Deficiency of adipocyte fatty-acid-binding protein alleviates myocardial ischaemia/reperfusion injury and diabetes-induced cardiac dysfunction

Clinical evidence shows that circulating levels of adipocyte fatty-acid-binding protein (A-FABP) are elevated in patients with diabetes and closely associated with ischaemic heart disease. Patients with diabetes are more susceptible to myocardial ischaemia/reperfusion (MI/R) injury. The experiments in the present study investigated the role of A-FABP in MI/R injury with or without diabetes. Non-diabetic and diabetic (streptozotocin-induced) A-FABP knockout and wild-type mice were subjected to MI/R or sham intervention. After MI/R, A-FABP knockout mice exhibited reductions in myocardial infarct size, apoptotic index, oxidative and nitrative stress, and inflammation. These reductions were accompanied by an improved left ventricular function compared with the relative controls under non-diabetic or diabetic conditions. After diabetes induction, A-FABP knockout mice exhibited a preserved cardiac function compared with that in wild-type mice. Endothelial cells, but not cardiomyocytes, were identified as the most likely source of cardiac A-FABP. Cardiac and circulating A-FABP levels were significantly increased in mice with diabetes or MI/R. Diabetes-induced superoxide anion production was significantly elevated in wild-type mice, but diminished in A-FABP knockout mice, and this elevation contributed to the exaggeration of MI/R-induced cardiac injury. Phosphorylation of endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) were enhanced in both diabetic and non-diabetic A-FABP knockout mice after MI/R injury, but diminished in wild-type mice. The beneficial effects of A-FABP deficiency on MI/R injury were abolished by the NOS inhibitor N(G)-nitro-L-arginine methyl ester. Thus, A-FABP deficiency protects mice against MI/R-induced and/or diabetes-induced cardiac injury at least partially through activation of the eNOS/NO pathway and reduction in superoxide anion production

Azapeptide activity-based probes for the SARS-CoV-2 main protease enable visualization of inhibition in infected cells

The COVID-19 pandemic has revealed the vulnerability of the modern, global society. With expected waves of future infections by SARS-CoV-2, treatment options for infected individuals will be crucial in order to decrease mortality and hospitalizations. The SARS-CoV-2 main protease is a validated drug target, for which the first inhibitor has been approved for use in patients. To facilitate future work on this drug target, we designed a solid-phase synthesis route towards azapeptide activity-based probes that are capped with a cysteine-reactive electrophile for covalent modification of the active site of Mpro. This design led to the most potent ABP for Mpro and one of the most potent inhibitors reported thus far. We demonstrate that this ABP can be used to visualize Mpro activity and target engagement by drugs in infected cells

Liquid Chromatography Electron Capture Dissociation Tandem Mass Spectrometry (LC-ECD-MS/MS) versus Liquid Chromatography Collision-induced Dissociation Tandem Mass Spectrometry (LC-CID-MS/MS) for the Identification of Proteins

Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins.Experiments were performed on a hybrid linear ion trap–Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin,lysozyme, cytochrome c, alcohol dehydrogenase, and β-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECDMS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags,providing greater confidence in protein assignment

Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors.

Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7-/-) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [3H]glutamate in P2rx7+/+ but not P2rx7-/- mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7-/- mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7-/- mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7-/- mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7-/- mice, which accompanied the increased uptake of [3H]5-HT and an elevated number of [3H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7-/- mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7-/- mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus

ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG

Effect of 6-months of physical exercise on the nitrate/nitrite levels in hypertensive postmenopausal women

<p>Abstract</p> <p>Background</p> <p>Evidences have showed that the incidence of arterial hypertension is greater in postmenopausal women as compared to premenopausal. Physical inactivity has been implicated as a major contributor to weight gain and abdominal obesity in postmenopausal women and the incidence of cardiovascular disease increases dramatically after menopause. Additionally, more women than men die each year of coronary heart disease and are twice as likely as men to die within the first year after a heart attack. A healthy lifestyle has been strongly associated with the regular physical activity and evidences have shown that physically active subjects have more longevity with reduction of morbidity and mortality. Nitric oxide (NO) produced by endothelial cells has been implicated in this beneficial effect with improvement of vascular relaxing and reduction in blood pressure in both laboratory animals and human. Although the effect of exercise training in the human cardiovascular system has been largely studied, the majority of these studies were predominantly conducted in men or young volunteers. Therefore, the aim of this work was to investigate the effects of 6 months of dynamic exercise training (ET) on blood pressure and plasma nitrate/nitrite concentration (NOx^-) in hypertensive postmenopausal women.</p> <p>Methods</p> <p>Eleven volunteers were submitted to the ET consisting in 3 days a week, each session of 60 minutes during 6 months at moderate intensity (50% of heart rate reserve). Anthropometric parameters, blood pressure, NOx^-concentration were measured at initial time and after ET.</p> <p>Results</p> <p>A significant reduction in both systolic and diastolic blood pressure values was seen after ET which was accompanied by markedly increase of NOx^-levels (basal: 10 ± 0.9; ET: 16 ± 2 μM). Total cholesterol was significantly reduced (basal: 220 ± 38 and ET: 178 ± 22 mg/dl), whereas triglycerides levels were not modified after ET (basal: 141 ± 89 and ET: 147 ± 8 mg/dl).</p> <p>Conclusion</p> <p>Our study shows that changing in lifestyle promotes reduction of arterial pressure which was accompanied by increase in nitrite/nitrate concentration. Therefore, 6-months of exercise training are an important approach in management arterial hypertension and play a protective effect in postmenopausal women.</p

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles

Background: This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles.Results: The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11).Conclusions: The current study demonstrated an absence of relationship between ZP-BF high/positive or low/negative score and nuclear and cytoplasmic in vitro maturation of oocytes from stimulation cycles

Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

<p>Abstract</p> <p>Background</p> <p>The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean^®Fecal DNA Isolation Kit, M; QIAamp^®DNA Stool Mini Kit, Q; FastDNA^®SPIN Kit, FSp; FastDNA^®SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.</p> <p>Method</p> <p>Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.</p> <p>Results</p> <p>Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.</p> <p>Conclusion</p> <p>We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.</p

Lipocalin-2 Deficiency Attenuates Insulin Resistance Associated With Aging and Obesity

OBJECTIVE - The proinflammatory cytokines/adipokines produced from adipose tissue act in an autocrine and/or endocrine manner to perpetuate local inflammation and to induce peripheral insulin resistance. The present study investigates whether lipocalin-2 deficiency or replenishment with this adipokine has any impact on systemic insulin sensitivity and the underlying mechanisms. METHODS AND RESULTS - Under conditions of aging or dietary-/genetic-induced obesity, lipocalin-2 knockout (Lcn2-KO) mice show significantly decreased fasting glucose and insulin levels and improved insulin sensitivity compared with their wild-type littermates. Despite enlarged fat mass, inflammation and the accumulation of lipid peroxidation products are significantly attenuated in the adipose tissues of Lcn2-KO mice. Adipose fatty acid composition of these mice varies significantly from that in wild-type animals. The amounts of arachidonic acid (C20:4 n6) are elevated by aging and obesity and are paradoxically further increased in adipose tissue, but not skeletal muscle and liver of Lcn2-KO mice. On the other hand, the expression and activity of 12-lipoxygenase, an enzyme responsible for metabolizing arachidonic acid, and the production of tumor necrosis factor-α (TNF-α), a critical insulin resistance-inducing factor, are largely inhibited by lipocalin-2 deficiency. Lipocalin-2 stimulates the expression and activity of 12-lipoxygenase and TNF-α production in fat tissues. Cinnamyl-3,4- dihydroxy-α-cyanocinnamate (CDC), an arachidonate lipoxygenase inhibitor, prevents TNF-α expression induced by lipocalin-2. Moreover, treatment with TNF-α neutralization antibody or CDC significantly attenuated the differences of insulin sensitivity between wild-type and Lcn2-KO mice. CONCLUSIONS - Lipocalin-2 deficiency protects mice from developing aging- and obesity-induced insulin resistance largely by modulating 12-lipoxygenase and TNF-α levels in adipose tissue. © 2010 by the American Diabetes Association.link_to_OA_fulltex

Hypertension Is Associated with Marked Alterations in Sphingolipid Biology: A Potential Role for Ceramide

Background Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. Methods and Findings In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p Conclusions Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone