Expression of type I GNRH receptor and in vivo and in vitro GNRH-I effects in corpora lutea of pseudopregnant rabbits

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24 h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F(2α) (PGF(2α)) secretion and cyclo-oxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE? at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP?, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF(2α) and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-l-arginine methyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directly CL progesterone production via PGF(2α) at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP?, DAG, PKC, COX-2, and NOS.     

Nephrotoxicity of uranium in drinking water from private drilled wells

Objectives

To investigate the association between uranium in drinking water from drilled wells and aspects of kidney function measured by sensitive urine tests.

Methods

Three hundred and one of 398 eligible subjects (75.6%) aged 18-74 years with daily drinking water supplies from private drilled wells located in uranium-rich bedrock (exposed group) volunteered to participate along with 153 of 271 local controls (56.4%) who used municipal water. Participants responded to a questionnaire on their water consumption and general health, and provided a morning urine sample and drinking water for analysis.

Results

The uranium content of well water samples (n=153) varied considerably (range <0.20-470 μg/l, median 6.7 μg/l, 5% >100 μg/l), while uranium levels in all samples of municipal water (n=14) were below the limit of quantification (0.2 μg/l). Urinary levels of uranium were more than eight times higher in exposed subjects than in controls (geometric means 38 and 4.3 ng/l, respectively; p<0.001), but their mean urine lead levels were not significantly different. There was a strong curvilinear correlation between uranium in drinking water and in urine (r²=0.66). Levels of albumin, β₂-microglobulin, protein HC as well as kappa and lambda immunoglobulin chains in urine from exposed and controls were similar. The N-acetyl-β-d-glucosaminidase (NAG) activity was significantly lower in the exposed group vs. controls, possibly secondary to differential storage duration of samples from the two groups. Even in regression models adjusting for gender, age and smoking no association of uranium in water and the kidney function parameters was observed. Using uranium in urine in the entire study group as a marker of exposure, however, a tendency of exposure-related increases of β₂-microglobulin, protein HC and kappa chains were noted. This tendency was enhanced after exclusion of subjects with diabetes mellitus from the analysis.

Conclusions

Uranium levels in urine were strongly correlated to levels in drinking water from drilled wells. There were no clear signs of nephrotoxicity from uranium in drinking water at levels recorded in this study, but some indications of an effect were observed using uranium in urine as a measure of overall uranium exposure. The clinical relevance of these findings remains unclear.     

The Death Receptor 3-TNF-like protein 1A pathway drives adverse bone pathology in inflammatory arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints that is associated with cartilage and bone destruction. Death Receptor 3 (DR3), a tumor necrosis factor (TNF) receptor superfamily member, has recently been associated with the pathogenesis of RA. We demonstrate that absence of DR3 confers resistance to the development of adverse bone pathology in experimental antigen-induced arthritis (AIA). DR3^ko mice exhibited a reduction in all histopathological hallmarks of AIA but, in particular, failed to develop subchondral bone erosions and were completely protected from this characteristic of AIA. In contrast, TNF-like protein 1A (TL1A), the ligand for DR3, exacerbated disease in a dose- and DR3-dependent fashion. Analysis of osteoclast number within AIA joint revealed a reduction in areas susceptible to bone erosion in DR3^ko mice, whereas in vitro osteoclastogenesis assays showed that TL1A could directly promote osteoclastogenesis in mouse and man. Treatment with antagonistic anti-TL1A mAb protected animals in a systemic model of RA disease collagen-induced arthritis. We therefore conclude that the DR3–TL1A pathway regulates joint destruction in two murine models of arthritis and represents a potential novel target for therapeutic intervention in inflammatory joint disease.     

Human CMV infection induces 5-lipoxygenase expression and leukotriene B4 production in vascular smooth muscle cells

Leukotrienes (LTs) are powerful proinflammatory lipid mediators that may play a central role in cardiovascular diseases, including arteriosclerosis, myocardial infarction, and stroke. Owing to restricted expression of 5-lipoxygenase (5-LO), the enzyme required for their synthesis, LTs are almost exclusively produced by myeloid cells. Here, we report that human cytomegalovirus (HCMV) infection of human vascular smooth muscle cells (SMCs) increases 5-LO mRNA levels by up to 170-fold in a dose- and time-dependent manner. Infected cells expressed 5-LO protein, as shown by immunohistochemistry, enabling them to synthesize bioactive LTB(4). HCMV-infected vascular SMCs expressing 5-LO protein were readily detected in tissue samples from CMV-infected patients with inflammatory bowel disease or AIDS. Thus, pathogen-induced LT production in HCMV-infected tissues may contribute to local inflammation, consistent with the ability of HCMV to control cellular and immunological functions. HCMV-induced LT biosynthesis in SMCs offers a molecular mechanism to explain HCMV-induced pathogenesis in inflammatory diseases.     

Arrhythmic risk evaluation during exercise at high altitude in healthy subjects: role of microvolt T-wave alternans

Background: Altitude‐induced sympathetic hyperactivity can elicit rhythm disturbances in healthy subjects, in particular during exercise. Aim: To asses the real susceptibility of healthy myocardium to malignant ventricular arrhythmias during exercise at high altitude using microvolt T‐wave alternans (MTWA). Methods: We evaluated eight healthy trained participants (one female, 42 ± 9 years) during a mountain climbing expedition on Gashembrum II (Pakistan, 8,150 m). MTWA and heart rate variability (HRV) were measured in each subject at sea level and at high altitude, both under rest conditions and during exercise. MTWA was determined with the modified moving average method. HRV was expressed as root mean square of successive differences. Results: Rest HRV at high altitude was significantly lower compared to rest HRV at sea level (36 ± 5 vs 56 ± 9 ms, P = 0.003). HRV during exercise was significantly lower with respect to rest condition both in normoxia (46 ± 7 vs 56 ± 9 ms, P = 0.0001) and hypoxia (27 ± 4 vs 36 ± 5 ms, P = 0.005). Moreover, HRV was significantly lower during exercise at high altitude compared to exercise at sea level (27 ± 4 vs 46 ± 7 ms, P = 0.0002) and arrhythmias were more frequent during exercise in hypoxia. Nevertheless, MTWA was absent under rest conditions both at sea level and at high altitude and minimally evoked during exercise in both conditions (22 ± 3 μV and 23 ± 3 μV, respectively, P = 0.2). Conclusions: In spite of an enhanced sympathetic activity, MTWA testing during exercise at high altitude was negative in all participants. Healthy trained subjects during exercise under hypoxia seem to be at low risk for dangerous arrhythmias.     

Influence of extrusion on the bioactive compounds and the antioxidant capacity of the bean/corn mixtures

The aim of this investigation was to examine the influence of extrusion on the bioactive compounds and the antioxidant capacity of bean/corn mixtures. Whole bean flour and nixtamalized corn were mixed in a 60:40 proportion and extrusion was performed in different moisture (14.5%, 15.4%, 17.1% and 18.0%) and temperature (150°C, 160°C, 170°C, 180°C and 190°C) conditions in order to find the optimal extrusion conditions. According to their functional properties and antioxidant status, the mixtures 142°C/16.3% H, 170°C/16.3% H and 198°C/16.3% H were defined as optimal, moderate and bad, respectively. Total polyphenols and flavonoids in the mixture of 142°C/16.3% H (15.09±1.7 mg gallic acid equivalent [GAE]/g dry weight [DW] and 1.57±0.2 mg catechin equivalent [CE]/g DW) were significantly higher (P<0.05) than in the sample 170°C/16.3% H (9.42±1.1 mg GAE/g DW and 1.4±0.1 mg CE/g DW) and the mixture 198°C/16.3% H (6.46±0.8 mg GAE/g DW and 0.78±0.1 mg CE/g DW). The antioxidant activity (37.02±3.8 and 25.01±2.5 μM Trolox equivalent [TE]/g DW) of mixture 142°C/16.3% H, determined by the cupric reducing antioxidant capacity with Trolox equivalent antioxidant capacity and β-carotene-linoleic acid (β-carotene,% of inhibition) assays, was significantly higher (P<0.05) than in 170°C/16.3% H (25.69±2.8 and 17.02±1.8 μM TE/g DW) and in mixture 198°C/16.3% H (13.93±1.5 and 8.94±0.9 μM TE/g DW), respectively. The free polyphenols, flavonoids and the antioxidant activities showed lower results than the hydrolyzed ones. The correlation coefficients between polyphenols, flavonoids, and cupric reducing antioxidant capacity capacities were between 0.93 and 0.99. In cereal proteins extracted and separated by electrophoresis, some differences were found in the sodium dodecyl sulfate-protein bands in the region from 36 to 45 kDa for 142°C/16.3% H, in comparison with other samples. Therefore, there is a need to find such conditions for the extrusion procedures that would take into consideration the contents of the bioactive compounds and the antioxidant capacity in the end product.     

Prolonged glucocorticoid treatment decreases cannabinoid CB1 receptor density in the hippocampus

Experimental studies indicate a bidirectional, functional relationship between glucocorticoids and the endocannabinoid system; however, the effects of repeated glucocorticoid treatment on the endocannabinoid system have not been examined. In this study, we treated male rats with either a single dose or a 21-day course of treatment with corticosterone (20 mg/kg) and measured hippocampal cannabinoid CB(1) receptor expression and endocannabinoid content. The 21-day, but not the single, administration of corticosterone significantly reduced both the binding site density and amount of protein of the hippocampal cannabinoid CB(1) receptor without affecting affinity for the CB(1) receptor agonist, [(3)H]CP55940. With regard to hippocampal endocannabinoid content, acute corticosterone treatment resulted in a significant reduction in anandamide but did not affect 2-arachidonylglycerol, while repeated corticosterone treatment did not alter content of either anandamide or 2-arachidonylglycerol. These data support the hypothesis that the cannabinoid CB(1) receptor is under negative regulation by glucocorticoids in the hippocampus, and suggest that hippocampal cannabinoid CB(1) receptor signaling could be reduced under conditions associated with hypersecretion of glucocorticoids, such as chronic stress.