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

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

Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles

Purpose: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. Methods: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). Results: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. Conclusions: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.

Strength Training with Blood Flow Restriction Diminishes Myostatin Gene Expression

LAURENTINO, G. C., C. UGRINOWITSCH, H. ROSCHEL, M. S. AOKI, A. G. SOARES, M. NEVES JR, A. Y. AIHARA, A. DA ROCHA CORREA FERNANDES, and V. TRICOLI. Strength Training with Blood Flow Restriction Diminishes Myostatin Gene Expression. Med. Sci. Sports Exerc., Vol. 44, No. 3, pp. 406-412, 2012. Purpose: The aim of the study was to determine whether the similar muscle strength and hypertrophy responses observed after either low-intensity resistance exercise associated with moderate blood flow restriction or high-intensity resistance exercise are associated with similar changes in messenger RNA (mRNA) expression of selected genes involved in myostatin (MSTN) signaling. Methods: Twenty-nine physically active male subjects were divided into three groups: low-intensity (20% one-repetition maximum (1RM)) resistance training (LI) (n = 10), low-intensity resistance exercise associated with moderate blood flow restriction (LIR) (n = 10), and high-intensity (80% 1RM) resistance exercise (HI) (n = 9). All of the groups underwent an 8-wk training program. Maximal dynamic knee extension strength (1RM), quadriceps cross-sectional area (CSA), MSTN, follistatin-like related genes (follistatin (FLST), follistatin-like 3 (FLST-3)), activin IIb, growth and differentiation factor-associated serum protein 1 (GASP-1), and MAD-related protein (SMAD-7) mRNA gene expression were assessed before and after training. Results: Knee extension 1RM significantly increased in all groups (LI = 20.7%, LIR = 40.1%, and HI = 36.2%). CSA increased in both the LIR and HI groups (6.3% and 6.1%, respectively). MSTN mRNA expression decreased in the LIR and HI groups (45% and 41%, respectively). There were no significant changes in activin IIb (P > 0.05). FLST and FLST-3 mRNA expression increased in all groups from pre- to posttest (P < 0.001). FLST-3 expression was significantly greater in the HI when compared with the LIR and LI groups at posttest (P = 0.024 and P = 0.018, respectively). GASP-1 and SMAD-7 gene expression significantly increased in both the LIR and HI groups. Conclusions: We concluded that LIR was able to induce gains in 1RM and quadriceps CSA similar to those observed after traditional HI. These responses may be related to the concomitant decrease in MSTN and increase in FLST isoforms, GASP-1, and SMAD-7 mRNA gene expression.

Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+ CD25+ Foxp3+ T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-gamma-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

Abstract Background Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. Methods We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. Results We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. Conclusions In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi.

Bovine Bone Matrix Action Associated With Morphogenetic Protein in Bone Defects in Rats Submitted to Alcoholism

Extended excessive alcohol use causes changes in bone tissue, thus affecting osteogenesis. The objective of this study was to evaluate if demineralized bone matrix (Gen-ox (R)) associated with bone morphogenetic protein (Gen-pro (R)) changes bone neoformation in rats submitted to experimental alcoholism. Forty male rats (Rattus norvegicus) were separated into 2 groups of 20 animals each: Group E1, which received ethyl alcohol at 25% and had the surgical cavity filled in only with blood clot; and Group E2. which received ethyl alcohol at 25% and had the surgical cavity filled in with demineralized bovine cortical bone associated with bone morphogenetic protein. The animals were submitted to a three-week period of gradual adaptation to alcohol, and then continued receiving alcohol at 25% for 90 days, when the surgical cavity was made. After the surgery, the animals continued consuming alcohol until reaching the sacrifice periods of 10, 20, 40, and 60 days, when the tibias were removed for histological processing. Results showed that surgical cavity repair and bone marrow reorganization occurred faster in Group E1 than in Group E2. At the end of the experiment, it was observed that animals in Group E2 had thick bony trabeculae surrounding the implanted material particles and a small area of connective tissue in the surface region. In conclusion, the implanted material did not accelerate bone neoformation, rather it served as a structure for osteogenesis.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Thioredoxin interacting protein genetic variation is associated with diabetes and hypertension in the Brazilian general population

Objective: To investigate the relationship between TXNIP polymorphisms, diabetes and hypertension phenotypes in the Brazilian general population. Methods: Five hundred seventy-six individuals randomly selected from the general urban population according to the MONICA-WHO project guidelines were phenotyped for cardiovascular risk factors. A second, independent, sample composed of 487 family-trios from a different site was also selected. Nine TXNIP polymorphisms were studied. The potential association between TXNIP variability and glucose-phenotypes in children was also explored. TXNIP expression was quantified by real-time PCR in 53 samples from human smooth muscle cells primary culture. Results: TXNIP rs7211 and rs7212 polymorphisms were significantly associated with glucose and blood pressure related phenotypes. In multivariate logistic regression models the studied markers remained associated with diabetes even after adjustment for covariates. TXNIP rs7211 T/rs7212 G haplotype (present in approximately 17% of individuals) was significantly associated to diabetes in both samples. In children, the TXNIP rs7211 T/rs7212 G haplotype was associated with fasting insulin concentrations. Finally, cells harboring TXNIP rs7212 G allele presented higher TXNIP expression levels compared with carriers of TXNIP rs7212 CC genotype (p = 0.02). Conclusion: Carriers of TXNIP genetic variants presented higher TXNIP expression, early signs of glucose homeostasis derangement and increased susceptibility to chronic metabolic conditions such as diabetes and hypertension. Our data suggest that genetic variation in the TXNIP gene may act as a "common ground" modulator of both traits: diabetes and hypertension. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

High levels of C-reactive protein are associated with reduced vagal modulation and low physical activity in young adults

The purpose of this study was to examine the relationship between cardiac autonomic control derived from heart rate variability (HRV), high-sensitivity C-reactive protein (hs-CRP) and physical activity (PA) levels measured using accelerometers. A total of 80 healthy university students volunteered to participate in this study (20.56 +/- 0.82 years, 1.36 +/- 1.5 mg/L of hs-CRP). The participants were divided into groups based on tertiles of hs-CRP. Analysis of covariance adjusted to PA was used to assess group differences in HRV. Associations between hs-CRP, HRV indices and PA were analyzed using Pearson's correlation. The participants at the highest tertile of hs-CRP (tertile 3) had lower cardiac vagal modulation (SDNN, tertile 1=78.05 +/- 5.9,tertile 2=82.43 +/- 5.9,tertile 3=56.03 +/- 6.1; SD1, tertile 1=61.27 +/- 5.3, tertile 2=62.93 +/- 5.4, tertile 3=40.03 +/- 5.5). In addition, vagal indices were inversely correlated with hs-CRP but positively correlated with PA (SDNN r=-0.320, SD1 r=-0.377; SDNN r=0.304, SD1 r=0.299; P<0.05). Furthermore, the most physically active subjects had lower levels of hs-CRP and the highest levels of vagal modulation.

ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - Reversal by a chemical chaperone

ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes. (C) 2012 Elsevier Ltd. All rights reserved.

Thioredoxin interacting protein (TXNIP) rs7212 polymorphism is associated with arterial stiffness in the Brazilian general population

Thioredoxin interacting protein plays a pivotal role in several important processes of cardiovascular homeostasis by functioning as a biological sensor for biomechanical and oxidative stress. However, the effects of genetic variants in the modulation of arterial stiffness are unknown. In this scenario, the present study evaluated the relationship between the TXNIP rs7212 polymorphism and arterial stiffness. In the overall sample and in the diabetic group, individuals carrying CG + GG genotypes had higher PWV values compared with CC genotype group ( 10.0 vs 9.8 ms(-1), P = 0.03; 12.3 vs 11.2 ms(-1), P = 0.01; respectively). Our findings indicated that the G allele may contribute to increased arterial stiffness in the Brazilian general population and suggest a possible interaction with diabetes.