Overexpression of sema3a in myocardial infarction border zone decreases vulnerability of ventricular tachycardia post‐myocardial infarction in rats

The expression of the chemorepellent Sema3a is inversely related to sympathetic innervation. We investigated whether overexpression of Sema3a in the myocardial infarction (MI) border zone could attenuate sympathetic hyper‐innervation and decrease the vulnerability to malignant ventricular tachyarrhythmia (VT) in rats. Survived MI rats were randomized to phosphate buffered saline (PBS, n = 12); mock lentivirus (MLV, n = 13) and lentivirus‐mediated overexpression of Sema3a (SLV, n = 13) groups. Sham‐operated rats served as control group (CON, n = 20). Cardiac function and electrophysiological study (PES) were performed at 1 week later. Blood and tissue samples were collected for histological analysis, epinephrine (EPI), growth‐associated factor 43 (GAP43) and tyrosine hydroxylase (TH) measurements. QTc intervals were significantly shorter in SLV group than in PBS and MLV groups (168.6 ± 7.8 vs. 178.1 ± 9.5 and 180.9 ± 8.2 ms, all P < 0.01). Inducibility of VT by PES was significantly lower in the SLV group [30.8% (4/13)] than in PBS [66.7% (8/12)] and MLV [61.5% (8/13)] groups (P < 0.05). mRNA and protein expressions of Sema3a were significantly higher and the protein expression of GAP43 and TH was significantly lower at 7 days after transduction in SLV group compared with PBS, MLV and CON groups. Myocardial EPI in the border zone was also significantly lower in SLV group than in PBS and MLV group (8.73 ± 1.30 vs. 11.94 ± 1.71 and 12.24 ± 1.54 μg/g protein, P < 0.001). Overexpression of Sema3a in MI border zone could reduce the inducibility of ventricular arrhythmias by reducing sympathetic hyper‐reinnervation after infarction.     

Effects of Cannabinoids on Caffeine Contractures in Slow and Fast Skeletal Muscle Fibers of the Frog

The effect of cannabinoids on caffeine contractures was investigated in slow and fast skeletal muscle fibers using isometric tension recording. In slow muscle fibers, WIN 55,212-2 (10 and 5 μM) caused a decrease in tension. These doses reduced maximum tension to 67.43 ± 8.07% (P = 0.02, n = 5) and 79.4 ± 14.11% (P = 0.007, n = 5) compared to control, respectively. Tension-time integral was reduced to 58.37 ± 7.17% and 75.10 ± 3.60% (P = 0.002, n = 5), respectively. Using the CB₁ cannabinoid receptor agonist ACPA (1 μM) reduced the maximum tension of caffeine contractures by 68.70 ± 11.63% (P = 0.01, n = 5); tension-time integral was reduced by 66.82 ± 6.89% (P = 0.02, n = 5) compared to controls. When the CB₁ receptor antagonist AM281 was coapplied with ACPA, it reversed the effect of ACPA on caffeine-evoked tension. In slow and fast muscle fibers incubated with the pertussis toxin, ACPA had no effect on tension evoked by caffeine. In fast muscle fibers, ACPA (1 μM) also decreased tension; the maximum tension was reduced by 56.48 ± 3.4% (P = 0.001, n = 4), and tension-time integral was reduced by 57.81 ± 2.6% (P = 0.006, n = 4). This ACPA effect was not statistically significant with respect to the reduction in tension in slow muscle fibers. Moreover, we detected the presence of mRNA for the cannabinoid CB₁ receptor on fast and slow skeletal muscle fibers, which was significantly higher in fast compared to slow muscle fiber expression. In conclusion, our results suggest that in the slow and fast muscle fibers of the frog cannabinoids diminish caffeine-evoked tension through a receptor-mediated mechanism.     

The effect of body weight on some welfare indicators in feedlot cattle in a hot environment

Heat stress has important effects on the welfare of livestock. The effects of heat stress in cattle include changes in biological functions and behaviors. The aim of this study was to investigate the behavioral differences between light and heavy feedlot cattle reared in a hot environment. Sixteen male Holstein feedlot cattle were allocated to light (353.8 ± 15.5 kg, n = 8) and heavy (737.1 ± 15.8 kg, n = 8) groups according to their live weight and were kept in a semi-open feedlot barn. The individual behavioral response variables measured were standing, lying, feeding, drinking, ruminating, locomotor activity and elimination (urinating and defecating). The effects of group, day, observation time, replicate and all interactions were included in an explanatory statistical (GLM) model. The data were analyzed using the PROC GLM procedure of SAS. Overall, the heavy cattle spent more time standing (P < 0.001), lying (P < 0.001), and eliminating (P < 0.05) compared to the light group. In contrast, the light group spent more time eating, drinking and ruminating (P < 0.001). Locomotor activity did not differ significantly between groups (P > 0.05). During the day, heavy cattle spent more time standing (at 1600 hours) and less time eating in comparison with the light cattle (P < 0.001) (at 1300 and 1600 hours). Light and heavy feedlot cattle behaved differently in a hot environment. The findings of the study indicate that the welfare of the heavy Holstein feedlot cattle was impacted negatively when the ambient temperature was high (at 1300 hours).     

Reactivity of isolated toad aortic rings to angiotension II: the role of nitric oxide

Little is known about the vascular actions of angiotensin II (Ang II) and nitric oxide (NO) in Amphibia. This study investigated (1) Ang II contractility, (2) NO concentrations, and (3) correlations between Ang II contractility, NO concentration and mean arterial pressure (MAP) in isolated Bufo arenarum toad aortic rings. Contractility was measured in isometric conditions, NO concentrations were determined by the Griess reaction, and MAP was determined by a direct method. In isolated toad aortic rings, Ang II produced a contractile response (292.7 ± 89.2 mg; n = 20). Furthermore, a contractile response to norepinephrine (NE) was also obtained. A significant correlation between both the Ang II and NE contractile responses was found (r = 0.89; n = 11; P < 0.01). Administration of Ang II increased MAP values (Basal 16.8 ± 1.7; n = 19 vs. Ang II 28.4 ± 1.8 mmHg; n = 19; P < 0.001), and the increase of MAP by Ang II was positively correlated with the Ang II contractile response (P < 0.01). Administration of L-NAME also increased MAP values, and this effect was higher in those toads that presented a lower pressure response to Ang II (Pearson r = −0.68; P < 0.05). NO was present in all aortic rings, and its concentrations were negatively related to the Ang II contractile response (P < 0.036) and pressure response (Pearson r = −7.08; P < 0.001). These findings suggest that, in the B. arenarum toad, the NO system contra-regulates both the contractile and pressure Ang II responses, although its action could be different in each specimen.     

Modulation of Glutamate and Glycine Transporters by Niflumic,Flufenamic and Mefenamic Acids

Three fenamates—niflumic, flufenamic and mefenamic acids—were tested for effects on substrate-induced currents of glutamate and glycine transporters (EAAT1, EAAT2, GLYT1b and GLYT2a) expressed in Xenopus laevis oocytes. All fenamates inhibited EAAT1 currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 53 ± 4% (P < 0.001). EAAT2 currents were less sensitive, but 100 μM flufenamic acid inhibited the I _max by 34 ± 5% (P = 0.006). All fenamates inhibited GLYT1b currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 61 ± 1% (P < 0.001). At 100 μM, effects on the GLYT2a I _max were mixed: 13 ± 2% inhibition by flufenamic acid (P = 0.002), 30 ± 6% enhancement by niflumic acid (P = 0.002), and no effect by mefenamic acid. Minor effects on substrate affinity suggested non-competitive mechanisms. These data could contribute to the development of selective transport modulators.     

Daily quadratic trend in basal monocyte expressed HSP72 in healthy human subjects

The inducible human stress protein heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. Recently it was shown over a 24 h period that basal HSP72 followed a diurnal variation. However, these results and previous literature demonstrate noticeable inter-subject variation in basal HSP72 expression. The notion of intra/inter-day variation in basal HSP72 expression has not been explored in detail. Basal monocyte expressed HSP72 was determined every 3 h, over a 9 h period in 12 healthy male subjects (20.2 ± 1.9 years, 178.7 ± 5.6 cm, 75.1 ± 6.0 kg) within a temperature controlled laboratory. A significant quadratic trend was observed for time (F = 26.0, P = 0.001, partial η² = 0.74), where HSP72 decreased between 0800 and 1100 hours (P < 0.001) and then increased between 1100 and 1400 hours (P = 0.015). The main effect for day (F = 2.6, P = 0.14) and the day × time interaction effect (F = 3.9, P = 0.08) were not significant. There was no correlation between serum and monocyte expressed HSP72, with no significant effect for time (F = 2.0, P = 0.21) in serum HSP72 expression. The results support findings by others that basal monocyte expressed HSP72 follows a diurnal variation which incorporates a quadratic trend, which is not compromised by any significant daily variation and that serum HSP72 expression has no endogenous circadian rhythm. The significant quadratic trend in basal monocyte HSP72 expression shown here highlights the need to tightly control variables, such as timing of sample collection, as it is known basal values influence the magnitude of HSP72 expression post-stressor/intervention.     

Effects of Chromium and Carnitine Co-supplementation on Body Weight and Metabolic Profiles in Overweight and Obese Women with Polycystic Ovary Syndrome: a Randomized,Double-Blind,Placebo-Controlled Trial

The primary aim of our study was to determine the influence of taking chromium plus carnitine on insulin resistance, with a secondary objective of evaluating the influences on lipid profiles and weight loss in overweight subjects with polycystic ovary syndrome (PCOS). In a 12-week randomized, double-blind, placebo-controlled clinical trial, 54 overweight women were randomly assigned to receive either supplements (200 μg/day chromium picolinate plus 1000 mg/day carnitine) or placebo (27/each group). Chromium and carnitine co-supplementation decreased weight (− 3.6 ± 1.8 vs. − 1.0 ± 0.7 kg, P < 0.001), BMI (− 1.3 ± 0.7 vs. − 0.3 ± 0.3 kg/m², P < 0.001), fasting plasma glucose (FPG) (− 5.1 ± 6.0 vs. − 1.1 ± 4.9 mg/dL, P = 0.01), insulin (− 2.0 ± 1.4 vs. − 0.2 ± 1.2 μIU/mL, P < 0.001), insulin resistance (− 0.5 ± 0.4 vs. − 0.04 ± 0.3, P < 0.001), triglycerides (− 18.0 ± 25.2 vs. + 5.5 ± 14.4 mg/dL, P < 0.001), total (− 17.0 ± 20.3 vs. + 3.6 ± 12.0 mg/dL, P < 0.001), and LDL cholesterol (− 13.3 ± 19.2 vs. + 1.4 ± 13.3 mg/dL, P = 0.002), and elevated insulin sensitivity (+ 0.007 ± 0.005 vs. + 0.002 ± 0.005, P < 0.001). In addition, co-supplementation upregulated peroxisome proliferator-activated receptor gamma (P = 0.02) and low-density lipoprotein receptor expression (P = 0.02). Overall, chromium and carnitine co-supplementation for 12 weeks to overweight women with PCOS had beneficial effects on body weight, glycemic control, lipid profiles except HDL cholesterol levels, and gene expression of PPAR-γ and LDLR. Clinical trial registration number: http://www.irct.ir: IRCT20170513033941N38.

     

Structure of vegetation patches in northwestern Patagonia, Argentina

Vegetation of arid and semiarid environments has in general a patchy distribution. Our objective was to (a) determine several qualitative and quantitative analytical characteristics of vegetation patches in an arid zone of Patagonia, Argentina, and (b) investigate relationships between them. Annual precipitation in this area was 200 mm during 1999–2005. Eight transects involving ten patches each were studied within a 15 × 15 km area. Mean (±1 SE) values (n = 80) in the vegetation patches were 315 ± 25 and 207 ± 16 cm for the greatest and lowest patch diameter, respectively; 23 ± 2 cm for mound height; 113 ± 12 cm for maximum vegetation height; and 170 ± 18 cm and 58 ± 2% for distance to the next vegetation patch and vegetation patch cover within a transect, respectively. Correlations between greatest and lowest diameters, mound height, maximum plant height and distance to the closest vegetation patch were all significant (P < 0.01; n = 80). In all vegetation patches, the greatest and lowest frequencies were found for the grass Stipa spp. (71.2%) and the shrub Grindelia chiloensis (Cornel.) Cabrera (12.5%). Stipa spp. and the shrub Atriplex lampa (Moq.) D. Dietr. showed the highest simultaneous frequency (50%). A reasonable association among species (>45%) was found for Stipa spp., Atriplex lampa and the shrubs Larrea divaricata Cav., Lycium chilense Miers ex Bertero and Junellia ligustrina (Lag.) Moldenke. Larrea divaricata and Atriplex lampa contributed more than 84% of the total patch standing crop (5,777 ± 435 g). Average patch size and specific diversity were 5.93 ± 0.33 m² and 1.31 ± 0.11, respectively. Aboveground standing crop of the two dominant shrubs decreased as plant species diversity increased (P < 0.05). Conservation of vegetation patches is crucial to prevent increased soil erosion and desertification in the study ecological system.     

Activation of tumor-specific T lymphocytes after laser-induced thermotherapy in patients with colorectal liver metastases

Purpose  To asses if laser-induced thermotherapy (LITT) induces a specific cytotoxic T cell response in patients treated with LITT for colorectal cancer liver metastases. Methods  Eleven patients with liver metastases of colorectal cancer underwent LITT. Blood was sampled before and after LITT. Peripheral T cell activation was assessed by an interferon gamma (IFNg) secretion assay and flow cytometry. Test antigens were autologous liver and tumor lysate obtained from each patient by biopsy. T cells were stained for CD3/CD4/CD8 and IFNg to detect activated T cells. The ratio of IFNg positive to IFNg negative T cells was determined as the stimulation index (SI). To assess cytolytic activity, T cells were co-incubated with human colorectal cancer cells (CaCo) and cytosolic adenylate kinase release was measured by a luciferase assay. Results  IFNg secretion assay: before LITT SI was 12.73 (±4.83) for CD3+, 4.36 (±3.32) for CD4+ and 3.64 (±1.77) for CD8+ T cells against autologous tumor tissue. Four weeks after LITT SI had increased to 92.09 (±12.04) for CD3+ (P < 0.001), 42.92 (±16.68) for CD4+ (P < 0.001) and 47.54 (±15.68) for CD8+ T cells (P < 0.001) against autologous tumor tissue. No increased SI was observed with normal liver tissue at any time point. Cytotoxicity assay: before LITT activity against the respective cancer cells was low, with RLU = 1,493 (±1,954.68), whereas after LITT cytolytic activity had increased to RLU = 7,260 [±3,929.76 (P < 0.001)]. Conclusion  Patients with liver metastases of colorectal cancer show a tumor-specific cytotoxic T cell stimulation and a significantly increased cytolytic activity of CD3+, CD4+ and CD8+ T cells after LITT against an allogenic tumor (CaCo cell line).     

Design and validation of an orally administrated active L. fermentum-L. acidophilus probiotic formulation using colorectal cancer Apc Min/+ mouse model

Probiotics have been shown to have beneficial properties in attenuating the risk of colorectal cancer (CRC) development. However, functional evidence to support such effects for some probiotic bacteria are relatively unknown. Here, we document a significant antioxidant, anti-proliferative and pro-apoptotic activities of Lactobacillus acidophilus ATCC 314 and Lactobacillus fermentum NCIMB 5221 on CRC cells, particularly when used in combination (La-Lf). Furthermore, a superior synergistic activity on the inhibition of tumor growth and modulation of cell proliferation and epithelial markers in the Apc ^Min/+ CRC mouse model was explored, based on the expression levels of Ki-67, E-cadherin, β-catenin, and cleaved caspase-3 (CC3) proteins. The anti-cancer activity of La-Lf co-culture was significantly enhanced in vitro with significant reduced proliferation (38.8 ± 6.9 %, P = 0.009) and increased apoptosis (413 RUL, P < 0.001) towards cancer cells, as well as significant protection of normal colon cell growth from toxic treatment (18.6 ± 9.8 %, P = 0.001). La-Lf formulation (10¹⁰cfu/animal/day) altered aspects of intestinal tumorigenesis by significantly reducing intestinal tumor multiplicity (1.7-fold, P = 0.016) and downregulating cellular proliferation markers, including β-catenin (P = 0.041) and Ki-67 (P = 0.008). In conclusion, La-Lf showed greater protection against intestinal tumorigenesis supporting a potential use as a biotherapeutic for the prevention of CRC.

     

Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts

Development of a transgenic porcine biomedical research model requires effective delivery of DNA into the donor cell followed by selection of genetically modified somatic cell lines to be used for nuclear transfer. The objective of the current study was 2-fold: (1) to compare the effectiveness of a single 1 ms pulse of different voltages (V; 100, 150, 200, 250, 300, 350) and multiple 1 ms pulses (1, 2, 3, 4 or 5) at 300 V for delivery and expression of super-coiled GFP vector in surviving cells of three fetal fibroblast cell lines, and (2) to determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies following G418 selection. Cell line (P < 0.001) and voltage (P < 0.001) affected DNA delivery into the cell as assessed by GFP expression while survival at 24 h was affected by voltage (P < 0.001) and not by cell line (P = 0.797). Using a single pulse while increasing voltage resulted in the percentage of GFP expressing cells increasing from 3.2 ± 0.8% to 43.0 ± 3.4% while survival decreased from 90.5 ± 8.0% to 44.8 ± 2.0%. The number of pulses at 300 V significantly affected survival (P < 0.001) and GFP expression (P < 0.001). Survival steadily decreased following 1–5 pulses from 63.2 ± 6.3% to 3.0 ± 0.3% with GFP expression of surviving cells increasing from 35.6 ± 2.67% to 71.4 ± 6.1%. Electroporation of a selectable marker at a 1:1 copy number ratio to a co-electroporated transgene resulted in 83% of G418 resistant colonies also being PCR positive for the secondary transgene. These electroporation conditions, specifically, three 1 ms pulses of 300 V to 200 μL of 1 × 10⁶ cells/mL in the presence of 12.5 μg DNA/mL effectively introduced DNA into somatic cells. The utilization of these conditions produced numerous transgenic fibroblast colonies following G418 selection that when used for somatic cell nuclear transfer resulted in the production of live offspring.     

The role of vascular endothelial growth factor +405 G/C polymorphism and albuminuria in patients with type 2 diabetes mellitus

Observations on the association between the vascular endothelial growth factor (VEGF) gene polymorphism and nephropathy have been inconsistent, which might be due to ethnic and geographical variations. Furthermore, the relationship between +405 G/C polymorphism and albuminuria in the diabetic population has not been sufficiently studied. The aim of this study was to evaluate for the first time the possible association between +405 G/C polymorphism and albuminuria in an population from Tehran of Iran. A total of 255 consecutive patients with type 2 diabetes and microalbuminuria (Group A) and 235 patients with type 2 diabetes and normoalbuminuria (Group B) were included. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to detect the VEGF alleles. In univariate analysis, the groups were statistically similar in all variables except for HbA1c (8.53 ± 1.7 in Group A vs. 8.2 ± 1.73 in Group B; P = 0.034), 24-h urinary albumin (201.33 ± 84.8 in Group A vs. 22.88 ± 3.5 in Group B; P < 0.001), and the frequency of GG genotype (31% in Group A vs. 18.7% in Group B; P = 0.006). The GG genotype was the independent predictor of albuminuria [P = 0.014, OR = 1.771, 95% confidence interval (CI) = 1.124–2.790]. Our study showed that the G allele was not associated with albuminuria, but the GG genotype in the VEGF gene is independently associated with development of nephropathy in the our diabetic population.     

Age and growth of early-life-stage Atlantic tarpon (Megalops atlanticus) from the northcentral Gulf of Mexico

Age and growth of early-life-stage Atlantic tarpon Megalops atlanticus collected from Mississippi coastal waters in the northcentral Gulf of Mexico (GOM) are described using otolith microstructure analysis. Tarpon leptocephali (n = 95, 16.0—27.8 mm standard length, L_S) collected from June throughOctober 2013—2018, ranged in age from 22 to 43 days (mean = 30.9 ± 0.5 days). Leptocephalus somatic growth rates ranged 0.46—1.24 mm day⁻¹ (mean = 0.76 ± 0.02 mm day⁻¹), and leptocephalus otolith growth rates ranged 1.78—3.97 μm day⁻¹ (mean = 2.58 ± 0.04 μm day⁻¹). Growth rates were inversely correlated to leptocephalus age, indicating the shrinkage phase associated with leptocephalus metamorphosis. Juvenile tarpon (n = 358, 50—359 mm fork length, L_F) were collected from August through December 2007—2018. Juveniles exhibited a positive allometric relationship (adjusted R² = 0.99, P < 0.001) between length and mass. The age of 100 juveniles (71—277 mm L_F) ranged from 76 to 174 days. Juvenile growth rate was estimated as 1.56 ± 0.11 mm day⁻¹. Significant (P < 0.001) linear relationships were found between juvenile age and otolith metrics, including otolith mass (R² = 0.81) and radius (R² = 0.68). Evaluation of the backcalculated hatch dates suggests that specimens in the collection hatched from late May through mid-September with slight peaks during July and August. A Rao's Spacing Test of Uniformity indicates the presence of significant lunar periodicity in leptocephalus hatch dates (n = 95, U = 250.1, P < 0.05), with 50% of the leptocephali hatched within 5 days (before or after) of the full moon. This study fills critical gaps in the scientific knowledge of tarpon and provides estimates of early-life-history metrics for an iconic game fish at the northernmost extent of its GOM range.     

Relationships between butterflyfish (Chaetodontidae) feeding rates and coral consumption on the Great Barrier Reef

This study explored differences in the feeding rate among 20 species of coral reef butterflyfishes (Chaetodontidae) from Lizard Island, Great Barrier Reef. Feeding rate, measured as bites per minute (b.p.m.), varied between 2.98 ± 0.65 and 12.29 ± 0.27 (mean ± SE) according to species and was positively related to the proportional consumption of coral (r ² = 0.40, n = 20, P < 0.01), independent of phylogeny (standardised independent contrasts r ² = 0.29, n = 19, P < 0.05). All species fed actively throughout the day, with obligate corallivores having a higher feeding rate at all times than either facultative corallivores or non-corallivores. The feeding rate of the obligate corallivores was also highest during the middle of the day. For eight of the species for which data was available, there was a positive correlation between bite rate and competitive dominance (r = 0.71, P < 0.05). Chaetodon ephippium was the only species for which the feeding rate of pairs was higher than for solitary individuals.     

Metabolic changes during carbon monoxide poisoning: An experimental study

Carbon monoxide (CO) is the leading cause of death by poisoning worldwide. The aim was to explore the effects of mild and severe poisoning on blood gas parameters and metabolites. Eleven pigs were exposed to CO intoxication and had blood collected before and during poisoning. Mild CO poisoning (carboxyhaemoglobin, COHb 35.2 ± 7.9%) was achieved at 32 ± 13 minutes, and severe poisoning (69.3 ± 10.2% COHb) at 64 ± 23 minutes from baseline (2.9 ± 0.5% COHb). Blood gas parameters and metabolites were measured on a blood gas analyser and nuclear magnetic resonance spectrometer, respectively. Unsupervised principal component, analysis of variance and Pearson's correlation tests were applied. A P-value ≤ .05 was considered statistically significant. Mild poisoning resulted in a 28.4% drop in oxyhaemoglobin (OHb) and 12-fold increase in COHb, while severe poisoning in a 65% drop in OHb and 24-fold increase in COHb. Among others, metabolites implicated in regulation of metabolic acidosis (lactate, P < .0001), energy balance (pyruvate, P < .0001; 3-hydroxybutyrc acid, P = .01), respiration (citrate, P = .007; succinate, P = .0003; fumarate, P < .0001), lipid metabolism (glycerol, P = .002; choline, P = .0002) and antioxidant-oxidant balance (glutathione, P = .03; hypoxanthine, P < .0001) were altered, especially during severe poisoning. Our study adds new insights into the deranged metabolism of CO poisoning and leads the way for further investigation.     

Hereditary haemochromatosis gene (HFE) H63D mutation shows an association with abnormal sperm motility

The aim of this study was to screen infertile men for HFE H63D mutation in correlation with clinical characteristics of infertile men (sperm concentration, sperm motility, morphology, testicular volume, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and total Testosterone levels) and find out if the HFE H63D mutation has an effect on male infertility. After excluding hormonal treatment, any scrotal pathology, having any systemic diseases such as diabetes mellitus, sickle cell anemia and microdeletions of the Y chromosome, a total of 148 infertile men with age range between 17 and 52-years-old (average age 29.6 ± 7.2) were enrolled into the study. Our analysis indicates that the mean FSH levels are significantly higher (6.3 ± 4.6 mIU/ml, P = 0.03), whereas sperm motility is significantly lower (36.6 ± 28.1%, P = 0.01) in the infertile men with the HFE H63D mutation compared with subjects lacking this mutation. Comparison of allele frequencies of the infertile men with Ts < 50% versus the infertile men with Ts > 50% revealed a significant difference as expected (P = 0.001, OR = 0.14, %95 CI = 0.04–0.44). Comparison of allele frequencies of infertile men with abnormal sperm motility versus infertile men with normal sperm motility revealed a highly significant difference (P = 0.005, OR = 3.11, %95 CI = 1.41–6.86). Thus, the HFE H63D mutation seems to be an important risk factor for impaired sperm motility and is clinically associated with male infertility.     

Mechanism of Post-Translational Modification by Tyrosine Phosphorylation of Apoptotic Proteins During Hypoxia in the Cerebral Cortex of Newborn Piglets

The present study aims to investigate the mechanism of phosphorylation of apoptotic proteins and tests the hypothesis that the hypoxia-induced increased tyrosine phosphorylation of apoptotic proteins Bcl-2 and Bcl-xl is Ca²⁺-influx-dependent. Piglets were divided in normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-pretreated with clonidine (Clo + Hx, n = 4) groups. Hypoxic animals were exposed to an FiO₂ of 0.06 for 1 h. Clonidine (12.5 μg/kg, IV) was administered to piglets 30 min prior to hypoxia. Hypoxia was confirmed by ATP and phosphocreatinine (PCr) levels. Cytosol was isolated and separated by 12% SDS–PAGE and probed with tyrosine phosphorylated (p) -Bax, Bad, Bcl-2 and Bcl-xl antibodies and bands were detected. The ATP levels (μmol/g brain) in the Nx, Hx, Clo + Hx were 4.3 ± 1.0 (P < 0.05 vs. Hx, Clo-Hx), 0.9 ± 0.8 and 1.5 ± 0.3, respectively. The PCr levels in the Nx, Hx, Clo + Hx were 2.7 ± 0.7 (P < 0.05 vs. Hx, Clo-Hx), 0.9 ± 0.2 and 0.9 ± 0.9, respectively. Ca²⁺-influx (pmoles/mg protein) was 4.96 ± 0.94 in Nx, 11.11 ± 2.38 in Hx, and 6.23 ± 2.07 in Clo + Hx (P < 0.05 Nx vs. Hx and Hx vs. Clo + Hx). p-Bcl-2 density was 21.1 ± 1.1 Nx, 58.9 ± 9.6 Hx and 29.5 ± 6.4 Clo + Hx (P < 0.05 vs. Hx). p-Bcl-xl density was 29.6 ± 1.5 Nx, 50.6 ± 7.4 Hx and 32.1 ± 0.1 Clo + Hx (P < 0.05 vs. Hx). p-Bax density was 38.6 ± 16.2 Nx, 46.1 ± 5.5 Hx and 41.6 ± 1.9 Clo + Hx groups (P = NS). p-Bad was 66.7 ± 12.8 Nx, 71.2 ± 6.8 Hx and 78.7 ± 22.5 Clo + Hx groups (P = NS). Results showed that clonidine administration prior to hypoxia prevents the hypoxia-induced increased nuclear Ca²⁺-influx and increased phosphorylation of Bcl-2 and Bcl-xl while phosphorylation of Bad and Bax was not altered. We conclude that post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl during hypoxia is nuclear Ca²⁺-influx-dependent. We propose that blockade of nuclear Ca²⁺-influx that prevents phosphorylation of antiapoptotic proteins may become a neuroprotective strategy.     

Hypoxia-induced Activation of Epidermal Growth Factor Receptor (EGFR) Kinase in the Cerebral Cortex of Newborn Piglets: The Role of Nitric Oxide

The present study aims to investigate the mechanism of EGFR kinase activation during hypoxia and tests the hypothesis that hypoxia-induced increased activation of EGFR kinase in the cerebral cortical membrane fraction of newborn piglets is mediated by nitric oxide (NO) derived from neuronal nitric oxide synthase (nNOS). Fifteen newborn piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-treated with nNOS inhibitor (Hx-nNOSi, n = 5). Hypoxia was induced by an FiO2 of 0.07 for 60 min. nNOS inhibitor I (selectivity >2,500 vs. endothelial NOS, eNOS, and >500 vs. inducible NOS, iNOS) was administered (0.4 mg/kg, i. v.) 30 min prior to hypoxia. EGFR kinase tyrosine phosphorylation at Tyr1173, an index of activation of EGFR kinase, was determined by Western blot analysis using an anti-phospho (pTyr¹¹⁷³)-EGFR kinase antibody. Protein bands were analyzed by imaging densitometry and expressed as absorbance (OD × mm²). EGFR kinase activity was determined radiochemically using immunopurified enzyme. EGFR kinase activity was expressed as pmols/mg protein/hr. Density of phosphor (pTyr¹¹⁷³)-EGFR kinase (OD × mm²) was 60.2 ± 9.8 in Nx, 177.0 ± 26.9 in Hx (P < 0.05 vs. Nx) and 79.9 ± 15.7 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Activity of EGFR kinase (pmoles/mg protein/hr) was 4,603 ± 155 in Nx, 8,493 ± 427 in Hx (P < 0.05 vs. Nx) and 4,516 ± 104 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Pretreatment with nNOS inhibitor prevented the hypoxia-induced increased phosphorylation and increased activity of EGFR kinase. We conclude that the mechanism of hypoxia-induced increased activation of EGFR kinase is mediated by nNOS-derived NO.     

Influence of Therapy with Metformin on the Concentration of Certain Divalent Cations in Patients with Non-insulin-Dependent Diabetes Mellitus

Research was performed on a group of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM), who never received antidiabetic medication before, and on a group of 17 healthy adults. The patients were administered treatment with metformin, 1,000 mg/day. Plasmatic and urinary concentration of magnesium have been measured, copper and zinc along with the concentrations of glucose, HDL, LDL, cholesterol, tryglicerides, HbA1c, and total erythrocyte magnesium, in advance and after 3 months of treatment. Data showed significant differences in the NIDDM group vs the control group: for plasma magnesium—1.95 ± 0.19 vs 2.20 ± 0.18 mg/dl, p < 0.001; urine magnesium—237.28 ± 34.51 vs 126.25 ± 38.22 mg/24 h, p < 0.001; erythrocyte magnesium—5.09 ± 0.63 vs 6.38 ± 0.75 mg/dl, p < 0.001; plasma zinc—67.56 ± 6.21 vs 98.41 ± 20.47 μg/dl, p < 0.001; urine zinc—1,347.54 ± 158.24 vs 851.65 ± 209.75 μg/24 h, p < 0.001; plasma copper—111.91 ± 20.98 vs 96.33 ± 8.56 μg/dl, p < 0.001; and urine copper—51.70 ± 23.79 vs 36.00 ± 11.70 μg/24 h, p < 0.05. Treatment with metformin for 3 months modified significant erythrocyte magnesium—5.75 ± 0.61 vs 5.09 ± 0.63 mg/dl, p < 0.001 and urine magnesium—198.27 ± 27.07 vs 237.28 ± 34.51 mg/24 h, p < 0.001, whereas it did not modify significant the plasmatic and urinary concentration of the other cations. The erythrocyte magnesium concentration was inversely correlated with HbA1c (r = −0.438, p = 0.015). The plasma level of copper was positively correlated with HbA1c (r = 0.517, p < 0.003), tryglicerides (r = 0.534, p < 0.003), and cholesterol (r = 0.440, p < 0.05), and the plasma level of zinc was inversely correlated with glycemia (r = −0.399, p = 0.029). Our data show a significant action of metformin therapy, by increasing the total intraerythrocyte magnesium concentration and decreasing the urinary magnesium elimination, positively correlated with the decrease of glycemia and HbA1c in NIDDM patients.     

Mechanism of Increased Tyrosine (Tyr<Superscript>99</Superscript>) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr⁹⁹ phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F_iO₂ of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr⁹⁹ and total) of calmodulin determined by Western blot using anti-phospho-(pTyr⁹⁹)-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr⁹⁹ calmodulin (ODxmm²) was 78.55 ± 10.76 in Nx, 165.05 ± 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 ± 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 ± 13.69 in Nx, 156.17 ± 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 ± 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr⁹⁹ phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr⁹⁹ phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr⁹⁹ phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr⁹⁹ phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.