired lysosomal and proteasomal degradation, metal ion accumulation, increased reactive oxygen species formation, and apoptosis. Critical Issues: It is still unclear to which extent protein oxidation is involved in the progression of aging and in the development of some age-related diseases. Future Directions: An extensive knowledge of the effects of protein oxidation on the aging process and its contribution to the development of age-related diseases could enable further strategies to reduce age-related impairments. Strategies aimed at lowering aggregate formation might be a straightforward intervention to reduce age-related malfunctions of organs. Antioxid. Redox Signal. 23, 239–255. PMID:25178482

  6. Oxidative modification of proteins: age-related changes.

    PubMed

    Chakravarti, Bulbul; Chakravarti, Deb N

    2007-01-01

    Aging is a complex biological phenomenon which involves progressive loss of different physiological functions of various tissues of living organisms. It is the inevitable fate of life and is a major risk factor for death and different pathological disorders. Based on a wide variety of studies performed in humans as well as in various animal models and microbial systems, reactive oxygen species (ROS) are believed to play a key role in the aging process. The production of ROS is influenced by cellular metabolic activities as well as environmental factors. ROS can react with all major biological macromolecules such as carbohydrates, nucleic acids, lipids, and proteins. Since, in general, proteins are the key molecules that play the ultimate role in various structural and functional aspects of living organisms, this review will focus on the age-related oxidative modifications of proteins as well as on mechanism for removal or repair of the oxidized proteins. The topics covered include protein oxidation as a marker of oxidative stress, experimental evidence indicating the role of ROS in protein oxidation, protein carbonyl content, enzymatic degradation of oxidized proteins, and effects of caloric restriction on protein oxidation in the context of aging. Finally, we will discuss different strategies which have been or can be undertaken to slow down the oxidative damage of proteins and the aging process.

  7. Immunization of AGE-modified albumin inhibits diabetic nephropathy progression in diabetic mice

    PubMed Central

    Mashitah, Musthika Wida; Azizah, Nurona; Samsu, Nur; Indra, Muhammad Rasjad; Bilal, Muhammad; Yunisa, Meti Verdian; Arisanti, Amildya Dwi

    2015-01-01

    Background Diabetic nephropathy (DN) is a serious vascular complication of diabetes and an important cause of end-stage renal disease. One mechanism by which hyperglycemia causes nephropathy is through the formation of advanced glycation end products (AGE). Development of vaccination would be a promising therapy for the future, while to date, anti-AGE therapy is based on medicines that are needed to be consumed lifelong. This study aimed to find out the effect of immunization of AGE-modified albumin against DN pathogenesis in streptozotocin-induced diabetic in mice. Methods We used 24 BALB/c male mice as experimental animals, which were divided into six groups, two nondiabetic groups (negative control and AGE-modified bovine serum albumin [BSA] preimmunized groups) and four streptozotocin-induced diabetic groups (diabetic control group and diabetic preimmunized groups for AGE-BSA, Keyhole limpet hemocyanin (KLH), and AGE-BSA-KLH, respectively). Results Diabetic preimmunized groups for AGE-BSA, KLH, and AGE-BSA-KLH showed amelioration in renal function and histopathology compared with the diabetic control group. Preimmunization also maintained nephrin intensity and decreased serum AGE level, kidney AGE deposition, and kidney cells apoptosis. Conclusion AGE-BSA and AGE-BSA-KLH immunizations inhibit the progression of DN. Our results strengthen the evidence that the anti-AGE antibodies have a protective role against diabetic vascular complication, especially DN. This study provides a basis for the development of DN-based immunotherapy with AGE immunization as a potential candidate. PMID:26346342

  8. Young age as a modifying factor in sports concussion management: what is the evidence?

    PubMed

    Foley, Cassidy; Gregory, Andrew; Solomon, Gary

    2014-01-01

    In 2008, the Concussion in Sport Group (CISG) published its third consensus statement and introduced 10 'modifying' factors that were presumed clinically to influence the investigation and management of concussions in sports. Young age was listed as one of the modifying factors. In some cases, these modifiers were thought to be predictive of prolonged or persistent symptoms. These same modifying factors were retained in the fourth iteration of the CISG consensus statement (2013), although mention was made of possible limitations of their efficacy. The CISG statements provided several empirical references regarding young age as a modifying factor. We reviewed the published sports concussion literature with the purpose of determining empirical studies that support or refute the inclusion of young age as a modifier of concussive injury in sports. We performed a systematic review of the PubMed database utilizing the keywords concussion, sports, mild traumatic brain injury, youth, adolescents, and children. English language studies were extracted by the authors and summarized for review. Multiple empirical studies were found indicating that younger athletes may take longer to recover from a sports-related concussion (SRC) than their older peers. However, studies did not indicate that younger athletes were at more risk for prolonged recovery (>4 wk). Empirical evidence supports the inclusion of young age as a modifying factor in sports concussion. However, the difference in recovery time seems relatively small (a few days) and young age does not predict prolonged recovery (>4 wk). The findings support the inclusion of young age as a specific modifier in the treatment of SRC and have implications for the clinical management of this common injury.

  9. Anorexia of aging: a modifiable risk factor for frailty.

    PubMed

    Martone, Anna Maria; Onder, Graziano; Vetrano, Davide Liborio; Ortolani, Elena; Tosato, Matteo; Marzetti, Emanuele; Landi, Francesco

    2013-10-14

    Anorexia of aging, defined as a loss of appetite and/or reduced food intake, affects a significant number of elderly people and is far more prevalent among frail individuals. Anorexia recognizes a multifactorial origin characterized by various combinations of medical, environmental and social factors. Given the interconnection between weight loss, sarcopenia and frailty, anorexia is a powerful, independent predictor of poor quality of life, morbidity and mortality in older persons. One of the most important goals in the management of older, frail people is to optimize their nutritional status. To achieve this objective it is important to identify subjects at risk of anorexia and to provide multi-stimulus interventions that ensure an adequate amount of food to limit and/or reverse weight loss and functional decline. Here, we provide a brief overview on the relevance of anorexia in the context of sarcopenia and frailty. Major pathways supposedly involved in the pathogenesis of anorexia are also illustrated. Finally, the importance of treating anorexia to achieve health benefits in frail elders is highlighted.

  10. Antifreeze proteins modify the freezing process in planta.

    PubMed

    Griffith, Marilyn; Lumb, Chelsey; Wiseman, Steven B; Wisniewski, Michael; Johnson, Robert W; Marangoni, Alejandro G

    2005-05-01

    During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 degrees C to 1.2 degrees C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.

  11. Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders.

    PubMed

    Poon, H Fai; Farr, Susan A; Thongboonkerd, Visith; Lynn, Bert C; Banks, William A; Morley, John E; Klein, Jon B; Butterfield, D Allan

    2005-01-01

    Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial

  12. Proteins modified by the lipid peroxidation aldehyde DODE in MCF7 breast cancer cells

    PubMed Central

    Slade, Peter G.; Williams, Michelle V.; Brahmbatt, Viral; Dash, Ajit; Wishnok, John S.; Tannenbaum, Steven R.

    2010-01-01

    The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE) which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were use to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by 2D-LC-MS/MS. Second, cells were treated with native 13-HPODE. Protein-carbonyls were biotinylated with an aldehyde reactive probe (ARP) and modified proteins enriched by neutravidin affinity and identified by 2D-LC-MS/MS. Third, using a newly developed DODE antibody, DODE modified proteins were located by 2D-SDS-PAGE and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress. PMID:20131800

  13. Evaluation of Thermal Oxidative Aging Effect on the Rheological Performance of Modified Asphalt Binders

    NASA Astrophysics Data System (ADS)

    Zhu, Cheng

    Modified asphalt binder, which is combined by base binder and additive modifier, has been implemented in pavement industry for more than 30 years. Recently, the oxidative aging mechanism of asphalt binder has been studied for several decades, and appreciable finding results of asphalt binder aging mechanism were achieved from the chemistry and rheological performance aspects. However, most of these studies were conducted with neat binders, the research of aging mechanism of modified asphalt binder was limited. Nowadays, it is still highly necessary to clarify how the asphalt binder aging happens with the modified asphalt binder, what is the effect of the different modifiers (additives) on the binder aging process, how the rheological performance changes under the thermal oxidative aging conditions and so on. The objective of this study was to investigate the effect of isothermal oxidative aging conditions on the rheological performance change of the modified and controlled asphalt binders. There were totally 14 different sorts of asphalt binders had been aged in the PAV pans in the air-force drafted ovens at 50°C, 60°C and 85°C for 0.5 day to 240 days. The Fourier-Transform Infrared Spectroscopy (FT-IR) and Dynamic Shear Rheometer (DSR) were used to perform the experiments. The analysis of rheological indices (Low shear viscosity-LSV, Crossover modulus-G*c, Glover-Rowe Parameter-G-R, DSR function-DSR Fn) as a function of carbonyl area (CA) was conducted. With the SBS modification, both of the hardening susceptibility of the rheological index-LSV and G-R decreases compared with the corresponding base binder. The TR increased the hardening susceptibility of all the rheological indexes. While for the G*c, SBS increases the slope of the most modified asphalt binders except A and B_TR_X series binders. The multiple linear regression statistical analysis results indicate that the oxidative aging conditions play an important role on the CA, and rheological performance

  14. (19)F-modified proteins and (19)F-containing ligands as tools in solution NMR studies of protein interactions.

    PubMed

    Sharaf, Naima G; Gronenborn, Angela M

    2015-01-01

    (19)F solution NMR is a powerful and versatile tool to study protein structure and protein-ligand interactions due to the favorable NMR characteristics of the (19)F atom, its absence in naturally occurring biomolecules, and small size. Protocols to introduce (19)F atoms into both proteins and their ligands are readily available and offer the ability to conduct protein-observe (using (19)F-labeled proteins) or ligand-observe (using (19)F-containing ligands) NMR experiments. This chapter provides two protocols for the (19)F-labeling of proteins, using an Escherichia coli expression system: (i) amino acid type-specific incorporation of (19)F-modified amino acids and (ii) site-specific incorporation of (19)F-modified amino acids using recombinantly expressed orthogonal amber tRNA/tRNA synthetase pairs. In addition, we discuss several applications, involving (19)F-modified proteins and (19)F-containing ligands.

  15. Age-related carbonyl stress and erythrocyte membrane protein carbonylation.

    PubMed

    Li, Guolin; Liu, Li; Hu, Hui; Zhao, Qiong; Xie, Fuxia; Chen, Keke; Liu, Shenglin; Chen, Yaqin; Shi, Wang; Yin, Dazhong

    2010-01-01

    Reactive carbonyl species (RCS) have been widely used as indicators of oxidative stress. However, the associations of carbonyl stress with aging process and biochemical alteration of erythrocyte are still poorly understood. Fresh blood samples in vacutainer tubes containing sodium heparinate were obtained from 874 volunteers who were divided into young, adult and old groups based on their age. Plasma RCS and thiols concentrations between different age groups and erythrocyte membrane protein carbonylation in the adult group were detected within 24h of the blood sampling. Results showed that the plasma thiols concentration decreased gradually during aging process, and the p-values between all three groups are less than 0.05. The plasma RCS concentration in different age groups showed a nonlinear association with age. The levels in the young group were slightly higher than the adult group (not significant) and lower than the old group (p < 0.01). The protein carbonylation of erythrocyte membrane was positively correlated with plasma RCS concentration (p < 0.01), but not plasma thiols concentration. We conclude that higher levels of RCS, not lower levels of thiols, in plasma are a direct risk factor for the protein carbonylation of erythrocyte membrane. Owing to the decrease of thiols levels and increase of RCS levels during aging process, a shift from RCS-related redox allostasis to carbonyl stress would contribute to age-related biological dysfunction and even aging process.

  16. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

    PubMed Central

    van der Goot, Annemieke T.; Zhu, Wentao; Vázquez-Manrique, Rafael P.; Seinstra, Renée I.; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C.; Ruiz Silva, Mariana; Thijssen, Karen L.; Kema, Ido P.; Neri, Christian; Oefner, Peter J.; Nollen, Ellen A. A.

    2012-01-01

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases. PMID:22927396

  17. ShcC proteins: brain aging and beyond.

    PubMed

    Sagi, Orli; Budovsky, Arie; Wolfson, Marina; Fraifeld, Vadim E

    2015-01-01

    To date, most studies of Shc family of signaling adaptor proteins have been focused on the near-ubiquitously expressed ShcA, indicating its relevance to age-related diseases and longevity. Although the role of the neuronal ShcC protein is much less investigated, accumulated evidence suggests its importance for neuroprotection against such aging-associated conditions as brain ischemia and oxidative stress. Here, we summarize more than decade of studies on the ShcC expression and function in normal brain, age-related brain pathologies and immune disorders with a focus on the interactions of ShcC with signaling proteins/pathways, and the possible implications of these interactions for changes associated with aging.

  18. Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans.

    PubMed

    Mathesius, C A; Barnett, J F; Cressman, R F; Ding, J; Carpenter, C; Ladics, G S; Schmidt, J; Layton, R J; Zhang, J X Q; Appenzeller, L M; Carlson, G; Ballou, S; Delaney, B

    2009-12-01

    Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (<30s) and intestinal fluid (<1min). The enzymatic activity was completely inactivated at 50 degrees C for 15 min demonstrating heat lability. The protein expressed in planta is not glycosylated and genetically modified soybeans expressing the GM-HRA protein produced similar protein/allergen profiles as its non-transgenic parental isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology.

  19. Aging Impairs Myocardial Fatty Acid and Ketone Oxidation and Modifies Cardiac Functional and Metabolic Responses to Insulin in Mice

    SciTech Connect

    Hyyti, Outi M.; Ledee, Dolena; Ning, Xue-Han; Ge, Ming; Portman, Michael A.

    2010-07-02

    Aging presumably initiates shifts in substrate oxidation mediated in part by changes in insulin sensitivity. Similar shifts occur with cardiac hypertrophy and may contribute to contractile dysfunction. We tested the hypothesis that aging modifies substrate utilization and alters insulin sensitivity in mouse heart when provided multiple substrates. In vivo cardiac function was measured with microtipped pressure transducers in the left ventricle from control (4–6 mo) and aged (22–24 mo) mice. Cardiac function was also measured in isolated working hearts along with substrate and anaplerotic fractional contributions to the citric acid cycle (CAC) by using perfusate containing 13C-labeled free fatty acids (FFA), acetoacetate, lactate, and unlabeled glucose. Stroke volume and cardiac output were diminished in aged mice in vivo, but pressure development was preserved. Systolic and diastolic functions were maintained in aged isolated hearts. Insulin prompted an increase in systolic function in aged hearts, resulting in an increase in cardiac efficiency. FFA and ketone flux were present but were markedly impaired in aged hearts. These changes in myocardial substrate utilization corresponded to alterations in circulating lipids, thyroid hormone, and reductions in protein expression for peroxisome proliferator-activated receptor (PPAR)α and pyruvate dehydrogenase kinase (PDK)4. Insulin further suppressed FFA oxidation in the aged. Insulin stimulation of anaplerosis in control hearts was absent in the aged. The aged heart shows metabolic plasticity by accessing multiple substrates to maintain function. However, fatty acid oxidation capacity is limited. Impaired insulin-stimulated anaplerosis may contribute to elevated cardiac efficiency, but may also limit response to acute stress through depletion of CAC intermediates.

  20. Investigation of aging processes of graphite tubes modified with iridium and rhodium used for atomic spectrometry

    NASA Astrophysics Data System (ADS)

    Bulska, Ewa; Piaścik, Marek; Katskov, Dmitri; Darangwa, Nicholas; Grotti, Marco

    2007-11-01

    UV spectrometry (187-380 nm) with charge coupled device (CCD) detection was used to study the evolution of absorption spectra during the vaporization of various species in the pyrocoated graphite furnace, with electrodeposited Ir and Rh as modifiers. In order to mimic a typical matrix composition, various salts of aluminum, manganese, copper, magnesium, sodium, and lead were used in microgram amounts. Changes in spectra and vapor release rate, along with aging of the tubes in the repetitive temperature cycles, were observed. Compared to the unmodified pyrocoated tubes, the presence of Ir or Rh causes a significant reduction in the vaporization efficiency, especially for microgram amounts of copper and aluminum introduced as nitrates, and manganese introduced as a sulfate. The vaporization efficiency, for magnesium and sodium as chlorides, and for lead as a sulfate, remained unchanged. Interestingly, the aging of the tubes was accompanied by partial restoration of the spectral characteristics for unmodified tubes. For example, with unmodified pyrocoated tubes, the vaporization spectrum, appearing as a consequence of the decomposition of aluminum nitrate, consisted of Al2O bands overlapped by Al atomic lines. In the freshly modified tubes, intensities of those lines and bands were substantially reduced, and in this case, the dominance of AlO molecules was observed. The efficiency of vaporization of aluminum species increased in the aged modified tubes. The scanning electron microscopy (SEM) images of the modified surfaces for the new and aged tubes indicated that aging of the tubes is accompanied by the destruction of the pyrocoating, formation of pyrographite shells around the areas where the modifier was electrodeposited, and finally, complete substitution of the metal on the graphite surface by pyrographite debris.

  1. Preparation and ageing-resistant properties of polyester composites modified with functional nanoscale additives.

    PubMed

    Guo, Gang; Shi, Qiwu; Luo, Yanbing; Fan, Rangrang; Zhou, Liangxue; Qian, Zhiyong; Yu, Jie

    2014-01-01

    This study investigated ageing-resistant properties of carboxyl-terminated polyester (polyethylene glycol terephthalate) composites modified with nanoscale titanium dioxide particles (nano-TiO2). The nano-TiO2 was pretreated by a dry coating method, with aluminate coupling agent as a functional grafting additive. The agglomeration resistance was evaluated, which exhibited significant improvement for the modified nanoparticles. Then, the effects of the modified nano-TiO2 on the crosslinking and ageing-resistant properties of the composites were studied. With a real-time Fourier transform infrared (FT-IR) measurement, the nano-TiO2 displayed promoting effect on the crosslinking of polyester resin with triglycidyl isocyanurate (TGIC) as crosslinking agent. Moreover, the gloss retention, colour aberration and the surface morphologies of the composites during accelerated UV ageing (1500 hours) were investigated. The results demonstrated much less degree of ageing degradation for the nanocomposites, indicating an important role of the nano-TiO2 in improving the ageing-resistant properties of synthetic polymer composites. PMID:24872802

  2. Preparation and ageing-resistant properties of polyester composites modified with functional nanoscale additives.

    PubMed

    Guo, Gang; Shi, Qiwu; Luo, Yanbing; Fan, Rangrang; Zhou, Liangxue; Qian, Zhiyong; Yu, Jie

    2014-01-01

    This study investigated ageing-resistant properties of carboxyl-terminated polyester (polyethylene glycol terephthalate) composites modified with nanoscale titanium dioxide particles (nano-TiO2). The nano-TiO2 was pretreated by a dry coating method, with aluminate coupling agent as a functional grafting additive. The agglomeration resistance was evaluated, which exhibited significant improvement for the modified nanoparticles. Then, the effects of the modified nano-TiO2 on the crosslinking and ageing-resistant properties of the composites were studied. With a real-time Fourier transform infrared (FT-IR) measurement, the nano-TiO2 displayed promoting effect on the crosslinking of polyester resin with triglycidyl isocyanurate (TGIC) as crosslinking agent. Moreover, the gloss retention, colour aberration and the surface morphologies of the composites during accelerated UV ageing (1500 hours) were investigated. The results demonstrated much less degree of ageing degradation for the nanocomposites, indicating an important role of the nano-TiO2 in improving the ageing-resistant properties of synthetic polymer composites.

  3. Preparation and ageing-resistant properties of polyester composites modified with functional nanoscale additives

    NASA Astrophysics Data System (ADS)

    Guo, Gang; Shi, Qiwu; Luo, Yanbing; Fan, Rangrang; Zhou, Liangxue; Qian, Zhiyong; Yu, Jie

    2014-05-01

    This study investigated ageing-resistant properties of carboxyl-terminated polyester (polyethylene glycol terephthalate) composites modified with nanoscale titanium dioxide particles (nano-TiO2). The nano-TiO2 was pretreated by a dry coating method, with aluminate coupling agent as a functional grafting additive. The agglomeration resistance was evaluated, which exhibited significant improvement for the modified nanoparticles. Then, the effects of the modified nano-TiO2 on the crosslinking and ageing-resistant properties of the composites were studied. With a real-time Fourier transform infrared (FT-IR) measurement, the nano-TiO2 displayed promoting effect on the crosslinking of polyester resin with triglycidyl isocyanurate (TGIC) as crosslinking agent. Moreover, the gloss retention, colour aberration and the surface morphologies of the composites during accelerated UV ageing (1500 hours) were investigated. The results demonstrated much less degree of ageing degradation for the nanocomposites, indicating an important role of the nano-TiO2 in improving the ageing-resistant properties of synthetic polymer composites.

  4. Preparation and ageing-resistant properties of polyester composites modified with functional nanoscale additives

    PubMed Central

    2014-01-01

    This study investigated ageing-resistant properties of carboxyl-terminated polyester (polyethylene glycol terephthalate) composites modified with nanoscale titanium dioxide particles (nano-TiO2). The nano-TiO2 was pretreated by a dry coating method, with aluminate coupling agent as a functional grafting additive. The agglomeration resistance was evaluated, which exhibited significant improvement for the modified nanoparticles. Then, the effects of the modified nano-TiO2 on the crosslinking and ageing-resistant properties of the composites were studied. With a real-time Fourier transform infrared (FT-IR) measurement, the nano-TiO2 displayed promoting effect on the crosslinking of polyester resin with triglycidyl isocyanurate (TGIC) as crosslinking agent. Moreover, the gloss retention, colour aberration and the surface morphologies of the composites during accelerated UV ageing (1500 hours) were investigated. The results demonstrated much less degree of ageing degradation for the nanocomposites, indicating an important role of the nano-TiO2 in improving the ageing-resistant properties of synthetic polymer composites. PMID:24872802

  5. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

    NASA Astrophysics Data System (ADS)

    Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

    2016-05-01

    Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

  6. Polycomb group proteins in hematopoietic stem cell aging and malignancies.

    PubMed

    Klauke, Karin; de Haan, Gerald

    2011-07-01

    Protection of the transcriptional "stemness" network is important to maintain a healthy hematopoietic stem cells (HSCs) compartment during the lifetime of the organism. Recent evidence shows that fundamental changes in the epigenetic status of HSCs might be one of the driving forces behind many age-related HSC changes and might pave the way for HSC malignant transformation and subsequent leukemia development, the incidence of which increases exponentially with age. Polycomb group (PcG) proteins are key epigenetic regulators of HSC cellular fate decisions and are often found to be misregulated in human hematopoietic malignancies. In this review, we speculate that PcG proteins balance HSC aging against the risk of developing cancer, since a disturbance in PcG genes and proteins affects several important cellular processes such as cell fate decisions, senescence, apoptosis, and DNA damage repair.

  7. New Horizons: Dietary protein, ageing and the Okinawan ratio.

    PubMed

    Le Couteur, David G; Solon-Biet, Samantha; Wahl, Devin; Cogger, Victoria C; Willcox, Bradley J; Willcox, D Craig; Raubenheimer, David; Simpson, Stephen J

    2016-07-01

    Nutrition has profound effects on ageing and lifespan. Caloric restriction is the major nutritional intervention that historically has been shown to influence lifespan and/or healthspan in many animal models. Studies have suggested that a reduction in protein intake can also increase lifespan, albeit not as dramatically as caloric restriction. More recent research based on nutritional geometry has attempted to define the effects of nutrition on ageing over a broad landscape of dietary macronutrients and energy content. Such studies in insects and mice indicate that animals with ad libitum access to low-protein, high-carbohydrate diets have longest lifespans. Remarkably, the optimum content and ratio of dietary protein to carbohydrates for ageing in experimental animals are almost identical to those in the traditional diets of the long-lived people on the island of Okinawa. PMID:27130207

  8. Ethanol Metabolism Modifies Hepatic Protein Acylation in Mice

    PubMed Central

    Fritz, Kristofer S.; Green, Michelle F.; Petersen, Dennis R.; Hirschey, Matthew D.

    2013-01-01

    Mitochondrial protein acetylation increases in response to chronic ethanol ingestion in mice, and is thought to reduce mitochondrial function and contribute to the pathogenesis of alcoholic liver disease. The mitochondrial deacetylase SIRT3 regulates the acetylation status of several mitochondrial proteins, including those involved in ethanol metabolism. The newly discovered desuccinylase activity of the mitochondrial sirtuin SIRT5 suggests that protein succinylation could be an important post-translational modification regulating mitochondrial metabolism. To assess the possible role of protein succinylation in ethanol metabolism, we surveyed hepatic sub-cellular protein fractions from mice fed a control or ethanol-supplemented diet for succinyl-lysine, as well as acetyl-, propionyl-, and butyryl-lysine post-translational modifications. We found mitochondrial protein propionylation increases, similar to mitochondrial protein acetylation. In contrast, mitochondrial protein succinylation is reduced. These mitochondrial protein modifications appear to be primarily driven by ethanol metabolism, and not by changes in mitochondrial sirtuin levels. Similar trends in acyl modifications were observed in the nucleus. However, comparatively fewer acyl modifications were observed in the cytoplasmic or the microsomal compartments, and were generally unchanged by ethanol metabolism. Using a mass spectrometry proteomics approach, we identified several candidate acetylated, propionylated, and succinylated proteins, which were enriched using antibodies against each modification. Additionally, we identified several acetyl and propionyl lysine residues on the same sites for a number of proteins and supports the idea of the overlapping nature of lysine-specific acylation. Thus, we show that novel post-translational modifications are present in hepatic mitochondrial, nuclear, cytoplasmic, and microsomal compartments and ethanol ingestion, and its associated metabolism, induce specific

  9. Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.

    PubMed

    Brekhman, Vera; Lugassie, Jennie; Zaffryar-Eilot, Shelly; Sabo, Edmond; Kessler, Ofra; Smith, Victoria; Golding, Hana; Neufeld, Gera

    2011-01-01

    Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -

  10. The Quality of Modified Atmosphere Packaged Meat from Lambs Slaughtered at 50 and 100 Days of Age

    PubMed Central

    Tański, Zenon; Stanisław, Milewski; Bożena, Zaleska

    2012-01-01

    The aim of this study was to determine the quality of modified atmosphere (MA) packaged meat from Pomeranian rams slaughtered at 50 and 100 days of age. Determined: chemical composition, physicochemical and sensory properties of meat, and the fatty acid profile of intramuscular fat. Meat from 100-d-old lambs was characterized by a significantly higher content of dry matter, total protein and crude fat, a darker color and higher physiological maturity (p≤0.01). Meat from younger lambs was marked by a better water-holding capacity (p≤0.05), a lower energy value and higher concentrations of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). An increase in the content of dry matter, total protein and ash, and energy value was noted in MA-stored meat. Meat pH decreased over MA storage and the sensory properties deteriorated. The studied meat remained safe from microbial contamination during storage. PMID:25049582

  11. Tocopherol-mediated modulation of age-related changes in microglial cells: turnover of extracellular oxidized protein material.

    PubMed

    Stolzing, Alexandra; Widmer, Rebecca; Jung, Tobias; Voss, Peter; Grune, Tilman

    2006-06-15

    Proteins accumulate during aging and form insoluble protein aggregates. Microglia are responsible for their removal from the brain. During aging, changes within the microglia might play a crucial role in the malfunctioning of these cells. Therefore, we isolated primary microglial cells from adult rats and compared their activation status and their ability to degrade proteins to that of microglial cells isolated from newborn animals. The ability of adult microglial cells to degrade proteins is substantially decreased. However, the preincubation of microglial cells with vitamin E improves significantly the degradation of such modified proteins. The degradation of proteins from apoptotic vesicles is decreased in microglia isolated from adult rats. This might be the result of a suppression of the CD36 receptor due to vitamin E treatment. We concluded that microglial cells isolated from adult organisms have different metabolic properties and seem to be a more valuable model to study age-related diseases.

  12. Phospho1 deficiency transiently modifies bone architecture yet produces consistent modification in osteocyte differentiation and vascular porosity with ageing.

    PubMed

    Javaheri, B; Carriero, A; Staines, K A; Chang, Y-M; Houston, D A; Oldknow, K J; Millan, J L; Kazeruni, Bassir N; Salmon, P; Shefelbine, S; Farquharson, C; Pitsillides, A A

    2015-12-01

    PHOSPHO1 is one of principal proteins involved in initiating bone matrix mineralisation. Recent studies have found that Phospho1 KO mice (Phospho1-R74X) display multiple skeletal abnormalities with spontaneous fractures, bowed long bones, osteomalacia and scoliosis. These analyses have however been limited to young mice and it remains unclear whether the role of PHOSPHO1 is conserved in the mature murine skeleton where bone turnover is limited. In this study, we have used ex-vivo computerised tomography to examine the effect of Phospho1 deletion on tibial bone architecture in mice at a range of ages (5, 7, 16 and 34 weeks of age) to establish whether its role is conserved during skeletal growth and maturation. Matrix mineralisation has also been reported to influence terminal osteoblast differentiation into osteocytes and we have also explored whether hypomineralised bones in Phospho1 KO mice exhibit modified osteocyte lacunar and vascular porosity. Our data reveal that Phospho1 deficiency generates age-related defects in trabecular architecture and compromised cortical microarchitecture with greater porosity accompanied by marked alterations in osteocyte shape, significant increases in osteocytic lacuna and vessel number. Our in vitro studies examining the behaviour of osteoblast derived from Phospho1 KO and wild-type mice reveal reduced levels of matrix mineralisation and modified osteocytogenic programming in cells deficient in PHOSPHO1. Together our data suggest that deficiency in PHOSPHO1 exerts modifications in bone architecture that are transient and depend upon age, yet produces consistent modification in lacunar and vascular porosity. It is possible that the inhibitory role of PHOSPHO1 on osteocyte differentiation leads to these age-related changes in bone architecture. It is also intriguing to note that this apparent acceleration in osteocyte differentiation evident in the hypomineralised bones of Phospho1 KO mice suggests an uncoupling of the interplay

  13. Rho-modifying bacterial protein toxins from Photorhabdus species.

    PubMed

    Jank, Thomas; Lang, Alexander E; Aktories, Klaus

    2016-06-15

    Photorhabdus bacteria live in symbiosis with entomopathogenic nematodes. The nematodes invade insect larvae, where they release the bacteria, which then produce toxins to kill the insects. Recently, the molecular mechanisms of some toxins from Photorhabdus luminescens and asymbiotica have been elucidated, showing that GTP-binding proteins of the Rho family are targets. The tripartite Tc toxin PTC5 from P. luminescens activates Rho proteins by ADP-ribosylation of a glutamine residue, which is involved in GTP hydrolysis, while PaTox from Photorhabdus asymbiotica inhibits the activity of GTPases by N-acetyl-glucosaminylation at tyrosine residues and activates Rho proteins indirectly by deamidation of heterotrimeric G proteins.

  14. Protein carbonylation and heat shock proteins in human skeletal muscle: relationships to age and sarcopenia.

    PubMed

    Beltran Valls, Maria R; Wilkinson, Daniel J; Narici, Marco V; Smith, Kenneth; Phillips, Bethan E; Caporossi, Daniela; Atherton, Philip J

    2015-02-01

    Aging is associated with a gradual loss of muscle mass termed sarcopenia, which has significant impact on quality-of-life. Because oxidative stress is proposed to negatively impact upon musculoskeletal aging, we investigated links between human aging and markers of oxidative stress, and relationships to muscle mass and strength in young and old nonsarcopenic and sarcopenic adults. Sixteen young and 16 old males (further subdivided into "old" and "old sarcopenic") were studied. The abundance of protein carbonyl adducts within skeletal muscle sarcoplasmic, myofibrillar, and mitochondrial protein subfractions from musculus vastus lateralis biopsies were determined using Oxyblot immunoblotting techniques. In addition, concentrations of recognized cytoprotective proteins (eg, heat shock proteins [HSP], αβ-crystallin) were also assayed. Aging was associated with increased mitochondrial (but not myofibrillar or sarcoplasmic) protein carbonyl adducts, independently of (stage-I) sarcopenia. Correlation analyses of all subjects revealed that mitochondrial protein carbonyl abundance negatively correlated with muscle strength ([1-repetition maximum], p = .02, r (2) = -.16), but not muscle mass (p = .13, r (2) = -.08). Abundance of cytoprotective proteins, including various HSPs (HSP 27 and 70), were unaffected by aging/sarcopenia. To conclude, these data reveal that mitochondrial protein carbonylation increases moderately with age, and that this increase may impact upon skeletal muscle function, but is not a hallmark of (stage-I) sarcopenia, per se.

  15. Characteristics and safety assessment of intractable proteins in genetically modified crops.

    PubMed

    Bushey, Dean F; Bannon, Gary A; Delaney, Bryan F; Graser, Gerson; Hefford, Mary; Jiang, Xiaoxu; Lee, Thomas C; Madduri, Krishna M; Pariza, Michael; Privalle, Laura S; Ranjan, Rakesh; Saab-Rincon, Gloria; Schafer, Barry W; Thelen, Jay J; Zhang, John X Q; Harper, Marc S

    2014-07-01

    Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.

  16. Protein Homeostasis and Aging: the importance of exquisite quality control

    PubMed Central

    Koga, Hiroshi; Kaushik, Susmita; Cuervo, Ana Maria

    2010-01-01

    All cells count on precise mechanisms that regulate protein homeostasis to maintain a stable and functional proteome. A progressive deterioration in the ability of cells to preserve the stability of their proteome occurs with age and contributes to the functional loss characteristic of old organisms. Molecular chaperones and the proteolytic systems are responsible for this cellular quality control by assuring continuous renewal of intracellular proteins. When protein damage occurs, such as during cellular stress, the coordinated action of these cellular surveillance systems allows detection and repair of the damaged structures or, in many instances, leads to the complete elimination of the altered proteins from inside cells. Dysfunction of the quality control mechanisms and intracellular accumulation of abnormal proteins in the form of protein inclusions and aggregates occur in almost all tissues of an aged organism. Preservation or enhancement of the activity of these surveillance systems until late in life improves their resistance to stress and is sufficient to slow down aging. In this work, we review recent advances on our understanding of the contribution of chaperones and proteolytic systems to the maintenance of cellular homeostasis, the cellular response to stress and ultimately to longevity. PMID:20152936

  17. Simultaneous detection of changes in protein expression and oxidative modification as function of age and APOE genotype in human APOE-targeted replacement mice

    EPA Science Inventory

    Background The purpose of this study was to improve the current method for detecting differentially-oxidized (carbonyl-modified) proteins by 2D-DIGE, while at the same time determining changes in total protein expression. Protein oxidation is a widely accepted model of aging and...

  18. Treatment of hides with tara-modified protein products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In prior research, we demonstrated that gelatin could be modified with quebracho to produce products whose physicochemical properties would enable them to be used effectively as fillers in leather processing, and that leather resulting from this treatment had improved subjective properties with litt...

  19. Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion

    PubMed Central

    Wall, Benjamin Toby; Gorissen, Stefan H.; Pennings, Bart; Koopman, René; Groen, Bart B. L.; Verdijk, Lex B.; van Loon, Luc J. C.

    2015-01-01

    Purpose Progressive loss of skeletal muscle mass with aging (sarcopenia) forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men. Procedures We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y) and 72 older (75±1 y) men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y) and 40 older (74±1 y) men. Findings Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion. Conclusions Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia. PMID:26536130

  20. Protein oxidation associated with aging is reduced by dietary restriction of protein or calories.

    PubMed Central

    Youngman, L D; Park, J Y; Ames, B N

    1992-01-01

    The accumulation of unrepaired oxidative damage products may be a major factor in cellular aging. Both oxidative lesions in DNA and oxidatively damaged proteins have been shown to accumulate during aging. The accumulation of oxidized proteins in Fischer 344 rats was compared for animals consuming protein-restricted and calorically restricted diets--both of which have been shown to extend lifespan. Rats were fed diets restricted in either protein (5% or 10% of the diet as compared with the normal 20% casein), or calories (25% or 40% less than normal), or total diet (40% less than normal). In addition, some of the rats fed a diet providing 5% or 20% protein were irradiated twice weekly (125 rads per exposure; 1 rad = 0.01 Gy). The level of oxidative damage to proteins (protein carbonyls) was determined in rats sacrificed at various times. The oxidative damage to proteins increased with aging and with radiation. Either protein or calorie restriction markedly inhibited the accumulation of oxidatively damaged proteins. Protein restriction reduced the accumulation of oxidatively damaged proteins during the oxidative stress of chronic irradiation. PMID:1409611

  1. Method For Determining And Modifying Protein/Peptide Solubilty

    DOEpatents

    Waldo, Geoffrey S.

    2005-03-15

    A solubility reporter for measuring a protein's solubility in vivo or in vitro is described. The reporter, which can be used in a single living cell, gives a specific signal suitable for determining whether the cell bears a soluble version of the protein of interest. A pool of random mutants of an arbitrary protein, generated using error-prone in vitro recombination, may also be screened for more soluble versions using the reporter, and these versions may be recombined to yield variants having further-enhanced solubility. The method of the present invention includes "irrational" (random mutagenesis) methods, which do not require a priori knowledge of the three-dimensional structure of the protein of interest. Multiple sequences of mutation/genetic recombination and selection for improved solubility are demonstrated to yield versions of the protein which display enhanced solubility.

  2. ASK1 and MAP2K6 as modifiers of age at onset in Huntington's disease.

    PubMed

    Arning, Larissa; Monté, Didier; Hansen, Wiebke; Wieczorek, Stefan; Jagiello, Peter; Akkad, Denis A; Andrich, Jürgen; Kraus, Peter H; Saft, Carsten; Epplen, Jörg T

    2008-04-01

    Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disease associated with abnormal expansions of a stretch of perfect CAG repeats in the HD gene. The number of repeat units is predictive for the age at onset (AO) of neurological symptoms. Part of the remaining variation in AO is attributed to modifier genes. In this study, genes involved in apoptosis were investigated as candidates for modulating AO in HD. A panel of 304 candidate genes was screened for allelic associations with motor AO via linked micro-satellite markers by pooling the DNAs of HD individuals from opposite ends of the AO distribution. After genotyping promising markers from the pooling experiment individually, markers revealed consolidated evidence for association in a candidate region comprising the genes MAP3K5 (ASK1)/PEX7 at 6q23.3 and in the gene MAP2K6 at 17q24.3. Fine-mapping of these candidate regions in a cohort of 250 Caucasian HD patients using single nucleotide polymorphism (SNP) markers delimitated the precise locations of association. Certain variations in an ASK1-PEX7 haplotype block explain 2.6% of additional variance in AO in our HD cohort. In males, 4.9% additional variance could be attributed to MAP2K6 genotype variations. Altogether, ASK1-PEX7 haplotypes and MAP2K2 genotype variations explain 6.3% additional variance in AO for HD. We hypothesise that sequence variations of ASK1 and MAP2K6 lead to partially sex-specific changes in the levels and/or phosphorylation states of p38 and p38-regulated proteins that might contribute to the observed delaying effects in the AO of HD.

  3. Sonochemical synthesis of (3-aminopropyl)triethoxysilane-modified monodispersed silica nanoparticles for protein immobilization

    SciTech Connect

    Shen, Shou-Cang; Ng, Wai Kiong; Chia, Leonard; Dong, Yuan-Cai; Tan, Reginald B.H.

    2011-10-15

    Graphical abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by rapid sonochemical co-condensation to achieve high capability for protein immobilization. Highlights: {yields} Amino-modified monodispersed silica nanoparticles were synthesized by rapid co-condensation. {yields} Strong positive charge was created by aminopropyl-modification. {yields} Capability for immobilization of negatively charged protein was enhanced. {yields} Electrostatic interaction between proteins and surface contributed to the enhanced adsorption. -- Abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by a rapid sonochemical co-condensation synthesis procedure. The chemical nature of surface organic modifier on the obtained modified silica nanoparticle was characterized by {sup 13}C and {sup 29}Si MAS Nuclear Magnetic Resonance (NMR) spectroscopies, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)- differential scanning calorimetry (DSC). Due to the strengthened positive surface charge of the silica nanoparticles by the modification with aminopropyl groups, the capability for bovine serum albumin (BSA) adsorption was significantly increased as compared with bare silica nanoparticles. 80 mg/g BSA was adsorbed on modified silica nanoparticles, whereas only 20 mg/g BSA could be loaded on pure silica nanoparticles. The enhanced positive surface charge repelled proteins with net positive charge and the modified silica nanoparticles exhibited negligible adsorption of lysozyme, thus a selective adsorption of proteins could be achieved.

  4. An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

    PubMed Central

    J Gingell, Joseph; Simms, John; Barwell, James; Poyner, David R; Watkins, Harriet A; Pioszak, Augen A; Sexton, Patrick M; Hay, Debbie L

    2016-01-01

    G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor

  5. Rho-modifying bacterial protein toxins from Photorhabdus species.

    PubMed

    Jank, Thomas; Lang, Alexander E; Aktories, Klaus

    2016-06-15

    Photorhabdus bacteria live in symbiosis with entomopathogenic nematodes. The nematodes invade insect larvae, where they release the bacteria, which then produce toxins to kill the insects. Recently, the molecular mechanisms of some toxins from Photorhabdus luminescens and asymbiotica have been elucidated, showing that GTP-binding proteins of the Rho family are targets. The tripartite Tc toxin PTC5 from P. luminescens activates Rho proteins by ADP-ribosylation of a glutamine residue, which is involved in GTP hydrolysis, while PaTox from Photorhabdus asymbiotica inhibits the activity of GTPases by N-acetyl-glucosaminylation at tyrosine residues and activates Rho proteins indirectly by deamidation of heterotrimeric G proteins. PMID:26026623

  6. CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells.

    PubMed

    Nakajou, Keisuke; Horiuchi, Seikoh; Sakai, Masakazu; Hirata, Kenshiro; Tanaka, Makiko; Takeya, Motohiro; Kai, Toshiya; Otagiri, Masaki

    2005-05-01

    Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.

  7. Influence of homology and node age on the growth of protein-protein interaction networks

    NASA Astrophysics Data System (ADS)

    Bottinelli, Arianna; Bassetti, Bruno; Lagomarsino, Marco Cosentino; Gherardi, Marco

    2012-10-01

    Proteins participating in a protein-protein interaction network can be grouped into homology classes following their common ancestry. Proteins added to the network correspond to genes added to the classes, so the dynamics of the two objects are intrinsically linked. Here we first introduce a statistical model describing the joint growth of the network and the partitioning of nodes into classes, which is studied through a combined mean-field and simulation approach. We then employ this unified framework to address the specific issue of the age dependence of protein interactions through the definition of three different node wiring or divergence schemes. A comparison with empirical data indicates that an age-dependent divergence move is necessary in order to reproduce the basic topological observables together with the age correlation between interacting nodes visible in empirical data. We also discuss the possibility of nontrivial joint partition and topology observables.

  8. Air Pollution modifies the association between successful and pathological aging throughout the frailty condition.

    PubMed

    Fougère, Bertrand; Vellas, Bruno; Billet, Sylvain; Martin, Perrine J; Gallucci, Maurizio; Cesari, Matteo

    2015-11-01

    The rapid growth in the number of older adults has many implications for public health, including the need to better understand the risks posed by environmental exposures. Aging leads to a decline and deterioration of functional properties at the cellular, tissue and organ level. This loss of functional properties yields to a loss of homeostasis and decreased adaptability to internal and external stress. Frailty is a geriatric syndrome characterized by weakness, weight loss, and low activity that is associated with adverse health outcomes. Frailty manifests as an age-related, biological vulnerability to stressors and decreased physiological reserves. Ambient air pollution exposure affects human health, and elderly people appear to be particularly susceptible to its adverse effects. The aim of this paper is to discuss the role of air pollution in the modulation of several biological mechanisms involved in aging. Evidence is presented on how air pollution can modify the bidirectional association between successful and pathological aging throughout the frailty conditions.

  9. Reversible and oriented immobilization of ferrocene-modified proteins.

    PubMed

    Yang, Lanti; Gomez-Casado, Alberto; Young, Jacqui F; Nguyen, Hoang D; Cabanas-Danés, Jordi; Huskens, Jurriaan; Brunsveld, Luc; Jonkheijm, Pascal

    2012-11-21

    Adopting supramolecular chemistry for immobilization of proteins is an attractive strategy that entails reversibility and responsiveness to stimuli. The reversible and oriented immobilization and micropatterning of ferrocene-tagged yellow fluorescent proteins (Fc-YFPs) onto β-cyclodextrin (βCD) molecular printboards was characterized using surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in combination with electrochemistry. The proteins were assembled on the surface through the specific supramolecular host-guest interaction between βCD and ferrocene. Application of a dynamic covalent disulfide lock between two YFP proteins resulted in a switch from monovalent to divalent ferrocene interactions with the βCD surface, yielding a more stable protein immobilization. The SPR titration data for the protein immobilization were fitted to a 1:1 Langmuir-type model, yielding K(LM) = 2.5 × 10(5) M(-1) and K(i,s) = 1.2 × 10(3) M(-1), which compares favorably to the intrinsic binding constant presented in the literature for the monovalent interaction of ferrocene with βCD self-assembled monolayers. In addition, the SPR binding experiments were qualitatively simulated, confirming the binding of Fc-YFP in both divalent and monovalent fashion to the βCD monolayers. The Fc-YFPs could be patterned on βCD surfaces in uniform monolayers, as revealed using fluorescence microscopy and atomic force microscopy measurements. Both fluorescence microscopy imaging and SPR measurements were carried out with the in situ capability to perform cyclic voltammetry and chronoamperometry. These studies emphasize the repetitive desorption and adsorption of the ferrocene-tagged proteins from the βCD surface upon electrochemical oxidation and reduction, respectively.

  10. Enhanced protein internalization and efficient endosomal escape using polyampholyte-modified liposomes and freeze concentration.

    PubMed

    Ahmed, Sana; Fujita, Satoshi; Matsumura, Kazuaki

    2016-09-21

    Here we show a new strategy for efficient freeze concentration-mediated cytoplasmic delivery of proteins, obtained via the endosomal escape property of polyampholyte-modified liposomes. The freeze concentration method successfully induces the efficient internalization of proteins simply by freezing cells with protein and nanocarrier complexes. However, the mechanism of protein internalization remains unclear. Here, we designed a novel protein delivery carrier by modifying liposomes through incorporating hydrophobic polyampholytes therein. These complexes were characterized for particle size, encapsulation efficiency, and cytotoxicity. Flow cytometry and microscopic analysis showed that the adsorption and internalization of protein-loaded polyampholyte-modified liposomes after freezing were enhanced compared with that observed in unfrozen complexes. Inhibition studies demonstrated that the internalization mechanism differs between unmodified and polyampholyte-modified liposomes. Furthermore, polyampholyte-modified liposomes exhibited high efficacy in facilitating endosomal escape to enhance protein delivery to the cytoplasm with low toxicity. These results strongly suggest that the freeze concentration-based strategy could be widely utilised for efficient cargo delivery into the cytoplasm in vitro not only in cancer treatment but also for gene therapy as well. PMID:27439774

  11. Protein- and Peptide-Modified Synthetic Polymeric Biomaterials

    PubMed Central

    Krishna, Ohm D.; Kiick, Kristi L.

    2015-01-01

    This review presents an overview on biohybrid approaches of integrating the structural and functional features of proteins and peptides with synthetic polymers and the resulting unique properties in such hybrids, with a focus on bioresponsive/bioactive systems with biomaterials applications. The review is divided in two broad sections. First, we describe several examples of biohybrids produced by combining versatile synthetic polymers with proteins/enzymes and drugs that have resulted in (1) hybrid materials based on responsive polymers, (2) responsive hydrogels based on enzyme-catalyzed reactions, protein–protein interactions and protein–drug sensing, and (3) dynamic hydrogels based on conformational changes of a protein. Next, we present hybrids produced by combining synthetic polymers with peptides, classified based on the properties of the peptide domain: (1) peptides with different conformations, such as α-helical, coiled-coil, and β-sheet; (2) peptides derived from structural protein domains such as silk, elastin, titin, and collagen; and (3) peptides with other biofunctional properties such as cell-binding domains and enzyme-recognized degradation domains. PMID:20091878

  12. Individually modifiable risk factors to ameliorate cognitive aging: a systematic review and meta-analysis.

    PubMed

    Lehert, P; Villaseca, P; Hogervorst, E; Maki, P M; Henderson, V W

    2015-10-01

    A number of health and lifestyle factors are thought to contribute to cognitive decline associated with age but cannot be easily modified by the individual patient. We identified 12 individually modifiable interventions that can be implemented during midlife or later with the potential to ameliorate cognitive aging. For ten of these, we used PubMed databases for a systematic review of long-duration (at least 6 months), randomized, controlled trials in midlife and older adults without dementia or mild cognitive impairment with objective measures of neuropsychological performance. Using network meta-analysis, we performed a quantitative synthesis for global cognition (primary outcome) and episodic memory (secondary outcome). Of 1038 publications identified by our search strategy, 24 eligible trials were included in the network meta-analysis. Results suggested that the Mediterranean diet supplemented by olive oil and tai chi exercise may improve global cognition, and the Mediterranean diet plus olive oil and soy isoflavone supplements may improve memory. Effect sizes were no more than small (standardized mean differences 0.11-0.22). Cognitive training may have cognitive benefit as well. Most individually modifiable risk factors have not yet been adequately studied. We conclude that some interventions that can be self-initiated by healthy midlife and older adults may ameliorate cognitive aging. PMID:26361790

  13. Individually modifiable risk factors to ameliorate cognitive aging: a systematic review and meta-analysis.

    PubMed

    Lehert, P; Villaseca, P; Hogervorst, E; Maki, P M; Henderson, V W

    2015-10-01

    A number of health and lifestyle factors are thought to contribute to cognitive decline associated with age but cannot be easily modified by the individual patient. We identified 12 individually modifiable interventions that can be implemented during midlife or later with the potential to ameliorate cognitive aging. For ten of these, we used PubMed databases for a systematic review of long-duration (at least 6 months), randomized, controlled trials in midlife and older adults without dementia or mild cognitive impairment with objective measures of neuropsychological performance. Using network meta-analysis, we performed a quantitative synthesis for global cognition (primary outcome) and episodic memory (secondary outcome). Of 1038 publications identified by our search strategy, 24 eligible trials were included in the network meta-analysis. Results suggested that the Mediterranean diet supplemented by olive oil and tai chi exercise may improve global cognition, and the Mediterranean diet plus olive oil and soy isoflavone supplements may improve memory. Effect sizes were no more than small (standardized mean differences 0.11-0.22). Cognitive training may have cognitive benefit as well. Most individually modifiable risk factors have not yet been adequately studied. We conclude that some interventions that can be self-initiated by healthy midlife and older adults may ameliorate cognitive aging.

  14. Molecular Design of Bisphosphonate-Modified Proteins for Efficient Bone Targeting In Vivo

    PubMed Central

    Katsumi, Hidemasa; Sano, Jun-ichi; Nishikawa, Makiya; Hanzawa, Keiko; Sakane, Toshiyasu; Yamamoto, Akira

    2015-01-01

    To establish a rational molecular design for bisphosphonate (BP)-modified proteins for efficient bone targeting, a pharmacokinetic study was performed using a series of alendronate (ALN), a nitrogen-containing BP, modified proteins with various molecular weights and varying degrees of modification. Four proteins with different molecular weight—yeast glutathione reductase (GR; MW: 112,000 Da), bovine serum albumin (BSA; MW: 67,000 Da), recombinant human superoxide dismutase (SOD; MW: 32,000 Da), and chicken egg white lysozyme (LZM; MW: 14,000 Da)—were modified with ALN to obtain ALN-modified proteins. Pharmacokinetic analysis of the tissue distribution of the ALN-modified and unmodified proteins was performed after radiolabeling them with indium-111 (111In) by using a bifunctional chelating agent. Calculation of tissue uptake clearances revealed that the bone uptake clearances of 111In-ALN-modified proteins were proportional to the degree of ALN modification. 111In-GR-ALN and BSA-ALN, the two high-molecular-weight proteins, efficiently accumulated in bones, regardless of the degree of ALN modification. Approximately 36 and 34% of the dose, respectively, was calculated to be delivered to the bones. In contrast, the maximum amounts taken up by bone were 18 and 13% of the dose for 111In-SOD-ALN(32) and LZM-ALN(9), respectively, because of their high renal clearance. 111In-SOD modified with both polyethylene glycol (PEG) and ALN (111In-PEG-SOD-ALN) was efficiently delivered to the bone. Approximately 36% of the dose was estimated to be delivered to the bones. In an experimental bone metastasis mouse model, treatment with PEG-SOD-ALN significantly reduced the number of tumor cells in the bone of the mice. These results indicate that the combination of PEG and ALN modification is a promising approach for efficient bone targeting of proteins with a high total-body clearance. PMID:26287482

  15. Rnq1: an epigenetic modifier of protein function in yeast.

    PubMed

    Sondheimer, N; Lindquist, S

    2000-01-01

    Two protein-based genetic elements (prions) have been identified in yeast. It is not clear whether other prions exist, nor is it understood how one might find them. We established criteria for searching protein databases for prion candidates and found several. The first examined, Rnq1, exists in distinct, heritable physical states, soluble and insoluble. The insoluble state is dominant and transmitted between cells through the cytoplasm. When the prion-like region of Rnq1 was substituted for the prion domain of Sup35, the protein determinant of the prion [PSI+], the phenotypic and epigenetic behavior of [PSI+] was fully recapitulated. These findings identity Rnq1 as a prion, demonstrate that prion domains are modular and transferable, and establish a paradigm for identifying and characterizing novel prions.

  16. Systemic delivery of recombinant proteins by genetically modified myoblasts

    SciTech Connect

    Barr, E.; Leiden, J.M. )

    1991-12-06

    The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with {beta}-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.

  17. Polyethyleneimine-modified graphene oxide nanocomposites for effective protein functionalization

    NASA Astrophysics Data System (ADS)

    Weng, Yejing; Jiang, Bo; Yang, Kaiguang; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2015-08-01

    A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions.A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions. Electronic supplementary information (ESI) available: Cell viability assay, enrichment of standard glycoprotein, pretreatment and analysis of real

  18. Chromatographic and traditional albumin isotherms on cellulose: a model for wound protein adsorption on modified cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Albumin is the most abundant protein found in healing wounds. Traditional and chromatogrpahic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressin...

  19. Modification of recombinant maize ChitA chitinase by fungal chitinase-modifying proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In commercial maize, there are at least two different alleles of the chiA gene that encode alloforms of ChitA chitinase, a protein that is abundant in developing seed. Both known alloforms are modified by Bz-cmp, a protein secreted by the fungal pathogen Bipolaris zeicola. One alloform (ChitA-B73) i...

  20. Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce.

    PubMed

    Sun, Hyeon-Jin; Cui, Min-Long; Ma, Biao; Ezura, Hiroshi

    2006-01-23

    Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein. PMID:16406368

  1. Biological Role of Anti-aging Protein Klotho

    PubMed Central

    Kim, Ji-Hee; Hwang, Kyu-Hee; Park, Kyu-Sang; Kong, In Deok; Cha, Seung-Kuy

    2015-01-01

    Klotho-deficient mice have accelerated aging phenotypes, whereas overexpression of Klotho in mice extends lifespan. Klotho is an anti-aging single-pass membrane protein predominantly produced in the kidney, with shedding of the amino-terminal extracellular domain into the systemic circulation. Circulating levels of soluble Klotho decrease with age, and the klotho gene is associated with increased risk of age-related diseases. The three forms of Klotho protein have distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors, functions as an obligatory co-receptor for FGF23, which is involved in aging and the development of chronic diseases via regulation of Pi and vitamin D metabolism. Secreted Klotho functions as a humoral factor with pleiotropic activities including regulation of oxidative stress, growth factor signaling, and ion homeostasis. Secreted Klotho is also involved in organ protection. The intracellular form of Klotho suppresses inflammation-mediated cellular senescence and mineral metabolism. Herein we provide a brief overview of the structure and function and recent research about Klotho. PMID:26528423

  2. Biocontainment of genetically modified organisms by synthetic protein design.

    PubMed

    Mandell, Daniel J; Lajoie, Marc J; Mee, Michael T; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E; Gregg, Christopher J; Stoddard, Barry L; Church, George M

    2015-02-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites. PMID:25607366

  3. Biocontainment of genetically modified organisms by synthetic protein design.

    PubMed

    Mandell, Daniel J; Lajoie, Marc J; Mee, Michael T; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E; Gregg, Christopher J; Stoddard, Barry L; Church, George M

    2015-02-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites.

  4. Biocontainment of genetically modified organisms by synthetic protein design

    NASA Astrophysics Data System (ADS)

    Mandell, Daniel J.; Lajoie, Marc J.; Mee, Michael T.; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E.; Gregg, Christopher J.; Stoddard, Barry L.; Church, George M.

    2015-02-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites.

  5. Biocontainment of genetically modified organisms by synthetic protein design

    PubMed Central

    Mandell, Daniel J.; Lajoie, Marc J.; Mee, Michael T.; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E.; Gregg, Christopher J.; Stoddard, Barry L.; Church, George M.

    2015-01-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient either because they impose evolutionary pressure on the organism to eject the safeguard, because they can be circumvented by environmentally available compounds, or because they can be overcome by horizontal gene transfer (HGT). Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code to confer metabolic dependence on nonstandard amino acids for survival. The resulting GMOs cannot metabolically circumvent their biocontainment mechanisms using environmentally available compounds, and they exhibit unprecedented resistance to evolutionary escape via mutagenesis and HGT. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by reliance on synthetic metabolites. PMID:25607366

  6. AGES AND METALLICITIES OF CLUSTER GALAXIES IN A779 USING MODIFIED STROeMGREN PHOTOMETRY

    SciTech Connect

    Sreedhar, Yuvraj Harsha; Rakos, Karl D.; Hensler, Gerhard; Zeilinger, Werner W.; Odell, Andrew P.

    2012-03-01

    In the quest for the formation and evolution of galaxy clusters, Rakos and co-workers introduced a spectrophotometric method using modified Stroemgren photometry, but with the considerable debate toward the project's abilities, we re-introduce the system by testing for the repeatability of the modified Stroemgren colors and compare them with the Stroemgren colors, and check for the reproducibility of the ages and metallicities (using the Principle Component Analysis (PCA) technique and the GALEV models) for the six common galaxies in all three A779 data sets. As a result, a fair agreement between two filter systems was found to produce similar colors (with a precision of 0.09 mag in (uz - vz), 0.02 mag in (bz - yz), and 0.03 mag in (vz - vz)) and the generated ages and metallicities are also similar (with an uncertainty of 0.36 Gyr and 0.04 dex from PCA and 0.44 Gyr and 0.2 dex using the GALEV models). We infer that the technique is able to relieve the age-metallicity degeneracy by separating the age effects from the metallicity effects, but it is still unable to completely eliminate it. We further extend this paper to re-study the evolution of galaxies in the low mass, dynamically poor A779 cluster (as it was not elaborately analyzed by Rakos and co-workers in their previous work) by correlating the luminosity (mass), density, and radial distance with the estimated age, metallicity, and the star formation history. Our results distinctly show the bimodality of the young, low-mass, metal-poor population with a mean age of 6.7 Gyr ({+-} 0.5 Gyr) and the old, high-mass, metal-rich galaxies with a mean age of 9 Gyr ({+-} 0.5 Gyr). The method also observes the color evolution of the blue cluster galaxies to red (Butcher-Oemler phenomenon), and the downsizing phenomenon. Our analysis shows that modified Stroemgren photometry is very well suited for studying low- and intermediate-z clusters, as it is capable of observing deeper with better spatial resolution at

  7. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men.

    PubMed

    Mitchell, Cameron J; McGregor, Robin A; D'Souza, Randall F; Thorstensen, Eric B; Markworth, James F; Fanning, Aaron C; Poppitt, Sally D; Cameron-Smith, David

    2015-10-21

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring (13)C₆ phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h(-1) in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h(-1) in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein.

  8. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

    PubMed Central

    Mitchell, Cameron J.; McGregor, Robin A.; D’Souza, Randall F.; Thorstensen, Eric B.; Markworth, James F.; Fanning, Aaron C.; Poppitt, Sally D.; Cameron-Smith, David

    2015-01-01

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein. PMID:26506377

  9. Common SIRT1 variants modify the effect of abdominal adipose tissue on aging-related lung function decline.

    PubMed

    Curjuric, Ivan; Imboden, Medea; Bridevaux, Pierre-Olivier; Gerbase, Margaret W; Haun, Margot; Keidel, Dirk; Kumar, Ashish; Pons, Marco; Rochat, Thierry; Schikowski, Tamara; Schindler, Christian; von Eckardstein, Arnold; Kronenberg, Florian; Probst-Hensch, Nicole M

    2016-06-01

    Lung function is an independent predictor of mortality and serves as an aging marker in never smokers. The protein sirtuin-1 of gene SIRT1 has profound anti-inflammatory effects and regulates metabolic pathways. Its suggested longevity effects on lower organisms remain poorly studied in humans. In 1132 never smokers of the population-based SAPALDIA cohort, we investigated associations between single nucleotide polymorphisms (SNPs; rs730821, rs10997868, rs10823116) of SIRT1 and aging-related lung function decline over 11 years in terms of change in forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and forced expiratory flow between 25 and 75 % of FVC (FEF25-75) using multiple linear regression models. Interactions between the SIRT1 SNPs and adiposity parameters (body mass index (BMI), its change and weight gain) were tested by including multiplicative interaction terms into the models. SIRT1 polymorphisms exhibited no main effects, but modified the association between obesity measures and FEV1/FVC and FEF25-75 decline (p = 0.009-0.046). Per risk allele, FEV1/FVC decline was accelerated up to -0.5 % (95 % CI -1.0 to 0 %) and -0.7 % (-1.3 to -0.2 %) over interquartile range increases in BMI (2.4 kg/m(2)) or weight (6.5 kg), respectively. For FEF25-75 decline, corresponding estimates were -57 mL/s (-117 to 4 mL/s) and -76 mL/s (-1429 to -9 mL/s). Interactions were not present in participants with genetically lowered C-reactive protein concentrations. Genetic variation in SIRT1 might therefore affect lung function and human longevity by modifying subclinical inflammation arising from abdominal adipose tissue. PMID:27125385

  10. Cognitive Reserve Modifies Age-Related Alterations in CSF Biomarkers of Alzheimer's Disease

    PubMed Central

    Almeida, Rodrigo P.; Schultz, Stephanie A.; Austin, Benjamin P.; Boots, Elizabeth A.; Dowling, N. Maritza; Gleason, Carey E.; Bendlin, Barbara B.; Sager, Mark; Hermann, Bruce P.; Zetterberg, Henrik; Carlsson, Cindy; Johnson, Sterling; Asthana, Sanjay; Okonkwo, Ozioma C.

    2015-01-01

    Importance Although advancing age is the strongest risk factor for the development of symptomatic Alzheimer's disease (AD), recent studies have shown that there are individual differences in susceptibility to age-related alterations in the biomarkers of AD pathophysiology. Objective In this study, we investigated whether cognitive reserve modifies the adverse influence of age on key cerebrospinal fluid (CSF) biomarkers of AD. Design, Setting, and Participants Cross-sectional cohort of 268 individuals (211 cognitively normal and 57 cognitively impaired) from the Wisconsin Registry for Alzheimer's Prevention and the Wisconsin Alzheimer's Disease Research Center participated in this study. They underwent lumbar puncture for collection of CSF samples, from which amyloid-β 42 (Aβ42), total tau (t-tau), and phosphorylated tau (p-tau) were immunoassayed. Additionally, we computed t-tau/Aβ42 and p-tau/Aβ42 ratios. Cognitive reserve was indexed by years of education, with ≥16 years taken to confer high reserve. Covariate-adjusted regression analyses were used to test whether the effect of age on CSF biomarkers was modified by cognitive reserve. Main outcome measures CSF levels of Aβ42, t-tau, p-tau, t-tau/Aβ42, and p-tau/Aβ42. Results There were significant age*cognitive reserve interactions for CSF t-tau (p=.019), p-tau (p=.009), t-tau/Aβ42 (p=.021), and p-tau/Aβ42 (p=.004). Specifically, with advancing age, individuals with high cognitive reserve exhibited attenuated adverse alterations in these CSF biomarkers compared with individuals with low cognitive reserve. This attenuation of age effects by cognitive reserve tended to be more pronounced in the cognitively-impaired group compared with the cognitively-normal group. Lastly, there was modest evidence of a dose response relationship such that the effect of age on the biomarkers was progressively attenuated given additional years of schooling. Conclusions and Relevance In a sample comprised of both cognitively

  11. Functional expression of miraculin, a taste-modifying protein in Escherichia coli.

    PubMed

    Matsuyama, Tomomi; Satoh, Makiko; Nakata, Rieko; Aoyama, Takashi; Inoue, Hiroyasu

    2009-04-01

    Miraculin isolated from red berries of Richadella dulcifica, a native shrub of West Africa, has the unusual property of modifying a sour taste into a sweet one. This homodimer protein consists of two glycosylated polypeptides that are cross-linked by a disulfide bond. Recently, functional expression of miraculin was reported in host cells with the ability to glycosylate proteins, such as lettuce, tomato and the microbe Aspergillus oryzae, but not Escherichia coli. Thus, a question remains as to whether glycosylation of miraculin is essential for its taste-modifying properties. Here we show that recombinant miraculin expressed in E. coli has taste-modifying properties as a homodimer, not as a monomer, indicating that glycosylation is not essential for the taste-modifying property. PMID:19122203

  12. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    PubMed

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

  13. Relationship between oxygen concentration, shear force and protein oxidation in modified atmosphere packaged pork.

    PubMed

    Bao, Yulong; Ertbjerg, Per

    2015-12-01

    Pork loins were stored at 5°C for 14 days to investigate the effect of oxygen concentration in modified atmosphere packaging (MAP) on shear force and oxidation of lipids and proteins. The modified atmosphere contained 0 to 80% O2, 20% CO2, and balanced with N2. The results showed that shear force and thiobarbituric acid-reactive substances (TBARS) values increased with increasing oxygen concentration. Protein oxidation when measured as loss of free thiol groups, was greater in meat packaged under oxygen (20-80%). Myosin heavy chain (MHC) cross-linking, another marker of protein oxidation, was greater in MAP with 80% oxygen than 0% and 20% oxygen. Desmin degradation was not affected by the presence of oxygen, suggesting that the mechanism of oxygen-induced toughening of meat is through protein oxidation leading to cross-linking of structural proteins rather than through inactivation of proteolytic enzymes leading to reduced proteolysis.

  14. Maltodextrin-modified magnetic microspheres for selective enrichment of maltose binding proteins.

    PubMed

    Zheng, Jin; Ma, Chongjun; Sun, Yangfei; Pan, Miaorong; Li, Li; Hu, Xiaojian; Yang, Wuli

    2014-03-12

    In this work, maltodextrin-modified magnetic microspheres Fe3O4@SiO2-Maltodextrin (Fe3O4@SiO2-MD) with uniform size and fine morphology were synthesized through a facile and low-cost method. As the maltodextrins on the surface of microspheres were combined with maltose binding proteins (MBP), the magnetic microspheres could be applied to enriching standard MBP fused proteins. Then, the application of Fe3O4@SiO2-MD in one-step purification and immobilization of MBP fused proteins was demonstrated. For the model protein we examined, Fe3O4@SiO2-MD showed excellent binding selectivity and capacity against other Escherichia coli proteins in the crude cell lysate. Additionally, the maltodextrin-modified magnetic microspheres can be recycled for several times without significant loss of binding capacity.

  15. Relationship between oxygen concentration, shear force and protein oxidation in modified atmosphere packaged pork.

    PubMed

    Bao, Yulong; Ertbjerg, Per

    2015-12-01

    Pork loins were stored at 5°C for 14 days to investigate the effect of oxygen concentration in modified atmosphere packaging (MAP) on shear force and oxidation of lipids and proteins. The modified atmosphere contained 0 to 80% O2, 20% CO2, and balanced with N2. The results showed that shear force and thiobarbituric acid-reactive substances (TBARS) values increased with increasing oxygen concentration. Protein oxidation when measured as loss of free thiol groups, was greater in meat packaged under oxygen (20-80%). Myosin heavy chain (MHC) cross-linking, another marker of protein oxidation, was greater in MAP with 80% oxygen than 0% and 20% oxygen. Desmin degradation was not affected by the presence of oxygen, suggesting that the mechanism of oxygen-induced toughening of meat is through protein oxidation leading to cross-linking of structural proteins rather than through inactivation of proteolytic enzymes leading to reduced proteolysis. PMID:26241463

  16. Ultrarush schedule of subcutaneous immunotherapy with modified allergen extracts is safe in paediatric age

    PubMed Central

    Arêde, Cristina; Sampaio, Graça; Borrego, Luis Miguel

    2016-01-01

    Background Traditional subcutaneous immunotherapy up dosing with allergenic extracts has been shown to be associated with frequent adverse reactions. In recent studies it has been demonstrated that using modified extracts, namely allergoids, it is a safe and effective procedure particularly on accelerated schedules. However data assessing its safety in paediatric age is scarce. Objective To evaluate the safety profile in paediatric population of using modified allergen extracts, in an ultrarush schedule, to reach the maintenance dose in the first day. Methods We included children undergoing treatment with subcutaneous immunotherapy during a five-year period, using modified aeroallergen extracts, depigmented, polymerized with glutaraldehyde and adsorbed on aluminium hydroxide using an ultrarush induction phase. The type of adverse reactions during the ultrarush protocol was recorded. Results We studied 100 paediatric patients (57 males) with a mean age of 11.6 years (5 to 18 years; standard deviation, 3.3), all with moderate to severe persistent rhinitis, with or without allergic conjunctivitis, asthma and atopic eczema, sensitized to mites and/or pollens. All reached the maintenance dose of 0.5 mL in the first day, except 1 child. During the ultrarush protocol the total number of injections was 199. There were 21 local adverse reactions in 11 patients, 11 immediate and 10 delayed; from those, had clinical relevance 1 immediate and 4 delayed. Systemic reactions were recorded in 2 cases, both immediate and mild. Conclusion The ultrarush protocol, without premedication, was a safe alternative to be used in paediatric age during the induction phase of subcutaneous immunotherapy using allergoid depigmented extracts. PMID:26844218

  17. Positive Lysosomal Modulation As a Unique Strategy to Treat Age-Related Protein Accumulation Diseases

    PubMed Central

    Wisniewski, Meagan L.; Butler, David

    2012-01-01

    Abstract Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ1–38 peptide corresponded with decreased levels of Aβ1–42, supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders

  18. Age modifies the genotype-phenotype relationship for the bitter receptor TAS2R38

    PubMed Central

    2010-01-01

    Background The purpose of this study was to investigate the effect of TAS2R38 haplotypes and age on human bitter taste perception. Results Children (3 to 10 yrs), adolescents (11 to 19 yrs) and adults (mostly mothers, 20 to 55 yrs (N = 980) were measured for bitter taste thresholds for 6-n-propylthiouracil (PROP) and genotyped for three polymorphisms of the AS2R38 gene (A49P, V262A, I296V). Subjects were grouped by haplotype and age, as well as sex and race/ethnicity, and compared for PROP thresholds. Subjects with the same haplotype were similar in bitter threshold regardless of race/ethnicity (all ages) or sex (children and adolescents; all p-values > 0.05) but age was a modifier of the genotype-phenotype relationship. Specifically, AVI/PAV heterozygous children could perceive a bitter taste at lower PROP concentrations than could heterozygous adults, with the thresholds of heterozygous adolescents being intermediate (p < 0.001). Similar age effects were not observed for subjects with the PAV/PAV or AVI/AVI homozygous haplotypes (p > 0.05) perhaps because there is less variation in taste perception among these homozygotes. Conclusions These data imply that the change in PROP bitter sensitivity which occurs over the lifespan (from bitter sensitive to less so) is more common in people with a particular haplotype combination, i.e., AVI/PAV heterozygotes. PMID:20594349

  19. Precipitation and mechanical properties of Nb-modified ferritic stainless steel during isothermal aging

    SciTech Connect

    Yan Haitao Bi Hongyun; Li Xin; Xu Zhou

    2009-03-15

    The influence of isothermal aging on precipitation behavior and mechanical properties of Nb-modified ferritic stainless steel was investigated using Thermo-calc software, scanning electron microscopy and transmission electron microscopy. It was observed that TiN, NbC and Fe{sub 2}Nb formed in the investigated steel and the experimental results agreed well with the results calculated by Thermo-calc software. During isothermal aging at 800 deg. C, the coarsening rate of Fe{sub 2}Nb is greater than that of NbC, and the calculated average sizes of NbC and Fe{sub 2}Nb of the aged specimen agreed with the experimental results. In addition, the tensile strength and micro-hardness of the ferritic stainless steel increased with increased aging time from 24 h to 48 h. But aging at 800 deg. C for 96 h caused the coarsening of the precipitation, which led to a decrease of tensile strength and micro-hardness.

  20. Dietary Protein to Maintain Muscle Mass in Aging: A Case for Per-meal Protein Recommendations.

    PubMed

    Murphy, C H; Oikawa, S Y; Phillips, S M

    2016-01-01

    It is well accepted that daily protein intake is an important dietary consideration to limit and treat age-related declines in muscle mass, strength, and function. Furthermore, we propose that there is a growing appreciation for the need to consider protein intake on a per-meal basis rather than simply focusing on the total daily protein intake. The existence of a saturable dose-response relationship between muscle protein synthesis (MPS) and the quantity of protein consumed in a single meal/bolus provides the rationale for promoting an even/balanced pattern of daily protein intake. We hypothesize that a balanced/even protein intake pattern with the ingestion a quantity of protein shown to optimally stimulate MPS at each meal may be an effective strategy to alleviate sarcopenic muscle loss. In this review we examine the available evidence supporting the influence of dietary protein intake pattern on muscle protein turnover, muscle mass, and muscle function. We present several practical considerations that, it is proposed, should be taken into account when translating a per-meal protein recommendation into dietary advice for older adults. PMID:26980369

  1. Dietary Protein to Maintain Muscle Mass in Aging: A Case for Per-meal Protein Recommendations.

    PubMed

    Murphy, C H; Oikawa, S Y; Phillips, S M

    2016-01-01

    It is well accepted that daily protein intake is an important dietary consideration to limit and treat age-related declines in muscle mass, strength, and function. Furthermore, we propose that there is a growing appreciation for the need to consider protein intake on a per-meal basis rather than simply focusing on the total daily protein intake. The existence of a saturable dose-response relationship between muscle protein synthesis (MPS) and the quantity of protein consumed in a single meal/bolus provides the rationale for promoting an even/balanced pattern of daily protein intake. We hypothesize that a balanced/even protein intake pattern with the ingestion a quantity of protein shown to optimally stimulate MPS at each meal may be an effective strategy to alleviate sarcopenic muscle loss. In this review we examine the available evidence supporting the influence of dietary protein intake pattern on muscle protein turnover, muscle mass, and muscle function. We present several practical considerations that, it is proposed, should be taken into account when translating a per-meal protein recommendation into dietary advice for older adults.

  2. Identification of a chitinase modifying protein from Fusarium verticillioides: truncation of a host resistance protein by a fungalysin metalloprotease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (cmps) are proteases, secreted by fungal pathogens, which truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. Cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. H...

  3. Physicochemical properties of protein-modified silver nanoparticles in seawater

    NASA Astrophysics Data System (ADS)

    Zhong, Hangyue

    2013-10-01

    This study investigated the physicochemical properties of silver nanoparticles stabilized with casein protein in seawater. UV?vis spectrometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were applied to measure the stability of silver nanoparticles in seawater samples. The obtained results show an increased aggregation tendency of silver nanoparticles in seawater, which could be attributed its relatively high cation concentration that could neutralize the negatively charges adsorbed on the surface of silver nanoparticles and reduce the electrostatic repulsion forces between nanoparticles. Similarly, due to the surface charge screening process, the zeta potential of silver nanoparticles in seawater decreased. This observation further supported the aggregation behavior of silver nanoparticles. This study also investigated the dissolution of silver nanoparticles in seawater. Result shows that the silver nanoparticle dissolution in DI water is lower than in seawater, which is attributed to the high Cl? concentration present in seawater. As Cl? can react with silver and form soluble AgCl complex, dissolution of silver nanoparticles was enhanced. Finally, this study demonstrated that silver nanoparticles are destabilized in seawater condition. These results may be helpful in understanding the environmental risk of discharged silver nanoparticles in seawater conditions.

  4. Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1

    PubMed Central

    Riechers, Sean-Patrick; Muehlenberg, Katja; Möller, Angeli; Reinhardt, Anita; Vinayagam, Arunachalam; Schaefer, Martin H.; Boutros, Michael; Tricoire, Hervé; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2012-01-01

    Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo. PMID:22916034

  5. Phospho1 deficiency transiently modifies bone architecture yet produces consistent modification in osteocyte differentiation and vascular porosity with ageing.

    PubMed

    Javaheri, B; Carriero, A; Staines, K A; Chang, Y-M; Houston, D A; Oldknow, K J; Millan, J L; Kazeruni, Bassir N; Salmon, P; Shefelbine, S; Farquharson, C; Pitsillides, A A

    2015-12-01

    PHOSPHO1 is one of principal proteins involved in initiating bone matrix mineralisation. Recent studies have found that Phospho1 KO mice (Phospho1-R74X) display multiple skeletal abnormalities with spontaneous fractures, bowed long bones, osteomalacia and scoliosis. These analyses have however been limited to young mice and it remains unclear whether the role of PHOSPHO1 is conserved in the mature murine skeleton where bone turnover is limited. In this study, we have used ex-vivo computerised tomography to examine the effect of Phospho1 deletion on tibial bone architecture in mice at a range of ages (5, 7, 16 and 34 weeks of age) to establish whether its role is conserved during skeletal growth and maturation. Matrix mineralisation has also been reported to influence terminal osteoblast differentiation into osteocytes and we have also explored whether hypomineralised bones in Phospho1 KO mice exhibit modified osteocyte lacunar and vascular porosity. Our data reveal that Phospho1 deficiency generates age-related defects in trabecular architecture and compromised cortical microarchitecture with greater porosity accompanied by marked alterations in osteocyte shape, significant increases in osteocytic lacuna and vessel number. Our in vitro studies examining the behaviour of osteoblast derived from Phospho1 KO and wild-type mice reveal reduced levels of matrix mineralisation and modified osteocytogenic programming in cells deficient in PHOSPHO1. Together our data suggest that deficiency in PHOSPHO1 exerts modifications in bone architecture that are transient and depend upon age, yet produces consistent modification in lacunar and vascular porosity. It is possible that the inhibitory role of PHOSPHO1 on osteocyte differentiation leads to these age-related changes in bone architecture. It is also intriguing to note that this apparent acceleration in osteocyte differentiation evident in the hypomineralised bones of Phospho1 KO mice suggests an uncoupling of the interplay

  6. Phospho1 deficiency transiently modifies bone architecture yet produces consistent modification in osteocyte differentiation and vascular porosity with ageing

    PubMed Central

    Javaheri, B.; Carriero, A.; Staines, K.A.; Chang, Y.-M.; Houston, D.A.; Oldknow, K.J.; Millan, J.L.; Kazeruni, Bassir N.; Salmon, P.; Shefelbine, S.; Farquharson, C.; Pitsillides, A.A.

    2015-01-01

    PHOSPHO1 is one of principal proteins involved in initiating bone matrix mineralisation. Recent studies have found that Phospho1 KO mice (Phospho1-R74X) display multiple skeletal abnormalities with spontaneous fractures, bowed long bones, osteomalacia and scoliosis. These analyses have however been limited to young mice and it remains unclear whether the role of PHOSPHO1 is conserved in the mature murine skeleton where bone turnover is limited. In this study, we have used ex-vivo computerised tomography to examine the effect of Phospho1 deletion on tibial bone architecture in mice at a range of ages (5, 7, 16 and 34 weeks of age) to establish whether its role is conserved during skeletal growth and maturation. Matrix mineralisation has also been reported to influence terminal osteoblast differentiation into osteocytes and we have also explored whether hypomineralised bones in Phospho1 KO mice exhibit modified osteocyte lacunar and vascular porosity. Our data reveal that Phospho1 deficiency generates age-related defects in trabecular architecture and compromised cortical microarchitecture with greater porosity accompanied by marked alterations in osteocyte shape, significant increases in osteocytic lacuna and vessel number. Our in vitro studies examining the behaviour of osteoblast derived from Phospho1 KO and wild-type mice reveal reduced levels of matrix mineralisation and modified osteocytogenic programming in cells deficient in PHOSPHO1. Together our data suggest that deficiency in PHOSPHO1 exerts modifications in bone architecture that are transient and depend upon age, yet produces consistent modification in lacunar and vascular porosity. It is possible that the inhibitory role of PHOSPHO1 on osteocyte differentiation leads to these age-related changes in bone architecture. It is also intriguing to note that this apparent acceleration in osteocyte differentiation evident in the hypomineralised bones of Phospho1 KO mice suggests an uncoupling of the interplay

  7. Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration

    PubMed Central

    Ebrahem, Quteba; Renganathan, Kutralanathan; Sears, Jonathan; Vasanji, Amit; Gu, Xiaorong; Lu, Liang; Salomon, Robert G.; Crabb, John W.; Anand-Apte, Bela

    2006-01-01

    Choroidal neovascularization (CNV), the advanced stage of age-related macular degeneration (AMD), accounts for >80% of vision loss in AMD. Carboxyethylpyrrole (CEP) protein modifications, uniquely generated from oxidation of docosahexaenoate-containing lipids, are more abundant in Bruch’s membrane from AMD eyes. We tested the hypothesis that CEP protein adducts stimulate angiogenesis and possibly contribute to CNV in AMD. Human serum albumin (HSA) or acetyl-Gly-Lys-O-methyl ester (dipeptide) were chemically modified to yield CEP-modified HSA (CEP-HSA) or CEP-dipeptide. The in vivo angiogenic properties of CEP-HSA and CEP-dipeptide were demonstrated by using the chick chorioallantoic membrane and rat corneal micropocket assays. Low picomole amounts of CEP-HSA and CEP-dipeptide stimulated neovascularization. Monoclonal anti-CEP antibody neutralized limbal vessel growth stimulated by CEP-HSA, whereas anti-VEGF antibody was found to only partially neutralize vessel growth. Subretinal injections of CEP-modified mouse serum albumin exacerbated laser-induced CNV in mice. In vitro treatments of human retinal pigment epithelial cells with CEP-dipeptide or CEP-HSA did not induce increased VEGF secretion. Overall, these results suggest that CEP-induced angiogenesis utilizes VEGF-independent pathways and that anti-CEP therapeutic modalities might be of value in limiting CNV in AMD. PMID:16938854

  8. Detection of AGEs as markers for carbohydrate metabolism and protein denaturation

    PubMed Central

    Nagai, Ryoji; Shirakawa, Jun-ichi; Fujiwara, Yukio; Ohno, Rei-ichi; Moroishi, Narumi; Sakata, Noriyuki; Nagai, Mime

    2014-01-01

    Approximately 100 years have passed since the Maillard reaction was first reported in the field of food chemistry as a condensation reaction between reducing sugars and amino acids. This reaction is thought to progress slowly primarily from glucose with proteins in vivo. An early-stage product, called the ”Amadori product”, is converted into advanced glycation end products. Those accumulate in the body in accordance with age, with such accumulation being enhanced by lifestyle-related diseases that result in the denaturation of proteins. Recent studies have demonstrated that intermediate carbonyls are generated by several pathways, and rapidly generate many glycation products. However, accurate quantification of glycation products in vivo is difficult due to instability and differences in physicochemical properties. In this connection, little is known about the relationship between the structure of glycation products and pathology. Furthermore, the interaction between proteins modified by glycation and receptors for advanced glycation end products is also known to induce the production of several inflammatory cytokines. Therefore, those inhibitors have been developed over the world to prevent lifestyle-related diseases. In this review, we describe the process of protein denaturation induced by glycation and discuss the possibility of using the process as a marker of age-related diseases. PMID:25120273

  9. Uterine Dysfunction and Genetic Modifiers in Centromere Protein B-deficient Mice

    PubMed Central

    Fowler, Kerry J.; Hudson, Damien F.; Salamonsen, Lois A.; Edmondson, Stephanie R.; Earle, Elizabeth; Sibson, Mandy C.; Choo, K.H. Andy

    2000-01-01

    Centromere protein B (CENP-B) binds constitutively to mammalian centromere repeat DNA and is highly conserved between humans and mouse. Cenpb null mice appear normal but have lower body and testis weights. We demonstrate here that testis-weight reduction is seen in male null mice generated on three different genetic backgrounds (denoted R1, W9.5, and C57), whereas body-weight reduction is dependent on the genetic background as well as the gender of the animals. In addition, Cenpb null females show 31%, 33%, and 44% reduced uterine weights on the R1, W9.5, and C57 backgrounds, respectively. Production of “revertant” mice lacking the targeted frameshift mutation but not the other components of the targeting construct corrected these differences, indicating that the observed phenotype is attributable to Cenpb gene disruption rather than a neighbouring gene effect induced by the targeting construct. The R1 and W9.5 Cenpb null females are reproductively competent but show age-dependent reproductive deterioration leading to a complete breakdown at or before 9 months of age. Reproductive dysfunction is much more severe in the C57 background as Cenpb null females are totally incompetent or are capable of producing no more than one litter. These results implicate a further genetic modifier effect on female reproductive performance. Histology of the uterus reveals normal myometrium and endometrium but grossly disrupted luminal and glandular epithelium. Tissue in situ hybridization demonstrates high Cenpb expression in the uterine epithelium of wild-type animals. This study details the first significant phenotype of Cenpb gene disruption and suggests an important role of Cenpb in uterine morphogenesis and function that may have direct implications for human reproductive pathology. PMID:10645947

  10. Age hardening and creep resistance of cast Al–Cu alloy modified by praseodymium

    SciTech Connect

    Bai, Zhihao; Qiu, Feng; Wu, Xiaoxue; Liu, Yingying; Jiang, Qichuan

    2013-12-15

    The effects of praseodymium on age hardening behavior and creep resistance of cast Al–Cu alloy were investigated. The results indicated that praseodymium facilitated the formation of the θ′ precipitates during the age process and improved the hardness of the Al–Cu alloy. Besides, praseodymium resulted in the formation of the Al{sub 11}Pr{sub 3} phase in the grain boundaries and among the dendrites of the modified alloy. Because of the good thermal stability of Al{sub 11}Pr{sub 3} phase, it inhibits grain boundary migration and dislocation movement during the creep process, which contributes to the improvement in the creep resistance of the modified alloy at elevated temperatures. - Highlights: • Pr addition enhances the hardness and creep resistance of the Al–Cu alloy. • Pr addition facilitates the formation of the θ′ precipitates. • Pr addition results in the formation of the Al11Pr3 phase in the Al–Cu alloy.

  11. The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients.

    PubMed

    Metzger, Silke; Walter, Carolin; Riess, Olaf; Roos, Raymund A C; Nielsen, Jørgen E; Craufurd, David; Nguyen, Huu Phuc

    2013-01-01

    The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the "REGISTRY" cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis.

  12. The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients.

    PubMed

    Metzger, Silke; Walter, Carolin; Riess, Olaf; Roos, Raymund A C; Nielsen, Jørgen E; Craufurd, David; Nguyen, Huu Phuc

    2013-01-01

    The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the "REGISTRY" cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis. PMID:23894380

  13. The V471A Polymorphism in Autophagy-Related Gene ATG7 Modifies Age at Onset Specifically in Italian Huntington Disease Patients

    PubMed Central

    Metzger, Silke; Walter, Carolin; Riess, Olaf; Roos, Raymund A. C.; Nielsen, Jørgen E.; Craufurd, David; Nguyen, Huu Phuc

    2013-01-01

    The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the “REGISTRY” cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis. PMID:23894380

  14. Treatment of proteins with dietary polyphenols lowers the formation of AGEs and AGE-induced toxicity.

    PubMed

    Zhang, Xinchen; Hu, Shuting; Chen, Feng; Wang, Mingfu

    2014-10-01

    Advanced glycation endproducts (AGEs) are a group of harmful compounds produced either endogenously or during thermal food processing. Once absorbed by humans via food intake, AGEs can cause oxidative cell damage and contribute to pathological development of various diseases. The AGE-inhibitory activity of dietary polyphenols in vitro has been extensively reported before, but the current study is pioneering in examining the antiglycation activity of five selected dietary polyphenols (phloretin, naringenin, epicatechin, chlorogenic acid, and rosmarinic acid) during the thermal protein glycation process. When added into the glucose-casein glycation model heated at 120 °C for 2 h, these polyphenols were capable of inhibiting the formation of both total fluorescent AGEs and nonfluorescent carboxymethyllysine (CML). The thermal stability and transformation of polyphenols are likely important factors affecting their antioxidant activity and inhibitory efficacy of reactive carbonyl species formation. Treatment with epicatechin would lower not only AGE formation but also AGE-induced cytotoxicity and oxidative stress to human retinal pigment epithelial (ARPE-19) cells. PMID:25208810

  15. Identification of oxidatively modified proteins in salt-stressed Arabidopsis: a carbonyl-targeted proteomics approach.

    PubMed

    Mano, Jun'ichi; Nagata, Mitsuaki; Okamura, Shoutarou; Shiraya, Takeshi; Mitsui, Toshiaki

    2014-07-01

    In plants, environmental stresses cause an increase in the intracellular level of reactive oxygen species (ROS), leading to tissue injury. To obtain biochemical insights into this damage process, we investigated the protein carbonyls formed by ROS or by the lipid peroxide-derived α,β-unsaturated aldehydes and ketones (i.e. reactive carbonyl species, or RCS) in the leaves of Arabidopsis thaliana under salt stress. A. thaliana Col-0 plants that we treated with 300 mM NaCl for 72 h under continuous illumination suffered irreversible leaf damage. Several RCS such as 4-hydroxy-(E)-2-nonenal (HNE) were increased within 12 h of this salt treatment. Immunoblotting using distinct antibodies against five different RCS, i.e. HNE, 4-hydroxy-(E)-2-hexenal, acrolein, crotonaldehyde and malondialdehyde, revealed that RCS-modified proteins accumulated in leaves with the progress of the salt stress treatment. The band pattern of Western blotting suggested that these different RCS targeted a common set of proteins. To identify the RCS targets, we collected HNE-modified proteins via an anti-HNE antiserum affinity trap and performed an isobaric tag for relative and absolute quantitation, as a quantitative proteomics approach. Seventeen types of protein, modified by 2-fold more in the stressed plants than in the non-stressed plants, were identified as sensitive RCS targets. With aldehyde-reactive probe-based affinity trapping, we collected the oxidized proteins and identified 22 additional types of protein as sensitive ROS targets. These RCS and ROS target proteins were distributed in the cytosol and apoplast, as well as in the ROS-generating organelles the peroxisome, chloroplast and mitochondrion, suggesting the participation of plasma membrane oxidation in the cellular injury. Possible mechanisms by which these modified targets cause cell death are discussed.

  16. Age-at-Onset in Late Onset Alzheimer Disease is Modified by Multiple Genetic Loci

    PubMed Central

    Naj, Adam C.; Jun, Gyungah; Reitz, Christiane; Kunkle, Brian W.; Perry, William; Park, YoSon; Beecham, Gary W.; Rajbhandary, Ruchita A.; Hamilton-Nelson, Kara L.; Wang, Li-San; Kauwe, John S.K.; Huentelman, Matthew J.; Myers, Amanda J.; Bird, Thomas D.; Boeve, Bradley F.; Baldwin, Clinton T.; Jarvik, Gail P.; Crane, Paul K.; Rogaeva, Ekaterina; Barmada, Michael M.; Demirci, F. Yesim; Cruchaga, Carlos; Kramer, Patricia; Ertekin-Taner, Nilufer; Hardy, John; Graff-Radford, Neill R.; Green, Robert C.; Larson, Eric B.; St George-Hyslop, Peter; Buxbaum, Joseph D.; Evans, Denis; Schneider, Julie A.; Lunetta, Kathryn L.; Kamboh, M. Ilyas; Saykin, Andrew J.; Reiman, Eric M.; De Jager, Philip L.; Bennett, David A.; Morris, John C.; Montine, Thomas J.; Goate, Alison M.; Blacker, Deborah; Tsuang, Debby W.; Hakonarson, Hakon; Kukull, Walter A.; Foroud, Tatiana M.; Martin, Eden R.; Haines, Jonathan L.; Mayeux, Richard; Farrer, Lindsay A.; Schellenberg, Gerard D.; Pericak-Vance, Margaret A.

    2015-01-01

    Importance As APOE locus variants contribute to both risk of late-onset Alzheimer disease and differences in age-at-onset, it is important to know if other established late-onset Alzheimer disease risk loci also affect age-at-onset in cases. Objectives To investigate the effects of known Alzheimer disease risk loci in modifying age-at-onset, and to estimate their cumulative effect on age-at-onset variation, using data from genome-wide association studies in the Alzheimer’s Disease Genetics Consortium (ADGC). Design, Setting and Participants The ADGC comprises 14 case-control, prospective, and family-based datasets with data on 9,162 Caucasian participants with Alzheimer’s occurring after age 60 who also had complete age-at-onset information, gathered between 1989 and 2011 at multiple sites by participating studies. Data on genotyped or imputed single nucleotide polymorphisms (SNPs) most significantly associated with risk at ten confirmed LOAD loci were examined in linear modeling of AAO, and individual dataset results were combined using a random effects, inverse variance-weighted meta-analysis approach to determine if they contribute to variation in age-at-onset. Aggregate effects of all risk loci on AAO were examined in a burden analysis using genotype scores weighted by risk effect sizes. Main Outcomes and Measures Age at disease onset abstracted from medical records among participants with late-onset Alzheimer disease diagnosed per standard criteria. Results Analysis confirmed association of APOE with age-at-onset (rs6857, P=3.30×10−96), with associations in CR1 (rs6701713, P=7.17×10−4), BIN1 (rs7561528, P=4.78×10−4), and PICALM (rs561655, P=2.23×10−3) reaching statistical significance (P<0.005). Risk alleles individually reduced age-at-onset by 3-6 months. Burden analyses demonstrated that APOE contributes to 3.9% of variation in age-at-onset (R2=0.220) over baseline (R2=0.189) whereas the other nine loci together contribute to 1.1% of

  17. Protein aggregates are associated with replicative aging without compromising protein quality control

    PubMed Central

    Saarikangas, Juha; Barral, Yves

    2015-01-01

    Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells. In budding yeast, various aging factors segregate to the aging (mother)-lineage, with poorly understood consequences. In this study, we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single, asymmetrically inherited deposit over the remaining lifespan. Surprisingly, deposit formation was not associated with stress or general decline in proteostasis. Rather, the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates. Deposit formation was dependent on Hsp42, which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery, including the prion Sup35. Importantly, loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell. Together, these data suggest that protein aggregation is an early aging-associated differentiation event in yeast, having a two-faceted role in organismal fitness. DOI: http://dx.doi.org/10.7554/eLife.06197.001 PMID:26544680

  18. Maize Seed Chitinase is Modified by a Protein Secreted by Bipolaris zeicola

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants contain defense mechanisms that prevent infection by most fungi. Some specialized fungi have the ability to overcome plant defenses. The Zea mays (maize) seed chitinase ChitA has been previously reported as an antifungal protein. Here we report that ChitA is converted to a modified form by...

  19. Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus.

    PubMed Central

    Tagawa, Y; Haritani, M; Ishikawa, H; Yuasa, N

    1993-01-01

    In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli. Images PMID:8478064

  20. Mass spectrometric analysis of protein tyrosine nitration in aging and neurodegenerative diseases.

    PubMed

    Yeo, Woon-Seok; Kim, Young Jun; Kabir, Mohammad Humayun; Kang, Jeong Won; Ahsan-Ul-Bari, Md; Kim, Kwang Pyo

    2015-01-01

    This review highlights the significance of protein tyrosine nitration (PTN) in signal transduction pathways, the progress achieved in analytical methods, and the implication of nitration in the cellular pathophysiology of aging and age-related neurodegenerative diseases. Although mass spectrometry of nitrated peptides has become a powerful tool for the characterization of nitrated peptides, the low stoichiometry of this modification clearly necessitates the use of affinity chromatography to enrich modified peptides. Analysis of nitropeptides involves identification of endogenous, intact modification as well as chemical conversion of the nitro group to a chemically reactive amine group and further modifications that enable affinity capture and enhance detectability by altering molecular properties. In this review, we focus on the recent progress in chemical derivatization of nitropeptides for enrichment and mass analysis, and for detection and quantification using various analytical tools. PTN participates in physiological processes, such as aging and neurodegenerative diseases. Accumulation of 3-nitrotyrosine has been found to occur during the aging process; this was identified through mass spectrometry. Further, there are several studies implicating the presence of nitrated tyrosine in age-related diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. PMID:24889964

  1. Translocator protein (TSPO) role in aging and Alzheimer's disease.

    PubMed

    Repalli, Jayanthi

    2014-01-01

    Cellular damage and deregulated apoptotic cell death lead to functional impairment, and a main consequence of these events is aging. Cellular damage is initiated by different stress/risk factors such as oxidative stress, inflammation, and heavy metals. These stress/risk factors affect the cellular homeostasis by altering methylation status of several aging and Alzheimer's disease associated genes; these effects can be manifested immediately after exposure to stress and at later stages of life. However, when cellular damage exceeds certain threshold levels apoptosis is initiated. This review discusses the stress factors involved in cellular damage and the role and potential of TSPO-mediated cell death in aging as well as in Alzheimer's disease, which is also characterized by extensive cell death. Mitochondrial-mediated apoptotic death through the release of cytochrome c is regulated by TSPO, and increased expression of this protein is observed in both elderly people and in patients with Alzheimer's disease. TSPO forms and mediates opening of the mitochondrial membrane pore, mPTP and oxidizes cardiolipin, and these events lead to the leakage of apoptotic death mediators, such as cytochrome c, resulting in cell death. However, TSPO has many proposed functions and can also increase steroid synthesis, which leads to inhibition of inflammation and inhibition of the release of apoptotic factors, thereby decreasing cell damage and promoting cell survival. Thus, TSPO mediates apoptosis and decreases the cell damage, which in turn dictates the process of aging as well as the functionality of organs such as the brain. TSPO modulation with ligands in the Alzheimer's disease mouse model showed improvement in behavioral symptoms, and studies in Drosophila species showed increased cell survival and prolonged lifespan in flies after TSPO inhibition. These data suggest that since effects/signs of stress can manifest at any time, prevention through change in lifestyle and TSPO

  2. Effects of extended aging and modified atmospheric packaging on beef top loin steak color.

    PubMed

    English, A R; Mafi, G G; VanOverbeke, D L; Ramanathan, R

    2016-04-01

    The objective of this study was to evaluate the effects of extended aging and modified atmospheric packaging on beef LM color. Using a randomized complete block design, each beef longissimus lumborum muscle ( = 10; USDA Choice, 3 d postmortem) was equally divided into 3 sections and randomly assigned to 1 of 3 aging periods (21, 42, or 62 d at 2°C). After respective aging, each loin section was cut into four 2.5-cm-thick steaks and randomly assigned to 1 of 3 packaging types (PVC, HiOx-MAP [80% oxygen and 20% carbon dioxide], or CO-MAP [0.4% carbon monoxide, 69.6% nitrogen, and 30% carbon dioxide]). The steaks were displayed under continuous fluorescent lighting for 6 d, and surface color was determined daily using a HunterLab Miniscan XE Plus spectrophotometer and a visual panel. The fourth steak was used to characterize oxygen consumption (OC), lipid oxidation, and metmyoglobin reducing activity (MRA) on 21, 42, and 62 d (before display). On d 6 display, MRA, OC, and lipid oxidation also were measured. An increase in aging time decreased ( < 0.0001) muscle pH. Loin sections aged for 42 and 62 d had a lower ( < 0.0002) pH compared with loin sections aged for 21 d. An aging period × packaging × display time interaction ( < 0.0001) resulted for a* values (redness), chroma, and visual color (muscle color and surface discoloration). As aging time increased, HiOx-MAP had the most discoloration ( < 0.0001) compared with other packaging types on d 6. At all aging periods, steaks packaged in CO-MAP had greater ( < 0.0001) MRA on d 6 than PVC and HiOx-MAP, whereas steaks packaged in HiOx-MAP had the least MRA ( < 0.0001). There were no differences ( = 0.34) in thiobarbituric acid reactive substances values between steaks aged for 21 and 42 d when steaks were packaged in CO-MAP and displayed for 6 d. However, steaks packaged in HiOx-MAP and displayed 6 d had greater ( < 0.0001) lipid oxidation than CO-MAP. Steaks packaged in HiOx-MAP had a lower ( < 0.0001) OC compared

  3. De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins.

    PubMed

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; Ambinder, Richard F; Hayward, S Diane

    2010-11-19

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line. PMID:20847044

  4. Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells.

    PubMed

    Eyckerman, Sven; Lemmens, Irma; Catteeuw, Dominiek; Verhee, Annick; Vandekerckhove, Joel; Lievens, Sam; Tavernier, Jan

    2005-06-01

    Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor-based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53. PMID:15908921

  5. Dietary protein modifies effect of plant extracts in the intestinal ecosystem of the pig at weaning.

    PubMed

    Manzanilla, E G; Pérez, J F; Martín, M; Blandón, J C; Baucells, F; Kamel, C; Gasa, J

    2009-06-01

    The plant extract mixture (XT) used in the present experiment, containing carvacrol, cinnamaldehyde, and capsicum oleoresin, has previously been shown to decrease diarrhea mortality and to modify the intestinal environment of pigs after weaning. However, results obtained among experiments have not been consistent. We hypothesized that dietary protein could be a main factor determining the effect of plant extracts on intestinal environment. Thus, in the present study we assessed the effects of XT in piglet diets with different protein sources and amounts. Pigs weaned at 20 +/- 1 d of age (n = 240) were allocated to 1 of 6 treatments, which followed a factorial arrangement, with 2 amounts (as-fed basis) of the XT (0 and 200 mg/kg) and 3 diets with various amounts of CP and protein sources. Diet FM18 contained 10% of low-temperature fish meal (LT-FM) and a CP level of 18%; diet SBM18 contained 5% of LT-FM plus 9% of full fat extruded soy and a CP level of 18%; and SBM20 diet contained 10% of LT-FM plus 6.3% of full fat extruded soy and a CP level of 20%. Growth performance of the animals was recorded for 14 d, but no differences were detected among treatments. Eight pigs per treatment were killed to examine variables describing aspects of gastrointestinal ecology. For diets containing 18% CP, FM18 and SBM18, XT tended to decrease ileal digestibility of OM (P = 0.064 and 0.071, respectively) and decreased starch digestibility (P = 0.032 and 0.014, respectively). It also reduced villi length (P = 0.003 and 0.013, respectively) and tended to decrease intraepithelial lymphocyte number (P = 0.051 and 0.100, respectively) in the proximal jejunum. The XT inclusion also increased ileal lactobacilli:enterobacteria (P = 0.017) ratio and decreased VFA production in the cecum (P = 0.045) for all diets. A decreased CP level appeared to favor the effects of the studied plant extracts in a positive or negative way depending on the variable measured. The microbial differences

  6. Atomic layer deposition modified track-etched conical nanochannels for protein sensing.

    PubMed

    Wang, Ceming; Fu, Qibin; Wang, Xinwei; Kong, Delin; Sheng, Qian; Wang, Yugang; Chen, Qiang; Xue, Jianming

    2015-08-18

    Nanopore-based devices have recently become popular tools to detect biomolecules at the single-molecule level. Unlike the long-chain nucleic acids, protein molecules are still quite challenging to detect, since the protein molecules are much smaller in size and usually travel too fast through the nanopore with poor signal-to-noise ratio of the induced transport signals. In this work, we demonstrate a new type of nanopore device based on atomic layer deposition (ALD) Al2O3 modified track-etched conical nanochannels for protein sensing. These devices show very promising properties of high protein (bovine serum albumin) capture rate with well time-resolved transport signals and excellent signal-to-noise ratio for the transport events. Also, a special mechanism involving transient process of ion redistribution inside the nanochannel is proposed to explain the unusual biphasic waveshapes of the current change induced by the protein transport.

  7. [The effect of modified nano-diamonds of detonation synthesis on the protein fractions of human blood].

    PubMed

    Botvich, Iu A; Olkhovskiĭ, I A; Baron, I I; Puzyr', A P; Baron, A V; Bondar', V S

    2013-11-01

    It is established that the modified nano-diamonds of detonation synthesis are able to bind serum proteins of human blood. The relative selectivity is established concerning the effect of modified nano-diamonds of detonation synthesis on beta2- and gamma-globulin fractions of serum. The evidence of concentration dependence of effect of modified nano-diamonds of detonation synthesis from serum proteins is established. The study results make it possible to consider modified nano-diamonds of detonation synthesis as a potential sorbent in technologies of hemodialysis, plasmapheresis, isolation of blood proteins and as a foundation for development of new systems of laboratory diagnostic.

  8. Estimating Modifying Effect of Age on Genetic and Environmental Variance Components in Twin Models.

    PubMed

    He, Liang; Sillanpää, Mikko J; Silventoinen, Karri; Kaprio, Jaakko; Pitkäniemi, Janne

    2016-04-01

    insights into age-specific heritability of BMI and evidence ofG×Einteractions. These findings highlight the fundamental importance and implication of the proposed models in facilitating twin studies to investigate the heritability specific to age and other modifying factors.

  9. Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging.

    PubMed

    Robinson, Renã A S; Evans, Adam R

    2012-06-01

    Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT(0), TMT(6), and iTRAQ(8) reagents and discuss limitations of the strategy. PMID:22509719

  10. DNA-modified Electrodes Fabricated using Copper-Free Click Chemistry for Enhanced Protein Detection

    PubMed Central

    Furst, Ariel L.; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development. PMID:24328347

  11. Folding behavior of ribosomal protein S6 studied by modified Go¯ -like model

    NASA Astrophysics Data System (ADS)

    Wu, L.; Zhang, J.; Wang, J.; Li, W. F.; Wang, W.

    2007-03-01

    Recent experimental and theoretical studies suggest that, although topology is the determinant factor in protein folding, especially for small single-domain proteins, energetic factors also play an important role in the folding process. The ribosomal protein S6 has been subjected to intensive studies. A radical change of the transition state in its circular permutants has been observed, which is believed to be caused by a biased distribution of contact energies. Since the simplistic topology-only Gō -like model is not able to reproduce such an observation, we modify the model by introducing variable contact energies between residues based on their physicochemical properties. The modified Gō -like model can successfully reproduce the Φ -value distributions, folding nucleus, and folding pathways of both the wild-type and circular permutants of S6. Furthermore, by comparing the results of the modified and the simplistic models, we find that the hydrophobic effect constructs the major force that balances the loop entropies. This may indicate that nature maintains the folding cooperativity of this protein by carefully arranging the location of hydrophobic residues in the sequence. Our study reveals a strategy or mechanism used by nature to get out of the dilemma when the native structure, possibly required by biological function, conflicts with folding cooperativity. Finally, the possible relationship between such a design of nature and amyloidosis is also discussed.

  12. Potential allergenicity research of Cry1C protein from genetically modified rice.

    PubMed

    Cao, Sishuo; He, Xiaoyun; Xu, Wentao; Luo, Yunbo; Ran, Wenjun; Liang, Lixing; Dai, Yunqing; Huang, Kunlun

    2012-07-01

    With the development of genetically modified crops, there has been a growing interest in available approaches to assess the potential allergenicity of novel gene products. We were not sure whether Cry1C could induce allergy. We examined the protein with three other proteins to determine the potential allergenicity of Cry1C protein from genetically modified rice. Female Brown Norway (BN) rats received 0.1 mg peanut agglutinin (PNA), 1mg potato acid phosphatase (PAP), 1mg ovalbumin (OVA) or 5 mg purified Cry1C protein dissolved in 1 mL water by daily gavage for 42 days to test potential allergenicity. Ten days after the last gavage, rats were orally challenged with antigens, and physiologic and immunologic responses were studied. In contrast to sensitization with PNA, PAP and OVA Cry1C protein did not induce antigen-specific IgG2a in BN rats. Cytokine expression, serum IgE and histamine levels and the number of eosinophils and mast cells in the blood of Cry1C group rats were comparable to the control group rats, which were treated with water alone. As Cry1C did not show any allergenicity, we make the following conclusion that the protein could be safety used in rice or other plants.

  13. Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature[W

    PubMed Central

    Armbruster, Ute; Labs, Mathias; Pribil, Mathias; Viola, Stefania; Xu, Wenteng; Scharfenberg, Michael; Hertle, Alexander P.; Rojahn, Ulrike; Jensen, Poul Erik; Rappaport, Fabrice; Joliot, Pierre; Dörmann, Peter; Wanner, Gerhard; Leister, Dario

    2013-01-01

    Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins. PMID:23839788

  14. Age-related variations of protein carbonyls in human saliva and plasma: is saliva protein carbonyls an alternative biomarker of aging?

    PubMed

    Wang, Zhihui; Wang, Yanyi; Liu, Hongchen; Che, Yuwei; Xu, Yingying; E, Lingling

    2015-06-01

    Free radical hypothesis which is one of the most acknowledged aging theories was developed into oxidative stress hypothesis. Protein carbonylation is by far one of the most widely used markers of protein oxidation. We studied the role of age and gender in protein carbonyl content of saliva and plasma among 273 Chinese healthy subjects (137 females and 136 males aged between 20 and 79) and discussed the correlation between protein carbonyl content of saliva and plasma. Protein carbonyl content of saliva and plasma were, respectively, 2.391 ± 0.639 and 0.838 ± 0.274 nmol/mg. Variations of saliva and plasma different age groups all reached significant differences in both male and female (all p < 0.05) while both saliva and plasma protein carbonyls were found to be significantly correlated with age (r = 0.6582 and r = 0.5176, all p < 0.001). Gender was discovered to be unrelated to saliva and plasma protein carbonyl levels (all p > 0.05). Saliva and plasma protein carbonyls were positively related (r = 0.4405, p < 0.001). Surprisingly, saliva and plasma protein carbonyls/ferric reducing ability of plasma (FRAP) ratios were proved to be significantly correlated with age (r = 0.7796 and r = 0.6938, all p < 0.001) while saliva protein carbonyls/FRAP ratio and plasma protein carbonyls/FRAP ratio were also correlated (r = 0.5573, p < 0.001). We concluded that saliva protein carbonyls seem to be an alternative biomarker of aging while the mechanisms of protein carbonylation and oxidative stress and the relationship between saliva protein carbonyls and diseases need to be further investigated.

  15. [Medical and biological evaluation of safety of protein concentrate from genetically-modified soybeans. Biochemical studies].

    PubMed

    Tutel'ian, V A; Kravchenko, L V; Lashneva, N V; Avren'eva, L I; Guseva, G V; Sorokina, E Iu; Chernysheva, O N

    1999-01-01

    The rats were fed with albuminous concentrate from the genetically modified soybean 40-3-2 ("Monsanto Co", USA) 1.25 g/rat/day for 5 months. Their blood, urea and liver were investigated to measure total protein and glucose levels, aminotransferase and alkaline phosphatase activities, pH, relative density and creatinine level in the urea, as well as hepatic enzyme activity of the I and II phases of xenobiotic metabolism, and the whole and non-sedimentated lysosomal enzyme activities. The lasting albuminous concentrate supplementation from the genetically modified soybean to the rat's diet has been shown to modify hepatocyte membrane function and enzymatic activity within physiological standards. It was not harmful to the adaptation systems.

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

    PubMed

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

    2015-08-01

    The aim of the present study was to investigate the differences in the expression of hippocampal proteins between normal control aged rats and aged rats with postoperative cognitive dysfunction (POCD). A total of 24 aged rats were randomly divided into a surgery group (n=12) and a control group (n=12). The rats in the surgery group were treated with 2 h isoflurane anesthesia and splenectomy, while the rats in the control group received 40% oxygen for 2 h without surgery. The cognitive functions of the two groups were examined using a Y-maze test. The protein expression profiles of the hippocampus of six aged rats (three rats with POCD and three from the normal control group) were assessed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. A total of three differential proteins were further confirmed between the POCD rats and normal rats using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The expression levels of 21 proteins in the rats with POCD were significantly different compared with the normal control rats. These proteins were functionally clustered to synaptic plasticity (three proteins), oxidative stress (four proteins), energy production (six proteins), neuroinflammation (three proteins) and glutamate metabolism (two proteins). In addition, three proteins (fatty acid binding protein 7, brain, glutamate dehydrogenase 1 and glutamine synthetase), associated with astrocytic function, were significantly different in the rats with POCD compared with those in the normal control (P<0.05). Similar changes in the mRNA expression levels of the three proteins in the hippocampi of POCD rats were also detected using RT-qPCR. Neuroinflammation, glutamate toxicity and oxidative stress were possibly involved in the pathological mechanism underlying POCD in aged rats. In addition, astrocytes may also be important in POCD in aged rats. PMID:25936412

  17. Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

    PubMed Central

    Arur, Swathi; Schedl, Tim

    2014-01-01

    Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

  18. Tuning of protein-surfactant interaction to modify the resultant structure.

    PubMed

    Mehan, Sumit; Aswal, Vinod K; Kohlbrecher, Joachim

    2015-09-01

    Small-angle neutron scattering and dynamic light scattering studies have been carried out to examine the interaction of bovine serum albumin (BSA) protein with different surfactants under varying solution conditions. We show that the interaction of anionic BSA protein (pH7) with surfactant and the resultant structure are strongly modified by the charge head group of the surfactant, ionic strength of the solution, and mixed surfactants. The protein-surfactant interaction is maximum when two components are oppositely charged, followed by components being similarly charged through the site-specific binding, and no interaction in the case of a nonionic surfactant. This interaction of protein with ionic surfactants is characterized by the fractal structure representing a bead-necklace structure of micellelike clusters adsorbed along the unfolded protein chain. The interaction is enhanced with ionic strength only in the case of site-specific binding of an anionic surfactant with an anionic protein, whereas it is almost unchanged for other complexes of cationic and nonionic surfactants with anionic proteins. Interestingly, the interaction of BSA protein with ionic surfactants is significantly suppressed in the presence of nonionic surfactant. These results with mixed surfactants thus can be used to fold back the unfolded protein as well as to prevent surfactant-induced protein unfolding. For different solution conditions, the results are interpreted in terms of a change in fractal dimension, the overall size of the protein-surfactant complex, and the number of micelles attached to the protein. The interplay of electrostatic and hydrophobic interactions is found to govern the resultant structure of complexes.

  19. Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

    PubMed

    Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno

    2016-06-01

    Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets.

  20. Retention of Proteins and Metalloproteins in Open Tubular Capillary Electrochromatography with Etched Chemically Modified Columns

    PubMed Central

    Pesek, Joseph J.; Matyska, Maria T.; Salgotra, Vasudha

    2010-01-01

    Etched chemically modified capillaries with two different bonded groups (pentyl and octadecyl) are compared for their migration behavior of several common proteins and metalloproteins as well as metalloproteinases. Migration times, efficiency and peak shape are evaluated over the pH range of 2.1 to 8.1 to determine any effects of the bonded group on the electrochromatographic behavior of these compounds. One goal was to determine if the relative hydrophobicity of the stationary phase has a significant effect on proteins in the open tubular format of capillary electrochromatography as it does in HPLC. Reproducibility of the migration times is also investigated. PMID:18850653

  1. [The investigation of presence of genetically modified protein in processed foodstuffs by ELISA test].

    PubMed

    Urbanek-Karłowska, Bogumiła; Sawilska-Rautenstrauch, Dorota; Jedra, Małgorzata

    2004-01-01

    The test based on immunoassay--TRAIT-RUR Toasted Soy Meal Kit was used for detection GMO-soy protein in the processed (heat treated) foodstuffs: bread, macaroni, sausages, ready-to-serve products and soya products (tofu, steaks). The threshold level is about 0,6% protein. The positive results were obtained for 27 from 106 investigated products. Only 5 foodstuffs were declared as containing GMO-soy in composition. The presence of genetically modified ingredients in foodstuffs must be controlled. The proper information should be labelled for the consumer.

  2. Predicting Cell Association of Surface-Modified Nanoparticles Using Protein Corona Structure - Activity Relationships (PCSAR).

    PubMed

    Kamath, Padmaja; Fernandez, Alberto; Giralt, Francesc; Rallo, Robert

    2015-01-01

    Nanoparticles are likely to interact in real-case application scenarios with mixtures of proteins and biomolecules that will absorb onto their surface forming the so-called protein corona. Information related to the composition of the protein corona and net cell association was collected from literature for a library of surface-modified gold and silver nanoparticles. For each protein in the corona, sequence information was extracted and used to calculate physicochemical properties and statistical descriptors. Data cleaning and preprocessing techniques including statistical analysis and feature selection methods were applied to remove highly correlated, redundant and non-significant features. A weighting technique was applied to construct specific signatures that represent the corona composition for each nanoparticle. Using this basic set of protein descriptors, a new Protein Corona Structure-Activity Relationship (PCSAR) that relates net cell association with the physicochemical descriptors of the proteins that form the corona was developed and validated. The features that resulted from the feature selection were in line with already published literature, and the computational model constructed on these features had a good accuracy (R(2)LOO=0.76 and R(2)LMO(25%)=0.72) and stability, with the advantage that the fingerprints based on physicochemical descriptors were independent of the specific proteins that form the corona.

  3. Computational allergenicity prediction of transgenic proteins expressed in genetically modified crops.

    PubMed

    Verma, Alok Kumar; Misra, Amita; Subash, Swarna; Das, Mukul; Dwivedi, Premendra D

    2011-09-01

    Development of genetically modified (GM) crops is on increase to improve food quality, increase harvest yields, and reduce the dependency on chemical pesticides. Before their release in marketplace, they should be scrutinized for their safety. Several guidelines of different regulatory agencies like ILSI, WHO Codex, OECD, and so on for allergenicity evaluation of transgenics are available and sequence homology analysis is the first test to determine the allergenic potential of inserted proteins. Therefore, to test and validate, 312 allergenic, 100 non-allergenic, and 48 inserted proteins were assessed for sequence similarity using 8-mer, 80-mer, and full FASTA search. On performing sequence homology studies, ~94% the allergenic proteins gave exact matches for 8-mer and 80-mer homology. However, 20 allergenic proteins showed non-allergenic behavior. Out of 100 non-allergenic proteins, seven qualified as allergens. None of the inserted proteins demonstrated allergenic behavior. In order to improve the predictability, proteins showing anomalous behavior were tested by Algpred and ADFS separately. Use of Algpred and ADFS softwares reduced the tendency of false prediction to a great extent (74-78%). In conclusion, routine sequence homology needs to be coupled with some other bioinformatic method like ADFS/Algpred to reduce false allergenicity prediction of novel proteins.

  4. The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins.

    PubMed

    Evans, T C; Benner, J; Xu, M Q

    1999-06-25

    Mini-inteins derived from Synechocystis sp. (Ssp DnaB intein) and Mycobacterium xenopi (Mxe GyrA intein) that have been modified to cleave peptide bonds at their C and N termini, respectively, were cloned in-frame to the N and C termini of a target protein. Peptide bond cleavage of the modified inteins generated an N-terminal cysteine and a C-terminal thioester on the same protein. These complementary reactive groups underwent intra- or intermolecular condensation to generate circular or polymeric protein species with a new peptide bond at the site of ligation. Three cyclic peptides, BBP, an organ specific localization peptide; RGD, an inhibitor of platelet aggregation; and CDR-H3/C2, which inhibits HIV-1 replication, were isolated using the two-intein system. BBP, RGD, and CDR-H3/C2 had masses of 977.1, 1119.9, and 2098.6 g/mol, respectively, as determined by matrix-assisted laser desorption-time of flight mass spectrometry, which agreed well with the values of 977.2, 1120.3, and 2098.3 g/mol, respectively, predicted for the cyclic species. This system was used to cyclize proteins as large as 395 amino acids. Furthermore, multimers of thioredoxin were formed upon concentration of the reactive species, indicating the potential to form novel biomaterials based on fibrous proteins.

  5. G-protein Coupled Receptor 30 Interacts with Receptor Activity Modifying Protein 3 and Confers Sex-Dependent Cardioprotection

    PubMed Central

    Lenhart, Patricia M.; Broselid, Stefan; Barrick, Cordelia J.; Leeb-Lundberg, L.M. Fredrik; Caron, Kathleen M.

    2013-01-01

    Receptor activity modifying protein 3 (RAMP3) is a single pass transmembrane protein known to interact with and affect the trafficking of several G-protein coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with G-protein coupled receptor 30 (GPR30), also known as G-protein estrogen receptor 1 (GPER1). GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Utilizing bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3−/− mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3−/− mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3- and sex-dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo, and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  6. Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin.

    PubMed

    Masuda, Y; Nirasawa, S; Nakaya, K; Kurihara, Y

    1995-08-19

    A cDNA clone encoding a taste-modifying protein, miraculin (MIR), was isolated and sequenced. The encoded precursor to MIR was composed of 220 amino acid (aa) residues, including a possible signal sequence of 29 aa. Northern blot analysis showed that the mRNA encoding MIR was already expressed in fruits of Richadella dulcifica at 3 weeks after pollination and was present specifically in the pulp. PMID:7665074

  7. Quantitative proteomics analysis of specific protein expression and oxidative modification in aged senescence-accelerated-prone 8 mice brain.

    PubMed

    Poon, H F; Castegna, A; Farr, S A; Thongboonkerd, V; Lynn, B C; Banks, W A; Morley, J E; Klein, J B; Butterfield, D A

    2004-01-01

    The senescence-accelerated mouse (SAM) is a murine model of accelerated senescence that was established using phenotypic selection. The SAMP series includes nine substrains, each of which exhibits characteristic disorders. SAMP8 is known to exhibit age-dependent learning and memory deficits. In our previous study, we reported that brains from 12-month-old SAMP8 have greater protein oxidation, as well as lipid peroxidation, compared with brains from 4-month-old SAMP8 mice. In order to investigate the relation between age-associated oxidative stress on specific protein oxidation and age-related learning and memory deficits in SAMP8, we used proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. We report here that in 12 month SAMP8 mice brains the expressions of neurofilament triplet L protein, lactate dehydrogenase 2 (LDH-2), heat shock protein 86, and alpha-spectrin are significantly decreased, while the expression of triosephosphate isomerase (TPI) is increased compared with 4-month-old SAMP8 brains. We also report that the specific protein carbonyl levels of LDH-2, dihydropyrimidinase-like protein 2, alpha-spectrin and creatine kinase, are significantly increased in the brain of 12-month-old SAMP8 mice when compared with the 4-month-old SAMP8 brain. These findings are discussed in reference to the effect of specific protein oxidation and changes of expression on potential mechanisms of abnormal alterations in metabolism and neurochemicals, as well as to the learning and memory deficits in aged SAMP8 mice.

  8. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    PubMed

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging

  9. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    PubMed

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

  10. Inhibition of x-box binding protein 1 reduces tunicamycin-induced apoptosis in aged murine macrophages.

    PubMed

    Song, Yang; Shen, Hua; Du, Wei; Goldstein, Daniel R

    2013-10-01

    Endoplasmic reticulum (ER) stress is induced by the accumulation of unfolded and misfolded proteins in the ER. Although apoptosis induced by ER stress has been implicated in several aging-associated diseases, such as atherosclerosis, it is unclear how aging modifies ER stress response in macrophages. To decipher this relationship, we assessed apoptosis in macrophages isolated from young (1.5-2 months) and aged (16-18 months) mice and exposed the cells to the ER stress inducer tunicamycin. We found that aged macrophages exhibited more apoptosis than young macrophages, which was accompanied by reduced activation of phosphorylated inositol-requiring enzyme-1 (p-IRE1α), one of the three key ER stress signal transducers. Reduced gene expression of x-box binding protein 1 (XBP1), a downstream effector of IRE1α, enhanced p-IRE1α levels and reduced apoptosis in aged, but not young macrophages treated with tunicamycin. These findings delineate a novel, age-dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggest an important protective role of IRE1α in aging-associated ER stress-induced apoptosis. This novel pathway may not only be important in our understanding of longevity, but may also have important implications for pathogenesis and potential treatment of aging-associated diseases in general.

  11. Age estimation by modified Demirjian's method (2004) and its applicability in Tibetan young adults: A digital panoramic study

    PubMed Central

    Bijjaragi, Shobha C; Sangle, Varsha A; Saraswathi, FK; Patil, Veerendra S; Ashwini Rani, SR; Bapure, Sunil K

    2015-01-01

    Context: Estimation of the age is a procedure adopted by anthropologists, archeologists and forensic scientists. Different methods have been undertaken. However none of them meet the standards as Demirjian's method since 1973. Various researchers have applied this method, in both original and modified form (Chaillet and Demirjian in 2004) in different ethnic groups and the results obtained were not satisfactory. Aims: To determine the applicability and accuracy of modified Demirjian's method of dental age estimation (AE) in 8–18 year old Tibetan young adults to evaluate the interrelationship between dental and chronological age and the reliability between intra- and inter observer relationship. Settings and Design: Clinical setting and computerized design. Subjects and Methods: A total of 300 Tibetan young adults with an age range from 8 to 18 years were recruited in the study. Digital panoramic radiographs (DPRs) were evaluated as per the modified Demirjian's method (2004). Statistical Analysis Used: Pearson correlation, paired t-test, linear regression analysis. Results: Inter -and intraobserver reliability revealed a strong agreement. A positive and strong association was found between chronological age and estimated dental age (r = 0.839) with P < 0.01. Modified Demirjian method (2004) overestimated the age by 0.04 years (2.04 months)in Tibetan young adults. Conclusions: Results suggest that, the modified Demirjian method of AE is not suitable for Tibetan young adults. Further studies: With larger sample size and comparision with different methods of AE in a given population would be an interesting area for future research. PMID:26097317

  12. Protein adsorption to poly(ethylenimine)-modified Sepharose FF: III. Comparison between different proteins.

    PubMed

    Hong, Yan; Liu, Na; Wei, Wei; Yu, Lin-Ling; Ma, Guanghui; Sun, Yan

    2014-05-16

    Previously, we studied bovine serum albumin (BSA) uptake to poly(ethylenimine) (PEI)-grafted Sepharose resins, and an ionic capacity (IC) range (600-740mmol/L) for steep increases of both protein capacity (qm) and effective pore diffusion coefficient (De) was found. In this work, seven PEI-grafted Sepharose FF resins at IC range of 270-1030mmol/L were synthesized to investigate the effect of protein properties on the adsorption and uptake kinetics using BSA and γ-globulin as two model proteins. For BSA, the change trends of qm and De values with IC were well consistent with the previous results. For γ-globulin, the qm values increased slowly till reaching a maximum value at IC=560mmol/L and then decreased rapidly at IC>560mol/L. The De values nearly kept unchanged at low ICs (IC<460mmol/L), and increased steeply at IC>460mmol/L till reaching a maximum at 680mmol/L (De/D0=0.48±0.01). After that increase, the De values for γ-globulin dropped quickly at IC>680mol/L, which was not observed for BSA. It is interesting to note that in the narrow IC range of 460-680mmol/L, the De values of γ-globulin increased dramatically for more than four folds. Moreover, it is notable that the IC range where the hopping of De values occurred for γ-globulin was earlier than that for BSA (460 vs. 560mmol/L). The earlier hopping of γ-globulin uptake rate was attributed to its larger size and less net charge, which facilitated the happenings of the "chain delivery" effect. The quick drops of both qm and De values for γ-globulin at IC>680mmol/L were considered due to its large size, which led to the significant decrease of its effective pore volume. The results indicate that both PEI layer and protein size played important roles in protein adsorption to PEI-grafted resins, and further prove the "chain delivery" effect did contributed significantly to the uptake rate hopping in the PEI-grafted resins. This work could also help the design and selection of resins based on protein

  13. Miraculin, a taste-modifying protein is secreted into intercellular spaces in plant cells.

    PubMed

    Hirai, Tadayoshi; Sato, Mayuko; Toyooka, Kiminari; Sun, Hyeon-Jin; Yano, Megumu; Ezura, Hiroshi

    2010-02-15

    A taste-modifying protein, miraculin, is highly accumulated in ripe fruit of miracle fruit (Richadella dulcifica) and the content can reach up to 10% of the total soluble protein in these fruits. Although speculated for decades that miraculin is secreted into intercellular spaces in miracle fruit, no evidence exists of its cellular localization. To study the cellular localization of miraculin in plant cells, using miracle fruit and transgenic tomato that constitutively express miraculin, immunoelectron microscopy, imaging GFP fusion proteins, and immunological detection of secreted proteins in culture medium of transgenic tomato were carried out. Immunoelectron microscopy showed the specific accumulation of miraculin in the intercellular layers of both miracle fruit and transgenic tomato. Imaging GFP fusion protein demonstrated that the miraculin-GFP fusion protein was accumulated in the intercellular spaces of tomato epidermal cells. Immunological detection of secreted proteins in culture medium of transgenic tomato indicated that miraculin was secreted from the roots of transgenic tomato expressing miraculin. This study firstly showed the evidences of the intercellular localization of miraculin, and provided a new insight of biological roles of miraculin in plants. PMID:19712996

  14. The Level of Protein in Milk Formula Modifies Ileal Sensitivity to LPS Later in Life in a Piglet Model

    PubMed Central

    Gras-Le Guen, Christèle; Lallès, Jean-Paul; Le Huërou-Luron, Isabelle; Boudry, Gaëlle

    2011-01-01

    Background Milk formulas have higher protein contents than human milk. This high protein level could modify the development of intestinal microbiota, epithelial barrier and immune functions and have long-term consequences. Methodology/Principal findings We investigated the effect of a high protein formula on ileal microbiota and physiology during the neonatal period and later in life. Piglets were fed from 2 to 28 days of age either a normoprotein (NP, equivalent to sow milk) or a high protein formula (HP, +40% protein). Then, they received the same solid diet until 160 days. During the formula feeding period ileal microbiota implantation was accelerated in HP piglets with greater concentrations of ileal bacteria at d7 in HP than NP piglets. Epithelial barrier function was altered with a higher permeability to small and large probes in Ussing chambers in HP compared to NP piglets without difference in bacterial translocation. Infiltration of T cells was increased in HP piglets at d28. IL-1β and NF-κB sub-units mRNA levels were reduced in HP piglets at d7 and d28 respectively; plasma haptoglobin also tended to be reduced at d7. Later in life, pro-inflammatory cytokines secretion in response to high doses of LPS in explants culture was reduced in HP compared to NP piglets. Levels of mRNA coding the NF-κB pathway sub-units were increased by the challenge with LPS in NP piglets, but not HP ones. Conclusions/Significance A high protein level in formula affects the postnatal development of ileal microbiota, epithelial barrier and immune function in piglets and alters ileal response to inflammatory mediators later in life. PMID:21573022

  15. G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection.

    PubMed

    Lenhart, Patricia M; Broselid, Stefan; Barrick, Cordelia J; Leeb-Lundberg, L M Fredrik; Caron, Kathleen M

    2013-01-01

    Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3(-/-) mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3(+)(/)(+) and Ramp3(-/-) mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  16. Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

    PubMed Central

    Davies, Douglas R.; Gelinas, Amy D.; Zhang, Chi; Rohloff, John C.; Carter, Jeffrey D.; O’Connell, Daniel; Waugh, Sheela M.; Wolk, Steven K.; Mayfield, Wesley S.; Burgin, Alex B.; Edwards, Thomas E.; Stewart, Lance J.; Gold, Larry; Janjic, Nebojsa; Jarvis, Thale C.

    2012-01-01

    Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets. PMID:23139410

  17. Pseudomonas aeruginosa PA1006 Is a Persulfide-Modified Protein That Is Critical for Molybdenum Homeostasis

    PubMed Central

    Tombline, Gregory; Schwingel, Johanna M.; Lapek, John D.; Friedman, Alan E.; Darrah, Thomas; Maguire, Michael; Van Alst, Nadine E.; Filiatrault, Melanie J.; Iglewski, Barbara H.

    2013-01-01

    A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) “top-down” analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein’s persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant. PMID:23409003

  18. CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection.

    PubMed

    Horká, Marie; Růzicka, Filip; Holá, Veronika; Slais, Karel

    2007-07-01

    The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.

  19. A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses*

    PubMed Central

    Song, Xuezheng; Yu, Hai; Chen, Xi; Lasanajak, Yi; Tappert, Mary M.; Air, Gillian M.; Tiwari, Vinod K.; Cao, Hongzhi; Chokhawala, Harshal A.; Zheng, Haojie; Cummings, Richard D.; Smith, David F.

    2011-01-01

    Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2–3- and α2–6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2–6- and α2–3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-d-glycero-d-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2–6-linked Neu5Ac9Lt or α2–6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions. PMID:21757734

  20. Controlled release of NELL-1 protein from chitosan/hydroxyapatite-modified TCP particles.

    PubMed

    Zhang, Yulong; Dong, Rui; Park, Yujin; Bohner, Marc; Zhang, Xinli; Ting, Kang; Soo, Chia; Wu, Benjamin M

    2016-09-10

    NEL-like molecule-1 (NELL-1) is a novel osteogenic protein that showing high specificity to osteochondral cells. It was widely used in bone regeneration research by loading onto carriers such as tricalcium phosphate (TCP) particles. However, there has been little research on protein controlled release from this material and its potential application. In this study, TCP was first modified with a hydroxyapatite coating followed by a chitosan coating to prepare chitosan/hydroxyapatite-coated TCP particles (Chi/HA-TCP). The preparation was characterized by SEM, EDX, FTIR, XRD, FM and Zeta potential measurements. The NELL-1 loaded Chi/HA-TCP particles and the release kinetics were investigated in vitro. It was observed that the Chi/HA-TCP particles prepared with the 0.3% (wt/wt) chitosan solution were able to successfully control the release of NELL-1 and maintain a slow, steady release for up to 28 days. Furthermore, more than 78% of the loaded protein's bioactivity was preserved in Chi/HA-TCP particles over the period of the investigation, which was significantly higher than that of the protein released from hydroxyapatite coated TCP (HA-TCP) particles. Collectively, this study suggests that the osteogenic protein NELL-1 showed a sustained release pattern after being encapsulated into the modified Chi/HA-TCP particles, and the NELL-1 integrated composite of Chi/HA-TCP showed a potential to function as a protein delivery carrier and as an improved bone matrix for use in bone regeneration research. PMID:27349789

  1. Quantification of Protein Expression Changes in the Aging Left Ventricle of Rattus norvegicus

    PubMed Central

    Grant, Jennifer E.; Bradshaw, Amy D.; Schwacke, John H.; Baicu, Catalin F.; Zile, Michael R.; Schey, Kevin L.

    2009-01-01

    As the heart ages, electrophysiological and biochemical changes can occur, and the ventricle in many cases loses distensibility, impairing diastolic function. How the proteomic signature of the aged ventricle is unique in comparison to young hearts is still under active investigation. We have undertaken a quantitative proteomics study of aging left ventricles (LVs) utilizing the isobaric Tagging for Relative and Absolute Quantification (iTRAQ) methodology. Differential protein expression was observed for 117 proteins including proteins involved in cell signaling, the immune response, structural proteins, and proteins mediating responses to oxidative stress. For many of these proteins, this is the first report of an association with the aged myocardium. Additionally, two proteins of unknown function were identified. This work serves as the basis for making future comparisons of the aged left ventricle proteome to that of left ventricles obtained from other models of disease and heart failure. PMID:19603826

  2. Molecular mechanisms of the action of miraculin, a taste-modifying protein.

    PubMed

    Misaka, Takumi

    2013-03-01

    Miraculin (MCL) is a homodimeric protein isolated from the fruits of Richadella dulcifica, a shrub native to West Africa. Although it is flat in taste at neutral pH, MCL has taste-modifying activity in which sour stimuli produce a sweet perception. Once MCL enters the mouth, strong sweetness can be detected for more than 1 h each time we taste a sour solution. While the human sweet taste receptor (hT1R2-hT1R3) has been identified, the molecular mechanisms underlying the taste-modifying activity of MCL remain unclear. Recently, experimental evidence has been published demonstrating the successful quantitative evaluation of the acid-induced sweetness of MCL using a cell-based assay system. The results strongly suggested that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH. Since sweet-tasting proteins may be used as low-calorie sweeteners because they contain almost no calories, it is expected that MCL will be used in the near future as a new low-calorie sweetener or to modify the taste of sour fruits. PMID:23466289

  3. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  4. ML3 Is a NEDD8- and Ubiquitin-Modified Protein1[C][W][OPEN

    PubMed Central

    Hakenjos, Jana P.; Bejai, Sarosh; Ranftl, Quirin; Behringer, Carina; Vlot, A. Corina; Absmanner, Birgit; Hammes, Ulrich; Heinzlmeir, Stephanie; Kuster, Bernhard; Schwechheimer, Claus

    2013-01-01

    NEDD8 (NEURAL PRECURSOR CELL-EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED PROTEIN8) is an evolutionarily conserved 8-kD protein that is closely related to ubiquitin and that can be conjugated like ubiquitin to specific lysine residues of target proteins in eukaryotes. In contrast to ubiquitin, for which a broad range of substrate proteins are known, only a very limited number of NEDD8 target proteins have been identified to date. Best understood, and also evolutionarily conserved, is the NEDD8 modification (neddylation) of cullins, core subunits of the cullin-RING-type E3 ubiquitin ligases that promote the polyubiquitylation of degradation targets in eukaryotes. Here, we show that Myeloid differentiation factor-2-related lipid-recognition domain protein ML3 is an NEDD8- as well as ubiquitin-modified protein in Arabidopsis (Arabidopsis thaliana) and examine the functional role of ML3 in the plant cell. Our analysis indicates that ML3 resides in the vacuole as well as in endoplasmic reticulum (ER) bodies. ER bodies are Brassicales-specific ER-derived organelles and, similar to other ER body proteins, ML3 orthologs can only be identified in this order of flowering plants. ML3 gene expression is promoted by wounding as well as by the phytohormone jasmonic acid and repressed by ethylene, signals that are known to induce and repress ER body formation, respectively. Furthermore, ML3 protein abundance is dependent on NAI1, a master regulator of ER body formation in Arabidopsis. The regulation of ML3 expression and the localization of ML3 in ER bodies and the vacuole is in agreement with a demonstrated importance of ML3 in the defense to herbivore attack. Here, we extend the spectrum of ML3 biological functions by demonstrating a role in the response to microbial pathogens. PMID:23903439

  5. [Studies on protein-based identification method of genetically modified capsicum].

    PubMed

    Liu, Jianjun; Deng, Pingjian; Fang, Shisong; Zhao, Jin

    2003-03-01

    The detection system based on protein is a method to evaluate the safety of genetically modified foods (GMF). Using cecropin BD gene in capsicum, a detecting method was set up. It is a system of evaluating the real expressive condition and safety of the foreign target protein of GMF. In this studies, with the preformative technic method, a satisfactory results by making use of hemolymph of immunized pupae of Antheraea pernyi as standard experimental material was achieved, comparing with the realities of the goal protein expressive condition of cecropin D gene in capsicum. The detecting steps were as following: the goal protein from material was extracted roughly, then with CM-Sepharose-FF ion-exchange chromatography twice, the goal protein was purified moderately. The purified product was identified by detecting the anti-bacterial activity, electrophoresis, biological auto-photography of the goal protein and MADDI-TOF mass spectrum. The results showed that the expressive foreign target protein in transgenic capsicum was in accordance with standard protein in the physical and chemical property, anti-bacterial activity and molecular weight. It indicated that expression of the target gene in capsicum is real, it corresponded to expected value. The separation, purification and identification methods of cecropin D were established in the study. By means of the comparative experiments about anti-bacterial activity and molecular weight of anti-bacterial peptide(ABP) from GM-capsicum and hemolymph of immunized pupae of Antheraea pernyi, the identification method of target protein from GM-capsicum was set up. The method is easy to be operated, fast and feasible.

  6. The coming of age of de novo protein design.

    PubMed

    Huang, Po-Ssu; Boyken, Scott E; Baker, David

    2016-01-01

    There are 20(200) possible amino-acid sequences for a 200-residue protein, of which the natural evolutionary process has sampled only an infinitesimal subset. De novo protein design explores the full sequence space, guided by the physical principles that underlie protein folding. Computational methodology has advanced to the point that a wide range of structures can be designed from scratch with atomic-level accuracy. Almost all protein engineering so far has involved the modification of naturally occurring proteins; it should now be possible to design new functional proteins from the ground up to tackle current challenges in biomedicine and nanotechnology. PMID:27629638

  7. Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin.

    PubMed

    Theerasilp, S; Hitotsuya, H; Nakajo, S; Nakaya, K; Nakamura, Y; Kurihara, Y

    1989-04-25

    The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 263, 11536-11539) was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor. PMID:2708331

  8. Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis.

    PubMed

    Han, Wenjuan; Yamauchi, Mika; Hasegawa, Urara; Noda, Masanori; Fukui, Kiichi; van der Vlies, André J; Uchiyama, Susumu; Uyama, Hiroshi

    2015-05-01

    Polymer-based monoliths with interconnected porous structure have attracted much attention as a high-performance stationary phase for online digestion liquid chromatography-mass spectrometry (LC-MS) system. In this study, a poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith prepared via thermally induced phase separation (TIPS) was used as a solid support to covalently immobilize pepsin. The PGM monolith was modified with aminoacetal to yield an aldehyde-bearing (PGM-CHO) monolith. Pepsin was immobilized onto the PGM-CHO monolith via reductive amination. The immobilized pepsin showed better pH and thermal stability compared with free pepsin. Furthermore, the PGM-CHO monolith modified with pepsin was applied for online protein digestion followed by LC-MS and LC-MS/MS analyses. As a result, a larger number of peptides are reproducibly identified compared to those by polystyrene/divinylbenzene particle (POROS)-based online pepsin column.

  9. Solution behavior of synthetic silk peptides and modified recombinant silk proteins

    NASA Astrophysics Data System (ADS)

    Foo, C. Wong Po; Bini, E.; Huang, J.; Lee, S. Y.; Kaplan, D. L.

    2006-02-01

    Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline β-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the α-helical structure and decreases in the β-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in β-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties.

  10. Aging characteristics of the Al-Si-Cu-Mg cast alloy modified with transition metals Zr, V and Ti

    NASA Astrophysics Data System (ADS)

    Czerwinski, F.; Shaha, S. K.; Kasprzak, W.; Friedman, J.; Chen, D. L.

    2016-03-01

    The hypoeutectic Al-7Si-1Cu-0.5Mg base alloy was modified with different contents of Zr, V and Ti. The wedge-shape samples with varying solidification rates during casting were subjected to isochronal aging at temperatures up to 500 °C. Moreover, as-cast and solution treated alloys were subjected to long-term isothermal aging at 150°C. As a reference, the A380 alloy, seen as commercial standard for the automotive application target, was used. The modified alloys exerted different aging characteristics than the A380 grade with higher peak hardness and lower temperature of alloy softening. Besides, the influence of the applied solidification rates on hardness after aging was less pronounced in modified alloys than in the A380 grade. For three combinations of Zr, V and Ti tested with contents of individual elements ranging from 0.14 to 0.47%, no essential differences in aging characteristics were recorded. The results are discussed in terms of the role of chemistry and heat treatment in generating precipitates contributing to the thermal stability of Al based alloys.

  11. The influence of thermal aging on the microstructure and fatigue properties of modified 9Cr-1Mo steel

    SciTech Connect

    Gieseke, B.G.; Brinkman, C.R.; Maziasz, P.J.

    1992-12-31

    Results of elevated-temperature low cycle fatigue and creep-fatigue tests are for one heat of modified 9Cr-1Mo steel in the normalized and tempered condition, after pre-aging 50,000 h at 538 and 593C, and after pre-aging for 75,000 h at 538C. These data show that pre-aging reduces the low cycle fatigue and creepfatigue lives in comparison to unaged material. The magnitude of these reductions are discussed along with the impact of pre-aging on the creep-fatigue damage diagrams. The effect of environment on creep-fatigue life of unaged modified 9Cr-1Mo steels is also addressed. Transmission electron microscopy explains changes in mechanical properties due to thermal aging. In the unaged alloy, TEM shows that dynamic recovery/recrystallization is occurring after significant strain-induced dislocation hardening around a stationary and stable array of as-tempered carbides during creep-fatigue. In contrast creep-fatigue testing of the pre-aged alloy produced a much coarser cellular subgrain structure and dislocation recovery without recrystallization. Aging causes as-tempered carbide dissolution and/or reprecipitation together with additional precipitation of Laves (Fe{sub 2}Mo) phase, which removes some of the precipitate-strengthening effects, and depletes solid-solution hardening effects on the dislocation networks and subgrain boundary structures.

  12. Genomewide linkage scan reveals novel loci modifying age of onset of Huntington's disease in the Venezuelan HD kindreds.

    PubMed

    Gayán, Javier; Brocklebank, Denise; Andresen, J Michael; Alkorta-Aranburu, Gorka; Zameel Cader, M; Roberts, Simone A; Cherny, Stacey S; Wexler, Nancy S; Cardon, Lon R; Housman, David E

    2008-07-01

    The age of onset of Huntington's disease (HD) is inversely correlated with the CAG length in the HD gene. The CAG repeat length accounts for 70% of the variability in HD age of onset. However, 90% of individuals worldwide with expanded alleles possess between 40 and 50 CAG repeat lengths in their HD gene. For these people, the size of their repeat only determines 44% of the variability in their age of onset. Once the effect of the CAG repeat has been accounted for, the residual variance in age of onset is a heritable trait. Targeted candidate gene studies and a genome scan have suggested some loci as potential modifiers of the age of onset of HD. We analyzed the large Venezuelan kindreds in which the HD gene was originally identified. These kindreds offer greater analytic power than standard sib-pair designs. We developed novel pedigree-member selection procedures to maximize power. Using a 5,858-single-nucleotide-polymorphism marker panel, we performed a genomewide linkage analysis. We discovered two novel loci on chromosome 2. Chromosome 2p25 (logarithm of the odds ratio (LOD)=4.29) and 2q35 (LOD=3.39) may contain genes that modify age of onset. A third linkage peak on chromosome 6q22 (LOD=2.48) may confirm the most promising locus from a previous genome scan. Two other candidate loci are suggestive on chromosome 5 (LOD=3.31 at 5p14 and LOD=3.14 at 5q32). All these regions harbor candidate genes that are potential HD modifier genes. Finding these modifier genes can reveal accessible and promising new therapeutic pathways and targets to ameliorate and cure HD.

  13. Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.

    PubMed

    Li, Yaojun; Shu, Yiwei; Peng, Changchao; Zhu, Lin; Guo, Guangyu; Li, Ning

    2012-08-01

    Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

  14. Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)*

    PubMed Central

    Weston, Cathryn; Lu, Jing; Li, Naichang; Barkan, Kerry; Richards, Gareth O.; Roberts, David J.; Skerry, Timothy M.; Poyner, David; Pardamwar, Meenakshi; Reynolds, Christopher A.; Dowell, Simon J.; Willars, Gary B.; Ladds, Graham

    2015-01-01

    The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development. PMID:26198634

  15. Protein Adsorption on Chemically Modified Block Copolymer Nanodomains: Influence of Charge and Flow.

    PubMed

    Silverstein, Joshua S; Casey, Brendan J; Kofinas, Peter; Dair, Benita J

    2016-02-01

    Understanding the interactions of biomacromolecules with nanoengineered surfaces is vital for assessing material biocompatibility. This study focuses on the dynamics of protein adsorption on nanopatterned block copolymers (BCPs). Poly(styrene)-block-poly(1,2-butadiene) BCPs functionalized with an acid, amine, amide, or captopril moieties were processed to produce nanopatterned films. These films were characterized using water contact angle measurements and atomic force microscopy in air and liquid to determine how the modification process affected. wettability and swelling. Protein adsorption experiments were conducted under static and dynamic conditions via a quartz crystal microbalance with dissipation. Proteins of various size, charge, and stability were investigated to determine whether their physical characteristics affected adsorption. Significantly decreased contact angles were caused by selective swelling of modified BCP domains. The results indicate that nanopatterned chemistry and experimental conditions strongly impact adsorption dynamics. Depending on the structural stability of the protein, polyelectrolyte surfaces significantly increased adsorption over controls. Further analysis suggested that protein stability may correlate with dissipation versus frequency plots. PMID:27433605

  16. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins

    PubMed Central

    Song, Jing; Durrin, Linda K.; Wilkinson, Thomas A.; Krontiris, Theodore G.; Chen, Yuan

    2004-01-01

    Posttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been established as an important regulatory mechanism. However, in most cases it is not clear how sumoylation regulates various cellular functions. Emerging evidence suggests that sumoylation may play a general role in regulating protein-protein interactions, as shown in RanBP2/Nup358 and RanGAP1 interaction. In this study, we have defined an amino acid sequence motif that binds SUMO. This motif, V/I-X-V/I-V/I, was identified by NMR spectroscopic characterization of interactions among SUMO-1 and peptides derived from proteins that are known to bind SUMO or sumoylated proteins. This motif binds all SUMO paralogues (SUMO-1-3). Using site-directed mutagenesis, we also show that this SUMO-binding motif in RanBP2/Nup358 is responsible for the interaction between RanBP2/Nup358 and sumoylated RanGAP1. The SUMO-binding motif exists in nearly all proteins known to be involved in SUMO-dependent processes, suggesting its general role in sumoylation-dependent cellular functions. PMID:15388847

  17. Changes in meat quality traits and sarcoplasmic proteins during aging in three different cattle breeds.

    PubMed

    Marino, R; Albenzio, M; della Malva, A; Caroprese, M; Santillo, A; Sevi, A

    2014-10-01

    The effects of breed and aging time (1, 7, 14, 21d) were evaluated on physical meat properties and on sarcoplasmic protein changes in 24 young bulls from Romagnola×Podolian, Podolian and Friesian breeds. Aging affects lightness showing an increase in all breeds while changes in redness varied according to the breed. Podolian breed showed meat with the darkest and the reddest color and the lowest drip loss compared to the other breeds. Extending aging to 21d reduced drip loss from meat. SDS-PAGE and 2DE showed that many changes in the sarcoplasmic proteins occurred among breeds and during aging. During post-mortem some sarcoplasmic proteins decline in intensity after 21d highlighting that they were susceptible to aging. Protein identification and western blotting showed the presence of myosin light chains, Troponin T and tropomyosin proteins during aging, suggesting a degradation of myofibers and a more intense proteolysis especially in the Podolian breed.

  18. The Validity of the Modified Sit-and-Reach Test in College-Age Students.

    ERIC Educational Resources Information Center

    Minkler, Sharin; Patterson, Patricia

    1994-01-01

    Reports a study that examined the criterion-related validity of the modified sit-and-reach test against criterion measures of hamstring and low back flexibility in college students. Results indicated the modified sit-and-reach test moderately related to hamstring flexibility, but its relation to low back flexibility was low. (SM)

  19. The effect of long-term aging on the impact properties of modified 9Cr-1Mo steel

    SciTech Connect

    Alexander, D.J.; Maziasz, P.J.; Brinkman, C.R.

    1992-12-31

    The Charpy impact and room-temperature tensile properties of two heats of modified 9Cr-1Mo steel have been examined after aging at temperatures from 482 to 704C for times up to 75,000 h. In general, aging at lower temperatures (482, 538, or 593C) resulted in little change in the room-temperature tensile properties, but rapid increases in the transition temperature, with the greatest increase for an aging time of 25,000 h. The upper-shelf energy level decreased, reaching a minimum at 25,000 h, followed by recovery at 50,000-h aging. At higher aging temperatures (649 and 704C) there was little change in the transition temperatures, but significant softening at room temperature, and large increases in the upper-shelf energy.

  20. Convergence Properties of Posttranslationally Modified Protein-Protein Switching Networks with Fast Decay Rates.

    PubMed

    Fan, Gaoyang; Cummins, Bree; Gedeon, Tomáš

    2016-06-01

    A significant conceptual difficulty in the use of switching systems to model regulatory networks is the presence of so-called "black walls," co-dimension 1 regions of phase space with a vector field pointing inward on both sides of the hyperplane. Black walls result from the existence of direct negative self-regulation in the system. One biologically inspired way of removing black walls is the introduction of intermediate variables that mediate the negative self-regulation. In this paper, we study such a perturbation. We replace a switching system with a higher-dimensional switching system with rapidly decaying intermediate proteins, and compare the dynamics between the two systems. We find that the while the individual solutions of the original system can be approximated for a finite time by solutions of a sufficiently close perturbed system, there are always solutions that are not well approximated for any fixed perturbation. We also study a particular example, where global basins of attraction of the perturbed system have a strikingly different form than those of the original system. We perform this analysis using techniques that are adapted to dealing with non-smooth systems. PMID:27271120

  1. 125I-labeled peptide mapping of some heat-modifiable proteins of the gonococcal outer membrane.

    PubMed Central

    Swanson, J

    1980-01-01

    Gonococci from opaque colonies have cell wall outer membrane proteins that are lacking from organisms which form transparent colonies. These "colony opacity-associated" proteins are among a group of "minor" proteins that exhibit heat modification of their apparent subunit molecular sizes, are easily extracted by deoxycholate, have apparent subunit molecular weights varying from 24,000 to 29,000 and are exposed on the surfaces of gonococci. Other minor proteins found on gonococci are the "leukocyte association proteins," whose presence correlates with reactivities of gonococci with human neutrophils. Several of the colony opacity-associated proteins and leukocyte association proteins were subjected to 125I-peptide mapping of protein bands separated by polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. The structural similarities and differences among these heat-modifiable surface proteins were studied, as well as their similarities with the major protein of the gonococcal outer membrane. A relatively high apparent degree of structural homology is found among the heat-modifiable proteins from different strains of opaque colony gonococcal forms. There is also some apparent structural homology for 125I-peptides of heat-modifiable versus major proteins of the gonococcal outer membrane. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:6769820

  2. Salivary antimicrobial proteins associate with age-related changes in streptococcal composition in dental plaque.

    PubMed

    Malcolm, J; Sherriff, A; Lappin, D F; Ramage, G; Conway, D I; Macpherson, L M D; Culshaw, S

    2014-12-01

    Secretion of antimicrobial proteins (AMPs) and salivary antibodies can modify biofilm formation at host body surfaces. In adolescents, associations have been reported between dental caries and salivary AMPs. AMPs demonstrate direct antimicrobial effects at high concentrations, and at lower more physiological concentrations they mediate changes in host cell defenses, which may alter the local environment and indirectly shape local biofilm formation. The expression of salivary AMPs in preschool children, at an age when the oral bacteria are known to change, has not been investigated. We sought to investigate salivary AMP expression in the context of previously well-documented changes in the oral cavities of this age group including salivary immunoglobulin A (IgA), oral bacteria and dental caries. Dental plaque and saliva were collected from 57 children aged 12-24 months at baseline, of whom 23 children were followed-up at 3 years of age. At each time, saliva was assessed for LL37, human neutrophil peptides 1-3, calprotectin, lactoferrin, salivary IgA, total plaque bacteria and Streptococcus mutans. Over time, concentrations of AMPs, S. mutans and bacteria-specific salivary IgA increased. Caries experience was also recorded when children were 3 years old. Concentrations of AMPs were highest in the saliva of 3-year-old children with the greatest burden of S. mutans. These data suggest that salivary AMPs are variable over time and between individuals, and are linked with bacterial colonization. At follow up, the majority of children remained caries free. Larger longitudinal studies are required to confirm whether salivary AMP levels are predictive of caries and whether their modulation offers therapeutic benefit.

  3. Carbohydrate-protein interactions investigated on plastic chips statically coated with hydrophobically modified hydroxyethylcellulose.

    PubMed

    Dang, Fuquan; Maeda, Eiki; Osafune, Tomo; Nakajima, Kazuki; Kakehi, Kazuaki; Ishikawa, Mitsuru; Baba, Yoshinobu

    2009-12-15

    We developed a novel method for rapid screening of carbohydrate-protein interactions using poly(methyl methacrylate) (PMMA) channels statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface intact by water allows rapid and reproducible separations of glycan samples using a 20 mM phosphate without HM-HEC. The underlying mechanism for dynamic and static coatings on the PMMA surface is discussed. Simultaneous analysis of the molecular interaction between a complex mixture of carbohydrates from alpha1-acid glycoprotein and proteins has been successfully achieved in PMMA channels statically coated with a SAM of HM-HEC.

  4. Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

    PubMed Central

    Holt, Matthew; Muir, Tom

    2016-01-01

    Histone proteins are subject to a host of posttranslational modifications (PTMs) that modulate chromatin structure and function. Such control is achieved by the direct alteration of the intrinsic physical properties of the chromatin fiber or by regulating the recruitment and activity of a host of trans-acting nuclear factors. The sheer number of histone PTMs presents a formidable barrier to understanding the molecular mechanisms at the heart of epigenetic regulation of eukaryotic genomes. One aspect of this multifarious problem, namely how to access homogeneously modified chromatin for biochemical studies, is well suited to the sensibilities of the organic chemist. Indeed, recent years have witnessed a critical role for synthetic protein chemistry methods in generating the raw materials needed for studying how histone PTMs regulate chromatin biochemistry. This review focuses on what is arguably the most powerful, and widely employed, of these chemical strategies, namely histone semisynthesis via the chemical ligation of peptide fragments. PMID:25784050

  5. Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin.

    PubMed

    Peichoto, María Elisa; Santoro, Marcelo Larami

    2016-09-01

    Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation. PMID:27390042

  6. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  7. Carboxymethylchitosan covalently modified capillary column for open tubular capillary electrochromatography of basic proteins and opium alkaloids.

    PubMed

    Zhou, Sunying; Tan, Jingjing; Chen, Qinhua; Lin, Xucong; Lü, Haixia; Xie, Zenghong

    2010-12-24

    A novel open tubular (OT) column covalently modified with hydrophilic polysaccharide, carboxymethylchitosan (CMC) as stationary phase has been developed, and employed for the separations of basic proteins and opium alkaloids by capillary electrochromatography (CEC). With the procedures including the silanization of 3-aminopropyltrimethoxysilane (APTS) and the combination of glutaraldehyde with amino-silylated silica surface and CMC, CMC was covalently bonded on the capillary inner wall and exhibited a remarkable tolerance and chemical stability against 0.1 mol/L HCl, 0.1 mol/L NaOH or some organic solvents. By varying the pH values of running buffer, a cathodic or anodic EOF could be gained in CMC modified column. With anodic EOF mode (pH<4.3), favorable separations of basic proteins (trypsin, ribonuclease A, lysozyme and cytochrome C) were successfully achieved with high column efficiencies ranging from 97,000 to 182,000 plates/m, and the undesired adsorptions of basic proteins on the inter-wall of capillary could be avoided. Good repeatability was gained with RSD of the migration time less than 1.3% for run-to-run (n=5) and less than 3.2% for day-to-day (n=3), RSD of peak area was less than 5.6% for run-to-run (n=5) and less than 8.8% for day-to-day (n=3). With cathodic EOF mode (pH>4.3), four opium alkaloids were also baseline separated in phosphate buffer (50 mmol/L, pH 6.0) with column efficiencies ranging from 92,000 to 132,000 plates/m. CMC-bonded OT capillary column might be used as an alternative medium for the further analysis of basic proteins and alkaline analytes.

  8. Ion-ion reactions with fixed-charge modified proteins to produce ions in a single, very high charge state

    NASA Astrophysics Data System (ADS)

    Frey, Brian L.; Krusemark, Casey J.; Ledvina, Aaron R.; Coon, Joshua J.; Belshaw, Peter J.; Smith, Lloyd M.

    2008-10-01

    Electrospray ionization (ESI) of denatured proteins produces a mass spectrum with a broad distribution of multiply charged ions. Attaching fixed positive charges, specifically quaternary ammonium groups, to proteins at their carboxylic acid groups generates substantially higher charge states compared to the corresponding unmodified proteins in positive-mode ESI. Ion-ion reactions of these modified proteins with reagent anions leads to charge reduction by proton transfer. These proton transfer reactions cannot remove charge from the quaternary ammonium groups, which do not have a proton to transfer to the anion. Thus, one might expect charge reduction to stop at a single charge state equal to the number of fixed charges on the modified protein. However, ion-ion reactions yield charge states lower than this number of fixed charges due to anion attachment (adduction) to the proteins. Charge reduction via ion-molecule reactions involving gas-phase bases also give adducts on the modified protein ions in low charge states. Such adducts are avoided by keeping the ions in charge states well above the number of fixed charges. In the present work protein ions were selectively "parked" within an ion trap mass spectrometer in a high charge state by mild radiofrequency excitation that dramatically slows their ion-ion reaction rate--a technique termed "ion parking". The combination of ion parking with the fixed-charge modified proteins permits generation of a large population of ions in a single, very high charge state.

  9. Theories of biological aging: genes, proteins, and free radicals.

    PubMed

    Rattan, Suresh I S

    2006-12-01

    Traditional categorization of theories of aging into programmed and stochastic ones is outdated and obsolete. Biological aging is considered to occur mainly during the period of survival beyond the natural or essential lifespan (ELS) in Darwinian terms. Organisms survive to achieve ELS by virtue of genetically determined longevity assuring maintenance and repair systems (MRS). Aging at the molecular level is characterized by the progressive accumulation of molecular damage caused by environmental and metabolically generated free radicals, by spontaneous errors in biochemical reactions, and by nutritional components. Damages in the MRS and other pathways lead to age-related failure of MRS, molecular heterogeneity, cellular dysfunctioning, reduced stress tolerance, diseases and ultimate death. A unified theory of biological aging in terms of failure of homeodynamics comprising of MRS, and involving genes, milieu and chance, is acquiring a definitive shape and wider acceptance. Such a theory also establishes the basis for testing and developing effective means of intervention, prevention and modulation of aging.

  10. Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

    PubMed

    Miodek, A; Castillo, G; Hianik, T; Korri-Youssoufi, H

    2014-06-15

    This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%. PMID:24480126

  11. Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

    PubMed

    Miodek, A; Castillo, G; Hianik, T; Korri-Youssoufi, H

    2014-06-15

    This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%.

  12. Amperometric immunosensor based on a protein A/deposited gold nanocrystals modified electrode for carbofuran detection.

    PubMed

    Sun, Xia; Zhu, Ying; Wang, Xiangyou

    2011-01-01

    In this paper, an amperometric immunosensor modified with protein A/deposited gold nanocrystals (DpAu) was developed for the ultrasensitive detection of carbofuran residues. First, DpAu were electrodeposited onto the Au electrode surface to absorb protein A (PA) and improve the electrode conductivity. Then PA was dropped onto the surface of DpAu film, used for binding antibody Fc fragments. Next, anti-carbofuran monoclonal antibody was immobilized on the PA modified electrode. Finally, bovine serum albumin (BSA) was employed to block the possible remaining active sites avoiding any nonspecific adsorption. The fabrication procedure of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. With the excellent electroconductivity of DpAu and the PA's oriented immobilization of antibodies, a highly efficient immuno-reaction and detection sensitivity could be achieved. The influences of the electrodeposition time of DpAu, pH of the detection solution and incubation time on the current response of the fabricated immunosensor were investigated. Under optimized conditions, the current response was proportional to the concentration of carbofuran which ranged from 1 to 100 ng/mL and 100 ng/mL to 100 μg/mL. The detection limit was 0.1924 ng/mL. The proposed carbofuran immnuosensor exhibited high specificity, reproducibility, stability and regeneration performance, which may open a new door for ultrasensitive detection of carbofuran residues in vegetables and fruits.

  13. Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

    PubMed

    Di Sansebastiano, Gian Pietro; Rizzello, Francesca; Durante, Miriana; Caretto, Sofia; Nisi, Rossella; De Paolis, Angelo; Faraco, Marianna; Montefusco, Anna; Piro, Gabriella; Mita, Giovanni

    2015-05-20

    Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

  14. Variants within the SP110 nuclear body protein modify risk of canine degenerative myelopathy

    PubMed Central

    Ivansson, Emma L.; Kozyrev, Sergey V.; Murén, Eva; Körberg, Izabella Baranowska; Swofford, Ross; Koltookian, Michele; Tonomura, Noriko; Zeng, Rong; Kolicheski, Ana L.; Hansen, Liz; Katz, Martin L.; Johnson, Gayle C.; Johnson, Gary S.; Coates, Joan R.; Lindblad-Toh, Kerstin

    2016-01-01

    Canine degenerative myelopathy (DM) is a naturally occurring neurodegenerative disease with similarities to some forms of amyotrophic lateral sclerosis (ALS). Most dogs that develop DM are homozygous for a common superoxide dismutase 1 gene (SOD1) mutation. However, not all dogs homozygous for this mutation develop disease. We performed a genome-wide association analysis in the Pembroke Welsh Corgi (PWC) breed comparing DM-affected and -unaffected dogs homozygous for the SOD1 mutation. The analysis revealed a modifier locus on canine chromosome 25. A haplotype within the SP110 nuclear body protein (SP110) was present in 40% of affected compared with 4% of unaffected dogs (P = 1.5 × 10−5), and was associated with increased probability of developing DM (P = 4.8 × 10−6) and earlier onset of disease (P = 1.7 × 10−5). SP110 is a nuclear body protein involved in the regulation of gene transcription. Our findings suggest that variations in SP110-mediated gene transcription may underlie, at least in part, the variability in risk for developing DM among PWCs that are homozygous for the disease-related SOD1 mutation. Further studies are warranted to clarify the effect of this modifier across dog breeds. PMID:27185954

  15. Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids

    PubMed Central

    Tookmanian, Elise M.; Fenlon, Edward E.; Brewer, Scott H.

    2015-01-01

    Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein. PMID:26478813

  16. Polydopamine assisted fabrication of titanium oxide nanoparticles modified column for proteins separation by capillary electrochromatography.

    PubMed

    Zhang, Yamin; Wang, Wentao; Ma, Xiangdong; Jia, Li

    2016-11-01

    Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO2) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO2 coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti(4+) to boost the nucleation and growth of TiO2 film. The as-prepared TiO2 coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO2 coating was investigated. The TiO2 coated OT column was used for successful separation of two variants of β-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white. PMID:27555440

  17. Quantitative Analysis of Age Specific Variation in the Abundance of Human Female Parotid Salivary Proteins

    PubMed Central

    Ambatipudi, Kiran S.; Lu, Bingwen; Hagen, Fred K; Melvin, James E.; Yates, John R.

    2010-01-01

    Summary Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20–30 and 55–65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high from low abundant proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g. lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women. PMID:19764810

  18. Surface forces and protein adsorption on dextran- and polyethylene glycol-modified polydimethylsiloxane.

    PubMed

    Farrell, Megan; Beaudoin, Stephen

    2010-12-01

    Dextran and polyethylene glycol (PEG) are often covalently bound to the surface of polydimethylsiloxane (PDMS) for the purpose of modifying its hydrophilicity and biocompatibility. In this work, the effects of the dextran and PEG on the morphology, wetting, and surface charge of the resulting surfaces were quantified and correlated with changes in the amount of fibrinogen and albumin adsorbed from aqueous solution. PDMS films were functionalized in a microwave oxygen plasma to create surface hydroxyl groups that were subsequently aminated by incubation in a (3-aminopropyl)trimethoxysilane (APTES) solution. Oxidized dextran and PEG-aldehyde were linked to the surface amines via reductive amination. This process resulted in low surface coverage of immobilized PEG in the end-on conformation and a more uniform and dense distribution of side-on immobilized dextran. The immobilized dextran reduced the contact angle of the PDMS film from 109° to 80° and neutralized the zeta potential over the pH range from 3 to 11. An atomic force microscope was used to measure the interaction force between the modified PDMS and a model hydrophobic surface (polystyrene latex) and a model hydrophilic surface (silica) in aqueous solution to show that van der Waals and hydrophobic attractive forces are the dominant forces for protein adsorption in this system. The PEG- and dextran-modified PDMS were exposed to BSA and fibrinogen to test their resistance to protein adsorption. The coatings were ineffective at reducing the adsorption of either molecule, and the dextran-modification of the PDMS caused more BSA to adsorb than in the case of the unmodified PDMS. PMID:20801620

  19. Age-related changes in the hippocampus (loss of synaptophysin and glial-synaptic interaction) are modified by systemic treatment with an NCAM-derived peptide, FGL.

    PubMed

    Ojo, Bunmi; Rezaie, Payam; Gabbott, Paul L; Davies, Heather; Colyer, Frances; Cowley, Thelma R; Lynch, Marina; Stewart, Michael G

    2012-07-01

    Altered synaptic morphology, progressive loss of synapses and glial (astrocyte and microglial) cell activation are considered as characteristic hallmarks of aging. Recent evidence suggests that there is a concomitant age-related decrease in expression of the presynaptic protein, synaptophysin, and the neuronal glycoprotein CD200, which, by interacting with its receptor, plays a role in maintaining microglia in a quiescent state. These age-related changes may be indicative of reduced neuroglial support of synapses. FG Loop (FGL) peptide synthesized from the second fibronectin type III module of neural cell adhesion molecule (NCAM), has previously been shown to attenuate age-related glial cell activation, and to 'restore' cognitive function in aged rats. The mechanisms by which FGL exerts these neuroprotective effects remain unclear, but could involve regulation of CD200, modifying glial-synaptic interactions (affecting neuroglial 'support' at synapses), or impacting directly on synaptic function. Light and electron microscopic (EM) analyses were undertaken to investigate whether systemic treatment with FGL (i) alters CD200, synaptophysin (presynaptic) and PSD-95 (postsynaptic) immunohistochemical expression levels, (ii) affects synaptic number, or (iii) exerts any effects on glial-synaptic interactions within young (4 month-old) and aged (22 month-old) rat hippocampus. Treatment with FGL attenuated the age-related loss of synaptophysin immunoreactivity (-ir) within CA3 and hilus (with no major effect on PSD-95-ir), and of CD200-ir specifically in the CA3 region. Ultrastructural morphometric analyses showed that FGL treatment (i) prevented age-related loss in astrocyte-synaptic contacts, (ii) reduced microglia-synaptic contacts in the CA3 stratum radiatum, but (iii) had no effect on the mean number of synapses in this region. These data suggest that FGL mediates its neuroprotective effects by regulating glial-synaptic interaction.

  20. The whole body cryostimulation modifies irisin concentration and reduces inflammation in middle aged, obese men.

    PubMed

    Dulian, Katarzyna; Laskowski, Radosław; Grzywacz, Tomasz; Kujach, Sylwester; Flis, Damian J; Smaruj, Mirosław; Ziemann, Ewa

    2015-12-01

    The anti-inflammatory effect induced by exposure to low temperature might trigger the endocrine function of muscle and fat tissue. Thus, the aim of this study was to investigate the influence of the whole body cryostimulation (CRY) on irisin, a myokine which activates oxygen consumption in fat cells as well as thermogenesis. In addition, the relationship between hepcidin (Hpc) - hormone regulating iron metabolism, and inflammation was studied. A group of middle aged men (n = 12, 38 ± 9 years old, BMI > 30 kg m(-2)) participated in the study. Subjects were exposed to a series of 10 sessions in a cryogenic chamber (once a day at 9:30 am, for 3 min, at temperature -110 °C). Blood samples were collected before the first cryostimulation and after completing the last one. Prior to treatment body composition and fitness level were determined. The applied protocol of cryostimulation lead to rise the blood irisin in obese non-active men (338.8 ± 42.2 vs 407.6 ± 118.5 ng mL(-1)), whereas has no effect in obese active men (371.5 ± 30.0 vs 343.3 ± 47.6 ng mL(-1)). Values recorded 24 h after the last cryo-session correlated significantly with the fat tissue, yet inversely with the skeletal muscle mass. Therefore, we concluded the subcutaneous fat tissue to be the main source of irisin in response to cold exposures. The applied cold treatment reduced the high sensitivity C-reactive protein (hsCRP) and Hpc concentration confirming its anti-inflammatory effect.

  1. Organic Chemistry Applied to Synthetic Proteins: Modifying the Vicinity of the Isopeptide Bond Revealed Differential Behavior of Ubiquitin Chains with Interacting Proteins

    PubMed Central

    Haj-Yahya, Najat; Haj-Yahya, Mahmood; Castañeda, Carlos A.; Spasser, Liat; Hemantha, Hosahalli P.; Jbara, Muhammad; Penner, Marlin; Ciechanover, Aaron; Fushman, David

    2013-01-01

    In Every Direction Chemical synthesis of proteins allowed the synthesis of ubiquitin chains modified in the vicinity of the isopeptide peptide to examine their behavior with deubiquitinases and ubiquitin binding domains. Our results set the ground for the generation of unique probes for studying the interactions of these chains with various ubiquitin-interacting proteins. PMID:24006204

  2. Evolution of the microstructure of unmodified and polymer modified asphalt binders with aging in an accelerated weathering tester.

    PubMed

    Menapace, Ilaria; Masad, Eyad

    2016-09-01

    This paper presents findings on the evolution of the surface microstructure of two asphalt binders, one unmodified and one polymer modified, directly exposed to aging agents with increasing durations. The aging is performed using an accelerated weathering tester, where ultraviolet radiation, oxygen and an increased temperature are applied to the asphalt binder surface. Ultraviolet and dark cycles, which simulated the succession of day and night, alternated during the aging process, and also the temperature varied, which corresponded to typical summer day and night temperatures registered in the state of Qatar. Direct aging of an exposed binder surface is more effective in showing microstructural modifications than previously applied protocols, which involved the heat treatment of binders previously aged with standardized methods. With the new protocol, any molecular rearrangements in the binder surface after aging induced by the heat treatment is prevented. Optical photos show the rippling and degradation of the binder surface due to aging. Microstructure images obtained by means of atomic force microscopy show gradual alteration of the surface due to aging. The original relatively flat microstructure was substituted with a profoundly different microstructure, which significantly protrudes from the surface, and is characterized by various shapes, such as rods, round structures and finally 'flower' or 'leaf' structures. PMID:27059404

  3. Evolution of the microstructure of unmodified and polymer modified asphalt binders with aging in an accelerated weathering tester.

    PubMed

    Menapace, Ilaria; Masad, Eyad

    2016-09-01

    This paper presents findings on the evolution of the surface microstructure of two asphalt binders, one unmodified and one polymer modified, directly exposed to aging agents with increasing durations. The aging is performed using an accelerated weathering tester, where ultraviolet radiation, oxygen and an increased temperature are applied to the asphalt binder surface. Ultraviolet and dark cycles, which simulated the succession of day and night, alternated during the aging process, and also the temperature varied, which corresponded to typical summer day and night temperatures registered in the state of Qatar. Direct aging of an exposed binder surface is more effective in showing microstructural modifications than previously applied protocols, which involved the heat treatment of binders previously aged with standardized methods. With the new protocol, any molecular rearrangements in the binder surface after aging induced by the heat treatment is prevented. Optical photos show the rippling and degradation of the binder surface due to aging. Microstructure images obtained by means of atomic force microscopy show gradual alteration of the surface due to aging. The original relatively flat microstructure was substituted with a profoundly different microstructure, which significantly protrudes from the surface, and is characterized by various shapes, such as rods, round structures and finally 'flower' or 'leaf' structures.

  4. Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis

    SciTech Connect

    Russell, Michael; Berardi, Philip; Gong Wei; Riabowol, Karl . E-mail: karl@ucalgary.ca

    2006-04-15

    The INhibitor of Growth (ING) family of plant homeodomain (PHD) proteins induce apoptosis and regulate gene expression through stress-inducible binding of phospholipids with subsequent nuclear and nucleolar localization. Relocalization occurs concomitantly with interaction with a subset of nuclear proteins, including PCNA, p53 and several regulators of acetylation such as the p300/CBP and PCAF histone acetyltransferases (HATs), as well as the histone deacetylases HDAC1 and hSir2. These interactions alter the localized state of chromatin compaction, subsequently affecting the expression of subsets of genes, including those associated with the stress response (Hsp70), apoptosis (Bax, MDM2) and cell cycle regulation (p21{sup WAF1}, cyclin B) in a cell- and tissue-specific manner. The expression levels and subcellular localization of ING proteins are altered in a significant number of human cancer types, while the expression of ING isoforms changes during cellular aging, suggesting that ING proteins may play a role in linking cellular transformation and replicative senescence. The variety of functions attributed to ING proteins suggest that this tumor suppressor serves to link the disparate processes of cell cycle regulation, cell suicide and cellular aging through epigenetic regulation of gene expression. This review examines recent findings in the ING field with a focus on the functions of protein-protein interactions involving ING family members and the mechanisms by which these interactions facilitate the various roles that ING proteins play in tumorigenesis, apoptosis and senescence.

  5. Loss of prion protein leads to age-dependent behavioral abnormalities and changes in cytoskeletal protein expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellular prion protein (PrPC) is a multifunctional protein, whose exact physiological role remains elusive. Since previous studies indicated a neuroprotective function of PrPC, we investigated whether Prnp knockout mice(Prnp0/0)display age-dependent behavioral abnormalities. Matched sets of Prnp0/0 ...

  6. Total protein, animal protein, and physical activity in relation to muscle mass in middle-aged and older Americans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resistance training is recognized as a good strategy for retarding age-related declines in muscle mass and strength. Recent studies have also highlighted the potential value of protein intakes in excess of current recommendations. The roles that leisure-time physical activity and protein quality mig...

  7. Neurotrophin receptor proteins immunoreactivity in the rat cerebellar cortex as a function of age.

    PubMed

    Torres, J M; Javier Naves, F; Esteban, I; Del Valle, M E; Vega, J A

    1995-08-31

    The influence of age on immunohistochemically demonstrable neurotrophin receptor proteins (p75, trkA-, trkB-, and trkC-proteins) was studied in the cerebellar cortex of Wistar male rats aged 3 (young), 12 (adult) and 24 (old) months. The number of Purkinje neurons displaying p75, trkA- and trkC-like proteins immunoreactivity (IR), as well as the intensity of p75 and trkA-like protein IR, were significantly reduced in aged rats in comparison with 3 and 12-month-old rats. The intensity of trkC-like protein in the cytoplasm of Purkinje neurons remained unchanged for all the period studied. Moreover, no significant age-dependent changes were observed in the density of p75 or trkC-like proteins IR in the granule neurons layer. The molecular layer showed faint p75 IR which decreased as a function of age. No immunolabelling for neuronal trkB-like proteins was observed, but trkB- and trkC-like proteins IR was found in non-neuronal cells. These results suggest that cerebellar cortex neurons are responsive to and/or dependent upon different neurotrophins. Moreover, the age-dependent impairment in the expression of some neurotrophin receptors in Purkinje neurons, but not in the granule neurons, lends support to a role for neurotrophins in cerebellar aging.

  8. Whole-genome sequencing suggests a chemokine gene cluster that modifies age at onset in familial Alzheimer's disease.

    PubMed

    Lalli, M A; Bettcher, B M; Arcila, M L; Garcia, G; Guzman, C; Madrigal, L; Ramirez, L; Acosta-Uribe, J; Baena, A; Wojta, K J; Coppola, G; Fitch, R; de Both, M D; Huentelman, M J; Reiman, E M; Brunkow, M E; Glusman, G; Roach, J C; Kao, A W; Lopera, F; Kosik, K S

    2015-11-01

    We have sequenced the complete genomes of 72 individuals affected with early-onset familial Alzheimer's disease caused by an autosomal dominant, highly penetrant mutation in the presenilin-1 (PSEN1) gene, and performed genome-wide association testing to identify variants that modify age at onset (AAO) of Alzheimer's disease. Our analysis identified a haplotype of single-nucleotide polymorphisms (SNPs) on chromosome 17 within a chemokine gene cluster associated with delayed onset of mild-cognitive impairment and dementia. Individuals carrying this haplotype had a mean AAO of mild-cognitive impairment at 51.0 ± 5.2 years compared with 41.1 ± 7.4 years for those without these SNPs. This haplotype thus appears to modify Alzheimer's AAO, conferring a large (~10 years) protective effect. The associated locus harbors several chemokines including eotaxin-1 encoded by CCL11, and the haplotype includes a missense polymorphism in this gene. Validating this association, we found plasma eotaxin-1 levels were correlated with disease AAO in an independent cohort from the University of California San Francisco Memory and Aging Center. In this second cohort, the associated haplotype disrupted the typical age-associated increase of eotaxin-1 levels, suggesting a complex regulatory role for this haplotype in the general population. Altogether, these results suggest eotaxin-1 as a novel modifier of Alzheimer's disease AAO and open potential avenues for therapy.

  9. A Mutation in DAOA Modifies the Age of Onset in PSEN1 E280A Alzheimer's Disease

    PubMed Central

    Vélez, Jorge I.; Rivera, Dora; Mastronardi, Claudio A.; Patel, Hardip R.; Tobón, Carlos; Villegas, Andrés; Cai, Yeping; Easteal, Simon; Arcos-Burgos, Mauricio

    2016-01-01

    We previously reported age of onset (AOO) modifier genes in the world's largest pedigree segregating early-onset Alzheimer's disease (AD), caused by the p.Glu280Ala (E280A) mutation in the PSEN1 gene. Here we report the results of a targeted analysis of functional exonic variants in those AOO modifier genes in sixty individuals with PSEN1 E280A AD who were whole-exome genotyped for ~250,000 variants. Standard quality control, filtering, and annotation for functional variants were applied, and common functional variants located in those previously reported as AOO modifier loci were selected. Multiloci linear mixed-effects models were used to test the association between these variants and AOO. An exonic missense mutation in the G72 (DAOA) gene (rs2391191, P = 1.94 × 10−4, PFDR = 9.34 × 10−3) was found to modify AOO in PSEN1 E280A AD. Nominal associations of missense mutations in the CLUAP1 (rs9790, P = 7.63 × 10−3, PFDR = 0.1832) and EXOC2 (rs17136239, P = 0.0325, PFDR = 0.391) genes were also found. Previous studies have linked polymorphisms in the DAOA gene with the occurrence of neuropsychiatric symptoms such as depression, apathy, aggression, delusions, hallucinations, and psychosis in AD. Our findings strongly suggest that this new conspicuous functional AOO modifier within the G72 (DAOA) gene could be pivotal for understanding the genetic basis of AD. PMID:26949549

  10. Integrative Transcriptome Profiling of Cognitive Aging and Its Preservation through Ser/Thr Protein Phosphatase Regulation.

    PubMed

    Park, C Sehwan; Valomon, Amandine; Welzl, Hans

    2015-01-01

    Environmental enrichment has been reported to delay or restore age-related cognitive deficits, however, a mechanism to account for the cause and progression of normal cognitive decline and its preservation by environmental enrichment is lacking. Using genome-wide SAGE-Seq, we provide a global assessment of differentially expressed genes altered with age and environmental enrichment in the hippocampus. Qualitative and quantitative proteomics in naïve young and aged mice was used to further identify phosphorylated proteins differentially expressed with age. We found that increased expression of endogenous protein phosphatase-1 inhibitors in aged mice may be characteristic of long-term environmental enrichment and improved cognitive status. As such, hippocampus-dependent performances in spatial, recognition, and associative memories, which are sensitive to aging, were preserved by environmental enrichment and accompanied by decreased protein phosphatase activity. Age-associated phosphorylated proteins were also found to correspond to the functional categories of age-associated genes identified through transcriptome analysis. Together, this study provides a comprehensive map of the transcriptome and proteome in the aging brain, and elucidates endogenous protein phosphatase-1 inhibition as a potential means through which environmental enrichment may ameliorate age-related cognitive deficits.

  11. Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

    NASA Astrophysics Data System (ADS)

    Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

    2005-04-01

    This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

  12. A genetically modified protein-based hydrogel for 3D culture of AD293 cells.

    PubMed

    Du, Xiao; Wang, Jingyu; Diao, Wentao; Wang, Ling; Long, Jiafu; Zhou, Hao

    2014-01-01

    Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence 'GRGDSP' to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery. PMID:25233088

  13. Modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin.

    PubMed

    Tian, Qing-Yun; Zhao, Yun-Peng; Liu, Chuan-ju

    2012-01-17

    Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel, extracellular matrix protein, and growth factor. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor.

  14. Extracellular Release and Signaling by Heat Shock Protein 27: Role in Modifying Vascular Inflammation

    PubMed Central

    Batulan, Zarah; Pulakazhi Venu, Vivek Krishna; Li, Yumei; Koumbadinga, Geremy; Alvarez-Olmedo, Daiana Gisela; Shi, Chunhua; O’Brien, Edward R.

    2016-01-01

    Heat shock protein 27 (HSP27) is traditionally viewed as an intracellular chaperone protein with anti-apoptotic properties. However, recent data indicate that a number of heat shock proteins, including HSP27, are also found in the extracellular space where they may signal via membrane receptors to alter gene transcription and cellular function. Therefore, there is increasing interest in better understanding how HSP27 is released from cells, its levels and composition in the extracellular space, and the cognate cell membrane receptors involved in effecting cell signaling. In this paper, the knowledge to date, as well as some emerging paradigms about the extracellular function of HSP27 is presented. Of particular interest is the role of HSP27 in attenuating atherogenesis by modifying lipid uptake and inflammation in the plaque. Moreover, the abundance of HSP27 in serum is an emerging new biomarker for ischemic events. Finally, HSP27 replacement therapy may represent a novel therapeutic opportunity for chronic inflammatory disorders, such as atherosclerosis. PMID:27507972

  15. Overexpression of a modified amaranth protein in Escherichia coli with minimal media and lactose as inducer.

    PubMed

    Morales-Camacho, Jocksan Ismael; Dominguez-Dominguez, Jorge; Paredes-Lopez, Octavio

    2013-04-01

    In this research it was attempted to overexpress the acidic subunit, from the 11S amaranth seed globulin termed amarantin, modified with antihypertensive peptides in Escherichia coli Rosetta (DE3) by manipulating some factors in batch fermenter such as growth medium composition, inducer (isopropyl β-D-thiogalactopyranoside [IPTG] or lactose), air flow, cultivation temperature, agitation speed and induction time. The possibility of using several minimal media and lactose as inducer to increase yields of the recombinant protein was investigated. Previous fermentations at flask level showed that two minimal culture media (A6 and A7) and 0.5% (w/v) lactose presented high yields of the engineered protein expression. Thus, the latter two media were tested at fermenter level, the lactose inducer, and different environmental conditions. Factors with significant effects were identified by Plackett-Burman design with center points and were adjusted at the level suggested and the yields of the recombinant protein were increased from 303.2 to 1,531 mg L(-1) in A6 and from 363.4 to 1,681 mg L(-1) in A7. Unlike some patents where the highest productivity was achieved at 24 h or afterwards, in this research the best productivity of the recombinant acidic subunit was attained at 4 and 6 h of induction using both media, respectively. PMID:23294401

  16. Protein Intake and Muscle Health in Old Age: From Biological Plausibility to Clinical Evidence

    PubMed Central

    Landi, Francesco; Calvani, Riccardo; Tosato, Matteo; Martone, Anna Maria; Ortolani, Elena; Savera, Giulia; D’Angelo, Emanuela; Sisto, Alex; Marzetti, Emanuele

    2016-01-01

    The provision of sufficient amounts of dietary proteins is central to muscle health as it ensures the supply of essential amino acids and stimulates protein synthesis. Older persons, in particular, are at high risk of insufficient protein ingestion. Furthermore, the current recommended dietary allowance for protein (0.8 g/kg/day) might be inadequate for maintaining muscle health in older adults, probably as a consequence of “anabolic resistance” in aged muscle. Older individuals therefore need to ingest a greater quantity of protein to maintain muscle function. The quality of protein ingested is also essential to promoting muscle health. Given the role of leucine as the master dietary regulator of muscle protein turnover, the ingestion of protein sources enriched with this essential amino acid, or its metabolite β-hydroxy β-methylbutyrate, is thought to offer the greatest benefit in terms of preservation of muscle mass and function in old age. PMID:27187465

  17. Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

    PubMed

    Jordheim, Lars Petter; Cros-Perrial, Emeline; Matera, Eva-Laure; Bouledrak, Karima; Dumontet, Charles

    2014-10-01

    Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions.

  18. Self-Assembled Modified Soy Protein/Dextran Nanogel Induced by Ultrasonication as a Delivery Vehicle for Riboflavin.

    PubMed

    Jin, Bei; Zhou, Xiaosong; Li, Xiangzhong; Lin, Weiqin; Chen, Guangbin; Qiu, Riji

    2016-01-01

    A simple and green approach was developed to produce a novel nanogel via self-assembly of modified soy protein and dextran, to efficiently deliver riboflavin. First, modified soy protein was prepared by heating denaturation at 60 °C for 30 min or Alcalase hydrolysis for 40 min. Second, modified soy protein was mixed with dextran and ultrasonicated for 70 min so as to assemble nanogels. The modified soy protein-dextran nanogels were characterized by Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) and ζ-potential studies to confirm the formation of NGs. Transmission electron microscopy (TEM) revealed the NGs to be spherical with core-shell structures, in the range of 32-40 nm size. The nanogels were stable against various environmental conditions. Furthermore, the particle size of the nanogels hardly changed with the incorporation of riboflavin. The encapsulation efficiency of nanogels was found to be up to 65.9% at a riboflavin concentration of 250 μg/mL. The nanogels exhibited a faster release in simulated intestine fluid (SIF) compared with simulated gastric fluid (SGF). From the results obtained it can be concluded that modified soy protein-dextran nanogels can be considered a promising carrier for drugs and other bioactive molecule delivery purposes. PMID:26999081

  19. Determination of disulfide array and subunit structure of taste-modifying protein, miraculin.

    PubMed

    Igeta, H; Tamura, Y; Nakaya, K; Nakamura, Y; Kurihara, Y

    1991-09-20

    The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity. PMID:1911854

  20. Measuring Subvisible Particles in Protein Formulations Using a Modified Light Obscuration Sensor with Improved Detection Capabilities.

    PubMed

    Ríos Quiroz, Anacelia; Québatte, Gabriela; Stump, Fabian; Finkler, Christof; Huwyler, Joerg; Schmidt, Roland; Mahler, Hanns-Christian; Koulov, Atanas V; Adler, Michael

    2015-06-16

    Although light obscuration is the "gold standard" for subvisible particle measurements in biopharmaceutical products, the current technology has limitations with respect to the detection of translucent proteinaceous particles and particles of sizes smaller and around 2 μm. Here, we describe the evaluation of a modified light obscuration sensor utilizing a novel measuring mode. Whereas standard light obscuration methodology monitors the height (amplitude) of the signal, the new approach monitors its length (width). Experimental evaluation demonstrated that this new detection mode leads to improved detection of subvisible particles of sizes smaller than 2 μm, reduction of artifacts during measurements especially of low concentrations of translucent protein particles, and higher counting accuracy as compared to flow imaging microscopy and standard light obscuration measurements.

  1. Approaches to assessment of the allergenic potential of novel proteins in food from genetically modified crops.

    PubMed

    Kimber, Ian; Dearman, Rebecca J

    2002-07-01

    The safety assessment of food derived from genetically modified plants continues to attract considerable attention. Among the important issues that need to be considered is whether the products of novel genes introduced into crop plants will have the potential to induce allergic sensitization or to elicit allergic disease. Hierarchical approaches to allergenicity testing have been proposed, and these incorporate evaluation of the structural and sequence homology and serological identity of novel proteins with known allergens, measurement of resistance to proteolytic digestion, and assessment of allergenic potential using animal models. Accounts of these approaches are available elsewhere, and it is not the purpose of this article to provide a detailed critique of specific methods. Our intention is rather to look more broadly at the strategy for assessment of allergenic potential, the challenges such assessments pose for the practicing toxicologist, and how some of these might best be addressed.

  2. Nucleolin targeting AS1411 modified protein nanoparticle for antitumor drugs delivery.

    PubMed

    Wu, Jinhui; Song, Chenchen; Jiang, Chenxiao; Shen, Xin; Qiao, Qian; Hu, Yiqiao

    2013-10-01

    Over recent years, cell surface nucleolin as an anticancer target has attracted many researchers' attentions. To improve the antitumor efficacy, we developed a nucleolin targeted protein nanoparticle (NTPN) delivery system in which human serum albumin (HSA) was used as drug carrier and a DNA aptamer named AS1411, which had high affinity to nucleolin, was used as a bullet. The HSA nanoparticles (NPs-PTX) were fabricated by a novel self-assembly method and then modified with AS1411 (Apt-NPs-PTX). The resulted Apt-NPs-PTX were spherical. Compared with NPs-PTX, the uptake of Apt-NPs-PTX displayed a significant increase in MCF-7 cells while there was a decrease in nontumor cell lines such as MCF-10A and 3T3 cells. In a cytotoxic study, Apt-NPs-PTX displayed an enhanced cytotoxicity in MCF-7 tumor cells while there was almost no cytotoxicity in MCF-10A cells. Endostatin, a nucleolin inhibitor, could significantly decrease the internalization of Apt-NPs-PTX, suggesting nucleolin mediates the transmembrane process of Apt-NPs-PTX. Therefore, the AS1411 modified NTPN delivery system might be a promising targeted drug delivery system. PMID:23679916

  3. Imaging murine NALT following intranasal immunization with flagellin-modified circumsporozoite protein malaria vaccines

    PubMed Central

    Nacer, Adéla; Carapau, Daniel; Mitchell, Robert; Meltzer, Abby; Shaw, Alan; Frevert, Ute; Nardin, Elizabeth H

    2013-01-01

    Intranasal (IN) immunization with a Plasmodium circumsporozoite (CS) protein conjugated to flagellin, a TLR5 agonist, was found to elicit antibody mediated protective immunity in our previous murine studies. To better understand IN elicited immune responses, we examined the nasopharynx-associated lymphoid tissue (NALT) in immunized mice and the interaction of flagellin-modified CS with murine dendritic cells (DC) in vitro. NALT of immunized mice contained a predominance of germinal center (GC) B cells and increased numbers of CD11c+ DC localized beneath the epithelium and within the GC T cell area. We detected microfold (M) cells distributed throughout the NALT epithelial cell layer and DC dendrites extending into the nasal cavity which could potentially function in luminal CS antigen uptake. Flagellin-modified CS taken up by DC in vitro was initially localized within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT APC populations will advance development of an efficacious needle-free vaccine for the 40% of the world's population at risk of malaria. PMID:23820750

  4. Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit.

    PubMed

    Theerasilp, S; Kurihara, Y

    1988-08-15

    The taste-modifying protein, miraculin, has the unusual property of modifying a sour taste into a sweet taste. Previous attempts to isolate miraculin from deeply colored alkaline extracts of the miracle fruit were unsuccessful. We found that miraculin is extracted with 0.5 M NaCl solution. The extracted solution is colorless and shows the strong sweet-inducing activity. Miraculin was purified from the extracted solution by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose affinity chromatography. The purified miraculin thus obtained gave a single sharp peak in reverse phase high performance liquid chromatography, indicating that it is highly pure. The sample also gave a single band having molecular weight 28,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was much lower than the values reported previously (40,000-48,000). The amino acid composition of the purified miraculin was determined. Sequence analysis of the purified miraculin indicated that it is composed of a pure single polypeptide and identified 20 amino-terminal amino acids. The purified miraculin contained as much as 13.9% of sugars, which consisted of glucosamine, mannose, galactose, xylose, and fucose in a molar ratio of 3.03:3.00:0.69:0.96:2.12. PMID:3403544

  5. Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties*

    PubMed Central

    Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.

    2016-01-01

    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function. PMID:27013657

  6. Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.

    PubMed

    Campos, Samuel K; Parrott, M Brandon; Barry, Michael A

    2004-06-01

    While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.

  7. Inefficient DNA Repair Is an Aging-Related Modifier of Parkinson's Disease.

    PubMed

    Sepe, Sara; Milanese, Chiara; Gabriels, Sylvia; Derks, Kasper W J; Payan-Gomez, Cesar; van IJcken, Wilfred F J; Rijksen, Yvonne M A; Nigg, Alex L; Moreno, Sandra; Cerri, Silvia; Blandini, Fabio; Hoeijmakers, Jan H J; Mastroberardino, Pier G

    2016-05-31

    The underlying relation between Parkinson's disease (PD) etiopathology and its major risk factor, aging, is largely unknown. In light of the causative link between genome stability and aging, we investigate a possible nexus between DNA damage accumulation, aging, and PD by assessing aging-related DNA repair pathways in laboratory animal models and humans. We demonstrate that dermal fibroblasts from PD patients display flawed nucleotide excision repair (NER) capacity and that Ercc1 mutant mice with mildly compromised NER exhibit typical PD-like pathological alterations, including decreased striatal dopaminergic innervation, increased phospho-synuclein levels, and defects in mitochondrial respiration. Ercc1 mouse mutants are also more sensitive to the prototypical PD toxin MPTP, and their transcriptomic landscape shares important similarities with that of PD patients. Our results demonstrate that specific defects in DNA repair impact the dopaminergic system and are associated with human PD pathology and might therefore constitute an age-related risk factor for PD. PMID:27210754

  8. Inefficient DNA Repair Is an Aging-Related Modifier of Parkinson's Disease.

    PubMed

    Sepe, Sara; Milanese, Chiara; Gabriels, Sylvia; Derks, Kasper W J; Payan-Gomez, Cesar; van IJcken, Wilfred F J; Rijksen, Yvonne M A; Nigg, Alex L; Moreno, Sandra; Cerri, Silvia; Blandini, Fabio; Hoeijmakers, Jan H J; Mastroberardino, Pier G

    2016-05-31

    The underlying relation between Parkinson's disease (PD) etiopathology and its major risk factor, aging, is largely unknown. In light of the causative link between genome stability and aging, we investigate a possible nexus between DNA damage accumulation, aging, and PD by assessing aging-related DNA repair pathways in laboratory animal models and humans. We demonstrate that dermal fibroblasts from PD patients display flawed nucleotide excision repair (NER) capacity and that Ercc1 mutant mice with mildly compromised NER exhibit typical PD-like pathological alterations, including decreased striatal dopaminergic innervation, increased phospho-synuclein levels, and defects in mitochondrial respiration. Ercc1 mouse mutants are also more sensitive to the prototypical PD toxin MPTP, and their transcriptomic landscape shares important similarities with that of PD patients. Our results demonstrate that specific defects in DNA repair impact the dopaminergic system and are associated with human PD pathology and might therefore constitute an age-related risk factor for PD.

  9. Development, characterization and applications of electrodes modified with conductive polymers, ionic liquids and proteins

    NASA Astrophysics Data System (ADS)

    Tang, Yijun

    My research involves both fundamental studies and applications of the electrodes whose surfaces are chemically modified. Conductive polymers are one of the major materials that are used to modify electrode surfaces. The thorough understanding of the behavior of conductive polymers in ionic liquids is interesting and important as the ionic liquids are becoming promising solvents. With poly(vinyl ferrocene) as the model conductive polymer, electrochemical studies were performed in various ionic liquid electrolytes. A theoretical square model and dynamic equilibrium were proposed to describe the interaction between conductive polymers and ionic liquids when the electrons transferred between the electrode and electrolyte. These findings were applied to enable and accelerate the structure relaxation of conductive polymers so that the conductive polymers were capable of delivering peptides efficiently. Incorporation of metallic nanoparticles to the conductive polymer matrix entitled new properties to the conductive polymer, increasing conductivity and providing catalytic abilities. This modification on electrode surface might bring potential uses in gas sensing, energy storage, energy conversion, etc. Conductive polymer coated electrodes produced unique double layer in ionic liquids and a fundamental study of quantum charging help to understand the double layer properties. I also studied the application of surface modified electrodes in chemo- and biosensing. A nonregeneration protocol was created to save the cost and the time in analyzing interfacial binding activities and to prevent the potential of deterioration caused to biological ligands by the conventional regeneration. In the study of carbohydrate/protein interactions, a "click" chemical reaction was first used in constructing a carbohydrate-based biosensor, which was capable of detecting and analyzing proteins specifically and accurately. In another biosensor design, the hydrogen bonding between the template and

  10. The morphology and ageing behaviour of δ-ferrite in a modified 9Cr-1Mo steel

    NASA Astrophysics Data System (ADS)

    Kishore, R.; Singh, R. N.; Sinha, T. K.; Kashyap, B. P.

    1992-10-01

    Dual phase (martensite + δ-ferrite) microstructures were developed in a modified 9Cr-1Mo steel, by austenitising at 1523-1623 K, followed by water-quenching. These duplex structures were thermally aged at 973 K for ageing periods varying from 30 min to 21 h. Morphological aspects of δ-ferrite phase and its response to age-hardening were studied by optical, scanning electron and transmission electron microscopy, X-ray diffraction, electron probe microanalysis and microhardness testing. It was observed that austenitizing at 1523 K produced fine, acicular δ-ferrite while the δ-ferrite formed by austenitising at higher temperatures (1573-1623 K) were massive, irregular-shaped and banded. Moreover the presence of 8-ferrite caused an abnormally strong (110) reflection, observed in X-ray diffraction patterns of martensite plus δ-ferrite structures. This behaviour is thought to be due to development of (110) texture in δ-ferrite phase. Thermal ageing at 973 K caused age-hardening of δ-ferrite with a peak hardness attained after 3.6 ks of ageing. Electron microscopic results suggest that the observed hardening was caused by the formation of Fe 2Mo Laves phase.

  11. Age- and Hypertension-Associated Protein Aggregates in Mouse Heart Have Similar Proteomic Profiles.

    PubMed

    Ayyadevara, Srinivas; Mercanti, Federico; Wang, Xianwei; Mackintosh, Samuel G; Tackett, Alan J; Prayaga, Sastry V S; Romeo, Francesco; Shmookler Reis, Robert J; Mehta, Jawahar L

    2016-05-01

    Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II-induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each P<0.05. One fifth of these proteins were previously associated with age-progressive neurodegenerative or cardiovascular diseases, or both (eg, ApoE, ApoJ, ApoAIV, clusterin, complement C3, and others involved in stress-response and protein-homeostasis pathways). Because fibrosis is a characteristic of both aged and hypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual.

  12. UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products

    PubMed Central

    Linetsky, Mikhail; Raghavan, Cibin T.; Johar, Kaid; Fan, Xingjun; Monnier, Vincent M.; Vasavada, Abhay R.; Nagaraj, Ram H.

    2014-01-01

    Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm2, 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans. PMID:24798334

  13. Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

    PubMed

    Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

    2006-09-01

    An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

  14. Translationally controlled tumor protein supplemented chitosan modified glass ionomer cement promotes osteoblast proliferation and function.

    PubMed

    Sangsuwan, Jiraporn; Wanichpakorn, Supreya; Kedjarune-Leggat, Ureporn

    2015-09-01

    The objective of this study was to evaluate the effect of translationally controlled tumor protein (TCTP) supplemented in a novel glass ionomer cement (BIO-GIC) on normal human osteoblasts (NHost cells). BIO-GIC was a glass ionomer cement (GIC) modified by adding chitosan and albumin to promote the release of TCTP. NHost cells were seeded on specimens of GIC, GIC+TCTP, BIO-GIC and BIO-GIC+TCTP. Cell proliferation was determined by BrdU assay. It was found that BIO-GIC+TCTP had significantly higher proliferation of cells than other specimens. Bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) gene expressions assessed by quantitative real time PCR and alkaline phosphatase (ALP) activity were used to determine cell differentiation. Bone cell function was investigated by calcium deposition using alizarin assay. Both BMP-2 and OPN gene expressions of cells cultured on specimens with added TCTP increased gradually up-regulation after day 1 and reached the highest on day 3 then down-regulation on day 7. The ALP activity of cells cultured on BIO-GIC+TCTP for 7 days and calcium content after 14 days were significantly higher than other groups. BIO-GIC+TCTP can promote osteoblast cells proliferation, differentiation and function.

  15. Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine

    PubMed Central

    Cappelletti, Martina; Perazzolli, Michele; Antonielli, Livio; Nesler, Andrea; Torboli, Esmeralda; Bianchedi, Pier L.; Pindo, Massimo; Puopolo, Gerardo; Pertot, Ilaria

    2016-01-01

    Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies. PMID:27486468

  16. KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling*

    PubMed Central

    DiStefano, Peter V.; Kuebel, Julia M.; Sarelius, Ingrid H.; Glading, Angela J.

    2014-01-01

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1. PMID:25320085

  17. Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection.

    PubMed

    Horká, Marie; Růzicka, Filip; Horký, Jaroslav; Holá, Veronika; Slais, Karel

    2006-09-01

    We suggest a method for the reproducible and efficient capillary isoelectric focusing of proteins and microorganisms in the pH gradient 3-10. The method involves the segmental injection of the simple ampholytes, the solution of the selected electrolytes, and the sample mixture of bioanalytes and carrier ampholytes to the fused silica capillaries dynamically modified by poly(ethylene glycol), PEG 4000, which is added to the catholyte, the anolyte and injected solutions. In order to receive the reproducible results, the capillaries were rinsed by the mixture of acetone/ethanol between analyses. For the tracing of the pH gradients the low-molecular-mass pI markers were used. The simple proteins and the mixed cultures of microorganisms, Saccharomyces cerevisiae CCM 8191, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Staphylococcus aureus, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224, Staphylococcus epidermidis CCM 4418 and Stenotrophomonas maltophilia, were focused and separated by the method suggested. The minimum detectable number of microbial cells was 5x10(2) to 1x10(3) with on-column UV detection at 280 nm.

  18. Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine.

    PubMed

    Cappelletti, Martina; Perazzolli, Michele; Antonielli, Livio; Nesler, Andrea; Torboli, Esmeralda; Bianchedi, Pier L; Pindo, Massimo; Puopolo, Gerardo; Pertot, Ilaria

    2016-01-01

    Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies. PMID:27486468

  19. MitoInteractome: Mitochondrial protein interactome database, and its application in 'aging network' analysis

    PubMed Central

    2009-01-01

    Background Mitochondria play a vital role in the energy production and apoptotic process of eukaryotic cells. Proteins in the mitochondria are encoded by nuclear and mitochondrial genes. Owing to a large increase in the number of identified mitochondrial protein sequences and completed mitochondrial genomes, it has become necessary to provide a web-based database of mitochondrial protein information. Results We present 'MitoInteractome', a consolidated web-based portal containing a wealth of information on predicted protein-protein interactions, physico-chemical properties, polymorphism, and diseases related to the mitochondrial proteome. MitoInteractome contains 6,549 protein sequences which were extracted from the following databases: SwissProt, MitoP, MitoProteome, HPRD and Gene Ontology database. The first general mitochondrial interactome has been constructed based on the concept of 'homologous interaction' using PSIMAP (Protein Structural Interactome MAP) and PEIMAP (Protein Experimental Interactome MAP). Using the above mentioned methods, protein-protein interactions were predicted for 74 species. The mitochondrial protein interaction data of humans was used to construct a network for the aging process. Analysis of the 'aging network' gave us vital insights into the interactions among proteins that influence the aging process. Conclusion MitoInteractome is a comprehensive database that would (1) aid in increasing our understanding of the molecular functions and interaction networks of mitochondrial proteins, (2) help in identifying new target proteins for experimental research using predicted protein-protein interaction information, and (3) help in identifying biomarkers for diagnosis and new molecular targets for drug development related to mitochondria. MitoInteractome is available at http://mitointeractome.kobic.kr/. PMID:19958484

  20. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  1. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  2. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  3. The maternal-age-associated risk of congenital heart disease is modifiable.

    PubMed

    Schulkey, Claire E; Regmi, Suk D; Magnan, Rachel A; Danzo, Megan T; Luther, Herman; Hutchinson, Alayna K; Panzer, Adam A; Grady, Mary M; Wilson, David B; Jay, Patrick Y

    2015-04-01

    Maternal age is a risk factor for congenital heart disease even in the absence of any chromosomal abnormality in the newborn. Whether the basis of this risk resides with the mother or oocyte is unknown. The impact of maternal age on congenital heart disease can be modelled in mouse pups that harbour a mutation of the cardiac transcription factor gene Nkx2-5 (ref. 8). Here, reciprocal ovarian transplants between young and old mothers establish a maternal basis for the age-associated risk in mice. A high-fat diet does not accelerate the effect of maternal ageing, so hyperglycaemia and obesity do not simply explain the mechanism. The age-associated risk varies with the mother's strain background, making it a quantitative genetic trait. Most remarkably, voluntary exercise, whether begun by mothers at a young age or later in life, can mitigate the risk when they are older. Thus, even when the offspring carry a causal mutation, an intervention aimed at the mother can meaningfully reduce their risk of congenital heart disease.

  4. Development of Cy5.5-Labeled Hydrophobically Modified Glycol Chitosan Nanoparticles for Protein Delivery

    NASA Astrophysics Data System (ADS)

    Chin, Amanda

    Therapeutic proteins are often highly susceptible to enzymatic degradation, thus restricting their in vivo stability. To overcome this limitation, delivery systems designed to promote uptake and reduce degradation kinetics have undergone a rapid shift from macro-scale systems to nanomaterial based carriers. Many of these nanomaterials, however, elicit immune responses and may have cytotoxic effects both in vitro and in vivo. The naturally derived polysaccharide chitosan has emerged as a promising biodegradable material and has been utilized for many biomedical applications; nevertheless, its function is often constrained by poor solubility. Glycol chitosan, a derivative of chitosan, can be hydrophobically modified to impart amphiphilic properties that enable the self-assembly into nanoparticles in aqueous media at neutral pH. This nanoparticle system has shown initial success as a therapeutic agent in several model cell culture systems, but little is known about its stability against enzymatic degradation. Therefore, the goal of this research was to investigate the resistance of hydrophobically modified glycol chitosan against enzyme-catalyzed degradation using an in vivo simulated system containing lysozyme. To synthesize the nanoparticles, hydrophobic cholanic acid was first covalently conjugated to glycol chitosan using of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Conjugates were purified by dialysis, lyophilized, and ultra-sonicated to form nanoparticles. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of 5beta-cholanic acid to the glycol chitosan. Particle size and stability over time were determined with dynamic light scattering (DLS), and particle morphology was evaluated by transmission electron microscopy (TEM). The average diameter of the nanoparticles was approximately 200 nm, which remained stable at 4°C for up to 10 days. Additionally, a near infrared fluorescent (NIRF) dye

  5. Reproductive Aging Drives Protein Accumulation in the Uterus and Limits Lifespan in C. elegans

    PubMed Central

    Zimmerman, Stephanie M.; Hinkson, Izumi V.; Elias, Joshua E.; Kim, Stuart K.

    2015-01-01

    Aging in Caenorhabditis elegans is characterized by widespread physiological and molecular changes, but the mechanisms that determine the rate at which these changes occur are not well understood. In this study, we identify a novel link between reproductive aging and somatic aging in C. elegans. By measuring global age-related changes in the proteome, we identify a previously uncharacterized group of secreted proteins in the adult uterus that dramatically increase in abundance with age. This accumulation is blunted in animals with an extended reproductive period and accelerated in sterile animals lacking a germline. Uterine proteins are not removed in old post-reproductive animals or in young vulvaless worms, indicating that egg-laying is necessary for their rapid removal in wild-type young animals. Together, these results suggest that age-induced infertility contributes to extracellular protein accumulation in the uterus with age. Finally, we show that knocking down multiple age-increased proteins simultaneously extends lifespan. These results provide a mechanistic example of how the cessation of reproduction contributes to detrimental changes in the soma, and demonstrate how the timing of reproductive decline can influence the rate of aging. PMID:26656270

  6. Reproductive Aging Drives Protein Accumulation in the Uterus and Limits Lifespan in C. elegans.

    PubMed

    Zimmerman, Stephanie M; Hinkson, Izumi V; Elias, Joshua E; Kim, Stuart K

    2015-12-01

    Aging in Caenorhabditis elegans is characterized by widespread physiological and molecular changes, but the mechanisms that determine the rate at which these changes occur are not well understood. In this study, we identify a novel link between reproductive aging and somatic aging in C. elegans. By measuring global age-related changes in the proteome, we identify a previously uncharacterized group of secreted proteins in the adult uterus that dramatically increase in abundance with age. This accumulation is blunted in animals with an extended reproductive period and accelerated in sterile animals lacking a germline. Uterine proteins are not removed in old post-reproductive animals or in young vulvaless worms, indicating that egg-laying is necessary for their rapid removal in wild-type young animals. Together, these results suggest that age-induced infertility contributes to extracellular protein accumulation in the uterus with age. Finally, we show that knocking down multiple age-increased proteins simultaneously extends lifespan. These results provide a mechanistic example of how the cessation of reproduction contributes to detrimental changes in the soma, and demonstrate how the timing of reproductive decline can influence the rate of aging.

  7. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats.

    PubMed

    Schrøder, Malene; Poulsen, Morten; Wilcks, Andrea; Kroghsbo, Stine; Miller, Andreas; Frenzel, Thomas; Danier, Jürgen; Rychlik, Michael; Emami, Kaveh; Gatehouse, Angharad; Shu, Qingyao; Engel, Karl-Heinz; Altosaar, Illimar; Knudsen, Ib

    2007-03-01

    An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15mg Bt toxin/kg and based on the average feed consumption the daily intake was 0.54mg Bt toxin/kg body weight. No adverse effects on animal behaviour or weight gain were observed during the study. Blood samples collected one week prior to sacrifice were analyzed and compared for standard haematological and biochemical parameters. A few parameters were significantly different, but all within the normal reference intervals for rats of this breed and age and not in relation to any other findings, thus not considered treatment related. Upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s).

  8. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra

    PubMed Central

    Cannistraci, Carlo Vittorio; Abbas, Ahmed; Gao, Xin

    2015-01-01

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet's performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis. PMID:25619991

  9. Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin.

    PubMed

    Yamashita, H; Theerasilp, S; Aiuchi, T; Nakaya, K; Nakamura, Y; Kurihara, Y

    1990-09-15

    A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed. PMID:2394746

  10. Proteomic profiling reveals a severely perturbed protein expression pattern in aged skeletal muscle.

    PubMed

    O'Connell, Kathleen; Gannon, Joan; Doran, Philip; Ohlendieck, Kay

    2007-08-01

    Extended longevity is often accompanied by frailty and increased susceptibility to a variety of crippling disorders. One of the most striking features of human aging is sarcopenia, which is defined as the age-related decline in skeletal muscle mass and strength. Although various metabolic and functional defects in aging muscle fibres have been described over the last decade, it is not known whether a pathophysiological hierarchy exists within degenerative pathways leading to muscle wasting. Hence, in order to identify novel biomarkers of age-dependent skeletal muscle degeneration, we have here applied mass spectrometry-based proteomics for studying global muscle protein expression patterns. As a model system of sarcopenia, we have employed crude extracts from senescent rat gastrocnemius muscle, as compared to young adult tissue preparations. Using the highly sensitive protein dye Deep Purple for the analysis of the 2-D separated muscle proteome and peptide mass fingerprinting for the identification of individual protein spots, a differential expression pattern was observed for contractile proteins, metabolic factors, regulatory components and heat shock elements. A drastic increase was shown for alpha B-crystallin, myosin light chain MLC-1, phosphoglycerate kinase, adenylate kinase, triosephosphate isomerase, albumin, aconitase and nucleoside-diphosphate kinase in aged fibres. In contrast, the expression of pyruvate kinase, aldolase, creatine kinase, transferrin, alpha-tropomyosin and myosin light chain MLC-3 was decreased in old skeletal muscle. Comparative 2-D immunoblotting of selected candidate proteins has confirmed the effect of aging on the skeletal muscle proteome. These findings demonstrate a severely perturbed protein expression pattern in aged skeletal muscle, which reflects the underlying molecular alterations causing a drastic decline of muscle strength in the senescent organism. In the long-term, the systematic deduction of abnormal protein expression

  11. MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age.

    PubMed

    Ferree, Andrew W; Trudeau, Kyle; Zik, Eden; Benador, Ilan Y; Twig, Gilad; Gottlieb, Roberta A; Shirihai, Orian S

    2013-11-01

    To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.

  12. A Mutation in DAOA Modifies the Age of Onset in PSEN1 E280A Alzheimer's Disease.

    PubMed

    Vélez, Jorge I; Rivera, Dora; Mastronardi, Claudio A; Patel, Hardip R; Tobón, Carlos; Villegas, Andrés; Cai, Yeping; Easteal, Simon; Lopera, Francisco; Arcos-Burgos, Mauricio

    2016-01-01

    We previously reported age of onset (AOO) modifier genes in the world's largest pedigree segregating early-onset Alzheimer's disease (AD), caused by the p.Glu280Ala (E280A) mutation in the PSEN1 gene. Here we report the results of a targeted analysis of functional exonic variants in those AOO modifier genes in sixty individuals with PSEN1 E280A AD who were whole-exome genotyped for ~250,000 variants. Standard quality control, filtering, and annotation for functional variants were applied, and common functional variants located in those previously reported as AOO modifier loci were selected. Multiloci linear mixed-effects models were used to test the association between these variants and AOO. An exonic missense mutation in the G72 (DAOA) gene (rs2391191, P = 1.94 × 10(-4), P FDR = 9.34 × 10(-3)) was found to modify AOO in PSEN1 E280A AD. Nominal associations of missense mutations in the CLUAP1 (rs9790, P = 7.63 × 10(-3), P FDR = 0.1832) and EXOC2 (rs17136239, P = 0.0325, P FDR = 0.391) genes were also found. Previous studies have linked polymorphisms in the DAOA gene with the occurrence of neuropsychiatric symptoms such as depression, apathy, aggression, delusions, hallucinations, and psychosis in AD. Our findings strongly suggest that this new conspicuous functional AOO modifier within the G72 (DAOA) gene could be pivotal for understanding the genetic basis of AD. PMID:26949549

  13. The SWIRM domain: a conserved module found in chromosomal proteins points to novel chromatin-modifying activities

    PubMed Central

    Aravind, L; Iyer, Lakshminarayan M

    2002-01-01

    Background Eukaryotic chromosomal components, especially histones, are subject to a wide array of covalent modifications and catalytic reorganization. These modifications have an important role in the regulation of chromatin structure and are mediated by large multisubunit complexes that contain modular proteins with several conserved catalytic and noncatalytic adaptor domains. Results Using computational sequence-profile analysis methods, we identified a previously uncharacterized, predicted α-helical domain of about 85 residues in chromosomal proteins such as Swi3p, Rsc8p, Moira and several other uncharacterized proteins. This module, termed the SWIRM domain, is predicted to mediate specific protein-protein interactions in the assembly of chromatin-protein complexes. In one group of proteins, which are highly conserved throughout the crown-group eukaryotes, the SWIRM domain is linked to a catalytic domain related to the monoamine and polyamine oxidases. Another human protein has the SWIRM domain linked to a JAB domain that is involved in protein degradation through the ubiquitin pathway. Conclusions Identification of the SWIRM domain could help in directed experimental analysis of specific interactions in chromosomal proteins. We predict that the proteins in which it is combined with an amino-oxidase domain define a novel class of chromatin-modifying enzymes, which are likely to oxidize either the amino group of basic residues in histones and other chromosomal proteins or the polyamines in chromatin, and thereby alter the charge distribution. Other forms, such as KIAA1915, may link chromatin modification to ubiquitin-dependent protein degradation. PMID:12186646

  14. Protein synthesis and antioxidant capacity in aging mice: effects of long-term voluntary exercise.

    PubMed

    Vaanholt, Lobke M; Speakman, John R; Garland, Theodore; Lobley, Gerald E; Visser, G Henk

    2008-01-01

    Exercise increases metabolic rate and the production of reactive oxygen species (ROS) but also elevates protein turnover. ROS cause damage to macromolecules (e.g., proteins) and thereby contribute to aging. Protein turnover removes and replaces damaged proteins. The balance between these two responses may underlie beneficial effects of physical activity on aging. Effects of lifelong exercise on antioxidant enzyme activities and fractional synthesis rate of protein (FSRP) were examined at various ages (2-26 mo) in heart, liver, and muscle of mice that had been selectively bred for high wheel-running activity, housed with (S+) or without (S-) a running wheel, and their random-bred controls (C+) housed with running wheels. FSRP decreased with age and increased in muscle of young, but not old, activity-selected mice. Enzyme activity of superoxide dismutase and glutathione peroxidase decreased with age and showed a peak at 10 mo of age in liver. Selection for wheel-running activity did not affect antioxidant enzyme activity. Daily energy expenditure correlated positively with antioxidant levels in liver. This might indicate that oxidative stress (ROS production) increases with metabolic rate, driving upregulation of antioxidant enzymes. Alternatively, the elevated energy expenditure may reflect the energetic cost of elevated protection, consistent with the disposable-soma hypothesis and with other studies showing positive links between energy expenditure and life span. Long-term elevations in voluntary exercise did not result in elevations in antioxidant enzyme activities or protein synthesis rates.

  15. Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

    PubMed Central

    Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

    2013-01-01

    Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

  16. Altered MCM protein levels and autophagic flux in aged and systemic sclerosis dermal fibroblasts.

    PubMed

    Dumit, Verónica I; Küttner, Victoria; Käppler, Jakob; Piera-Velazquez, Sonsoles; Jimenez, Sergio A; Bruckner-Tuderman, Leena; Uitto, Jouni; Dengjel, Jörn

    2014-09-01

    Aging is a common risk factor of many disorders. With age, the level of insoluble extracellular matrix increases leading to increased stiffness of a number of tissues. Matrix accumulation can also be observed in fibrotic disorders, such as systemic sclerosis (SSc). Although the intrinsic aging process in skin is phenotypically distinct from SSc, here we demonstrate similar behavior of aged and SSc skin fibroblasts in culture. We have used quantitative proteomics to characterize the phenotype of dermal fibroblasts from healthy subjects of various ages and from patients with SSc. Our results demonstrate that proteins involved in DNA and RNA processing decrease with age and in SSc, whereas those involved in mitochondrial and other metabolic processes behave the opposite. Specifically, minichromosome maintenance (MCM) helicase proteins are less abundant with age and SSc, and they exhibit an altered subcellular distribution. We observed that lower levels of MCM7 correlate with reduced cell proliferation, lower autophagic capacity, and higher intracellular protein abundance phenotypes of aged and SSc cells. In addition, we show that SSc fibroblasts exhibit higher levels of senescence compared with their healthy counterparts, suggesting further similarities between the fibrotic disorder and the aging process. Hence, at the molecular level, SSc fibroblasts exhibit intrinsic characteristics of fibroblasts from aged skin.

  17. Protein repair L-isoaspartyl methyltransferase in plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley seeds.

    PubMed Central

    Mudgett, M B; Lowenson, J D; Clarke, S

    1997-01-01

    Protein L-isoaspartate (D-aspartate) O-methyltransferases (MTs; EC 2.1.1.77) can initiate the conversion of detrimental L-isoaspartyl residues in spontaneously damaged proteins to normal L-aspartyl residues. We detected this enzyme in 45 species from 23 families representing most of the divisions of the plant kingdom. MT activity is often localized in seeds, suggesting that it has a role in their maturation, quiescence, and germination. The relationship among MT activity, the accumulation of abnormal protein L-isoaspartyl residues, and seed viability was explored in barley (Hordeum vulgare cultivar Himalaya) seeds, which contain high levels of MT. Natural aging of barley seeds for 17 years resulted in a significant reduction in MT activity and in seed viability, coupled with increased levels of "unrepaired" L-isoaspartyl residues. In seeds heated to accelerate aging, we found no reduction of MT activity, but we did observe decreased seed viability and the accumulation of isoaspartyl residues. Among populations of accelerated aged seed, those possessing the highest levels of L-isoaspartyl-containing proteins had the lowest germination percentages. These results suggest that the MT present in seeds cannot efficiently repair all spontaneously damaged proteins containing altered aspartyl residues, and their accumulation during aging may contribute to the loss of seed viability. PMID:9414558

  18. The aging process modifies the distensibility of elastic but not muscular arteries.

    PubMed

    Bortolotto, L A; Hanon, O; Franconi, G; Boutouyrie, P; Legrain, S; Girerd, X

    1999-10-01

    Aging decreases the distensibility of large elastic arteries; however, the effects of age on the functional parameters of muscular, medium-sized arteries are not well determined. This study evaluated the consequences of aging on the functional parameters of the carotid and radial arteries in normotensive men. A total of 62 elderly subjects (aged 74+/-2 years) were compared with 87 young subjects (aged 35+/-3 years). Internal diameter and intima-media thickness (IMT) were measured by a high-resolution echo-tracking system to calculate distensibility and incremental elastic modulus (Einc). Although in the normal range, systolic and diastolic blood pressure levels were statistically different in the 2 groups at 128+/-19 and 74+/-13 mm Hg versus 121+/-27 and 71+/-18 mm Hg in the young and elderly subjects, respectively (P<0.05). At the carotid artery level, elderly subjects exhibited a greater IMT (742+/-144 versus 469+/-132 microm; P<0.01) and internal diameter (7067+/-828 versus 6062+/-1026 microm; P<0.01) than young subjects; elderly subjects also had lower distensibility (12+/-2 versus 21+/-2 kPa(-1) x 10(-3); P<0.01) and higher Einc (0.9+/-0.2 versus 0.7+/-0.3 kPa x 10(3); P<0.01). At the radial artery level, both IMT (240+/-42 versus 218+/-51 microm; P<0.01) and internal diameter (2685+/-432 versus 2491+/-444 microm; P<0.01) were greater in elderly subjects, but no differences in distensibility and Einc were observed between the 2 groups. All differences remained significant, even after adjusting for mean blood pressure. These results indicate that the increase of the internal diameter and IMT observed during the aging process can have opposite effects on the functional parameters of large elastic or medium-sized muscular arteries.

  19. Effect of hydrodynamic pressure processing and aging on sarcoplasmic proteins of beef strip loins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study evaluated the effects of hydrodynamic pressure processing (HDP) and aging on the sarcoplasmic proteins of beef strip loins. Loins (n=12) were halved at 48 h postmortem and assigned to HDP or control treatments. Following treatment, each half was divided into three portions for aging (0, 5...

  20. The MTHFR C677T polymorphism modifies age at onset in Parkinson's disease.

    PubMed

    Vallelunga, Annamaria; Pegoraro, Valentina; Pilleri, Manuela; Biundo, Roberta; De Iuliis, Angela; Marchetti, Mauro; Facchini, Silvia; Formento Dojot, Patrizia; Antonini, Angelo

    2014-01-01

    Hyperhomocysteinemia is a risk factor for Parkinson's disease (PD) and may result from genetic mutations or/and environmental factors. 5,10-methylenetetrahydrofolate reductase (MTHFR) is a folate-dependent enzyme that catalyzed remethylation of homocysteine (Hcy) and the MTHFR C677T polymorphism makes the MTHFR enzyme thermolabile causing hyperhomocysteinemia. In this study we analyzed whether two functional polymorphisms of MTHFR gene, A1298C and C677T, affect age of onset in PD. We enrolled 120 patients with sporadic PD. Patients were divided into three groups based on MTHFR C677T polymorphisms: (a) homozygotes wild type (CC) (b) heterozygotes (CT) and (c) homozygotes carriers of mutation (TT). MTHFR SNPs were analyzed using High-Resolution Melt analysis and ANOVA was performed to assess whether polymorphisms of MTHFR gene could influence age of onset. The MTHFR A1298C polymorphism had no effect on PD age at onset (p = 1.0) while there was a significant association with MTHFR C677T (p = 0.019 Bonferroni-adjusted post hoc) showing an earlier onset in CC as compared with TT. (p = 0.024). No differences were found for vascular load assessed with magnetic resonance imaging, pharmacological therapy and cognitive state for two MTHFR SNPs. Our results suggest a possible association of MTHFR C677T with age at onset of PD and may have important implications regarding the role of MTHFR. PMID:24052451

  1. Age modifies effect of body size on fecundity in Culex quinquefasciatus Say (Diptera: Culicidae).

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fecundity of mosquitoes can vary with many factors, and can have a strong effect on population growth. This study reports the effects of body size, blood meal size and age on reproductive output of nulliparous Culex quinquefasciatus, a vector of arboviruses and other pathogens. Mated adult female m...

  2. Atherosclerosis and Vascular Aging as Modifiers of Tumor Progression, Angiogenesis, and Responsiveness to Therapy

    PubMed Central

    Klement, Halka; St. Croix, Brad; Milsom, Chloe; May, Linda; Guo, Qing; Yu, Joanne L.; Klement, Petr; Rak, Janusz

    2007-01-01

    It is rarely considered that age-related common vascular co-morbidities may affect therapeutic outcomes of antiangiogenic therapy in cancer. Indeed, the accepted model of human disease consists of 4- to 8-week-old (young) tumor-bearing, but otherwise healthy, experimental mice, yet human cancers are diagnosed and treated in later decades of life when atherosclerosis and vascular diseases are highly prevalent. Here we present evidence that tumor growth and angiogenesis are profoundly altered in mice affected by natural aging and with genetically induced atherosclerosis (in ApoE−/− mice). Thus, transplantable tumors (Lewis lung carcinoma and B16F1) grew at higher rates in young (4 to 8 weeks old) ApoE+/+ and ApoE−/− nonatherosclerotic syngeneic recipients than in their old (12 to 18 months old) or atherosclerotic (old/ApoE−/−) counterparts. These age-related changes were paralleled by reduced tumor vascularity, lower expression of tumor endothelial marker 1, increased acute tumor hypoxia, depletion of circulating CD45−/VEGFR+ cells, and impaired endothelial sprouting ex vivo. Exposure of tumor-bearing mice to metronomic therapy with cyclophosphamide exerted antimitotic effects on tumors in young hosts, but this effect was reduced in atherosclerotic mice. Collectively, our results suggest that vascular aging and disease may affect tumor progression, angiogenesis, and responses to therapy. PMID:17823292

  3. Total protein, animal protein and physical activity in relation to muscle mass in middle-aged and older Americans.

    PubMed

    Morris, Martha Savaria; Jacques, Paul F

    2013-04-14

    Resistance training is recognised as a good strategy for retarding age-related declines in muscle mass and strength. Recent studies have also highlighted the potential value of protein intakes in excess of present recommendations. The roles that leisure-time physical activity and protein quality play in the preservation of skeletal muscle during ageing, and how such influences interact in free-living people are unclear. We sought to clarify these issues using data collected on 2425 participants aged ≥ 50 years in the US National Health and Nutrition Examination Survey (2003-2006). We estimated subjects' usual intakes of total protein and beef from two 24 h diet recalls and computed the appendicular skeletal muscle mass index from anthropometric measures. Participants self-reported their physical activity levels. Analyses accounted for demographic factors and smoking. The association between muscle-strengthening activity and the appendicular skeletal muscle mass index varied with protein intake. Furthermore, among obese subjects with protein intakes < 70 g/d, those who performed such activities had a lower appendicular skeletal muscle mass index than those who were physically inactive. Protein intakes above the present recommendations were associated with benefits to obese subjects only. The appendicular skeletal muscle mass index of non-obese subjects who performed vigorous aerobic activities was consistently high; in obese subjects, it varied with protein intake. High-protein intake was associated with a modest increase in the appendicular skeletal muscle mass index in non-obese, physically inactive subjects. The present findings reinforce the idea that muscle-strengthening exercise preserves muscle when combined with adequate dietary protein. Vigorous aerobic activity may also help.

  4. Preliminary study on the effects of ageing cold oxygen plasma treated PET/PP with respect to protein adsorption.

    PubMed

    Chen, Rui; Bayon, Yves; Hunt, John A

    2012-08-01

    Surfaces of polyethylene terephthalate (PET) and polypropylene (PP) have been modified by oxygen plasma. The surface hydrophilicity and changes in topography during up to 90 days storage in water and in dry air in a desiccator were analysed by dynamic contact angle test and atomic force microscopy (AFM). Clear ageing effects on the plasma treated surface were observed as increases in contact angle and changes in roughness as functions of increasing storage time. However, the effect of oxygen plasma treatment to increase the hydrophilicity of surface was still evident on the treated surfaces even after 90 days storage either in dry air or in water. In protein adsorption experiments, human serum albumin (HSA) and fibrinogen (Fg) were adsorbed on untreated and oxygen plasma treated PET and PP surfaces. The quantified ATR-FTIR results showed that both HSA and Fg adsorption on PET and PP surfaces decreased after oxygen plasma treatment, with the effect most evident for HSA. Although for both proteins adsorption increased with ageing, the amount of adsorbed proteins was still lower than untreated surface at 30 days. This suggests the shelf life of oxygen plasma treated samples could be as long as 30 days. PMID:22521680

  5. A sustainable slashing industry using biodegradable sizes from modified soy protein to replace petro-based poly(vinyl alcohol).

    PubMed

    Zhao, Yi; Zhao, Yuzhu; Xu, Helan; Yang, Yiqi

    2015-02-17

    Biodegradable sizing agents from triethanolamine (TEA) modified soy protein could substitute poly(vinyl alcohol)(PVA) sizes for high-speed weaving of polyester and polyester/cotton yarns to substantially decrease environmental pollution and impel sustainability of textile industry. Nonbiodegradable PVA sizes are widely used and mainly contribute to high chemical oxygen demand (COD) in textile effluents. It has not been possible to effectively degrade, reuse or replace PVA sizes so far. Soy protein with good biodegradability showed potential as warp sizes in our previous studies. However, soy protein sizes lacked film flexibility and adhesion for required high-speed weaving. Additives with multiple hydroxyl groups, nonlinear molecule, and electric charge could physically modify secondary structure of soy protein and lead to about 23.6% and 43.3% improvement in size adhesion and ability of hair coverage comparing to unmodified soy protein. Industrial weaving results showed TEA-soy protein had relative weaving efficiency 3% and 10% higher than PVA and chemically modified starch sizes on polyester/cotton fabrics, and had relative weaving efficiency similar to PVA on polyester fabrics, although with 3- 6% lower add-on. In addition, TEA-soy sizes had a BOD5/COD ratio of 0.44, much higher than 0.03 for PVA, indicating that TEA-soy sizes were easily biodegradable in activated sludge.

  6. From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants.

    PubMed

    Hiwasa-Tanase, Kyoko; Hirai, Tadayoshi; Kato, Kazuhisa; Duhita, Narendra; Ezura, Hiroshi

    2012-03-01

    The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use. PMID:22160133

  7. Aggregate structure and effect of phthalic anhydride modified soy protein on the mechanical properties of styrene-butadiene copolymer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aggregate structure of phthalic anhydride (PA) modified soy protein isolate (SPI) was investigated by estimating its fractal dimension from the equilibrated dynamic strain sweep experiments. The estimated fractal dimensions of the filler aggregates were less than 2, indicating that these partic...

  8. From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants.

    PubMed

    Hiwasa-Tanase, Kyoko; Hirai, Tadayoshi; Kato, Kazuhisa; Duhita, Narendra; Ezura, Hiroshi

    2012-03-01

    The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.

  9. Mesona Chinensis Benth extract prevents AGE formation and protein oxidation against fructose-induced protein glycation in vitro

    PubMed Central

    2014-01-01

    Background Mesona chinensis Benth (Chinese Mesona), an economically significant agricultural plant, is the most widely consumed as an herbal beverage in Southeast Asia and China. The objective of this study was to evaluate the inhibitory activity of Mesona chinensis (MC) extract on the formation of advanced glycation end products (AGEs) and protein oxidation in an in vitro model of fructose-mediated protein glycation. Methods The content of total polyphenolic compounds was measured by using Folin–Ciocalteu assay. Antiglycation activity was determined using the formation of AGE fluorescence intensity, Nϵ-(carboxymethyl)lysine (CML), the level of fructosamine, and the formation of amyloid cross β-structure. The protein oxidation was examined using the level of protein carbonyl content and thiol group. Results Our results revealed that the content of total polyphenolic compound in MC extract was 212.4 ± 5.6 mg gallic acid equivalents/g dried extract. MC extract (0.25-1.00 mg/mL) significantly inhibited the formation of fluorescence AGEs in fructose-glycated bovine serum albumin (BSA) during 4 weeks of study. Furthermore, MC extract also decreased the level of Nϵ-CML, fructosamine, and amyloid cross β-structure in fructose-glycated BSA. While the total thiol group was elevated and the protein carbonyl content was decreased in BSA incubated with fructose and MC extract. Conclusions The extract of MC inhibits fructose-mediated protein glycation and protein oxidation. This edible plant could be a natural rich source of antiglycation agent for preventing AGE-mediated diabetic complication. PMID:24708679

  10. Biochemical and Structural Studies of Conserved Maf Proteins Revealed Nucleotide Pyrophosphatases with a Preference for Modified Nucleotides

    PubMed Central

    Tchigvintsev, Anatoli; Tchigvintsev, Dmitri; Flick, Robert; Popovic, Ana; Dong, Aiping; Xu, Xiaohui; Brown, Greg; Lu, Wenyun; Wu, Hong; Cui, Hong; Dombrowski, Ludmila; Joo, Jeong Chan; Beloglazova, Natalia; Min, Jinrong; Savchenko, Alexei; Caudy, Amy A.; Rabinowitz, Joshua D.; Murzin, Alexey G.; Yakunin, Alexander F.

    2013-01-01

    Summary Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning. PMID:24210219

  11. Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein.

    PubMed

    Ohkubo, Takayuki; Tamiya, Minoru; Abe, Keiko; Ishiguro, Masaji

    2015-01-01

    Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness. PMID:26010443

  12. Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein

    PubMed Central

    Ohkubo, Takayuki; Tamiya, Minoru; Abe, Keiko; Ishiguro, Masaji

    2015-01-01

    Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness. PMID:26010443

  13. Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging.

    PubMed

    Laimer, Martin; Kocher, Thomas; Chiocchetti, Andreas; Trost, Andrea; Lottspeich, Friedrich; Richter, Klaus; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

    2010-10-01

    Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).

  14. Protein biogenesis machinery is a driver of replicative aging in yeast

    PubMed Central

    Janssens, Georges E; Meinema, Anne C; González, Javier; Wolters, Justina C; Schmidt, Alexander; Guryev, Victor; Bischoff, Rainer; Wit, Ernst C; Veenhoff, Liesbeth M; Heinemann, Matthias

    2015-01-01

    An integrated account of the molecular changes occurring during the process of cellular aging is crucial towards understanding the underlying mechanisms. Here, using novel culturing and computational methods as well as latest analytical techniques, we mapped the proteome and transcriptome during the replicative lifespan of budding yeast. With age, we found primarily proteins involved in protein biogenesis to increase relative to their transcript levels. Exploiting the dynamic nature of our data, we reconstructed high-level directional networks, where we found the same protein biogenesis-related genes to have the strongest ability to predict the behavior of other genes in the system. We identified metabolic shifts and the loss of stoichiometry in protein complexes as being consequences of aging. We propose a model whereby the uncoupling of protein levels of biogenesis-related genes from their transcript levels is causal for the changes occurring in aging yeast. Our model explains why targeting protein synthesis, or repairing the downstream consequences, can serve as interventions in aging. DOI: http://dx.doi.org/10.7554/eLife.08527.001 PMID:26422514

  15. Physical growth and bone age of survivors of protein energy malnutrition.

    PubMed Central

    Alvear, J; Artaza, C; Vial, M; Guerrero, S; Muzzo, S

    1986-01-01

    Early postnatal malnutrition produces delay in growth and developmental processes, and children from a low socioeconomical level where undernutrition is prevalent are shorter than those from higher socioeconomic levels. We examined the effects of severe and early protein energy malnutrition on growth and bone maturation. We studied 40 preschool children who had been admitted to hospital in infancy with protein energy malnutrition and 38 children from the same socioeconomic level, paired for age and sex, who had never been malnourished. Growth measurements were made over a period of 4-6 years, and bone age was determined in a subgroup through wrist roentgenograms. Results showed a correlation between protein energy malnutrition, birth weight of infants, and mother's height and head circumference. The group with protein energy malnutrition showed a significant delay in stature after four years, especially the girls (p less than 0.001). Weight:height ratio was reduced in boys compared with controls but not in girls. Both groups showed a delay in bone maturation, but there were no significant differences between them. We found a positive correlation between bone age and arm fat area in control boys and between bone age and height for age in boys with protein energy malnutrition. The finding that rehabilitated children were shorter than the control group but had similar bone age at follow up suggests that genetic or prenatal factors were important in their later poor growth, and this suggestion is supported by their smaller birth size and the smaller size of their mothers. PMID:3083790

  16. Vitamin D–Binding Protein Modifies the Vitamin D–Bone Mineral Density Relationship

    PubMed Central

    Powe, Camille E; Ricciardi, Catherine; Berg, Anders H; Erdenesanaa, Delger; Collerone, Gina; Ankers, Elizabeth; Wenger, Julia; Karumanchi, S Ananth; Thadhani, Ravi; Bhan, Ishir

    2011-01-01

    Studies examining the relationship between total circulating 25-hydroxyvitamin D [25(OH)D] levels and bone mineral density (BMD) have yielded mixed results. Vitamin D–binding protein (DBP), the major carrier protein for 25(OH)D, may alter the biologic activity of circulating vitamin D. We hypothesized that free and bioavailable 25(OH)D, calculated from total 25(OH)D, DBP, and serum albumin levels, would be more strongly associated with BMD than levels of total 25(OH)D. We measured total 25(OH)D, DBP, and serum albumin levels in 49 healthy young adults enrolled in the Metabolic Abnormalities in College-Aged Students (MACS) study. Lumbar spine BMD was measured in all subjects using dual-energy X-ray absorptiometry. Clinical, diet, and laboratory information also was gathered at this time. We determined free and bioavailable (free + albumin-bound) 25(OH)D using previously validated formulas and examined their associations with BMD. BMD was not associated with total 25(OH)D levels (r = 0.172, p = .236). In contrast, free and bioavailable 25(OH)D levels were positively correlated with BMD (r = 0.413, p = .003 for free, r = 0.441, p = .002 for bioavailable). Bioavailable 25(OH)D levels remained independently associated with BMD in multivariate regression models adjusting for age, sex, body mass index, and race (p = .03). It is concluded that free and bioavailable 25(OH)D are more strongly correlated with BMD than total 25(OH)D. These findings have important implications for vitamin D supplementation in vitamin D–deficient states. Future studies should continue to explore the relationship between free and bioavailable 25(OH)D and health outcomes. © 2011 American Society for Bone and Mineral Research. PMID:21416506

  17. C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging.

    PubMed

    van Ham, Tjakko J; Thijssen, Karen L; Breitling, Rainer; Hofstra, Robert M W; Plasterk, Ronald H A; Nollen, Ellen A A

    2008-03-01

    Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders. PMID:18369446

  18. The effect of aging on mitochondrial proteins in germinating soybean embryonic axes

    SciTech Connect

    Furman, K.C.

    1988-01-01

    Aging-induced deterioration is a major problem associated with seed storage. Impairment of mitochondrial function is one of the first effects of aging. The composition and synthesis of nuclear and mito-coded mitochondrial proteins from soybean (Glycine max. L. Merr.) embryonic axes were studied to elucidate the cause of impaired respiratory development during germination of aged seeds. Axes excised from high vigor (HV) seeds and aged or low vigor (LV) seeds were protected from imbibition injury and germinated for various times, or excised from developing seeds, and then radiolabeled for one hour in ({sup 35}S)methionine. Mitochondria were then isolated and total mitochondrial protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by quantitative staining and fluorography of labeled polypeptides. Alternatively, an original two-dimensional native-to-denaturing gel electrophoretic technique was used to analyze native protein associations and to purify a 23 kD polypeptide.

  19. Evaluating age-associated phenotypes in a mouse model of protein dyshomeostasis.

    PubMed

    Min, Jin-Na; Patterson, Cam

    2011-03-01

    Proteotoxicity caused by an imbalanced protein quality control surveillance system is believed to contribute to the phenotypes associated with aging as well as many neurodegenerative diseases. Understanding and monitoring the impact of proteotoxicity in these processes offers researchers keen insight into the biology of aging, as well as other conditions that share similar pathological etiologies. In Section 2, we present various technical approaches that can be used to calculate and characterize the phenotypes associated with aging that are linked to increased proteotoxicity. Methods such as the measurement of oligomer protein expression and the capacity of proteasome function are useful tools in observing both aging phenotypes and neurodegenerative diseases, both of which share the phenomenon of impaired protein homeostasis.

  20. Loss of prion protein leads to age-dependent behavioral abnormalities and changes in cytoskeletal protein expression.

    PubMed

    Schmitz, Matthias; Greis, Catharina; Ottis, Philipp; Silva, Christopher J; Schulz-Schaeffer, Walter J; Wrede, Arne; Koppe, Katharina; Onisko, Bruce; Requena, Jesús R; Govindarajan, Nambirajan; Korth, Carsten; Fischer, Andre; Zerr, Inga

    2014-12-01

    The cellular prion protein (PrPC) is a highly conserved protein whose exact physiological role remains elusive. In the present study, we investigated age-dependent behavioral abnormalities in PrPC-knockout (Prnp0/0) mice and wild-type (WT) controls. Prnp0/0 mice showed age-dependent behavioral deficits in memory performance, associative learning, basal anxiety, and nest building behavior. Using a hypothesis-free quantitative proteomic investigation, we found that loss of PrPC affected the levels of neurofilament proteins in an age-dependent manner. In order to understand the biochemical basis of these observations, we analyzed the phosphorylation status of neurofilament heavy chain (NF-H). We found a reduction in NF-H phosphorylation in both Prnp0/0 mice and in PrPC-deficient cells. The expression of Fyn and phospho-Fyn, a potential regulator for NF phosphorylation, was associated with PrPC ablation. The number of β-tubulin III-positive neurons in the hippocampus was diminished in Prnp0/0 mice relative to WT mice. These data indicate that PrPC plays an important role in cytoskeletal organization, brain function, and age-related neuroprotection. Our work represents the first direct biochemical link between these proteins and the observed behavioral phenotypes.

  1. Structural and Functional Properties of Soy Protein Isolates Modified by Soy Soluble Polysaccharides.

    PubMed

    Xu, Yan-Teng; Liu, Ling-Ling

    2016-09-28

    Aiming to achieve the modification to soy protein isolate (SPI) by soy soluble polysaccharides (SSPS), electrically driven complex systems were first established in the environment of pH 3.0, and then reconstituted SPI particles with different SPI-SSPS ratios were obtained under freeze-drying process. Through this treatment, the structures of SPI particles were partly unfolded and adsorbed SSPS mainly via hydrophobic interactions and hydrogen bonding with larger particle sizes. The adherence of SSPS decreased the surface hydrophobicity of reconstituted SPI particles, but exerted not much influence on the emulsifying and foaming activities and increased the corresponding stabilities due to enhancing the unfolded extent of structure and improving the conformation flexibility. Reconstituted SPI-SSPS particles might rearrange and link each other due to the presence of SSPS on the air-water interface to better stabilize these systems. At SPI-SSPS ratio of 10:1, lower temperature was required to form gels with lower gel intensity and porous structure. The findings provide a further comprehension to the relationship between structures and functional properties of SPI modified by SSPS and the feasibility of applying these reconstituted particles to needed areas. PMID:27608266

  2. Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin.

    PubMed

    Takahashi, N; Hitotsuya, H; Hanzawa, H; Arata, Y; Kurihara, Y

    1990-05-15

    The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text) PMID:2335505

  3. Age-dependent decline of nogo-a protein in the mouse cerebrum.

    PubMed

    Kumari, Anita; Thakur, M K

    2014-11-01

    Nogo-A, a myelin-associated neurite growth inhibitory protein, is implicated in synaptic plasticity. It binds to its receptor namely the Nogo-66 receptor1 (NgR1) and regulates filamentous (F) actin dynamics via small GTPases of the Rho family, RhoA kinase (ROCK), LimK and cofilin. These proteins are associated with the structural plasticity, one of the components of synaptic plasticity, which is known to decline with normal aging. So, the level of Nogo-A and its receptor NgR1 are likely to vary during normal brain aging. However, it is not clearly understood how the levels of Nogo-A and its receptor NgR1 change in the cerebrum during aging. Several studies show an age- and gender-dependent decline in synaptic plasticity. Therefore, the present study was planned to analyze the relative changes in the mRNA and protein levels of Nogo-A and NgR1 in both male and female mice cerebrum during normal aging. Western blot analysis has shown decrease in Nogo-A protein level during aging in both male and female mice cerebrum. This was further confirmed by immunofluorescence analysis. RT-PCR analysis of Nogo-A mRNA showed no significant difference in the above-mentioned groups. This was also supported by in situ hybridization. NgR1 protein and its mRNA expression levels showed no significant alteration with aging in the cerebrum of both male and female mice. Taken together, we speculate that the downregulation of Nogo-A protein might have a role in the altered synaptic plasticity during aging.

  4. Physical Stability of Octenyl Succinate-Modified Polysaccharides and Whey Proteins for Potential Use as Bioactive Carriers in Food Systems.

    PubMed

    Puerta-Gomez, Alex F; Castell-Perez, M Elena

    2015-06-01

    The high cost and potential toxicity of biodegradable polymers like poly(lactic-co-glycolic)acid (PLGA) has increased the interest in natural and modified biopolymers as bioactive carriers. This study characterized the physical stability (water sorption and state transition behavior) of selected starch and proteins: octenyl succinate-modified depolymerized waxy corn starch (DWxCn), waxy rice starch (DWxRc), phytoglycogen, whey protein concentrate (80%, WPC), whey protein isolate (WPI), and α-lactalbumin (α-L) to determine their potential as carriers of bioactive compounds under different environmental conditions. After enzyme modification and particle size characterization, glass transition temperature and moisture isotherms were used to characterize the systems. DWxCn and DWxRc had increased water sorption compared to native starch. The level of octenyl succinate anhydrate (OSA) modification (3% and 7%) did not reduce the water sorption of the DWxCn and phytoglycogen samples. The Guggenheim-Andersen-de Boer model indicated that native waxy corn had significantly (P < 0.05) higher water monolayer capacity followed by 3%-OSA-modified DWxCn, WPI, 3%-OSA-modified DWxRc, α-L, and native phytoglycogen. WPC had significantly lower water monolayer capacity. All Tg values matched with the solid-like appearance of the biopolymers. Native polysaccharides and whey proteins had higher glass transition temperature (Tg) values. On the other hand, depolymerized waxy starches at 7%-OSA modification had a "melted" appearance when exposed to environments with high relative humidity (above 70%) after 10 days at 23 °C. The use of depolymerized and OSA-modified polysaccharides blended with proteins created more stable blends of biopolymers. Hence, this biopolymer would be suitable for materials exposed to high humidity environments in food applications.

  5. Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins.

    PubMed

    Dyawanapelly, Sathish; Koli, Uday; Dharamdasani, Vimisha; Jain, Ratnesh; Dandekar, Prajakta

    2016-08-01

    The main aim of the present study was to compare mucoadhesion and cellular uptake efficiency of chitosan (CS) and chitosan oligosaccharide (COS) surface-modified polymer nanoparticles (NPs) for mucosal delivery of proteins. We have developed poly (D, L-lactide-co-glycolide) (PLGA) NPs, surface-modified COS-PLGA NPs and CS-PLGA NPs, by using double emulsion solvent evaporation method, for encapsulating bovine serum albumin (BSA) as a model protein. Surface modification of NPs was confirmed using physicochemical characterization methods such as particle size and zeta potential, SEM, TEM and FTIR analysis. Both surface-modified PLGA NPs displayed a slow release of protein compared to PLGA NPs. Furthermore, we have explored the mucoadhesive property of COS as a material for modifying the surface of polymeric NPs. During in vitro mucoadhesion test, positively charged COS-PLGA NPs and CS-PLGA NPs exhibited enhanced mucoadhesion, compared to negatively charged PLGA NPs. This interaction was anticipated to improve the cell interaction and uptake of NPs, which is an important requirement for mucosal delivery of proteins. All nanoformulations were found to be safe for cellular delivery when evaluated in A549 cells. Moreover, intracellular uptake behaviour of FITC-BSA loaded NPs was extensively investigated by confocal laser scanning microscopy and flow cytometry. As we hypothesized, positively charged COS-PLGA NPs and CS-PLGA NPs displayed enhanced intracellular uptake compared to negatively charged PLGA NPs. Our results demonstrated that CS- and COS-modified polymer NPs could be promising carriers for proteins, drugs and nucleic acids via nasal, oral, buccal, ocular and vaginal mucosal routes. PMID:27106502

  6. Protein adsorption capability on polyurethane and modified-polyurethane membrane for periodontal guided tissue regeneration applications.

    PubMed

    Sheikh, Zeeshan; Khan, Abdul Samad; Roohpour, Nima; Glogauer, Michael; Rehman, Ihtesham U

    2016-11-01

    Periodontal disease if left untreated can result in creation of defects within the alveolar ridge. Barrier membranes are frequently used with or without bone replacement graft materials for achieving periodontal guided tissue regeneration (GTR). Surface properties of barrier membranes play a vital role in their functionality and clinical success. In this study polyetherurethane (PEU) membranes were synthesized by using 4,4'-methylene-diphenyl diisocyanate (MDI), polytetramethylene oxide (PTMO) and 1,4-butane diol (BDO) as a chain extender via solution polymerization. Hydroxyl terminated polydimethylsiloxane (PDMS) due to having inherent surface orientation towards air was used for surface modification of PEU on one side of the membranes. This resulting membranes had one surface being PEU and the other being PDMS coated PEU. The prepared membranes were treated with solutions of bovine serum albumin (BSA) in de-ionized water at 37°C at a pH of 7.2. The surface protein adsorptive potential of PEU membranes was observed using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Raman spectroscopy and Confocal Raman spectroscopy. The contact angle measurement, tensile strength and modulus of prepared membranes were also evaluated. PEU membrane (89.86±1.62°) exhibited less hydrophobic behavior than PEU-PDMS (105.87±3.16°). The ultimate tensile strength and elastic modulus of PEU (27±1MPa and 14±2MPa) and PEU-PDMS (8±1MPa and 26±1MPa) membranes was in required range. The spectral analysis revealed adsorption of BSA proteins on the surface of non PDMS coated PEU surface. The PDMS modified PEU membranes demonstrated a lack of BSA adsorption. The non PDMS coated side of the membrane which adsorbs proteins could potentially be used facing towards the defect attracting growth factors for periodontal tissue regeneration. Whereas, the PDMS coated side could serve as an occlusive barrier for preventing gingival epithelial cells from

  7. Protein adsorption capability on polyurethane and modified-polyurethane membrane for periodontal guided tissue regeneration applications.

    PubMed

    Sheikh, Zeeshan; Khan, Abdul Samad; Roohpour, Nima; Glogauer, Michael; Rehman, Ihtesham U

    2016-11-01

    Periodontal disease if left untreated can result in creation of defects within the alveolar ridge. Barrier membranes are frequently used with or without bone replacement graft materials for achieving periodontal guided tissue regeneration (GTR). Surface properties of barrier membranes play a vital role in their functionality and clinical success. In this study polyetherurethane (PEU) membranes were synthesized by using 4,4'-methylene-diphenyl diisocyanate (MDI), polytetramethylene oxide (PTMO) and 1,4-butane diol (BDO) as a chain extender via solution polymerization. Hydroxyl terminated polydimethylsiloxane (PDMS) due to having inherent surface orientation towards air was used for surface modification of PEU on one side of the membranes. This resulting membranes had one surface being PEU and the other being PDMS coated PEU. The prepared membranes were treated with solutions of bovine serum albumin (BSA) in de-ionized water at 37°C at a pH of 7.2. The surface protein adsorptive potential of PEU membranes was observed using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Raman spectroscopy and Confocal Raman spectroscopy. The contact angle measurement, tensile strength and modulus of prepared membranes were also evaluated. PEU membrane (89.86±1.62°) exhibited less hydrophobic behavior than PEU-PDMS (105.87±3.16°). The ultimate tensile strength and elastic modulus of PEU (27±1MPa and 14±2MPa) and PEU-PDMS (8±1MPa and 26±1MPa) membranes was in required range. The spectral analysis revealed adsorption of BSA proteins on the surface of non PDMS coated PEU surface. The PDMS modified PEU membranes demonstrated a lack of BSA adsorption. The non PDMS coated side of the membrane which adsorbs proteins could potentially be used facing towards the defect attracting growth factors for periodontal tissue regeneration. Whereas, the PDMS coated side could serve as an occlusive barrier for preventing gingival epithelial cells from

  8. A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC.

    PubMed

    Ng, J; Hart, C M; Morgan, K; Simon, J A

    2000-05-01

    The extra sex combs (ESC) and Enhancer of zeste [E(Z)] proteins, members of the Polycomb group (PcG) of transcriptional repressors, interact directly and are coassociated in fly embryos. We report that these two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, we show that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, we present evidence that ESC is phosphorylated in vivo and that this modified ESC is preferentially associated in the complex with E(Z). Modified ESC accumulates between 2 and 6 h of embryogenesis, which is the developmental time when esc function is first required. We find that mutations in E(z) reduce the ratio of modified to unmodified ESC in vivo. We have also generated germ line transformants that express ESC proteins bearing site-directed mutations that disrupt ESC-E(Z) binding in vitro. These mutant ESC proteins fail to provide esc function, show reduced levels of modification in vivo, and are still assembled into complexes. Taken together, these results suggest that ESC phosphorylation normally occurs after assembly into ESC-E(Z) complexes and that it contributes to the function or regulation of these complexes. We discuss how biochemically separable ESC-E(Z) and PC-PH complexes might work together to provide PcG repression. PMID:10757791

  9. Direct analysis of in-gel proteins by carbon nanotubes-modified paper spray ambient mass spectrometry.

    PubMed

    Han, Feifei; Yang, Yuhan; Ouyang, Jin; Na, Na

    2015-02-01

    The in situ and direct extraction, desorption and ionization of in-gel intact proteins after electrophoresis has been achieved by carbon nanotubes (CNTs)-modified paper spray mass spectrometry at ambient conditions. Characteristics of CNTs (including larger surface area, smaller pore diameter and enhanced conductivity) were endowed to the porous filter paper substrate by uniformly dispersing the CNTs on the filter paper. Upon applying electric potential to the CNTs-modified paper, the in-gel proteins were extracted from the gel and subsequently migrated to the tip of the filter paper by electrophoresis-like behavior for paper spray ionization, which was monitored by extracted ion chronograms. The characterizations of modified filter papers and CNTs nanoparticles further confirmed the role of CNTs in in-gel protein extraction, protein migration as well as spray ionization at the paper tip. Under optimized conditions, a mixture of cytochrome c, lysozyme and myoglobin was successfully separated by native electrophoresis and subsequently analysed by the present method, showing a limit of detection of 10 ng per gel band. The present strategy offers a new pathway for the direct detection of in-gel intact proteins at ambient conditions without any pre-treatment (e.g. digestion, chemical extraction and desalting), which exhibits potential applications in top-down proteomics.

  10. Comprehensive Analysis of Maillard Protein Modifications in Human Lenses: Effect of Age and Cataract

    PubMed Central

    Smuda, Mareen; Henning, Christian; Raghavan, Cibin T.; Johar, Kaid; Vasavada, Abhay R.; Nagaraj, Ram H.; Glomb, Marcus A.

    2015-01-01

    In human lens proteins, advanced glycation endproducts (AGEs) originate from the reaction of glycating agents, e.g., vitamin C and glucose. AGEs have been considered to play a significant role in lens aging and cataract formation. Although several AGEs have been detected in the human lens, the contribution of individual glycating agents to their formation remains unclear. A highly sensitive liquid chromatography–tandem mass spectrometry multimethod was developed that allowed us to quantitate 21 protein modifications in normal and cataractous lenses, respectively. N6-Carboxymethyl lysine, N6-carboxyethyl lysine, N7-carboxyethyl arginine, methylglyoxal hydroimidazolone 1, and N6-lactoyl lysine were found to be the major Maillard protein modifications among these AGEs. The novel vitamin C specific amide AGEs, N6-xylonyl and N6-lyxonyl lysine, but also AGEs from glyoxal were detected, albeit in minor quantities. Among the 21 modifications, AGEs from the Amadori product (derived from the reaction of glucose and lysine) and methylglyoxal were dominant. PMID:25849437

  11. Deficiency of circadian protein CLOCK reduces lifespan and increases age-related cataract development in mice.

    PubMed

    Dubrovsky, Yulia V; Samsa, William E; Kondratov, Roman V

    2010-12-01

    Circadian clock is implicated in the regulation of aging. The transcription factor CLOCK, a core component of the circadian system, operates in complex with another circadian clock protein BMAL1. Recently it was demonstrated that BMAL1 deficiency results in premature aging in mice. Here we investigate the aging of mice deficient for CLOCK protein. Deficiency of the CLOCK protein significantly affects longevity: the average lifespan of Clock-/- mice is reduced by 15% compared with wild type mice, while maximum lifespan is reduced by more than 20%. CLOCK deficiency also results in the development of two age-specific pathologies in these mice, cataracts and dermatitis, at a much higher rate than in wild type mice. In contrast to BMAL1 deficient animals, Clock-/- mice do not develop a premature aging phenotype and do not develop the multiple age-associated pathologies characteristic of BMAL1 deficiency. Thus, although CLOCK and BMAL1 form a transcriptional complex, the physiological result of their deficiency is different. Our results suggest that CLOCK plays an important role in aging, specifically; CLOCK activity is critical for the regulation of normal physiology and aging of the lens and skin.

  12. Age modifies the immunologic response and clinical presentation of American tegumentary leishmaniasis.

    PubMed

    Carvalho, Augusto M; Amorim, Camila F; Barbosa, Juliana L S; Lago, Alexsandro S; Carvalho, Edgar M

    2015-06-01

    Leishmania (Viannia) braziliensis is the main causal agent of American tegumentary leishmaniasis (ATL) that may present as cutaneous, mucosal, or disseminated cutaneous leishmaniasis. The disease is highly prevalent in young males and there is a lack of studies of ATL in the elderly. Herein, we compared clinical manifestations, immunologic response, and response to antimony therapy between patients > 60 years of age (N = 58) and patients who were 21-30 years of age (N = 187). The study was performed in Corte de Pedra, Bahia, Brazil, a well-known area of L. braziliensis transmission. Cytokine production by cultured peripheral blood mononuclear cells stimulated with soluble Leishmania antigen was performed. Elderly subjects more frequently had a previous history of cutaneous leishmaniasis, large lesions, or mucosal leishmaniasis, and they were less likely to have lymphadenopathy. There was no difference regarding gender and response to therapy. Peripheral blood mononuclear cells from elderly subjects produced a similar amount of tumor necrosis factor than young patients but they produced less interferon-gamma and more interleukin-10 than young subjects. We concluded that elderly patients with cutaneous leishmaniasis should be searched for mucosal or disseminated leishmaniasis. The decreased interferon-gamma production and increase in interleukin-10 observed in elderly patients may contribute to parasite persistence and L. braziliensis infection dissemination.

  13. Age Modifies the Immunologic Response and Clinical Presentation of American Tegumentary Leishmaniasis

    PubMed Central

    Carvalho, Augusto M.; Amorim, Camila F.; Barbosa, Juliana L. S.; Lago, Alexsandro S.; Carvalho, Edgar M.

    2015-01-01

    Leishmania (Viannia) braziliensis is the main causal agent of American tegumentary leishmaniasis (ATL) that may present as cutaneous, mucosal, or disseminated cutaneous leishmaniasis. The disease is highly prevalent in young males and there is a lack of studies of ATL in the elderly. Herein, we compared clinical manifestations, immunologic response, and response to antimony therapy between patients > 60 years of age (N = 58) and patients who were 21–30 years of age (N = 187). The study was performed in Corte de Pedra, Bahia, Brazil, a well-known area of L. braziliensis transmission. Cytokine production by cultured peripheral blood mononuclear cells stimulated with soluble Leishmania antigen was performed. Elderly subjects more frequently had a previous history of cutaneous leishmaniasis, large lesions, or mucosal leishmaniasis, and they were less likely to have lymphadenopathy. There was no difference regarding gender and response to therapy. Peripheral blood mononuclear cells from elderly subjects produced a similar amount of tumor necrosis factor than young patients but they produced less interferon-gamma and more interleukin-10 than young subjects. We concluded that elderly patients with cutaneous leishmaniasis should be searched for mucosal or disseminated leishmaniasis. The decreased interferon-gamma production and increase in interleukin-10 observed in elderly patients may contribute to parasite persistence and L. braziliensis infection dissemination. PMID:25918209

  14. Polyethylenimine modified poly(ethylene terephthalate) capillary channeled-polymer fibers for anion exchange chromatography of proteins.

    PubMed

    Jiang, Liuwei; Jin, Yi; Marcus, R Kenneth

    2015-09-01

    Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been previously studied as stationary phases for reversed phase and affinity protein separations. In this study, surface modified PET C-CP fibers were evaluated for the anion exchange separation of proteins. The native PET C-CP fibers were aminated using polyethylenimine (PEI) followed by a 1,4-butanediol diglycidyl ether (BUDGE) cross-linking step. Subsequent PEI/BUDGE treatments can be employed to further develop the polyamine layer on the fiber surfaces. The PEI densities of the modified fibers were quantified through the ninhydrin reaction, yielding values of 0.43-0.89μmolg(-1). The surface modification impact on column permeability was found to be 0.66×10(-11) to 1.33×10(-11)m(2), depending on the modification time and conditions. The dynamic binding capacities of the modified fiber media were determined to be 1.99-8.54mgmL(-1) bed volume, at linear velocities of 88-438cmmin(-1) using bovine serum albumin as the model protein. It was found that increasing the mobile phase linear velocity (up to 438cmmin(-1)) had no effect on the separation quality for a synthetic protein mixture, reflecting the lack of van Deemter C-term effects for the C-CP fiber phase. The low-cost, easy modification method and the capability of fast protein separation illustrate great potential in the use of PEI/BUDGE-modified PET C-CP fibers for high-throughput protein separation and downstream processing. PMID:26253835

  15. Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.

    PubMed

    Tan, C; Prescott, J F; Patterson, M C; Nicholson, V M

    1995-01-01

    Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen. PMID:7704843

  16. Identification of a protein with antioxidant activity that is important for the protection against beer ageing.

    PubMed

    Wu, Ming J; Clarke, Frank M; Rogers, Peter J; Young, Paul; Sales, Narelle; O'Doherty, Patrick J; Higgins, Vincent J

    2011-01-01

    This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

  17. Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

    PubMed Central

    Wu, Ming J.; Clarke, Frank M.; Rogers, Peter J.; Young, Paul; Sales, Narelle; O’Doherty, Patrick J.; Higgins, Vincent J.

    2011-01-01

    This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process. PMID:22016646

  18. Alterations in lenticular proteins during ageing and selenite-induced cataractogenesis in Wistar rats

    PubMed Central

    Sakthivel, Muniyan; Elanchezhian, Rajan; Thomas, Philip A.

    2010-01-01

    Purpose To determine putative alterations in the major lenticular proteins in Wistar rats of different ages and to compare these alterations with those occurring in rats with selenite-induced cataract. Methods Lenticular transparency was determined by morphological examination using slit-lamp biomicroscopy. Alterations in lenticular protein were determined by sodium dodecyl sulfate-PAGE (SDS–PAGE) and confirmed immunologically by western blot. Results Morphological examination did not reveal observable opacities in the lenses of the rats of different age groups; however, dense nuclear opacities were noted in lenses of rats in the selenite-cataract group. Western blot assays revealed age-related changes in soluble and urea-soluble lenticular proteins. Decreased αA- and βB1-crystallins in the soluble fraction and aggregation of αA-crystallin, in addition to the degraded fragment of βB1-crystallin, in the urea-soluble fraction appeared to occur in relation to increasing age of the rats from which the lenses were taken; similarly, cytoskeletal proteins appeared to decline with increasing age. The lenses from rats in the selenite-cataract group exhibited similar changes, except that there was also high molecular weight aggregation of αA-crystallin. Conclusions The results of this study suggest that there is loss, as well as aggregation, of αA-crystallin in the aging rat lens, although there is no accompanying loss of lenticular transparency. PMID:20300567

  19. Design and evaluation of solid lipid nanoparticles modified with peptide ligand for oral delivery of protein drugs.

    PubMed

    Fan, Tingting; Chen, Chunhui; Guo, Han; Xu, Juan; Zhang, Jian; Zhu, Xi; Yang, Yang; Zhou, Zhou; Li, Lian; Huang, Yuan

    2014-10-01

    Designing feasible and effective peptide ligand modified solid lipid nanoparticles (SLNs) to improve oral bioavailability of protein drugs and evaluating the influence of mucus remains important. In the present work, two kinds of peptide ligand modified SLNs loaded with salmon calcitonin (sCT), namely, sCT CSK-SLNs and sCT IRQ-SLNs, were prepared by coupling the peptide ligand CSKSSDYQC (CSK) which was reported to show affinity with goblet cells, or IRQRRRR (IRQ), a cell penetrating peptide, to polyoxyethylene (40) stearate (SA-PEG2000). Compared with unmodified SLNs, CSK or IRQ modified SLNs with better drug protection ability could facilitate the internalization of drug on Caco-2/HT29-MTX co-cultured cells and permeation in excised rat duodenum mucosa. The internalization mechanism of two kinds of peptide ligand modified SLNs was mainly active transport via both clathrin- and caveolae-dependent endocytosis. Although mucus was an impediment to the transport of SLNs, the peptide ligand modified SLNs still showed improved drug absorption. The absolute bioavailability of sCT CSK-SLNs (12.41 ± 3.65%) and sCT IRQ-SLNs (10.05 ± 5.10%) raised to 2.45-fold and 1.98-fold compared with unmodified SLNs (5.07 ± 0.54%), implying the feasibility and effectiveness of CSK and IRQ peptide modification for the enhancement of the oral bioavailability of protein drugs. In summary, the nanoparticles modified with CSK or IRQ peptide ligand could be the potential carriers for the transport of protein drugs across intestinal barriers.

  20. The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones

    PubMed Central

    Truttmann, Matthias C.; Guo, Xuanzong; Engert, Christoph; Schwartz, Thomas U.; Ploegh, Hidde L.

    2016-01-01

    Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans. PMID:27138431

  1. Mortality Benefits of Antibiotic Computerised Decision Support System: Modifying Effects of Age

    NASA Astrophysics Data System (ADS)

    Chow, Angela L. P.; Lye, David C.; Arah, Onyebuchi A.

    2015-11-01

    Antibiotic computerised decision support systems (CDSSs) are shown to improve antibiotic prescribing, but evidence of beneficial patient outcomes is limited. We conducted a prospective cohort study in a 1500-bed tertiary-care hospital in Singapore, to evaluate the effectiveness of the hospital’s antibiotic CDSS on patients’ clinical outcomes, and the modification of these effects by patient factors. To account for clustering, we used multilevel logistic regression models. One-quarter of 1886 eligible inpatients received CDSS-recommended antibiotics. Receipt of antibiotics according to CDSS’s recommendations seemed to halve mortality risk of patients (OR 0.54, 95% CI 0.26-1.10, P = 0.09). Patients aged ≤65 years had greater mortality benefit (OR 0.45, 95% CI 0.20-1.00, P = 0.05) than patients that were older than 65 (OR 1.28, 95% CI 0.91-1.82, P = 0.16). No effect was observed on incidence of Clostridium difficile (OR 1.02, 95% CI 0.34-3.01), and multidrug-resistant organism (OR 1.06, 95% CI 0.42-2.71) infections. No increase in infection-related readmission (OR 1.16, 95% CI 0.48-2.79) was found in survivors. Receipt of CDSS-recommended antibiotics reduced mortality risk in patients aged 65 years or younger and did not increase the risk in older patients. Physicians should be informed of the benefits to increase their acceptance of CDSS recommendations.

  2. Mortality Benefits of Antibiotic Computerised Decision Support System: Modifying Effects of Age

    PubMed Central

    Chow, Angela L. P.; Lye, David C.; Arah, Onyebuchi A.

    2015-01-01

    Antibiotic computerised decision support systems (CDSSs) are shown to improve antibiotic prescribing, but evidence of beneficial patient outcomes is limited. We conducted a prospective cohort study in a 1500-bed tertiary-care hospital in Singapore, to evaluate the effectiveness of the hospital’s antibiotic CDSS on patients’ clinical outcomes, and the modification of these effects by patient factors. To account for clustering, we used multilevel logistic regression models. One-quarter of 1886 eligible inpatients received CDSS-recommended antibiotics. Receipt of antibiotics according to CDSS’s recommendations seemed to halve mortality risk of patients (OR 0.54, 95% CI 0.26–1.10, P = 0.09). Patients aged ≤65 years had greater mortality benefit (OR 0.45, 95% CI 0.20–1.00, P = 0.05) than patients that were older than 65 (OR 1.28, 95% CI 0.91–1.82, P = 0.16). No effect was observed on incidence of Clostridium difficile (OR 1.02, 95% CI 0.34–3.01), and multidrug-resistant organism (OR 1.06, 95% CI 0.42–2.71) infections. No increase in infection-related readmission (OR 1.16, 95% CI 0.48–2.79) was found in survivors. Receipt of CDSS-recommended antibiotics reduced mortality risk in patients aged 65 years or younger and did not increase the risk in older patients. Physicians should be informed of the benefits to increase their acceptance of CDSS recommendations. PMID:26617195

  3. Autosomal Dominant Retinal Dystrophies Caused by a Founder Splice Site Mutation, c.828+3A>T, in PRPH2 and Protein Haplotypes in trans as Modifiers

    PubMed Central

    Shankar, Suma P.; Hughbanks-Wheaton, Dianna K.; Birch, David G.; Sullivan, Lori S.; Conneely, Karen N.; Bowne, Sara J.; Stone, Edwin M.; Daiger, Stephen P.

    2016-01-01

    Purpose We determined the phenotypic variation, disease progression, and potential modifiers of autosomal dominant retinal dystrophies caused by a splice site founder mutation, c.828+3A>T, in the PRPH2 gene. Methods A total of 62 individuals (19 families) harboring the PRPH2 c.828+3A>T mutation, had phenotype analysis by fundus appearance, electrophysiology, and visual fields. The PRPH2 haplotypes in trans were sequenced for potential modifying variants and generalized estimating equations (GEE) used for statistical analysis. Results Several distinct phenotypes caused by the PRPH2 c.828+3A>T mutation were observed and fell into two clinical categories: Group I (N = 44) with mild pattern dystrophies (PD) and Group II (N = 18) with more severe cone-rod dystrophy (CRD), retinitis pigmentosa (RP), and central areolar chorioretinal dystrophy (CACD). The PRPH2 Gln304-Lys310-Asp338 protein haplotype in trans was found in Group I only (29.6% vs. 0%), whereas the Glu304-Lys310-Gly338 haplotype was predominant in Group II (94.4% vs. 70.4%). Generalized estimating equations analysis for PD versus the CRD/CACD/RP phenotypes in individuals over 43 years alone with the PRPH2 haplotypes in trans and age as predictors, adjusted for correlation within families, confirmed a significant effect of haplotype on severity (P = 0.03) with an estimated odds ratio of 7.16 (95% confidence interval [CI] = [2.8, 18.4]). Conclusions The PRPH2 c.828+3A>T mutation results in multiple distinct phenotypes likely modified by protein haplotypes in trans; the odds of having the CACD/RP-like phenotype (versus the PD phenotype) are 7.16 times greater with a Glu304-Lys310-Gly338 haplotype in trans. Further functional studies of the modifying haplotypes in trans and PRPH2 splice variants may offer therapeutic targets. PMID:26842753

  4. Protein profiling reveals consequences of lifestyle choices on predicted biological aging

    PubMed Central

    Enroth, Stefan; Enroth, Sofia Bosdotter; Johansson, Åsa; Gyllensten, Ulf

    2015-01-01

    Ageing is linked to a number of changes in how the body and its organs function. On a molecular level, ageing is associated with a reduction of telomere length, changes in metabolic and gene-transcription profiles and an altered DNA-methylation pattern. Lifestyle factors such as smoking or stress can impact some of these molecular processes and thereby affect the ageing of an individual. Here we demonstrate by analysis of 77 plasma proteins in 976 individuals, that the abundance of circulating proteins accurately predicts chronological age, as well as anthropometrical measurements such as weight, height and hip circumference. The plasma protein profile can also be used to identify lifestyle factors that accelerate and decelerate ageing. We found smoking, high BMI and consumption of sugar-sweetened beverages to increase the predicted chronological age by 2–6 years, while consumption of fatty fish, drinking moderate amounts of coffee and exercising reduced the predicted age by approximately the same amount. This method can be applied to dried blood spots and may thus be useful in forensic medicine to provide basic anthropometrical measures for an individual based on a biological evidence sample. PMID:26619799

  5. Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles

    PubMed Central

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Whitwell, Harry; Elgy, Christine; Ding, Ping; Mahajan, Sumeet; Morgan, Cliff; Griffiths, Mark; Clark, Howard; Madsen, Jens

    2015-01-01

    Abstract The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A−/− mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages. PMID:25676620

  6. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    NASA Astrophysics Data System (ADS)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-02-01

    An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU-PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU-PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU-PVP (6.0 h) film reduced greatly to 0.08 μg/cm2, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  7. Comparative proteomic analysis of plasma proteins in patients with age-related macular degeneration

    PubMed Central

    Xu, Xin-Rong; Zhong, Lu; Huang, Bing-Lin; Wei, Yuan-Hua; Zhou, Xin; Wang, Ling; Wang, Fu-Qiang

    2014-01-01

    AIM To find the significant altered proteins in age-related macular degeneration (AMD) patients as potential biomarkers of AMD. METHODS A comparative analysis of the protein pattern of AMD patients versus healthy controls was performed by means of proteomic analysis using two-dimensional gel electrophoresis followed by protein identification with MALDI TOF/TOF mass spectrometry. RESULTS We identified 28 proteins that were significantly altered with clinical relevance in AMD patients. These proteins were involved in a wide range of biological functions including immune responses, growth cytokines, cell fate determination, wound healing, metabolism, and anti-oxidance. CONCLUSION These results demonstrate the capacity of proteomic analysis of AMD patient plasma. In addition to the utility of this approach for biomarker discovery, identification of alterations in endogenous proteins in the plasma of AMD patient could improve our understanding of the disease pathogenesis. PMID:24790867

  8. Using nonlinear ultrasound measurements to track thermal aging in modified 9%Cr ferritic martensitic steel

    NASA Astrophysics Data System (ADS)

    Marino, Daniel; Kim, Jin-Yeon; Jacobs, Laurence J.; Ruiz, Alberto; Joo, Young-Sang

    2015-03-01

    This study investigates early thermal aging in 9%Cr ferritic martensitic (FM) steel, which is caused by the formation of second phases during high temperature exposure. This study employs a recently developed nonlinear ultrasonic technique to explore the sensitivity of the nonlinearity parameter. Experimental results show that the nonlinearity parameter is sensitive to certain changes in material's properties such as thermal embrittlement and hardness changes; therefore, it can be used as an indicator of the thermal damage. The specimens investigated are heat treated for different holding times ranging from 200h to 3000h at 650°C. Nonlinear ultrasonic experiments are conducted for each specimen using a wedge transducer to generate and an air-coupled transducer to detect Raleigh surface waves. The amplitudes of the first and second order harmonics are measured at different propagation distances and these amplitudes are used to obtain the relative nonlinearity parameter for each specimen with a different holding time. The nonlinear ultrasonic results are compared with independent mechanical measurements and metallographic images. This research proposes the nonlinear ultrasonic technique as a nondestructive evaluation tool not only to detect thermal damage in early stages, and also to qualitatively assess the stage of thermal damage.

  9. Cryptic residual GALT activity is a potential modifier of scholastic outcome in school age children with classic galactosemia.

    PubMed

    Ryan, Emily L; Lynch, Mary Ellen; Taddeo, Elles; Gleason, Tyler J; Epstein, Michael P; Fridovich-Keil, Judith L

    2013-11-01

    Classic galactosemia is a potentially lethal disorder that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. Although early diagnosis and rigorous dietary restriction of galactose prevent or resolve the potentially lethal acute symptoms, patients are at markedly increased risk of long-term complications including significant cognitive, speech, and behavioral difficulties, among other problems. The mechanisms that underlie these long-term complications remain unclear, as do the factors that modify their severity. Here we explored the scholastic and behavioral outcomes experienced by a cohort of 54 school age children with classic galactosemia. Data collected included survey responses from parents and teachers, school records including standardized test scores, and GALT genotype data used to estimate predicted residual GALT activity based on a yeast expression system. As expected, many but not all of the children in our study demonstrated speech, scholastic, and behavioral difficulties. Perhaps most striking, we found that predicted cryptic residual GALT activity, often below the threshold of detection of clinical assays, appeared to modify scholastic outcome. These data raise the intriguing possibility that cryptic GALT activity might also influence the severity of other long-term complications in classic galactosemia. PMID:23319291

  10. Protein quality control in time and space - links to cellular aging.

    PubMed

    Nyström, Thomas; Liu, Beidong

    2014-02-01

    The evolutionary theory of aging regards aging as an evolved characteristic of the soma, and proponents of the theory state that selection does not allow the evolution of aging in unicellular species lacking a soma-germ demarcation. However, the life history of some microorganisms, reproducing vegetatively by either budding or binary fission, has been demonstrated to encompass an ordered, polar-dependent, segregation of damage leading to an aging cell lineage within the clonal population. In the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli, such segregation is under genetic control and includes an asymmetrical inheritance of protein aggregates and inclusions. Herein, the ultimate and proximate causation for such an asymmetrical inheritance, with special emphasis on damaged/aggregated proteins in budding yeast, is reviewed.

  11. Protein quality control in time and space - links to cellular aging.

    PubMed

    Nyström, Thomas; Liu, Beidong

    2014-02-01

    The evolutionary theory of aging regards aging as an evolved characteristic of the soma, and proponents of the theory state that selection does not allow the evolution of aging in unicellular species lacking a soma-germ demarcation. However, the life history of some microorganisms, reproducing vegetatively by either budding or binary fission, has been demonstrated to encompass an ordered, polar-dependent, segregation of damage leading to an aging cell lineage within the clonal population. In the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli, such segregation is under genetic control and includes an asymmetrical inheritance of protein aggregates and inclusions. Herein, the ultimate and proximate causation for such an asymmetrical inheritance, with special emphasis on damaged/aggregated proteins in budding yeast, is reviewed. PMID:24103195

  12. Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart.

    PubMed

    Kerner, Janos; Yohannes, Elizabeth; Lee, Kwangwon; Virmani, Ashraf; Koverech, Aleardo; Cavazza, Claudio; Chance, Mark R; Hoppel, Charles

    2015-01-01

    Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein, we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitine's effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial β-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. Two-dimensional fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a two-fold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins. PMID:25660059

  13. Fe0 nanoparticles remain mobile in porous media after aging due to slow desorption of polymeric surface modifiers.

    PubMed

    Kim, Hye-Jin; Phenrat, Tanapon; Tilton, Robert D; Lowry, Gregory V

    2009-05-15

    Nanosized zerovalent iron (nZVI) is used for in situ remediation of contaminated groundwater. Polyelectrolyte surface coatings are used to inhibit nZVI aggregation and enhance mobility in the subsurface for emplacement. The fate of nZVI is of interest given the uncertainties regarding the effects of nanomaterials on the environment, and depends in part on the stability of these surface coatings against desorption and biodegradation. This study measured the rate and extent of desorption of polyelectrolyte coatings used to stabilize nZVI, including polyaspartate (PAP MW = 2.5 kg/mol and 10 kg/mol), carboxymethyl cellulose (CMC MW= 90 kg/nol and 700 kg/ mol), and polystyrene sulfonate (PSS MW = 70 kg/mol and 1000 kg/mol). The initial adsorbed mass of polyelectrolyte ranged from 0.85 to 3.71 mg/m2 depending on the type and molecular weight (MW). Polyelectrolyte adsorption was confirmed by an increase in nZVI electrophoretic mobility. In all cases, desorption of polyelectrolyte was slow, with less than 30 wt% desorbed after 4 months. The higher MW polyelectrolyte had a greater adsorbed mass and a slower desorption rate for PAP and CMC. nZVI mobility in sand columns after 8 month of desorption was similar to freshly modified nZVI, and significantly greater than unmodified nZVI aged for the same time under identical conditions. Based on these results, polyelectrolyte modified nanoparticles will remain more mobile than their unmodified counterparts even after-aging. Other factors potentially affecting the fate of coated nZVI must be evaluated, especially the potential for biodegradation of coatings.

  14. Why is there a modifying effect of gestational age on risk factors for cerebral palsy?

    PubMed Central

    Greenwood, C; Yudkin, P; Sellers, S; Impey, L; Doyle, P

    2005-01-01

    Objective: To investigate risk factors for cerebral palsy in relation to gestational age. Design: Three case-control studies within a geographically defined cohort. Setting: The former Oxfordshire Health Authority. Participants: A total of 235 singleton children with cerebral palsy not of postnatal origin, born between 1984 and 1993, identified from the Oxford Register of Early Childhood Impairment; 646 controls matched for gestation in three bands: ⩽32 weeks; 33–36 weeks; ⩾37 weeks. Results: Markers of intrapartum hypoxia and infection were associated with an increased risk of cerebral palsy in term and preterm infants. The odds ratio (OR) for hypoxia was 12.2 (95% confidence interval 1.2 to 119) at ⩽32 weeks and 146 (7.4 to 3651) at ⩾37 weeks. Corresponding ORs for neonatal sepsis were 3.1 (1.8 to 5.4) and 10.6 (2.1 to 51.9). In contrast, pre-eclampsia carried an increased risk of cerebral palsy at ⩾37 weeks (OR 5.1 (2.2 to 12.0)) but a decreased risk at ⩽32 weeks (OR 0.4 (0.2 to 1.0)). However, all infants ⩽32 weeks with maternal pre-eclampsia were delivered electively, and their risk of cerebral palsy was no lower than that of other electively delivered ⩽32 week infants (OR 0.9 (0.3 to 2.7)). Nearly 60% of ⩽32 week controls were delivered after spontaneous preterm labour, itself an abnormal event. Conclusion: Inflammatory processes, including pre-eclampsia, are important in the aetiology of cerebral palsy. The apparent reduced risk of cerebral palsy associated with pre-eclampsia in very preterm infants is driven by the characteristics of the gestation matched control group. Use of the term "protective" in this context should be abandoned. PMID:15724038

  15. Physical exercise as a preventive or disease-modifying treatment of dementia and brain aging.

    PubMed

    Ahlskog, J Eric; Geda, Yonas E; Graff-Radford, Neill R; Petersen, Ronald C

    2011-09-01

    A rapidly growing literature strongly suggests that exercise, specifically aerobic exercise, may attenuate cognitive impairment and reduce dementia risk. We used PubMed (keywords exercise and cognition) and manuscript bibliographies to examine the published evidence of a cognitive neuroprotective effect of exercise. Meta-analyses of prospective studies documented a significantly reduced risk of dementia associated with midlife exercise; similarly, midlife exercise significantly reduced later risks of mild cognitive impairment in several studies. Among patients with dementia or mild cognitive impairment, randomized controlled trials (RCTs) documented better cognitive scores after 6 to 12 months of exercise compared with sedentary controls. Meta-analyses of RCTs of aerobic exercise in healthy adults were also associated with significantly improved cognitive scores. One year of aerobic exercise in a large RCT of seniors was associated with significantly larger hippocampal volumes and better spatial memory; other RCTs in seniors documented attenuation of age-related gray matter volume loss with aerobic exercise. Cross-sectional studies similarly reported significantly larger hippocampal or gray matter volumes among physically fit seniors compared with unfit seniors. Brain cognitive networks studied with functional magnetic resonance imaging display improved connectivity after 6 to 12 months of exercise. Animal studies indicate that exercise facilitates neuroplasticity via a variety of biomechanisms, with improved learning outcomes. Induction of brain neurotrophic factors by exercise has been confirmed in multiple animal studies, with indirect evidence for this process in humans. Besides a brain neuroprotective effect, physical exercise may also attenuate cognitive decline via mitigation of cerebrovascular risk, including the contribution of small vessel disease to dementia. Exercise should not be overlooked as an important therapeutic strategy.

  16. Aging of the brain, dementias, role of infectious proteins: facts and theories.

    PubMed

    Morinet, Frédéric

    2014-01-01

    Neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases are a major and growing public health issue for aging populations as aging is the greatest risk factor for neurodegeneration. Protein misfolding and spreading are common to these neurodegenerative diseases. There are many high-quality reviews concerning these diseases; also in this brief chapter, I have tried to give a summary of the principal points involved in the pathogenesis of these three clinical entities. PMID:24862020

  17. Electrochemical impedance spectroscopy for graphene surface modification and protein translocation through the chemically modified graphene nanopore

    NASA Astrophysics Data System (ADS)

    Tiwari, Purushottam; Shan, Yuping; Wang, Xuewen; Darici, Yesim; He, Jin

    2014-03-01

    The multilayer graphene surface has been modified using mercaptohexadecanoic acid (MHA) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (DPPE-PEG750). The surface modifications are evaluated using electrochemical impedance spectroscopy (EIS). EIS measurements show the better graphene surface passivation with DPPE-PEG750 than with MHA. After modification with ferritin, the MHA modified surface shows greater charge transfer resistance (Rct) change than DPPE-PEG750 modified surface. Based on these results the translocations of ferritin through modified graphene nanopore with diameter 5-20 nm are studied. The translocation is more successful through DPPE-PEG750 modified graphene nanopore. This concludes that that the attachment of ferritin to DPPE-PEG750 modified graphene nanopore is not significant compared to MHA modified pore for the ferritin translocation hindrance. These results nicely correlate with the EIS data for respective Rct change of ferritin modified surfaces. P. Tiwari would like to thank FIU School of Integrated Science & Humanity, College Arts & Sciences for the research assistantship.

  18. Rapeseed and soybean products as protein sources for growing turkeys of different ages.

    PubMed

    Palander, S; Näsi, M; Ala-Fossi, I

    2004-10-01

    (1) Apparent ileal and total tract protein digestibilities of rapeseed meal and cake and soybean meal and cake were assayed in growing turkeys at 4, 8 and 12 weeks of age. (2) In addition, the effect of killing technique on apparent ileal protein digestibility values obtained by a slaughter method and effect of rapeseed feeding on size of specific organs were studied. (3) Protein digestibility coefficients of rapeseed products were mostly 0.10 to 0.15 units lower than those of soybean products. Ileal digestibility of protein increased slightly or remained unchanged from 4 to 8 weeks and decreased thereafter. No effect of feed processing method (meal vs cake) on ileal digestibility was observed. (4) Killing the birds by carbon dioxide inhalation and bleeding led to slightly lower ileal digestibility values than mechanical stunning and neck dislocation. (5) Total tract digestibility of protein decreased from 4 to 8 weeks of age for soybean meal and rapeseed meal but increased for soybean cake and rapeseed cake. From 8 to 12 weeks of age total tract digestibility of protein decreased for all the products tested. (6) Feed containing rapeseed led to enlargement of thyroid glands and hearts, but did not affect liver size or mortality.

  19. Protein-Modified-Paramagnetic-Particles as a Tool for Detection of Silver(I) Ions

    NASA Astrophysics Data System (ADS)

    Kizek, R.; Krizkova, S.; Adam, V.; Huska, D.; Hubalek, J.; Trnkova, L.

    2009-04-01

    incorporated mixed with carbon paste were stable. Even after 14 days we did not determine change in the peak height higher than 5 %. Further linkage of MT with polyclonal chicken antibodies incorporated in carbon paste electrode was determined by SWV. Two signals were observed in voltammograms, cysMT corresponding to -SH moieties of MT and Wa corresponding to tryptophan residues of chicken antibodies. We optimized time of interaction (300 s) and concentration of MT (125 µg/ml) to suggest silver(I) ions biosensor. Biosensor (MT-antibody-modified CPE) prepared under the optimized conditions was utilized for silver(I) ions detection. The detection limit (3 S/N) for silver(I) ions were estimated as 100 nM. The proposed biosensor was tested by detection of silver(I) ions spiked in various water samples (from very pure distilled water to rainwater). Recoveries varied from 74 to 104 %. MT, low molecular mass proteins rich cysteine, play important role in the processes of heavy metals ions metabolism. Due to their unique physico-chemical properties they are able to bind heavy metals with high affinity. We used this feature to suggest simple biosensor based on immobilization of MT on the surface of carbon paste electrode via chicken antibodies against MT. The suggested biosensor was further successfully employed to detect silver(I) ions. The main advantage of the biosensor is that it can be easily miniaturized, whereas carbon nanostructures with immobilized MT should be used as working electrodes. Acknowledgements Financial support from INCHEMBIOL MSMT 0021622412 and GA CR 526/07/0674 is highly acknowledged.

  20. Proteolytic changes of myofibrillar proteins in Podolian meat during aging: focusing on tenderness.

    PubMed

    Marino, R; Della Malva, A; Albenzio, M

    2015-03-01

    The aim of this study was to understand the relationship between changes in postmortem degradation of myofibrillar proteins and tenderness development in 3 different muscles from 10 Podolian young bulls aged 1, 7, 14, and 21 d. Psoas major (PM), longissimus dorsi (LD), and semitendinosus muscle () were removed from each half carcass 24 h postmortem. Meat chemical composition, Warner-Bratzler shear force (WBSF), myofibril fragmentation index (MFI), total collagen content, and changes in myofibrillar proteins were estimated in triplicate at each aging time. A significant muscle effect was found on meat chemical composition. Semitendinosus muscle showed the lowest intramuscular fat percentage ( < 0.01) and the highest total collagen content ( < 0.01) with respect to LD and PM. Warner-Bratzler shear force decreased during aging in all muscles ( < 0.001), and semitendinosus was the toughest muscle whereas PM was the most tender ( < 0.001). Myofibril fragmentation index significantly increased ( < 0.001) in LD and PM meat throughout aging time whereas in semitendinosus it increased from 14 d of aging. Proteolysis was investigated by SDS-PAGE, western blotting, and 2-dimensional electrophoresis. Throughout postmortem aging, some structural proteins changed in intensity in all muscles analyzed. The blotting profile highlighted that desmin and troponin-T (TnT) bands were affected by both muscle and aging effects. Desmin degradation was more intense and faster in LD muscle than in semitendinosus and PM muscles. A progressive increase of the degraded isoforms of TnT (33 and 30 kDa polypeptides) was found during aging in LD, while, in PM these bands appeared earlier showing a greater intensity from 1 d. During aging, 2-dimensional gel electrophoresis (2DE) image analyses results showed a significant increase of the total number of spots reaching the highest value in the LD muscle at 21 d of aging ( < 0.01). Proteins separation also revealed differences in the spot number and

  1. Solid-phase extraction based on ground methacrylate monolith modified with gold nanoparticles for isolation of proteins.

    PubMed

    Vergara-Barberán, María; Lerma-García, María Jesús; Simó-Alfonso, Ernesto Francisco; Herrero-Martínez, José Manuel

    2016-04-21

    In this study, a novel polymeric material functionalized with gold nanoparticles (AuNPs) was prepared as solid-phase extraction (SPE) sorbent for isolation of proteins. The sorbent was synthesized from a powdered poly(glycidyl-co-ethylene dimethacrylate) monolith, and modified with ammonia, followed by immobilization of AuNPs on the pore surface of the material. To evaluate the performance of this SPE support, proteins were selected as test solutes, being the extraction conditions and other parameters (loading capacity and regenerative ability of sorbent) established. The results indicated that this sorbent could be employed to selectively capture proteins according to their pI, on the basis of the strong affinity of these biomacromolecules towards to AuNPs surface. The applicability of this sorbent was demonstrated by isolating protein species of interest (bovine serum albumin, cytochrome c and lectins in European mistletoe leaves), followed by SDS-PAGE analysis. PMID:27026598

  2. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    NASA Astrophysics Data System (ADS)

    Kasálková, Nikola Slepičková; Slepička, Petr; Kolská, Zdeňka; Hodačová, Petra; Kučková, Štěpánka; Švorčík, Václav

    2014-04-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly- l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly- l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation.

  3. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    PubMed Central

    2014-01-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly-l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly-l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation. PMID:24708858

  4. Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein

    PubMed Central

    Kajikawa, Akinobu; Zhang, Lin; LaVoy, Alora; Bumgardner, Sara; Klaenhammer, Todd R.; Dean, Gregg A.

    2015-01-01

    Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides. PMID:26509697

  5. Solid-phase extraction based on ground methacrylate monolith modified with gold nanoparticles for isolation of proteins.

    PubMed

    Vergara-Barberán, María; Lerma-García, María Jesús; Simó-Alfonso, Ernesto Francisco; Herrero-Martínez, José Manuel

    2016-04-21

    In this study, a novel polymeric material functionalized with gold nanoparticles (AuNPs) was prepared as solid-phase extraction (SPE) sorbent for isolation of proteins. The sorbent was synthesized from a powdered poly(glycidyl-co-ethylene dimethacrylate) monolith, and modified with ammonia, followed by immobilization of AuNPs on the pore surface of the material. To evaluate the performance of this SPE support, proteins were selected as test solutes, being the extraction conditions and other parameters (loading capacity and regenerative ability of sorbent) established. The results indicated that this sorbent could be employed to selectively capture proteins according to their pI, on the basis of the strong affinity of these biomacromolecules towards to AuNPs surface. The applicability of this sorbent was demonstrated by isolating protein species of interest (bovine serum albumin, cytochrome c and lectins in European mistletoe leaves), followed by SDS-PAGE analysis.

  6. A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging.

    PubMed

    Patterson, Victoria L; Thompson, Brian S; Cherry, Catherine; Wang, Shao-Bin; Chen, Bo; Hoh, Josephine

    2016-01-01

    Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results

  7. Cholesterol removal from adult skeletal muscle impairs excitation-contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins.

    PubMed

    Barrientos, Genaro; Llanos, Paola; Hidalgo, Jorge; Bolaños, Pura; Caputo, Carlo; Riquelme, Alexander; Sánchez, Gina; Quest, Andrew F G; Hidalgo, Cecilia

    2015-01-01

    Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction (E-C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca(2+) transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na(+)/K(+)-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47(phox) and gp91(phox)), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E-C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.

  8. Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

    PubMed

    Li, D L; Huang, Y; Chang, L L; DU, Q; Chen, Y; Wang, T T; Luo, X M; Zhao, X M; Tong, D W

    2016-01-01

    Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2. PMID:27640437

  9. Scavenger receptors on sinusoidal liver endothelial cells are involved in the uptake of aldehyde-modified proteins.

    PubMed

    Duryee, Michael J; Freeman, Thomas L; Willis, Monte S; Hunter, Carlos D; Hamilton, Bartlett C; Suzuki, Hiroshi; Tuma, Dean J; Klassen, Lynell W; Thiele, Geoffrey M

    2005-11-01

    Scavenger receptors on sinusoidal liver endothelial cells (SECs) eliminate potentially harmful modified proteins circulating through the liver. It was shown recently that aldehyde-modified proteins bind to scavenger receptors and are associated with the development/progression of alcoholic liver diseases. For these studies, rat livers were perfused in situ with 125I-formaldehyde-bovine serum albumin (f-Alb) or 125I-malondialdehyde-acetaldehyde-bovine serum albumin (MAA-Alb) in the presence of known scavenger receptor ligands as inhibitors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and scavenger receptor Type A (SRA) knock-out mice were used to assess the role of these receptors in mediating immune responses. The degradation of 125I-f-Alb or 125I-MAA-Alb in whole livers and isolated SECs can be inhibited by known scavenger receptor ligands, including f-Alb, maleylated bovine albumin, and fucoidan. 125I-f-Alb could not be completely inhibited by MAA-Alb. In contrast, 125I-MAA-Alb was only partially inhibited with advanced glycosylated endproduct albumin. RT-PCR data show the presence of a number of scavenger receptors on SECs that may be responsible for the binding of MAA-modified proteins. SRA seems to be one of these receptors involved in the effects mediated by MAA-modified proteins. In a study using SRA knockout mice, it was shown that a decreased antibody response to MAA-Alb resulted. By RT-PCR, CD36, LOX-1, and SR-AI are the scavenger receptors most likely involved in the degradation of MAA-Alb.

  10. Scavenger receptors on sinusoidal liver endothelial cells are involved in the uptake of aldehyde-modified proteins.

    PubMed

    Duryee, Michael J; Freeman, Thomas L; Willis, Monte S; Hunter, Carlos D; Hamilton, Bartlett C; Suzuki, Hiroshi; Tuma, Dean J; Klassen, Lynell W; Thiele, Geoffrey M

    2005-11-01

    Scavenger receptors on sinusoidal liver endothelial cells (SECs) eliminate potentially harmful modified proteins circulating through the liver. It was shown recently that aldehyde-modified proteins bind to scavenger receptors and are associated with the development/progression of alcoholic liver diseases. For these studies, rat livers were perfused in situ with 125I-formaldehyde-bovine serum albumin (f-Alb) or 125I-malondialdehyde-acetaldehyde-bovine serum albumin (MAA-Alb) in the presence of known scavenger receptor ligands as inhibitors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and scavenger receptor Type A (SRA) knock-out mice were used to assess the role of these receptors in mediating immune responses. The degradation of 125I-f-Alb or 125I-MAA-Alb in whole livers and isolated SECs can be inhibited by known scavenger receptor ligands, including f-Alb, maleylated bovine albumin, and fucoidan. 125I-f-Alb could not be completely inhibited by MAA-Alb. In contrast, 125I-MAA-Alb was only partially inhibited with advanced glycosylated endproduct albumin. RT-PCR data show the presence of a number of scavenger receptors on SECs that may be responsible for the binding of MAA-modified proteins. SRA seems to be one of these receptors involved in the effects mediated by MAA-modified proteins. In a study using SRA knockout mice, it was shown that a decreased antibody response to MAA-Alb resulted. By RT-PCR, CD36, LOX-1, and SR-AI are the scavenger receptors most likely involved in the degradation of MAA-Alb. PMID:16105988

  11. Subproteomic analysis of basic proteins in aged skeletal muscle following offgel pre-fractionation.

    PubMed

    Gannon, Joan; Ohlendieck, Kay

    2012-04-01

    The progressive loss of skeletal muscle mass is a serious pathophysiological problem in the elderly, which warrants detailed biochemical studies into the underlying mechanism of age-related fiber degeneration. Over the last few years, mass spectrometry (MS)-based proteomics has identified a considerable number of new biomarkers of muscle aging in humans and animal models of sarcopenia. However, interpretation of the proteomic findings is often complicated by technical and biological limitations. Although gel electrophoresis-based approaches represent a highly sensitive analytical way for the large-scale and high-throughput survey of global changes in skeletal muscle proteins during aging, often the presence of components with an isoelectric point in the basic range is underestimated. We, therefore, carried out a comparative subproteomic study of young versus aged rat muscle focusing on potential changes in muscle proteins with an alkaline isoelectric point, using a combination of offgel electrophoresis and two-dimensional (2D) slab gel electrophoresis. Offgel electrophoresis was successfully applied as a prefractionation step to enrich basic protein species from crude tissue extracts representing young adult versus senescent muscle specimens. Proteomics has demonstrated alterations in a small cohort of basic proteins during muscle aging. The mass spectrometric identification of altered proteins and immunoblotting revealed a decrease in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a concomitant increase in mitochondrial creatine kinase (CK) and ubiquinol cytochrome‑c reductase. This agrees with the idea of a glycolytic-to-oxidative shift during muscle aging, which is indicative of an overall fast-to-slow transition process in senescent rat muscle. Thus, alterations in the abundance of metabolic enzymes appear to play a central role in the molecular pathogenesis of age‑dependent muscle wasting. PMID:22267262

  12. An autoantibody against N{sup {epsilon}}-(carboxyethyl)lysine (CEL): Possible involvement in the removal of CEL-modified proteins by macrophages

    SciTech Connect

    Mera, Katsumi; Nagai, Ryoji; Takeo, Kazuhiro; Izumi, Miyoko; Maruyama, Toru; Otagiri, Masaki

    2011-04-08

    Highlights: {yields} A higher amount of autoantibody against CEL than that of other AGEs was observed in human plasma. {yields} The purified human anti-CEL autoantibody specifically reacted with CEL. {yields} Anti-CEL antibody accelerated the uptake of {sup 125}I-CEL-HSA by macrophage in vitro. {yields} Endocytic uptake of {sup 125}I-CEL-HSA by mice liver was accelerated in the presence of anti-CEL antibody. -- Abstract: Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N{sup {epsilon}}-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when {sup 125}I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of {sup 125}I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.

  13. Protein synthesis rates in rat brain regions and subcellular fractions during aging

    SciTech Connect

    Avola, R.; Condorelli, D.F.; Ragusa, N.; Renis, M.; Alberghina, M.; Giuffrida Stella, A.M.; Lajtha, A.

    1988-04-01

    In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

  14. Understanding the Role of Heat Shock Protein Isoforms in Male Fertility, Aging and Apoptosis

    PubMed Central

    Purandhar, Kaveri; Jena, Prasant Kumar; Prajapati, Bhumika; Rajput, Parth

    2014-01-01

    Heat shock proteins (HSPs) play a role in the homeostasis, apoptosis regulation and the maintenance of the various other physiological processes. Aging is accompanied by a decrease in the resistance to environmental stress, while mitochondria are primary targets in the process of aging, their expression decreasing with age. Mitochondrion also plays a significant role in the process of spermatogenesis. HSPs have been shown to be involved in apoptosis with some of acting as apoptotic inhibitors and are involved in cytoprotection. In this review we discuss the roles of Hsp 27, 60, 70, and 90 in aging and male infertility and have concluded that these particular HSPs can be used as a molecular markers for mitochondrially- mediated apoptosis, aging and male infertility. PMID:25606560

  15. Experience Modulates the Effects of Histone Deacetylase Inhibitors on Gene and Protein Expression in the Hippocampus: Impaired Plasticity in Aging

    PubMed Central

    Sewal, Angila S.; Patzke, Holger; Perez, Evelyn J.; Park, Pul; Lehrmann, Elin; Zhang, Yongqing; Becker, Kevin G.; Fletcher, Bonnie R.; Long, Jeffrey M.

    2015-01-01

    The therapeutic potential of histone deacetylase inhibitor (HDACi) treatment has attracted considerable attention in the emerging area of cognitive neuroepigenetics. The possibility that ongoing cognitive experience importantly regulates the cell biological effects of HDACi administration, however, has not been systematically examined. In an initial experiment addressing this issue, we tested whether water maze training influences the gene expression response to acute systemic HDACi administration in the young adult rat hippocampus. Training powerfully modulated the response to HDACi treatment, increasing the total number of genes regulated to nearly 3000, including many not typically linked to neural plasticity, compared with <300 following HDACi administration alone. Although water maze training itself also regulated nearly 1800 genes, the specific mRNAs, gene networks, and biological pathways involved were largely distinct when the same experience was provided together with HDACi administration. Next, we tested whether the synaptic protein response to HDACi treatment is similarly dependent on recent cognitive experience, and whether this plasticity is altered in aged rats with memory impairment. Whereas synaptic protein labeling in the young hippocampus was selectively increased when HDACi administration was provided in conjunction with water maze training, combined treatment had no effect on synaptic proteins in the aged hippocampus. Our findings indicate that ongoing experience potently regulates the molecular consequences of HDACi treatment and that the interaction of recent cognitive experience with histone acetylation dynamics is disrupted in the aged hippocampus. SIGNIFICANCE STATEMENT The possibility that interventions targeting epigenetic regulation could be effective in treating a range of neurodegenerative disorders has attracted considerable interest. Here we demonstrate in the rat hippocampus that ongoing experience powerfully modifies the molecular

  16. Exploring Genetic Factors Involved in Huntington Disease Age of Onset: E2F2 as a New Potential Modifier Gene.

    PubMed

    Valcárcel-Ocete, Leire; Alkorta-Aranburu, Gorka; Iriondo, Mikel; Fullaondo, Asier; García-Barcina, María; Fernández-García, José Manuel; Lezcano-García, Elena; Losada-Domingo, José María; Ruiz-Ojeda, Javier; Álvarez de Arcaya, Amaia; Pérez-Ramos, José María; Roos, Raymund A C; Nielsen, Jørgen E; Saft, Carsten; Zubiaga, Ana M; Aguirre, Ana

    2015-01-01

    Age of onset (AO) of Huntington disease (HD) is mainly determined by the length of the CAG repeat expansion (CAGexp) in exon 1 of the HTT gene. Additional genetic variation has been suggested to contribute to AO, although the mechanism by which it could affect AO is presently unknown. The aim of this study is to explore the contribution of candidate genetic factors to HD AO in order to gain insight into the pathogenic mechanisms underlying this disorder. For that purpose, two AO definitions were used: the earliest age with unequivocal signs of HD (earliest AO or eAO), and the first motor symptoms age (motor AO or mAO). Multiple linear regression analyses were performed between genetic variation within 20 candidate genes and eAO or mAO, using DNA and clinical information of 253 HD patients from REGISTRY project. Gene expression analyses were carried out by RT-qPCR with an independent sample of 35 HD patients from Basque Country Hospitals. We found suggestive association signals between HD eAO and/or mAO and genetic variation within the E2F2, ATF7IP, GRIN2A, GRIN2B, LINC01559, HIP1 and GRIK2 genes. Among them, the most significant was the association between eAO and rs2742976, mapping to the promoter region of E2F2 transcription factor. Furthermore, rs2742976 T allele patient carriers exhibited significantly lower lymphocyte E2F2 gene expression, suggesting a possible implication of E2F2-dependent transcriptional activity in HD pathogenesis. Thus, E2F2 emerges as a new potential HD AO modifier factor.

  17. LIFESTYLE DETERMINANTS OF C-REACTIVE PROTEIN IN MIDDLE-AGED, URBAN CHINESE MEN

    PubMed Central

    Villegas, Raquel; Xiang, Yong-Bing; Cai, Hui; Elasy, Tom; Cai, Qiuyin; Zhang, Xianglan; Fazio, Sergio; Linton, MacRae; Li, Honglan; Xu, Wang Hong; Yang, Gong; Zheng, Wei; Shu, Xiao-Ou

    2011-01-01

    Background Increased levels of C-reactive protein (CRP), common in aging populations, are associated with higher risk for chronic diseases, including diabetes and coronary heart disease. The aim of this study was to investigate associations between lifestyle factors and high CRP among middleaged men living in Shanghai, China. PMID:21111583

  18. C-reactive protein and genetic variants and cognitive decline in old age: The PROSPER Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasma concentrations of C-reactive protein (CRP), a marker of chronic inflammation, have been associated with cognitive impairment in old age. However, it is unknown whether CRP is causally linked to cognitive decline. Within the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER) tri...

  19. Effects of postmortem aging and hydrodynamic pressure processing on sarcoplasmic proteins and beef tenderness

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to determine the effects of hydrodynamic pressure processing (HDP) and postmortem aging on the sarcoplasmic protein fraction of beef strip loins. Loins (n=12) were halved at 48 h postmortem and assigned to HDP or control treatments. Following treatment, each half was ...

  20. Alterations in the sarcoplasmic protein fraction of beef muscle with postmortem aging and hydrodynamic pressure processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Capillary electrophoresis (CE) and reversed-phase high performance liquid chromatography (RP-HPLC) analysis were utilized to detect differences in the sarcoplasmic protein profiles of beef strip loins subjected to aging and hydrodynamic pressure processing (HDP) treatments. At 48 h postmortem, stri...

  1. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    PubMed

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  2. XRCC1 haploinsufficiency in mice has little effect on aging, but adversely modifies exposure-dependent susceptibility

    PubMed Central

    McNeill, Daniel R.; Lin, Ping-Chang; Miller, Marshall G.; Pistell, Paul J.; de Souza-Pinto, Nadja C.; Fishbein, Kenneth W.; Spencer, Richard G.; Liu, Yie; Pettan-Brewer, Christina; Ladiges, Warren C.; Wilson, David M.

    2011-01-01

    Oxidative DNA damage plays a role in disease development and the aging process. A prominent participant in orchestrating the repair of oxidative DNA damage, particularly single-strand breaks, is the scaffold protein XRCC1. A series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice were examined. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of ~26 months and a nearly identical maximal life expectancy of ~37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated—weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity—HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure. PMID:21737425

  3. Drosophila melanogaster Hsp22: a mitochondrial small heat shock protein influencing the aging process

    PubMed Central

    Morrow, Geneviève; Tanguay, Robert M.

    2015-01-01

    Mitochondria are involved in many key cellular processes and therefore need to rely on good protein quality control (PQC). Three types of mechanisms are in place to insure mitochondrial protein integrity: reactive oxygen species scavenging by anti-oxidant enzymes, protein folding/degradation by molecular chaperones and proteases and clearance of defective mitochondria by mitophagy. Drosophila melanogaster Hsp22 is part of the molecular chaperone axis of the PQC and is characterized by its intra-mitochondrial localization and preferential expression during aging. As a stress biomarker, the level of its expression during aging has been shown to partially predict the remaining lifespan of flies. Since over-expression of this small heat shock protein increases lifespan and resistance to stress, Hsp22 most likely has a positive effect on mitochondrial integrity. Accordingly, Hsp22 has recently been implicated in the mitochondrial unfolding protein response of flies. This review will summarize the key findings on D. melanogaster Hsp22 and emphasis on its links with the aging process. PMID:25852752

  4. Roles for the Ubiquitin-Proteasome Pathway in Protein Quality Control and Signaling in the Retina: Implications in the Pathogenesis of Age-Related Macular Degeneration

    PubMed Central

    Shang, Fu; Taylor, Allen

    2012-01-01

    The accumulation of damaged or postsynthetically modified proteins and dysregulation of inflammatory responses and angiogenesis in the retina/RPE are thought be etiologically related to formation of drusen and choroidal neovascularization (CNV), hallmarks of age-related macular degeneration (AMD). The ubiquitin proteasome pathway (UPP) plays crucial roles in protein quality control, cell cycle control and signal transduction. Selective degradation of aberrant proteins by the UPP is essential for timely removal of potentially cytotoxic damaged or otherwise abnormal proteins. Proper function of the UPP is thought to be required for cellular function. In contrast, age- or stress induced- impairment the UPP or insufficient UPP capacity may contribute to the accumulation of abnormal proteins, cytotoxicity in the retina, and AMD. Crucial roles for the UPP in eye development, regulation of signal transduction, and antioxidant responses are also established. Insufficient UPP capacity in retina and RPE can result in dysregulation of signal transduction, abnormal inflammatory responses and CNV. There are also interactions between the UPP and lysosomal proteolytic pathways (LPP). Means that modulate the proteolytic capacity are making their way into new generation of pharmacotherapies for delaying age-related diseases and may augment the benefits of adequate nutrition, with regard to diminishing the burden of AMD. PMID:22521794

  5. Ethylene glycol assisted preparation of Ti(4+)-modified polydopamine coated magnetic particles with rough surface for capture of phosphorylated proteins.

    PubMed

    Ma, Xiangdong; Ding, Chun; Yao, Xin; Jia, Li

    2016-07-27

    The reversible protein phosphorylation is very important in regulating almost all aspects of cell life, while the enrichment of phosphorylated proteins still remains a technical challenge. In this work, polydopamine (PDA) modified magnetic particles with rough surface (rPDA@Fe3O4) were synthesized by introduction of ethylene glycol in aqueous solution. The PDA coating possessing a wealth of catechol hydroxyl groups could serve as an active medium to immobilize titanium ions through the metal-catechol chelation, which makes the fabrication of titanium ions modified rPDA@Fe3O4 particles (Ti(4+)-rPDA@Fe3O4) simple and very convenient. The spherical Ti(4+)-rPDA@Fe3O4 particles have a surface area of 37.7 m(2) g(-1) and superparamagnetism with a saturation magnetization value of 38.4 emu g(-1). The amount of Ti element in the particle was measured to be 3.93%. And the particles demonstrated good water dispersibility. The particles were used as adsorbents for capture of phosphorylated proteins and they demonstrated affinity and specificity for phosphorylated proteins due to the specific binding sites (Ti(4+)). Factors affecting the adsorption of phosphorylated proteins on Ti(4+)-rPDA@Fe3O4 particles were investigated. The adsorption capacity of Ti(4+)-rPDA@Fe3O4 particles for κ-casein was 1105.6 mg g(-1). Furthermore, the particles were successfully applied to isolate phosphorylated proteins in milk samples, which demonstrated that Ti(4+)-rPDA@Fe3O4 particles had potential application in selective separation of phosphorylated proteins.

  6. Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology.

    PubMed

    McLean, Michael D; Chen, Rongji; Yu, Deqiang; Mah, Kor-Zheng; Teat, John; Wang, Haifeng; Zaplachinski, Steve; Boothe, Joseph; Hall, J Christopher

    2012-12-01

    Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation. PMID:22382463

  7. A new subgroup of lectin-bound biliary proteins binds to cholesterol crystals, modifies crystal morphology, and inhibits cholesterol crystallization.

    PubMed Central

    Busch, N; Lammert, F; Marschall, H U; Matern, S

    1995-01-01

    Biliary proteins inhibiting or promoting cholesterol crystallization are assumed to play a major role in cholesterol gallstone pathogenesis. We now report a new group of biliary proteins that bind to cholesterol crystals, modify crystal morphology, and inhibit cholesterol crystallization. Various glycoprotein mixtures were extracted from abnormal human gallbladder bile using lectin affinity chromatography on concanavalin A, lentil, and Helix pomatia columns and were added to supersaturated model bile. Independent of the protein mixtures added, from the cholesterol crystals harvested, the same four GPs were isolated having molecular masses of 16, 28, 63, and 74 kD, respectively. Each protein was purified using preparative SDS-PAGE, and influence on cholesterol crystallization in model bile was tested at 10 microg/ml. Crystal growth was reduced by 76% (GP63), 65% (GP16), 55% (GP74), and 40% (GP28), respectively. Thus, these glycoproteins are the most potent biliary inhibitors of cholesterol crystallization known so far. Evidence that the inhibiting effect on cholesterol crystallization is mediated via protein-crystal interaction was further provided from scanning electron microscopy studies. Crystals grown in presence of inhibiting proteins showed significantly more ordered structures. Incidence of triclinic crystals and regular aggregates was shifted from 30 to 70% compared with controls. These observations may have important implications for understanding the role of biliary proteins in cholesterol crystallization and gallstone pathogenesis. Images PMID:8675674

  8. Identification of O-Linked N-Acetylglucosamine (O-GlcNAc)-modified Osteoblast Proteins by Electron Transfer Dissociation Tandem Mass Spectrometry Reveals Proteins Critical for Bone Formation*

    PubMed Central

    Nagel, Alexis K.; Schilling, Michael; Comte-Walters, Susana; Berkaw, Mary N.; Ball, Lauren E.

    2013-01-01

    The nutrient-responsive β-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGFβ-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation. PMID:23443134

  9. Interaction of RecA protein with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene and 4-hydroxyaminoquinoline 1-oxide.

    PubMed Central

    Kojima, M; Suzuki, M; Morita, T; Ogawa, T; Ogawa, H; Tada, M

    1990-01-01

    Interaction of RecA protein of Escherichia coli with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and 4-hydroxyaminoquinoline 1-oxide (4HAQO) was investigated. RecA protein bound more efficiently to modified DNA than to unmodified DNA as judged by filter-binding and gel electrophoresis assay. The binding of RecA protein with modified DNA resulted in the stimulation of ATPase activity and the activation for RecA protein to stimulate the repressor cleavage. These abilities of RecA protein were increased proportionally to the number of adducts in the plasmid DNA (0-5 adducts). Apurinic and alkylated DNA did not activate RecA protein. We suggest that modification of DNA by N-OH-AAF and 4HAQO provides binding sites for RecA protein and may act as an activation signal for SOS response. Images PMID:2140154

  10. A Model Sea Urchin Spicule Matrix Protein Self-Associates To Form Mineral-Modifying Protein Hydrogels.

    PubMed

    Jain, Gaurav; Pendola, Martin; Rao, Ashit; Cölfen, Helmut; Evans, John Spencer

    2016-08-01

    In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as the SpSM30A-F isoforms. However, because of limited abundance, it has been difficult to pursue extensive biochemical studies of the SpSM30 proteins and deduce their role in spicule formation and mineralization. To circumvent these problems, we expressed a model recombinant spicule matrix protein, rSpSM30B/C, which possesses the key sequence attributes of isoforms "B" and "C". Our findings indicate that rSpSM30B/C is expressed in insect cells as a single polypeptide containing variations in glycosylation that create microheterogeneity in rSpSM30B/C molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic mono- and bisialylated and mono- and bisulfated monosaccharides on the protein molecules and enhance its aggregation propensity. Bioinformatics and biophysical experiments confirm that rSpSM30B/C is an intrinsically disordered, aggregation-prone protein that forms porous protein hydrogels that control the in vitro mineralization process in three ways: (1) increase the time interval for prenucleation cluster formation and transiently stabilize an ACC polymorph, (2) promote and organize single-crystal calcite nanoparticles, and (3) promote faceted growth and create surface texturing of calcite crystals. These features are also common to mollusk shell nacre proteins, and we conclude that rSpSM30B/C is a spiculogenesis protein that exhibits traits found in other calcium carbonate mineral modification proteins. PMID:27426695

  11. Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group.

    PubMed

    Nunoura, Takuro; Takaki, Yoshihiro; Kakuta, Jungo; Nishi, Shinro; Sugahara, Junichi; Kazama, Hiromi; Chee, Gab-Joo; Hattori, Masahira; Kanai, Akio; Atomi, Haruyuki; Takai, Ken; Takami, Hideto

    2011-04-01

    The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea. PMID:21169198

  12. Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group

    PubMed Central

    Nunoura, Takuro; Takaki, Yoshihiro; Kakuta, Jungo; Nishi, Shinro; Sugahara, Junichi; Kazama, Hiromi; Chee, Gab-Joo; Hattori, Masahira; Kanai, Akio; Atomi, Haruyuki; Takai, Ken; Takami, Hideto

    2011-01-01

    The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the ‘Thaumarchaeota’ and ‘Korarchaeota’. Here, we show the genome sequence of Candidatus ‘Caldiarchaeum subterraneum’ that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea. PMID:21169198

  13. Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation.

    PubMed

    Cheng, R; Lin, B; Lee, K W; Ortwerth, B J

    2001-07-27

    Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R(f) values for the long wavelength-absorbing fluorophores. Glycation with [U-(14)C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A(330)-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow

  14. Purification of glycosylphosphatidylinositol-anchored proteins by modified triton X-114 partitioning and preparative gel electrophoresis.

    PubMed

    Ko, Y G; Thompson, G A

    1995-01-01

    Triton X-114 phase partitioning has frequently been used to obtain preparations enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and other hydrophobic proteins from crude cellular homogenates. We have developed a new modification of this phase-partitioning technique which allows two distinct GPI-anchored proteins of Tetrahymena mimbres to be separated from other hydrophobic as well as hydrophilic proteins and recovered in approximately 90% yield. The unique feature of the new method is a 24-h incubation of the first Triton X-114 extract at -20 degrees C. This improves the partitioning of GPI-anchored proteins into the detergent phase while promoting the aggregation of other hydrophobic proteins. Individual GPI-anchored proteins in the detergent phase are then purified to near homogeneity by one-step preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By substituting Triton X-114/water mixtures for the conventional SDS buffer used to collect proteins eluting from the preparative gel, the purified GPI-anchored protein can be rapidly concentrated from relatively large volumes of eluate by phase partitioning at 32 degrees C. The method is also effective in separating mammalian GPI-anchored alkaline phosphatase from other proteins. It is likely to be of general utility in characterizing the GPI anchor structures associated with nonabundant and abundant GPI-anchored proteins coexisting within the same cell type.

  15. Long-Term Estrogen Receptor Beta Agonist Treatment Modifies the Hippocampal Transcriptome in Middle-Aged Ovariectomized Rats

    PubMed Central

    Sárvári, Miklós; Kalló, Imre; Hrabovszky, Erik; Solymosi, Norbert; Rodolosse, Annie; Liposits, Zsolt

    2016-01-01

    Estradiol (E2) robustly activates transcription of a broad array of genes in the hippocampal formation of middle-aged ovariectomized rats via estrogen receptors (ERα, ERβ, and G protein-coupled ER). Selective ERβ agonists also influence hippocampal functions, although their downstream molecular targets and mechanisms are not known. In this study, we explored the effects of long-term treatment with ERβ agonist diarylpropionitrile (DPN, 0.05 mg/kg/day, sc.) on the hippocampal transcriptome in ovariectomized, middle-aged (13 month) rats. Isolated hippocampal formations were analyzed by Affymetrix oligonucleotide microarray and quantitative real-time PCR. Four hundred ninety-seven genes fulfilled the absolute fold change higher than 2 (FC > 2) selection criterion. Among them 370 genes were activated. Pathway analysis identified terms including glutamatergic and cholinergic synapse, RNA transport, endocytosis, thyroid hormone signaling, RNA degradation, retrograde endocannabinoid signaling, and mRNA surveillance. PCR studies showed transcriptional regulation of 58 genes encoding growth factors (Igf2, Igfb2, Igf1r, Fgf1, Mdk, Ntf3, Bdnf), transcription factors (Otx2, Msx1), potassium channels (Kcne2), neuropeptides (Cck, Pdyn), peptide receptors (Crhr2, Oprm1, Gnrhr, Galr2, Sstr1, Sstr3), neurotransmitter receptors (Htr1a, Htr2c, Htr2a, Gria2, Gria3, Grm5, Gabra1, Chrm5, Adrb1), and vesicular neurotransmitter transporters (Slc32a1, Slc17a7). Protein-protein interaction analysis revealed networking of clusters associated with the regulation of growth/troph factor signaling, transcription, translation, neurotransmitter and neurohormone signaling mechanisms and potassium channels. Collectively, the results reveal the contribution of ERβ-mediated processes to the regulation of transcription, translation, neurogenesis, neuromodulation, and neuroprotection in the hippocampal formation of ovariectomized, middle-aged rats and elucidate regulatory channels responsible for

  16. Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level.

    PubMed

    Bahrami, Soroush Alaghehband; Bakhtiari, Nuredin

    2016-08-01

    We previously reported that Ursolic Acid (UA) ameliorates skeletal muscle performance through satellite cells proliferation and cellular energy status. In studying the potential role of the hypothalamus in aging, we developed a strategy to pursue UA effects on the hypothalamus anti-aging proteins such as; SIRT1, SIRT6, PGC-1β and α-Klotho. In this study, we used a model of aging animals (C57BL/6). UA dissolved in Corn oil (20mg/ml) and then administrated (200mg/Kg i.p injection) to mice, twice daily for 7days. After treatment times, the mice perfused and the hypothalamus isolated for preparing of tissue to Immunofluorescence microscopy. The data illustrated that UA significantly increased SIRT1 (∼3.5±0.3 folds) and SIRT-6 (∼1.5±0.2 folds) proteins overexpression (P<0.001). In addition, our results showed that UA enhanced α-Klotho (∼3.3±0.3) and PGC-1β (∼2.6±0.2 folds) proteins levels (P<0. 01). In this study, data were analyzed using SPSS 16 (ANOVA test). To the best of our knowledge, it seems that UA through enhancing of anti-aging biomarkers (SIRT1 and SIRT6) and PGC-1β in hypothalamus regulates aging-process and attenuates mitochondrial-related diseases. In regard to the key role of α-Klotho in aging, our data indicate that UA may be on the horizon to forestall diseases of aging. PMID:27470332

  17. Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level.

    PubMed

    Bahrami, Soroush Alaghehband; Bakhtiari, Nuredin

    2016-08-01

    We previously reported that Ursolic Acid (UA) ameliorates skeletal muscle performance through satellite cells proliferation and cellular energy status. In studying the potential role of the hypothalamus in aging, we developed a strategy to pursue UA effects on the hypothalamus anti-aging proteins such as; SIRT1, SIRT6, PGC-1β and α-Klotho. In this study, we used a model of aging animals (C57BL/6). UA dissolved in Corn oil (20mg/ml) and then administrated (200mg/Kg i.p injection) to mice, twice daily for 7days. After treatment times, the mice perfused and the hypothalamus isolated for preparing of tissue to Immunofluorescence microscopy. The data illustrated that UA significantly increased SIRT1 (∼3.5±0.3 folds) and SIRT-6 (∼1.5±0.2 folds) proteins overexpression (P<0.001). In addition, our results showed that UA enhanced α-Klotho (∼3.3±0.3) and PGC-1β (∼2.6±0.2 folds) proteins levels (P<0. 01). In this study, data were analyzed using SPSS 16 (ANOVA test). To the best of our knowledge, it seems that UA through enhancing of anti-aging biomarkers (SIRT1 and SIRT6) and PGC-1β in hypothalamus regulates aging-process and attenuates mitochondrial-related diseases. In regard to the key role of α-Klotho in aging, our data indicate that UA may be on the horizon to forestall diseases of aging.

  18. Role of interfacial protein membrane in oxidative stability of vegetable oil substitution emulsions applicable to nutritionally modified sausage.

    PubMed

    Jiang, Jiang; Xiong, Youling L

    2015-11-01

    The potential health risk associated with excessive dietary intake of fat and cholesterol has led to a renewed interest in replacing animal fat with nutritionally-balanced unsaturated oil in processed meats. However, as oils are more fluid and unsaturated than fats, one must overcome the challenge of maintaining both physical and chemical (oxidative) stabilities of prepared emulsions. Apart from physical entrapments, an emulsion droplet to be incorporated into a meat protein gel matrix (batter) should be equipped with an interactive protein membrane rather than a small surfactant, and the classical DLVO stabilization theory becomes less applicable. This review paper describes the steric effects along with chemical roles (radical scavenging and metal ion chelation) of proteins and their structurally modified derivatives as potential interface-building materials for oxidatively stable meat emulsions.

  19. Role of interfacial protein membrane in oxidative stability of vegetable oil substitution emulsions applicable to nutritionally modified sausage.

    PubMed

    Jiang, Jiang; Xiong, Youling L

    2015-11-01

    The potential health risk associated with excessive dietary intake of fat and cholesterol has led to a renewed interest in replacing animal fat with nutritionally-balanced unsaturated oil in processed meats. However, as oils are more fluid and unsaturated than fats, one must overcome the challenge of maintaining both physical and chemical (oxidative) stabilities of prepared emulsions. Apart from physical entrapments, an emulsion droplet to be incorporated into a meat protein gel matrix (batter) should be equipped with an interactive protein membrane rather than a small surfactant, and the classical DLVO stabilization theory becomes less applicable. This review paper describes the steric effects along with chemical roles (radical scavenging and metal ion chelation) of proteins and their structurally modified derivatives as potential interface-building materials for oxidatively stable meat emulsions. PMID:26008711

  20. Methoprene application and diet protein supplementation to male melon fly, Bactrocera cucurbitae, modifies female remating behavior

    PubMed Central

    ul Haq, Ihsan; Vreysen, Marc J B; Teal, P E A; Hendrichs, Jorge

    2014-01-01

    Methoprene (an analogue of juvenile hormone) application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae's ability to inhibit female remating. While 14-d-old females were fed on protein diet, 6-d-old males were exposed to one of the following treatments: (i) topical application of methoprene and fed on a protein diet; (ii) no methoprene but fed on a protein diet; (iii) methoprene and sugar-fed only; and (iv) sugar-fed, 14-d-old males acted as controls. Treatments had no effect on a male's ability to depress the female remating receptivity in comparison to the control. Females mated with protein-deprived males showed higher remating receptivity than females first mated with protein-fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar-fed males remated with protein-fed males and females first mated with methoprene treated and protein-fed males were more likely to remate with similarly treated males. Females mating latency (time to start mating) was significantly shorter with protein-fed males, and mating duration was significantly longer with protein-fed males compared with protein-deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area-wide control of melon fly integrating the sterile insect technique (SIT). PMID:24376160

  1. Epidemic Spreading Model to Characterize Misfolded Proteins Propagation in Aging and Associated Neurodegenerative Disorders

    PubMed Central

    Iturria-Medina, Yasser; Sotero, Roberto C.; Toussaint, Paule J.; Evans, Alan C.

    2014-01-01

    Misfolded proteins (MP) are a key component in aging and associated neurodegenerative disorders. For example, misfolded Amyloid-ß (Aß) and tau proteins are two neuropathogenic hallmarks of Alzheimer's disease. Mechanisms underlying intra-brain MP propagation/deposition remain essentially uncharacterized. Here, is introduced an epidemic spreading model (ESM) for MP dynamics that considers propagation-like interactions between MP agents and the brain's clearance response across the structural connectome. The ESM reproduces advanced Aß deposition patterns in the human brain (explaining 46∼56% of the variance in regional Aß loads, in 733 subjects from the ADNI database). Furthermore, this model strongly supports a) the leading role of Aß clearance deficiency and early Aß onset age during Alzheimer's disease progression, b) that effective anatomical distance from Aß outbreak region explains regional Aß arrival time and Aß deposition likelihood, c) the multi-factorial impact of APOE e4 genotype, gender and educational level on lifetime intra-brain Aß propagation, and d) the modulatory impact of Aß propagation history on tau proteins concentrations, supporting the hypothesis of an interrelated pathway between Aß pathophysiology and tauopathy. To our knowledge, the ESM is the first computational model highlighting the direct link between structural brain networks, production/clearance of pathogenic proteins and associated intercellular transfer mechanisms, individual genetic/demographic properties and clinical states in health and disease. In sum, the proposed ESM constitutes a promising framework to clarify intra-brain region to region transference mechanisms associated with aging and neurodegenerative disorders. PMID:25412207

  2. Epidemic spreading model to characterize misfolded proteins propagation in aging and associated neurodegenerative disorders.

    PubMed

    Iturria-Medina, Yasser; Sotero, Roberto C; Toussaint, Paule J; Evans, Alan C

    2014-11-01

    Misfolded proteins (MP) are a key component in aging and associated neurodegenerative disorders. For example, misfolded Amyloid-ß (Aß) and tau proteins are two neuropathogenic hallmarks of Alzheimer's disease. Mechanisms underlying intra-brain MP propagation/deposition remain essentially uncharacterized. Here, is introduced an epidemic spreading model (ESM) for MP dynamics that considers propagation-like interactions between MP agents and the brain's clearance response across the structural connectome. The ESM reproduces advanced Aß deposition patterns in the human brain (explaining 46∼56% of the variance in regional Aß loads, in 733 subjects from the ADNI database). Furthermore, this model strongly supports a) the leading role of Aß clearance deficiency and early Aß onset age during Alzheimer's disease progression, b) that effective anatomical distance from Aß outbreak region explains regional Aß arrival time and Aß deposition likelihood, c) the multi-factorial impact of APOE e4 genotype, gender and educational level on lifetime intra-brain Aß propagation, and d) the modulatory impact of Aß propagation history on tau proteins concentrations, supporting the hypothesis of an interrelated pathway between Aß pathophysiology and tauopathy. To our knowledge, the ESM is the first computational model highlighting the direct link between structural brain networks, production/clearance of pathogenic proteins and associated intercellular transfer mechanisms, individual genetic/demographic properties and clinical states in health and disease. In sum, the proposed ESM constitutes a promising framework to clarify intra-brain region to region transference mechanisms associated with aging and neurodegenerative disorders.

  3. Aging results in an unusual expression of Drosophila heat shock proteins

    SciTech Connect

    Fleming, J.E.; Walton, J.K.; Dubitsky, R.; Bensch, K.G. )

    1988-06-01

    The authors used high-resolution two-dimensional polyacrylamide gel electrophoresis to evaluate the effect of aging on the heat shock response in Drosophila melanogaster. Although the aging process is not well understood at the molecular level, recent observations suggest that quantitative changes in gene expression occur as these fruit flies approach senescence. Such genetic alterations are in accord with our present data, which clearly show marked differences in the synthesis of heat shock proteins between young and old fruit flies. In 10-day-old flies, a heat shock of 20 min results in the expression of 14 new proteins as detectable by two-dimensional electrophoresis of ({sup 35}S)methionine-labeled polypeptides, whereas identical treatment of 45-day-old flies leads to the expression of at least 50 new or highly up-regulated proteins. In addition, there is also a concomitant increase in the rate of synthesis of a number of the normal proteins in the older animals. Microdensitometric determinations of the low molecular weight heat shock polypeptides on autoradiographs of five age groups revealed that their maximum expression occurs at 47 days for a population of flies with a mean life span of 33.7 days. Moreover, a heat shock effect similar to that observed in senescent flies occurs in young flies fed canavanine, an arginine analogue, before heat shock.

  4. Proteomic identification of age-dependent protein nitration in rat skeletal muscle.

    PubMed

    Kanski, Jaroslaw; Alterman, Michail A; Schöneich, Christian

    2003-11-15

    Age-related protein nitration was studied in skeletal muscle of Fisher 344 and Fisher 344/Brown Norway (BN) F1 rats by a proteomic approach. Proteins from young (4 months) and old (24 months) Fisher 344 rats and young (6 months) and old (34 months) Fisher 344/BN F1 animals were separated by 2-D gel electrophoresis. Western blot showed an age-related increase in the nitration of a few specific proteins, which were identified by MALDI-TOF MS and ESI-MS/MS. We identified age-dependent apparent nitration of beta-enolase, alpha-fructose aldolase, and creatine kinase, which perform important functions in muscle energy metabolism, suggesting that the nitration of such key proteins can be, in part, responsible for the decline of muscle motor function of the muscle. Furthermore, we have identified the apparent nitration of succinate dehydrogenase, rab GDP dissociation inhibitor beta (GdI-2), triosephosphate isomerase, troponin I, alpha-crystallin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

  5. Proteins immobilization on the surface of modified plant viral particles coated with hydrophobic polycations.

    PubMed

    Nikitin, Nikolai A; Malinin, Andrei S; Trifonova, Ekaterina A; Rakhnyanskaya, Anna A; Yaroslavov, Aleksandr A; Karpova, Olga V; Atabekov, Joseph G

    2014-01-01

    Two hydrophobic cations based on poly-N-ethyl-vinylpyridine were used to produce biologically active complexes. The complexes obtained from tobacco mosaic virus (TMV) spherical particles (SPs), hydrophobic polycation, and a model protein were stable and did not aggregate in solution, particularly at high ionic strengths. The nucleic acid-free SPs were generated by thermal remodeling of the TMV (helical rod-shaped plant virus). The model protein preserved its antigenic activity in the ternary complex (SP-polycation-protein). Immobilization of proteins on the surface of SPs coated with hydrophobic cation is a promising approach to designing biologically active complexes used in bionanotechnologies. PMID:25121344

  6. Physical, Heritable and Age-Related Factors as Modifiers of Radiation Cancer Risk in Patched Heterozygous Mice

    SciTech Connect

    Pazzaglia, Simonetta Pasquali, Emanuela M.Sc.; Tanori, Mirella; Mancuso, Mariateresa; Leonardi, Simona; Di Majo, Vincenzo; Rebessi, Simonetta; Saran, Anna

    2009-03-15

    Purpose: To address the tumorigenic potential of exposure to low/intermediate doses of ionizing radiation and to identify biological factors influencing tumor response in a mouse model highly susceptible to radiogenic cancer. Methods and Materials: Newborn Ptc1 heterozygous mice were exposed to X-ray doses of 100, 250, and 500 mGy, and tumor development was monitored for their lifetime. Additional groups were irradiated with the same doses and sacrificed at fixed times for determination of short-term endpoints, such as apoptosis and early preneoplastic lesions in cerebellum. Finally, groups of Ptc1 heterozygous mice were bred on the C57BL/6 background to study the influence of common variant genes on radiation response. Results: We have identified a significant effect of low-intermediate doses of radiation (250 and 500 mGy) in shortening mean survival and inducing early and more progressed stages of tumor development in the cerebellum of Ptc1{sup +/-} mice. In addition, we show that age at exposure and heritable factors are potent modifiers of radiation-related cancer risk. Conclusions: The Ptc1 knockout mouse model offers a highly sensitive system that may potentially help to improve understanding and quantification of risk at low doses, such as doses experienced in occupational and medical exposures, and clarify the complex interactions between genetic and environmental factors underlying cancer susceptibility.

  7. Age-Dependent Impairment of Eyeblink Conditioning in Prion Protein-Deficient Mice

    PubMed Central

    Kishimoto, Yasushi; Hirono, Moritoshi; Atarashi, Ryuichiro; Sakaguchi, Suehiro; Yoshioka, Tohru; Katamine, Shigeru; Kirino, Yutaka

    2013-01-01

    Mice lacking the prion protein (PrPC) gene (Prnp), Ngsk Prnp0/0 mice, show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrPC-like protein (PrPLP/Dpl). Because PrPC is highly expressed in cerebellar neurons (including PCs and granule cells), it may be involved in cerebellar synaptic function and cerebellar cognitive function. However, no studies have been conducted to investigate the possible involvement of PrPC and/or PrPLP/Dpl in cerebellum-dependent discrete motor learning. Therefore, the present cross-sectional study was designed to examine cerebellum-dependent delay eyeblink conditioning in Ngsk Prnp0/0 mice in adulthood (16, 40, and 60 weeks of age). The aims of the present study were two-fold: (1) to examine the role of PrPC and/or PrPLP/Dpl in cerebellum-dependent motor learning and (2) to confirm the age-related deterioration of eyeblink conditioning in Ngsk Prnp0/0 mice as an animal model of progressive cerebellar degeneration. Ngsk Prnp0/0 mice aged 16 weeks exhibited intact acquisition of conditioned eyeblink responses (CRs), although the CR timing was altered. The same result was observed in another line of PrPc-deficient mice, ZrchI PrnP0/0 mice. However, at 40 weeks of age, CR incidence impairment was observed in Ngsk Prnp0/0 mice. Furthermore, Ngsk Prnp0/0 mice aged 60 weeks showed more significantly impaired CR acquisition than Ngsk Prnp0/0 mice aged 40 weeks, indicating the temporal correlation between cerebellar PC degeneration and motor learning deficits. Our findings indicate the importance of the cerebellar cortex in delay eyeblink conditioning and suggest an important physiological role of prion protein in cerebellar motor learning. PMID:23593266

  8. Dietary soy protein isolate modifies hepatic retinoic acid receptor-beta proteins and inhibits their DNA binding activity in rats.

    PubMed

    Xiao, Chao Wu; Mei, Jie; Huang, Wenxin; Wood, Carla; L'abbé, Mary R; Gilani, G Sarwar; Cooke, Gerard M; Curran, Ivan H

    2007-01-01

    Retinoic acid receptors (RAR) belong to the same nuclear receptor superfamily as thyroid hormone receptors (TR) that were previously shown to be modulated by dietary soy protein isolate (SPI). This study has examined the effect of dietary SPI and isoflavones (ISF) on hepatic RAR gene expression and DNA binding activity. In Expt. 1, Sprague-Dawley rats were fed diets containing 20% casein or 20% alcohol-washed SPI in the absence or presence of increasing amounts of ISF (5-1250 mg/kg diet) for 70, 190, or 310 d. In Expt. 2, weanling Sprague-Dawley rats were fed diets containing 20% casein with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10, and 20%) for 90 d. Intake of soy proteins significantly elevated hepatic RARbeta2 protein content dose-dependently compared with a casein diet, whereas supplemental ISF had no consistent effect. Neither RARbeta protein in the other tissues measured nor the other RAR (RARalpha and RARgamma) in the liver were affected by dietary SPI, indicating a tissue and isoform-specific effect of SPI. RARbeta2 mRNA abundances were not different between dietary groups except that its expression was markedly suppressed in male rats fed SPI for 310 d. DNA binding activity of nuclear RARbeta was significantly attenuated and the isoelectric points of RARbeta2 were shifted by dietary SPI. Overall, these results show for the first time, to our knowledge, that dietary soy proteins affect hepatic RARbeta2 protein content and RARbeta DNA binding activity, which may contribute to the suppression of retinoid-induced hypertriglyceridemia by SPI as reported.

  9. Cell separation by immunoaffinity partitioning with polyethylene glycol-modified Protein A in aqueous polymer two-phase systems

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Van Alstine, James M.; Snyder, Robert S.; Shafer, Steven G.; Harris, J. Milton

    1988-01-01

    Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG-dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work it is shown that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, the paper provides a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.

  10. Ageing of solid-state protein films: Behavior of azurin at ambient conditions

    NASA Astrophysics Data System (ADS)

    Pompa, P. P.; Bramanti, A.; Maruccio, G.; Mercato, L. L. del; Cingolani, R.; Rinaldi, R.

    2005-03-01

    We report on the folding properties of the protein azurin, deposited onto SiO 2 surfaces and subsequently dehydrated. The molecular films have been maintained at ambient conditions through several days, and the ageing effects have been investigated by fluorescence spectroscopy. The experimental results show a modest initial conformational rearrangement, followed by long-term stability. Interestingly, upon rehydration of the biomolecular films at the end of the investigated period (approximately one month), azurin returns to exhibit a native-like conformation. This study indicates a rather surprising resilience of proteins to ambient conditions and sheds a somewhat unexpected positive light on reliability in biomolecular electronics.

  11. Protein-bound advanced glycation endproducts (AGEs) as bioactive amino acid derivatives in foods.

    PubMed

    Henle, T

    2005-12-01

    The Maillard reaction or nonenzymatic browning is of outstanding importance for the formation of flavour and colour of heated foods. Corresponding reactions, also referred to as "glycation", are known from biological systems, where the formation of advanced glycation endproducts (AGEs) shall play an important pathophysiological role in diabetes and uremia. In this review, pathways leading to the formation of individual protein-bound lysine and arginine derivatives in foods are described and nutritional consequences resulting from this posttranslational modifications of food proteins are discussed. PMID:15997413

  12. Effects of vacuum and modified atmosphere on textural parameters and structural proteins of cultured meagre (Argyrosomus regius) fillets.

    PubMed

    Sáez, María I; Martínez, Tomás F; Cárdenas, Salvador; Suárez, María D

    2015-09-01

    The influence of two preservation strategies (vacuum package and modified atmosphere package) on the post-mortem changes of textural parameters, pH, water holding capacity, sarcoplasmic and myofibrillar proteins, and collagen content of meagre (Argyrosomus regius) fillets was studied. Fillets were stored in a cold room in aerobic (control, C), vacuum (V) and modified atmosphere (MA) package. Samples were withdrawn at six sampling points throughout 15-day storage, and post-mortem changes were assessed. The textural parameters were significantly enhanced in V and MA compared to C. Both V and MA treatments reduced the intensity of a group of myofibrillar protein fractions (140-195 kDa) and increased insoluble collagen compared to C. Consequently, the post-mortem flesh softening in C was attributed to increased proteolysis in both intracellular and extracellular structural proteins. The preservation of the textural and biochemical characteristics of meagre fillets subjected to V and MA treatments makes these two treatments highly recommendable for the commercialization of meagre fillets.

  13. Expression of PAT and NPT II proteins during the developmental stages of a genetically modified pepper developed in Korea.

    PubMed

    Kim, Hyo Jin; Lee, Si Myung; Kim, Jae Kwang; Ryu, Tae Hun; Suh, Seok Cheol; Cho, Hyun Suk

    2010-10-27

    Estimation of the protein levels introduced in a biotechnology-derived product is conducted as part of an overall safety assessment. An enzyme-linked immunosorbent assay (ELISA) was used to analyze phosphinothricin acetyltransferase (PAT) and neomycin phosphotransferase II (NPT II) protein expression in a genetically modified (GM) pepper plant developed in Korea. PAT and NPT II expression levels, based on both dry weight and fresh weight, were variable among different plant generations and plant sections from isolated genetically modified organism (GMO) fields at four developmental stages. PAT expression was highest in leaves at anthesis (11.44 μg/gdw and 2.17 μg/gfw) and lowest in roots (0.12 μg/gdw and 0.01 μg/gfw). NPT II expression was also highest in leaves at anthesis (17.31 μg/gdw and 3.41 μg/gfw) and lowest in red pepper (0.65 μg/gdw and 0.12 μg/gfw). In pollen, PAT expression was 0.59-0.62 μg/gdw, while NPT II was not detected. Both PAT and NPT II showed a general pattern of decreased expression with progression of the growing season. As expected, PAT and NPT II protein expression was not detectable in control pepper plants.

  14. Effects of vacuum and modified atmosphere on textural parameters and structural proteins of cultured meagre (Argyrosomus regius) fillets.

    PubMed

    Sáez, María I; Martínez, Tomás F; Cárdenas, Salvador; Suárez, María D

    2015-09-01

    The influence of two preservation strategies (vacuum package and modified atmosphere package) on the post-mortem changes of textural parameters, pH, water holding capacity, sarcoplasmic and myofibrillar proteins, and collagen content of meagre (Argyrosomus regius) fillets was studied. Fillets were stored in a cold room in aerobic (control, C), vacuum (V) and modified atmosphere (MA) package. Samples were withdrawn at six sampling points throughout 15-day storage, and post-mortem changes were assessed. The textural parameters were significantly enhanced in V and MA compared to C. Both V and MA treatments reduced the intensity of a group of myofibrillar protein fractions (140-195 kDa) and increased insoluble collagen compared to C. Consequently, the post-mortem flesh softening in C was attributed to increased proteolysis in both intracellular and extracellular structural proteins. The preservation of the textural and biochemical characteristics of meagre fillets subjected to V and MA treatments makes these two treatments highly recommendable for the commercialization of meagre fillets. PMID:25082978

  15. Review of animal models designed to predict the potential allergenicity of novel proteins in genetically modified crops.

    PubMed

    Ladics, G S; Knippels, L M J; Penninks, A H; Bannon, G A; Goodman, R E; Herouet-Guicheney, C

    2010-03-01

    The safety assessment of genetically modified crops involves the evaluation of the potential allergenicity of novel proteins by using several in silico and in vitro endpoints. In this publication, the variables and questions associated with the development of in vivo models are examined and several unpublished results are presented. Both rodent and non-rodent (dog and pig) models have been investigated using various routes of administration with purified proteins or food extracts, with or without the use of an adjuvant. The ideal model should be simple, reproducible across laboratories over time, specific and sensitive enough for distinguishing a threshold beyond which relevant allergenicity would be predicted and, for ranking proteins correlated with the allergic responses in humans, and acceptable under animal care. Preliminary data suggest that a few appear promising; however, further evaluation of these models is required. In particular, more extensive validation testing with additional allergenic and non-allergenic material should be performed before using them in the safety assessment of genetically modified crops.

  16. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  17. Distribution of oxidized and HNE-modified proteins in U87 cells.

    PubMed

    Jung, Tobias; Engels, Martina; Kaiser, Barbara; Grune, Tilman

    2005-01-01

    Protein modification is one of the important processes during oxidative stress. This modification of proteins is either due to direct oxidation of proteins by various oxidants or due to secondary modification by lipid peroxidation products, e.g. 4-hydroxynonenal. In the here presented work we compare the intracellular distribution of protein modification products after treatment of human U87 astrocytoma cells with hydrogen peroxide or HNE. The treatment with hydrogen peroxide leads mainly to a cytosolic formation of oxidized proteins whereas HNE treatment is forming HNE-adducts throughout the cell. Therefore, we concluded that HNE diffusion distance in cells enables this lipid peroxidation product to act as a second messenger within the cell and on the other hand is the reason for the genotoxic properties of this compound.

  18. Prokaryotic expression and allergenicity assessment of hygromycin B phosphotransferase protein derived from genetically modified plants.

    PubMed

    Lu, Y; Xu, W; Kang, A; Luo, Y; Guo, F; Yang, R; Zhang, J; Huang, K

    2007-09-01

    The hygromycin B phosphotransferase gene (hpt) has been widely used in the process of plant genetic engineering to produce plants that can secrete the HPT protein. As part of a safety assessment, sufficient quantities of the protein were produced in Escherichia coli to conduct in vitro digestibility and animal studies. Western blotting analysis showed that the HPT protein was digested by simulated gastric fluid within 40 s. ELISA demonstrated that the protein did not induce detectable levels of specific IgE antibodies or histamine in test animals. Alignment of the amino acid sequence of HPT with those of known allergens did not produce evidence of sequence similarities between these allergens and the HPT protein. We conclude that HPT has a low probability to induce allergenicity.

  19. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors*

    PubMed Central

    Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J.; Mobarec, Juan Carlos; Woodlock, David A.; Reynolds, Christopher A.; Poyner, David R.; Watkins, Harriet A.; Ladds, Graham

    2016-01-01

    The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. PMID:27566546

  20. Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents

    PubMed Central

    Rohloff, John C; Gelinas, Amy D; Jarvis, Thale C; Ochsner, Urs A; Schneider, Daniel J; Gold, Larry; Janjic, Nebojsa

    2014-01-01

    Limited chemical diversity of nucleic acid libraries has long been suspected to be a major constraining factor in the overall success of SELEX (Systematic Evolution of Ligands by EXponential enrichment). Despite this constraint, SELEX has enjoyed considerable success over the past quarter of a century as a result of the enormous size of starting libraries and conformational richness of nucleic acids. With judicious introduction of functional groups absent in natural nucleic acids, the “diversity gap” between nucleic acid–based ligands and protein-based ligands can be substantially bridged, to generate a new class of ligands that represent the best of both worlds. We have explored the effect of various functional groups at the 5-position of uracil and found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers). Such modified nucleotides create unique intramolecular motifs and make direct contacts with proteins. Importantly, SOMAmers engage their protein targets with surfaces that have significantly more hydrophobic character compared with conventional aptamers, thereby increasing the range of epitopes that are available for binding. These improvements have enabled us to build a collection of SOMAmers to over 3,000 human proteins encompassing major families such as growth factors, cytokines, enzymes, hormones, and receptors, with additional SOMAmers aimed at pathogen and rodent proteins. Such a large and growing collection of exquisite affinity reagents expands the scope of possible applications in diagnostics and therapeutics. PMID:25291143

  1. MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age

    PubMed Central

    Ferree, Andrew W; Trudeau, Kyle; Zik, Eden; Benador, Ilan Y; Twig, Gilad; Gottlieb, Roberta A; Shirihai, Orian S

    2013-01-01

    To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport. PMID:24149000

  2. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis modified Cry3A... of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.505...

  3. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis modified Cry3A... of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.505...

  4. Strengthening of the DNA-protein complex during stationary phase aging of cell cultures

    SciTech Connect

    Khokhlov, A.N.; Chirkova, E.Yu.; Gorin, A.I.

    1986-09-01

    The possibility of accumulation of cross-linkages in the DNA-protein complex was studied during stationary phase aging of cells in culture. Chinese hamster cells were used in the experiments, along with human fibroblasts. /sup 3/H-thymidine, /sup 14/C-valine, and /sup 14/C-leucine were added to the medium. The quantity of protein firmly bound with DNA was judged from the value of the coefficient /sup 14/C//sup 3/H determined with allowance for penetration of counting from the /sup 14/C-channel into the /sup 3/H-channel. The authors maintain that the results presented in this paper provide further evidence of the value of stationary phase cell cultures for the study of the mechanisms of aging and also of some of the general principles underlying hereditary pathology.

  5. Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

    NASA Astrophysics Data System (ADS)

    Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

    2001-05-01

    In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

  6. IN-MACA-MCC: Integrated Multiple Attractor Cellular Automata with Modified Clonal Classifier for Human Protein Coding and Promoter Prediction

    PubMed Central

    Pokkuluri, Kiran Sree; Inampudi, Ramesh Babu; Nedunuri, S. S. S. N. Usha Devi

    2014-01-01

    Protein coding and promoter region predictions are very important challenges of bioinformatics (Attwood and Teresa, 2000). The identification of these regions plays a crucial role in understanding the genes. Many novel computational and mathematical methods are introduced as well as existing methods that are getting refined for predicting both of the regions separately; still there is a scope for improvement. We propose a classifier that is built with MACA (multiple attractor cellular automata) and MCC (modified clonal classifier) to predict both regions with a single classifier. The proposed classifier is trained and tested with Fickett and Tung (1992) datasets for protein coding region prediction for DNA sequences of lengths 54, 108, and 162. This classifier is trained and tested with MMCRI datasets for protein coding region prediction for DNA sequences of lengths 252 and 354. The proposed classifier is trained and tested with promoter sequences from DBTSS (Yamashita et al., 2006) dataset and nonpromoters from EID (Saxonov et al., 2000) and UTRdb (Pesole et al., 2002) datasets. The proposed model can predict both regions with an average accuracy of 90.5% for promoter and 89.6% for protein coding region predictions. The specificity and sensitivity values of promoter and protein coding region predictions are 0.89 and 0.92, respectively. PMID:25132849

  7. IN-MACA-MCC: Integrated Multiple Attractor Cellular Automata with Modified Clonal Classifier for Human Protein Coding and Promoter Prediction.

    PubMed

    Pokkuluri, Kiran Sree; Inampudi, Ramesh Babu; Nedunuri, S S S N Usha Devi

    2014-01-01

    Protein coding and promoter region predictions are very important challenges of bioinformatics (Attwood and Teresa, 2000). The identification of these regions plays a crucial role in understanding the genes. Many novel computational and mathematical methods are introduced as well as existing methods that are getting refined for predicting both of the regions separately; still there is a scope for improvement. We propose a classifier that is built with MACA (multiple attractor cellular automata) and MCC (modified clonal classifier) to predict both regions with a single classifier. The proposed classifier is trained and tested with Fickett and Tung (1992) datasets for protein coding region prediction for DNA sequences of lengths 54, 108, and 162. This classifier is trained and tested with MMCRI datasets for protein coding region prediction for DNA sequences of lengths 252 and 354. The proposed classifier is trained and tested with promoter sequences from DBTSS (Yamashita et al., 2006) dataset and nonpromoters from EID (Saxonov et al., 2000) and UTRdb (Pesole et al., 2002) datasets. The proposed model can predict both regions with an average accuracy of 90.5% for promoter and 89.6% for protein coding region predictions. The specificity and sensitivity values of promoter and protein coding region predictions are 0.89 and 0.92, respectively. PMID:25132849

  8. Assessing the allergenicity of proteins introduced into genetically modified crops using specific human IgE assays.

    PubMed

    Goodman, Richard E; Leach, John N

    2004-01-01

    Global commercial production of genetically modified (GM) crops has grown to over 67 million hectares annually, primarily of herbicide-tolerant and insect protection crop varieties. GM crops are produced by the insertion of specific genes that either encode a protein, or a regulatory RNA sequence. A comprehensive safety evaluation is conducted for each new commercial GM crop, including an assessment of the potential allergenicity of any newly introduced protein. If the gene was derived from an allergenic organism, or the protein sequence is highly similar to a known allergen, immunoassays, e.g., Western blot assays and enzyme-linked immunosorbent assay tests, are performed to identify protein-specific IgE binding by sera of individuals allergic to the gene source, or the source of the sequence-matched allergen. Although such assays are commonly used to identify previously unknown allergens, criteria have not been established to demonstrate that a protein is unlikely to cause allergic reactions. This review discusses factors that affect the predictive value of these tests, including clinical selection criteria for serum donors, selection of blocking reagents to reduce nonspecific antibody binding, inhibition assays to verify specificity of binding, and scientifically justified limits of detection (sensitivity) in the absence of information regarding biological thresholds.

  9. Protein-based biomemory device consisting of the cysteine-modified azurin

    NASA Astrophysics Data System (ADS)

    Choi, Jeong-Woo; Oh, Byung-Keun; Kim, Young Jun; Min, Junhong

    2007-12-01

    We demonstrated a protein-based memory device using recombinant Pseudomonas aeruginosa azurin (azurin), a metalloprotein with a redox property. Azurin was recombined with a cysteine residue to enhance the stability of the self-assembled protein on the gold surface. The memory device characteristics, including the "read," "write," and "erase" functions of the self-assembled azurin layer, were well demonstrated with three distinct electrical states of azurin layers by cyclic voltammetry. The robustness of the protein-based biomemory device was validated by the repeated electrochemical performance of 500000cycles.

  10. Determination of protein-ligand interactions using accelerator mass spectrometry: modified crosslinking assay.

    PubMed

    Hah, Sang Soo

    2009-05-01

    A highly sensitive detection method for the determination of protein-ligand interactions has been developed. Radiocarbon-labeled 17beta-estradiol was incubated with estrogen receptor-alpha; as a selective binding partner, and covalently attached using crosslinking agents, to form covalently linked protein-ligand complexes. After separation using a denaturing gel, the (14)C content in the sliced gels was identified by accelerator mass spectrometry. The obtained data demonstrated specific binding of the small molecule to its binding partner. In theory, this method can be applied to most protein-ligand interaction studies.

  11. Age-Related Changes of Myelin Basic Protein in Mouse and Human Auditory Nerve

    PubMed Central

    Xing, Yazhi; Samuvel, Devadoss J.; Stevens, Shawn M.; Dubno, Judy R.; Schulte, Bradley A.; Lang, Hainan

    2012-01-01

    Age-related hearing loss (presbyacusis) is the most common type of hearing impairment. One of the most consistent pathological changes seen in presbyacusis is the loss of spiral ganglion neurons (SGNs). Defining the cellular and molecular basis of SGN degeneration in the human inner ear is critical to gaining a better understanding of the pathophysiology of presbyacusis. However, information on age-related cellular and molecular alterations in the human spiral ganglion remains scant, owing to the very limited availably of human specimens suitable for high resolution morphological and molecular analysis. This study aimed at defining age-related alterations in the auditory nerve in human temporal bones and determining if immunostaining for myelin basic protein (MBP) can be used as an alternative approach to electron microscopy for evaluating myelin degeneration. For comparative purposes, we evaluated ultrastructural alternations and changes in MBP immunostaining in aging CBA/CaJ mice. We then examined 13 temporal bones from 10 human donors, including 4 adults aged 38–46 years (middle-aged group) and 6 adults aged 63–91 years (older group). Similar to the mouse, intense immunostaining of MBP was present throughout the auditory nerve of the middle-aged human donors. Significant declines in MBP immunoreactivity and losses of MBP+ auditory nerve fibers were observed in the spiral ganglia of both the older human and aged mouse ears. This study demonstrates that immunostaining for MBP in combination with confocal microscopy provides a sensitive, reliable, and efficient method for assessing alterations of myelin sheaths in the auditory nerve. The results also suggest that myelin degeneration may play a critical role in the SGN loss and the subsequent decline of the auditory nerve function in presbyacusis. PMID:22496821

  12. X-box-binding protein 1-modified neural stem cells for treatment of Parkinson's disease.

    PubMed

    Si, Lihui; Xu, Tianmin; Wang, Fengzhang; Liu, Qun; Cui, Manhua

    2012-04-01

    X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson's disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson's disease in rats.

  13. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing

    PubMed Central

    Gehwolf, Renate; Wagner, Andrea; Lehner, Christine; Bradshaw, Amy D.; Scharler, Cornelia; Niestrawska, Justyna A.; Holzapfel, Gerhard A.; Bauer, Hans-Christian; Tempfer, Herbert; Traweger, Andreas

    2016-01-01

    Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc−/− tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc−/− tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons. PMID:27586416

  14. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

    PubMed

    Gehwolf, Renate; Wagner, Andrea; Lehner, Christine; Bradshaw, Amy D; Scharler, Cornelia; Niestrawska, Justyna A; Holzapfel, Gerhard A; Bauer, Hans-Christian; Tempfer, Herbert; Traweger, Andreas

    2016-01-01

    Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons. PMID:27586416

  15. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

    PubMed

    Gehwolf, Renate; Wagner, Andrea; Lehner, Christine; Bradshaw, Amy D; Scharler, Cornelia; Niestrawska, Justyna A; Holzapfel, Gerhard A; Bauer, Hans-Christian; Tempfer, Herbert; Traweger, Andreas

    2016-09-02

    Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

  16. Separation of macromolecular proteins and removal of humic acid by cellulose acetate modified UF membranes.

    PubMed

    Kanagaraj, P; Nagendran, A; Rana, D; Matsuura, T

    2016-08-01

    Surface modifying macromolecules (SMMs) were synthesized with various polyurethane pre polymers end-capped with different groups and blended into the casting solution of cellulose acetate (CA) to prepare surface modified ultra-filtration (UF) membranes for water filtration applications. The surface modification of the CA membranes was confirmed by the FTIR and static contact angle (SCA) measurements. The membranes so prepared had the typical characteristics of UF membranes as confirmed by scanning electron microscopy (SEM). Membrane properties were studied in terms of membrane compaction, percentage water content (%WC), pure water flux (PWF), membrane hydraulic resistance (Rm), molecular weight cut-off (MWCO), average pore size and porosity. The result showed that PWF, %WC, MWCO and pore size increased whereas the Rm decreased by the addition of SMMs. The significant effect of SMMs on the fouling by humic acid (HA) was also observed. It was found that the cSMM-3 membrane, in which SMM was synthesized with diethylene glycol (DEG) and hydroxyl benzene sulfonate (HBS) was blended, had the highest flux recovery ratio FRR (84.6%), as well as the lowest irreversible fouling (15.4%), confirming their improved antifouling properties. Thus, the SMM modified CA membranes had proven, to play an important role in the water treatment by UF. PMID:27118046

  17. Separation of macromolecular proteins and removal of humic acid by cellulose acetate modified UF membranes.

    PubMed

    Kanagaraj, P; Nagendran, A; Rana, D; Matsuura, T

    2016-08-01

    Surface modifying macromolecules (SMMs) were synthesized with various polyurethane pre polymers end-capped with different groups and blended into the casting solution of cellulose acetate (CA) to prepare surface modified ultra-filtration (UF) membranes for water filtration applications. The surface modification of the CA membranes was confirmed by the FTIR and static contact angle (SCA) measurements. The membranes so prepared had the typical characteristics of UF membranes as confirmed by scanning electron microscopy (SEM). Membrane properties were studied in terms of membrane compaction, percentage water content (%WC), pure water flux (PWF), membrane hydraulic resistance (Rm), molecular weight cut-off (MWCO), average pore size and porosity. The result showed that PWF, %WC, MWCO and pore size increased whereas the Rm decreased by the addition of SMMs. The significant effect of SMMs on the fouling by humic acid (HA) was also observed. It was found that the cSMM-3 membrane, in which SMM was synthesized with diethylene glycol (DEG) and hydroxyl benzene sulfonate (HBS) was blended, had the highest flux recovery ratio FRR (84.6%), as well as the lowest irreversible fouling (15.4%), confirming their improved antifouling properties. Thus, the SMM modified CA membranes had proven, to play an important role in the water treatment by UF.

  18. A Modular Approach To Study Protein Adsorption on Surface Modified Hydroxyapatite.

    PubMed

    Ozhukil Kollath, Vinayaraj; Van den Broeck, Freya; Fehér, Krisztina; Martins, José C; Luyten, Jan; Traina, Karl; Mullens, Steven; Cloots, Rudi

    2015-07-13

    Biocompatible inorganic nano- and microcarriers can be suitable candidates for protein delivery. This study demonstrates facile methods of functionalization by using nanoscale linker molecules to change the protein adsorption capacity of hydroxyapatite (HA) powder. The adsorption capacity of bovine serum albumin as a model protein has been studied with respect to the surface modifications. The selected linker molecules (lysine, arginine, and phosphoserine) can influence the adsorption capacity by changing the electrostatic nature of the HA surface. Qualitative and quantitative analyses of linker-molecule interactions with the HA surface have been performed by using NMR spectroscopy, zeta-potential measurements, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Additionally, correlations to theoretical isotherm models have been calculated with respect to Langmuir and Freundlich isotherms. Lysine and arginine increased the protein adsorption, whereas phosphoserine reduced the protein adsorption. The results show that the adsorption capacity can be controlled with different functionalization, depending on the protein-carrier selections under consideration. The scientific knowledge acquired from this study can be applied in various biotechnological applications that involve biomolecule-inorganic material interfaces. PMID:26096378

  19. Pepsin degradation of Cry1A(b) protein purified from genetically modified maize (Zea mays).

    PubMed

    de Luis, Ruth; Lavilla, María; Sánchez, Lourdes; Calvo, Miguel; Pérez, María D

    2010-02-24

    The aim of this work was to study the in vitro digestion of Cry1A(b) protein by pepsin. To perform this work, a protein fraction purified from transgenic maize by immunoadsorption was employed. The undigested fraction showed several bands of molecular weight ranging between 14 and 70 kDa when assayed by SDS-PAGE. These bands were identified as corresponding to Cry1A(b) protein by immunochemical techniques and mass spectrometry. The rate of degradation of the purified fraction by pepsin estimated by ELISA was found to be about 75% within 30 min, and the protein concentration remained constant up to 4 h. In all treated samples, the full-length protein and fragments present in Cry1A(b) fraction were absent and peptides of less than 8.5 kDa were mainly found by SDS-PAGE and mass spectrometry. These peptides did not react with antiserum against Cry1A(b) protein by Western blotting. These results suggest that Cry1A(b) fraction purified from transgenic maize is rapidly and extensively degraded by pepsin, giving peptides of low molecular mass.

  20. A Modular Approach To Study Protein Adsorption on Surface Modified Hydroxyapatite.

    PubMed

    Ozhukil Kollath, Vinayaraj; Van den Broeck, Freya; Fehér, Krisztina; Martins, José C; Luyten, Jan; Traina, Karl; Mullens, Steven; Cloots, Rudi

    2015-07-13

    Biocompatible inorganic nano- and microcarriers can be suitable candidates for protein delivery. This study demonstrates facile methods of functionalization by using nanoscale linker molecules to change the protein adsorption capacity of hydroxyapatite (HA) powder. The adsorption capacity of bovine serum albumin as a model protein has been studied with respect to the surface modifications. The selected linker molecules (lysine, arginine, and phosphoserine) can influence the adsorption capacity by changing the electrostatic nature of the HA surface. Qualitative and quantitative analyses of linker-molecule interactions with the HA surface have been performed by using NMR spectroscopy, zeta-potential measurements, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Additionally, correlations to theoretical isotherm models have been calculated with respect to Langmuir and Freundlich isotherms. Lysine and arginine increased the protein adsorption, whereas phosphoserine reduced the protein adsorption. The results show that the adsorption capacity can be controlled with different functionalization, depending on the protein-carrier selections under consideration. The scientific knowledge acquired from this study can be applied in various biotechnological applications that involve biomolecule-inorganic material interfaces.

  1. Unbiased proteomic screen for binding proteins to modified lysines on histone H3

    PubMed Central

    Chan, Doug W.; Wang, Yi; Wu, Meng; Wong, Jiemin; Qin, Jun; Zhao, Yingming

    2010-01-01

    We report a sensitive peptide pull-down approach in combination with protein identification by LC-MS/MS and qualitative abundance measurements by spectrum counting to identify proteins binding to histone H3 tail containing dimethyl lysine 4 (H3K4me2), dimethyl lysine 9 (H3K9me2), or acetyl lysine 9 (H3K9ac). Our study identified 86 nuclear proteins that associate with the histone H3 tail peptides examined, including seven known direct binders and 16 putative direct binders with conserved PHD finger, bromodomain, and WD40 domains. The reliability of our proteomic screen is supported by the fact that more than one-third of the proteins identified were previously described to associate with histone H3 tail directly or indirectly. To our knowledge, the results presented here are the most comprehensive analysis of H3K4me2, H3K9me2, and H3K9ac associated proteins and will provide a useful resource for researchers studying the mechanisms of histone code effector proteins. PMID:19337993

  2. Age-dependent degradation of the protein adsorption capacity of titanium.

    PubMed

    Hori, N; Att, W; Ueno, T; Sato, N; Yamada, M; Saruwatari, L; Suzuki, T; Ogawa, T

    2009-07-01

    Reported bone-implant contact percentages are far below the ideal 100%. We tested a hypothesis that the protein adsorption capability of titanium, which is critical to the process of osseointegration, changes over time before its use. Machined, acid-etched, and sandblasted surfaces were prepared and stored under dark ambient conditions for 3 days, 1 week, or 4 weeks. For all surfaces, protein adsorption decreased as the storage time increased, and their decreasing rates were dependent on titanium topography. After 4 weeks, the amounts of albumin and fibronectin adsorbed by the acid-etched surface were only 20% and 35%, respectively, of that adsorbed by the fresh surface after 2 hours of incubation, and remained substantially low even after 24 hours. This time-dependent degradation in protein adsorption of titanium correlated with its naturally decreasing hydrophilicity, which was not observed for the nickel and chromium surfaces, indicating a titanium-specific biological aging.

  3. On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters.

    PubMed

    Wang, Tao; Kulkarni, Nikita; D'Souza, Gerard G M; Petrenko, Valery A; Torchilin, Vladimir P

    2011-10-01

    The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with cocultured, nontarget cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency. PMID:21675738

  4. On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters

    PubMed Central

    Wang, Tao; Kulkarni, Nikita; D’Souza, Gerard G.M.; Petrenko, Valery A.; Torchilin, Vladimir P.

    2011-01-01

    The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with co-cultured, non-target cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency. PMID:21675738

  5. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans

    PubMed Central

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-01

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development. PMID:26791749

  6. The impact of low-protein high-carbohydrate diets on aging and lifespan.

    PubMed

    Le Couteur, David G; Solon-Biet, Samantha; Cogger, Victoria C; Mitchell, Sarah J; Senior, Alistair; de Cabo, Rafael; Raubenheimer, David; Simpson, Stephen J

    2016-03-01

    Most research on nutritional effects on aging has focussed on the impact of manipulating single dietary factors such as total calorie intake or each of the macronutrients individually. More recent studies using a nutritional geometric approach called the Geometric Framework have facilitated an understanding of how aging is influenced across a landscape of diets that vary orthogonally in macronutrient and total energy content. Such studies have been performed using ad libitum feeding regimes, thus taking into account compensatory feeding responses that are inevitable in a non-constrained environment. Geometric Framework studies on insects and mice have revealed that diets low in protein and high in carbohydrates generate longest lifespans in ad libitum-fed animals while low total energy intake (caloric restriction by dietary dilution) has minimal effect. These conclusions are supported indirectly by observational studies in humans and a heterogeneous group of other types of interventional studies in insects and rodents. Due to compensatory feeding for protein dilution, low-protein, high-carbohydrate diets are often associated with increased food intake and body fat, a phenomenon called protein leverage. This could potentially be mitigated by supplementing these diets with interventions that influence body weight through physical activity and ambient temperature.

  7. Role of forkhead box protein A3 in age-associated metabolic decline

    PubMed Central

    Ma, Xinran; Xu, Lingyan; Gavrilova, Oksana; Mueller, Elisabetta

    2014-01-01

    Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown. Here we report that during aging, levels of Foxa3 are significantly and selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter. Overall, our data demonstrate a role for Foxa3 in energy expenditure and in age-associated metabolic disorders. PMID:25225406

  8. Absorption-Mode Fourier Transform Mass Spectrometry: The Effects of Apodization and Phasing on Modified Protein Spectra

    NASA Astrophysics Data System (ADS)

    Qi, Yulin; Li, Huilin; Wills, Rebecca H.; Perez-Hurtado, Pilar; Yu, Xiang; Kilgour, David P. A.; Barrow, Mark P.; Lin, Cheng; O'Connor, Peter B.

    2013-06-01

    The method of phasing broadband Fourier transform ion cyclotron resonance (FT-ICR) spectra allows plotting the spectra in the absorption-mode; this new approach significantly improves the quality of the data at no extra cost. Herein, an internal calibration method for calculating the phase function has been developed and successfully applied to the top-down spectra of modified proteins, where the peak intensities vary by 100×. The result shows that the use of absorption-mode spectra allows more peaks to be discerned within the recorded data, and this can reveal much greater information about the protein and modifications under investigation. In addition, noise and harmonic peaks can be assigned immediately in the absorption-mode.

  9. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    NASA Astrophysics Data System (ADS)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  10. Absorption-mode Fourier transform mass spectrometry: the effects of apodization and phasing on modified protein spectra.

    PubMed

    Qi, Yulin; Li, Huilin; Wills, Rebecca H; Perez-Hurtado, Pilar; Yu, Xiang; Kilgour, David P A; Barrow, Mark P; Lin, Cheng; O'Connor, Peter B

    2013-06-01

    The method of phasing broadband Fourier transform ion cyclotron resonance (FT-ICR) spectra allows plotting the spectra in the absorption-mode; this new approach significantly improves the quality of the data at no extra cost. Herein, an internal calibration method for calculating the phase function has been developed and successfully applied to the top-down spectra of modified proteins, where the peak intensities vary by 100×. The result shows that the use of absorption-mode spectra allows more peaks to be discerned within the recorded data, and this can reveal much greater information about the protein and modifications under investigation. In addition, noise and harmonic peaks can be assigned immediately in the absorption-mode.

  11. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    PubMed Central

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-01-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8–9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies. PMID:10884430

  12. Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

    PubMed Central

    Zajac, María Paula Del Médico; Taboga, Oscar Alberto; Calamante, Gabriela

    2012-01-01

    A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry. PMID:22705743

  13. Redox proteomics analysis of HNE-modified proteins in Down syndrome brain: clues for understanding the development of Alzheimer disease.

    PubMed

    Di Domenico, Fabio; Pupo, Gilda; Tramutola, Antonella; Giorgi, Alessandra; Schininà, Maria Eugenia; Coccia, Raffaella; Head, Elizabeth; Butterfield, D Allan; Perluigi, Marzia

    2014-06-01

    Down syndrome (DS) is the most common genetic cause of intellectual disability, due to partial or complete triplication of chromosome 21. DS subjects are characterized by a number of abnormalities including premature aging and development of Alzheimer disease (AD) neuropathology after approximately 40 years of age. Several studies show that oxidative stress plays a crucial role in the development of neurodegeneration in the DS population. Increased lipid peroxidation is one of the main events causing redox imbalance within cells through the formation of toxic aldehydes that easily react with DNA, lipids, and proteins. In this study we used a redox proteomics approach to identify specific targets of 4-hydroxynonenal modifications in the frontal cortex from DS cases with and without AD pathology. We suggest that a group of identified proteins followed a specific pattern of oxidation in DS vs young controls, probably indicating characteristic features of the DS phenotype; a second group of identified proteins showed increased oxidation in DS/AD vs DS, thus possibly playing a role in the development of AD. The third group of comparison, DS/AD vs old controls, identified proteins that may be considered specific markers of AD pathology. All the identified proteins are involved in important biological functions including intracellular quality control systems, cytoskeleton network, energy metabolism, and antioxidant response. Our results demonstrate that oxidative damage is an early event in DS, as well as dysfunctions of protein-degradation systems and cellular protective pathways, suggesting that DS subjects are more vulnerable to oxidative damage accumulation that might contribute to AD development. Further, considering that the majority of proteins have been already demonstrated to be oxidized in AD brain, our results strongly support similarities with AD in DS.

  14. Role of Myofibrillar Protein Catabolism in Development of Glucocorticoid Myopathy: Aging and Functional Activity Aspects

    PubMed Central

    Seene, Teet; Kaasik, Priit

    2016-01-01

    Muscle weakness in corticosteroid myopathy is mainly the result of the destruction and atrophy of the myofibrillar compartment of fast-twitch muscle fibers. Decrease of titin and myosin, and the ratio of nebulin and MyHC in myopathic muscle, shows that these changes of contractile and elastic proteins are the result of increased catabolism of the abovementioned proteins in skeletal muscle. Slow regeneration of skeletal muscle is in good correlation with a decreased number of satellite cells under the basal lamina of muscle fibers. Aging causes a reduction of AMP-activated protein kinase (AMPK) activity as the result of the reduced function of the mitochondrial compartment. AMPK activity increases as a result of increased functional activity. Resistance exercise causes anabolic and anticatabolic effects in skeletal muscle: muscle fibers experience hypertrophy while higher myofibrillar proteins turn over. These changes are leading to the qualitative remodeling of muscle fibers. As a result of these changes, possible maximal muscle strength is increasing. Endurance exercise improves capillary blood supply, increases mitochondrial biogenesis and muscle oxidative capacity, and causes a faster turnover rate of sarcoplasmic proteins as well as qualitative remodeling of type I and IIA muscle fibers. The combination of resistance and endurance exercise may be the fastest way to prevent or decelerate muscle atrophy due to the anabolic and anticatabolic effects of exercise combined with an increase in oxidative capacity. The aim of the present short review is to assess the role of myofibrillar protein catabolism in the development of glucocorticoid-caused myopathy from aging and physical activity aspects. PMID:27187487

  15. Heat-induced changes in the properties of modified skim milks with different casein to whey protein ratios.

    PubMed

    Singh, Mandeep Jeswan; Chandrapala, Jayani; Udabage, Punsandani; McKinnon, Ian; Augustin, Mary Ann

    2015-05-01

    The heat-induced changes in pH, Ca activity and viscosity after heating at 90 °C for 10 min of five modified skim milks were studied as a function of the initial pH of the milks at 25 °C. The milks had (i) different ratios of casein : whey protein (0.03, 1.74, 3.97, 5.27 and 7.25), (ii) the same total solids concentration (9% w/w) and (iii) prior to the adjustment of the pH, similar values of pH (6.67-6.74), concentration of serum calcium, and calcium activity, suggesting that the sera have similar mineral composition. The total protein concentrations of the milks differ (2.8-4.0%, w/w). The pH decrease in situ upon heating from 25-90 °C was similar for all the modified skim milks with the same starting pH, suggesting that the pH changes to milk on heating were primarily mediated by the initial mineral composition of the serum and were unaffected by the casein : whey protein ratio or the total protein content of the milk. The heat-induced changes in pH and calcium activity were largely reversible on cooling. The two milks with the lowest ratios of casein to whey protein gelled on heating to 90 °C for 10 min and cooling to 25 °C when the pH was adjusted to pH = 6.2 prior to heating. The viscosities of all other milks with casein to whey protein ratio of 3.97, 5.27 and 7.25 and/or pH ≥6.7 prior to heating did not change significantly. The effect of casein : whey protein ratio and the pH are the dominant factors in controlling the susceptibility to thickening of the milks on heating in this study.

  16. A novel methyltransferase (Hmt1p) modifies poly(A)+-RNA-binding proteins.

    PubMed Central

    Henry, M F; Silver, P A

    1996-01-01

    RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA. As such, they demonstrate a myriad of dynamic behaviors and modifications. In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells. We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A)+-RNA-binding protein termed Npl3p. We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus. New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele. Four alleles of the same gene were identified in this manner. Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase. HMT1 is not required for normal cell viability except when NPL3 is also defective. The Hmt1 protein is located in the nucleus. We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro. These findings now allow further exploration of the function of this previously uncharacterized class of enzymes. PMID:8668183

  17. Chemically modified diamond-like carbon (DLC) for protein enrichment and profiling by MALDI-MS.

    PubMed

    Najam-ul-Haq, M; Rainer, M; Huck, C W; Ashiq, M N; Bonn, G K

    2012-08-01

    The development of new high throughput methods based on different materials with chemical modifications for protein profiling of complex mixtures leads towards biomarkers; used particularly for early diagnosis of a disease. In this work, diamond-like carbon (DLC) is developed and optimized for serum protein profiling by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). This study is carried out in connection with a material-based approach, termed as material-enhanced laser desorption ionization mass spectrometry. DLC is selected as carrier surface which provides large surface to volume ratio and offers high sensitivity. DLC has a dual role of working as MALDI target while acting as an interface for protein profiling by specifically binding peptides and proteins out of serum samples. Serum constituents are bound through immobilized metal ion affinity chromatography (IMAC) functionality, created through glycidyl methacrylate polymerization under ultraviolet light followed by further derivatization with iminodiacetic acid and copper ion loading. Scanning electron microscopy highlights the morphological characteristics of DLC surface. It could be demonstrated that IMAC functionalized DLC coatings represent a powerful material in trapping biomolecules for their further analysis by MALDI-MS resulting in improved sensitivity, specificity and capacity in comparison to other protein-profiling methods.

  18. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  19. Selective cleavage of thioether linkage in proteins modified with 4-hydroxynonenal.

    PubMed Central

    Uchida, K; Stadtman, E R

    1992-01-01

    The peroxidation of polyunsaturated fatty acids leads to numerous products, including 4-hydroxynonenal (HNE). That 4-hydroxy-2-alkenal compounds react with sulfhydryl groups of proteins to form thioether adducts possessing a carbonyl function has been established [Schauenstein, E. & Esterbauer, H. (1979) Ciba Found. Symp. 67, 225-244]. Taking advantage of the fact that Raney nickel catalyzes cleavage of thioether bonds, we have developed a procedure to quantitate the amount of HNE moiety bound to protein by means of a thioether linkage. Adducts of HNE with N-acetylcysteine and glutathione were prepared, labeled with NaB[3H]H4, and then treated with Raney nickel. The 3H-labeled product was recovered in 85-90% yield from both HNE-N-acetylcysteine and HNE-glutathione adducts in a solvent [10% (vol/vol) methanol/chloroform]-estractable form. Treatment of proteins with HNE led to the disappearance of protein sulfhydryl groups. However, less than 10% of the labeled adducts obtained after subsequent reduction with NaB[3H]H4 could be released in a solvent-extractable form upon treatment with Raney nickel. This and the observation that HNE reacts with proteins lacking a sulfhydryl group attests to the fact that HNE can react with amino acid residues other than cysteinyl residues. Images PMID:1608970

  20. Impact of aging on heat shock protein expression in the substantia nigra and striatum of the female rat.

    PubMed

    Gleixner, A M; Pulugulla, S H; Pant, D B; Posimo, J M; Crum, T S; Leak, R K

    2014-07-01

    Many heat shock proteins are chaperones that help refold or degrade misfolded proteins and battle apoptosis. Because of their capacity to protect against protein misfolding, they may help keep diseases of aging at bay. A few reports have examined heat shock proteins (eg. Hsp25, Hsp60, Hsp70, and heat shock cognate 70 or Hsc70) as a function of age in the striatum and nigra. In the present study, we examined the impact of aging on Hsp25, heme oxygenase 1 (HO1 or Hsp32), Hsp40, Hsp60, Hsc70, Hsc/Hsp70 interacting protein (Hip), 78 kDa glucose-regulated protein (GRP78), Hsp90, and ubiquitinated proteins in the nigra and striatum of the female rat by infrared immunoblotting. Female animals are not typically examined in aging studies, adding further to the novelty of our study. Striatal HO1 and Hsp40 were both higher in middle-aged females than in the oldest group. Hsp60 levels were also highest in middle age in the nigra, but were highest in the oldest animals in the striatum. Striatal levels of Hsc70 and the co-chaperone Hip were lower in the oldest group relative to the youngest animals. In contrast, Hsp25 rose with advancing age in both regions. Hsp25 was also colocalized with tyrosine hydroxylase in nigral neurons. Ubiquitinated proteins exhibited a trend to rise in the oldest animals in both regions, and K48 linkage-specific ubiquitin rose significantly from 4-6 to 16-19 months in the striatum. Our study reveals a complex array of age-related changes in heat shock proteins. Furthermore, the age-related rises in some proteins, such as Hsp25, may reflect endogenous adaptations to cellular stress.

  1. Genetic Modifiers of Age at Onset in Carriers of the G206A Mutation in PSEN1 With Familial Alzheimer Disease Among Caribbean Hispanics

    PubMed Central

    Lee, Joseph H.; Cheng, Rong; Vardarajan, Badri; Lantigua, Rafael; Reyes-Dumeyer, Dolly; Ortmann, Ward; Graham, Robert R.; Bhangale, Tushar; Behrens, Timothy W.; Medrano, Martin; Jiménez-Velázquez, Ivonne Z.; Mayeux, Richard

    2016-01-01

    Importance The present study identified potential genetic modifiers that may delay or accelerate age at onset of familial Alzheimer disease (AD) by examining age at onset in PSEN1 mutation carrier families, and further investigation of these modifiers may provide insight into the pathobiology of AD and potential therapeutic measures. Objective To identify genetic variants that modify age at onset of AD. Design, Setting, And Participants Using a subset of Caribbean Hispanic families that carry the PSEN1 p.G206A mutation, we performed a 2-stage genome study. The mutation carrier families from an ongoing genetic study served as a discovery set, and the cohort of those with LOAD served as a confirmation set. To identify candidate loci, we performed linkage analysis using 5 p.G206A carrier families (n = 56), and we also performed whole-exome association analysis using 31 p.G206A carriers from 26 families. To confirm the genetic modifiers identified from the p.G206A carrier families, we analyzed the GWAS data for 2888 elderly individuals with LOAD. All study participants were Caribbean Hispanics. Main Outcomes and Measures Age at onset of AD. Results Linkage analysis of AD identified the strongest linkage support at 4q35 (LOD [logarithm of odds] score, 3.69), and the GWAS of age at onset identified variants on 1p13.1, 2q13, 4q25, and 17p11. In the confirmation stage, genewise analysis identified SNX25, PDLIM3, and 3 SH3 domain genes (SORBS2, SH3RF3, and NPHP1) to be significantly associated with LOAD. Subsequent allelic association analysis confirmed SNX25, PDLIM3, and SORBS2 as genetic modifiers of age at onset of EOAD and LOAD and provided modest support for SH3RF3 and NPHP1. Conclusions and Relevance Our 2-stage analysis revealed that SNX25, PDLIM3, and SORBS2 may serve as genetic modifiers of age at onset in both EOAD and LOAD. PMID:26214276

  2. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    PubMed

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy. PMID:26356810

  3. Impact of PEG and PEG-b-PAGE modified PLGA on nanoparticle formation, protein loading and release.

    PubMed

    Rietscher, René; Czaplewska, Justyna A; Majdanski, Tobias C; Gottschaldt, Michael; Schubert, Ulrich S; Schneider, Marc; Lehr, Claus-Michael

    2016-03-16

    The effect of modifying the well-established pharmaceutical polymer PLGA by different PEG-containing block-copolymers on the preparation of ovalbumin (OVA) loaded PLGA nanoparticles (NPs) was studied. The used polymers contained poly(d,l-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG) and poly(allyl glycidyl ether) (PAGE) as building blocks. The double emulsion technique yielded spherical NPs in the size range from 170 to 220 nm (PDI<0.15) for all the differently modified polymers, allowing to directly compare protein loading of and release. PEGylation is usually believed to increase the hydrophilic character of produced particles, favoring encapsulation of hydrophilic substances. However, in this study simple PEGylation of PLGA had only a slight effect on protein release. In contrast, incorporating a PAGE block between the PEG and PLGA units, also eventually enabling active targeting introducing a reactive group, led to a significantly higher loading (+25%) and release rate (+100%), compared to PLGA and PEG-b-PLGA NPs. PMID:26784983

  4. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    PubMed

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy.

  5. A receptor-binding protein of Campylobacter jejuni bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino-modified pseudaminic acid.

    PubMed

    Javed, Muhammad Afzal; van Alphen, Lieke B; Sacher, Jessica; Ding, Wen; Kelly, John; Nargang, Cheryl; Smith, David F; Cummings, Richard D; Szymanski, Christine M

    2015-01-01

    Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.

  6. Transport of DNA into the nuclei of xenopus oocytes by a modified VirE2 protein of Agrobacterium.

    PubMed Central

    Guralnick, B; Thomsen, G; Citovsky, V

    1996-01-01

    We used Agrobacterium T-DNA nuclear transport to examine the specificity of nuclear targeting between plants and animals and the nuclear import of DNA by a specialized transport protein. Two karyophilic Agrobacterium virulence (Vir) proteins, VirD2 and VirE2, which presumably associate with the transported T-DNA and function in many plant species, were microinjected into Drosophila embryos and Xenopus oocytes. In both animal systems, VirD2 localized to the cell nuclei and VirE2 remained exclusively cytoplasmic, suggesting that VirE2 nuclear localization signals may be plant specific. Repositioning one amino acid residue within VirE2 nuclear localization signals enabled them to function in animal cells. The modified VirE2 protein bound DNA and actively transported it into the nuclei of Xenopus oocytes. These observations suggest a functional difference in nuclear import between animals and plants and show that DNA can be transported into the cell nucleus via a protein-specific pathway. PMID:8721747