Selective immunoneutralization of luteinizing hormone results in the apoptotic cell death of pachytene spermatocytes and spermatids in the rat testis

The selective withdrawal of pituitary gonadotropins through specific antibodies is known to cause disruption of spermatogenesis. The cellular mechanism responsible for the degenerative changes under isolated effect of luteinizing hormone (LH) deprivation is not clear. Using antibodies specific to LH we have investigated the effect of immunoneutralization of LH on apoptotic cell death in the testicular cells of the immature and the adult rats. Specific neutralization of LH resulted in apoptotic cell death of germ cells, both in the immature and the adult rats. The germ cells from control animals showed predominantly high molecular weight DNA, while the antiserum treated group showed DNA cleavage into low molecular weight DNA ladder characteristic of apoptosis. This pattern could be observed within 24 h of a/s administration and the effect could be reversed by testosterone. The germ cells were purified by centrifugal elutriation and the vulnerability of germ cell types to undergo apoptosis under LH deprivation was investigated. The round spermatids and the pachytene spermatocytes were found to be the most sensitive germ cells to lack of LH and underwent apoptosis. Interestingly, spermatogonial cells were found to be the least sensitive germ cells to the lack of LH in terms of apoptotic cell death. Results show that LH, in addition to being involved in the germ cell differentiation, is also involved in cell survival and prevent degeneration of germ cells during spermatogenesis. Apoptotic DNA fragmentation may serve as a useful marker for the study of hormonal regulation of spermatogenesis and the specific neutralization of gonadotropic hormones can be a reliable model for the study of the molecular mechanism of apoptosis.

DNA Condensation by the Rat Spermatidal Protein TP2 Shows GC-Rich Sequence Preference and Is Zinc Dependent

Transition protein-2 (TP2), isolated from rat testes, was recently shown to be a zinc metalloprotein. We have now carried out a detailed analysis of the DNA condensing properties of TP2 with various polynucleotides using circular dichroism spectroscopy. The condensation of the alternating copolymers by TP2 (incubated with 10 mu M ZnSO4), namely, poly(dG-dC). poly(dG-dC) and poly(dA-dT). poly(dA-dT), was severalfold higher than condensation of either of the homoduplexes poly(dG). poly-(dC) and poly(dA). poly(dT) or rat oligonucleosomal DNA. Between the two alternating copolymers, poly(dG-dC). poly(dG-dC) was condensed 3.2-fold more effectively than poly(dA-dT). poly(dA-dT). Preincubation of TP2 with 5 mM EDTA significantly reduced its DNA-condensing property. Interestingly, condensation of the alternating copolymer poly(dI-dC). poly(dI-dC) by TP2 was much less as compared to that of poly(dG-dC). poly(dG-dC). The V8 protease-derived N-terminal fragment (88 aa) condensed poly(dA-dT). poly(dA-dT) to a very small extent but did not have any effect on poly(dG-dC). poly-(dG-dC). The C-terminal fragment (28 aa) was able to condense poly(dA-dT) . poly(dA-dT) more effectively than poly(dG-dC). poly(dG-dC). These results suggest that TP2 in its zinc-coordinated form condenses GC-rich polynucleotides much more effectively than other types of polynucleotides. Neither the N-terminal two-thirds of TP2 which is the zinc-binding domain nor the C-terminal basic domain are as effective as intact TP2 in bringing about condensation of DNA.

Influence of cadmium on lipid peroxidation and antioxidant enzymes in isolated rat hepatocytes

Cadmium (Cd) influences lipid peroxidation (LPO) by enhancing peroxidation of membrane lipids and by disturbing the antioxidant system of cells. In isolated rat hepatocytes, LPO was observed in cells incubated with Cd (50-250 mu M) for various time periods up to 90 min. The antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were inhibited along with depletion of glutathione (GSH) in hepatocytes treated with Cd. The results show that Cd influences LPO in rat hepatocytes due to decrease in antioxidant status.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

Background and purpose of the study: Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods: Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results: The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion: Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1).

Subclinical autonomic neuropathy in alcoholics: Decreased heart rate variability

Cardiac autonomic neuropathy is known to occur in alcoholics but the extent of its subclinical form is not usually recognized, Heart Rate Variability (HRV) analysis can detect subclinical autonomic neuropathy. In this study the HRV parameters were compared in 20 neurologically asymptomatic alcoholics, 20 age-matched normals and 16 depressives. All were males, ECG was recorded in a quiet room for four minutes in supine position. Time and Frequency domain parameters of HRV were computed by a researcher blind to clinical details. Alcoholics had significantly smaller Coefficient of Variation of R-R intervals (CVR-R) on time domain analysis and smaller HF band (0.15-0.5 Hz) power on spectral analysis. The decreased Heart Rate Variability indicates cardiac autonomic dysfunction.

Protection against UVB inactivation (in vitro) of rat lens enzymes by natural antioxidants

Oxidative damage, through increased production of free radicals, is believed to be involved in UV-induced cataractogenesis (eye lens opacification). The possibility of UVB radiation causing damage to important lenticular enzymes was assessed by irradiating 3 months old rat lenses (in RPMI-1640 medium) at 300 nm (100 mu Wcm(-2)) for 24 h, in the absence and presence of ascorbic acid, alpha-tocopherol acetate and beta-carotene. UVB irradiation resulted in decreased activities of hexokinase, glucose-6-phosphate dehydrogenase, aldose reductase, and Na, K- ATPase by 42, 40, 44 and 57% respectively. While endopeptidase activity (229%) and lipid peroxidation (156%) were increased, isocitrate dehydrogenase activity was not altered on irradiation. In the presence of externally added ascorbic acid, tocopherol and beta-carotene (separately) to the medium, the changes in enzyme activities (except endopeptidase) and increased lipid peroxidation, due to UVB exposure, were prevented. These results suggest that UVB radiation exerts oxidative damage on lens enzymes and antioxidants were protective against this damage.

Regulation of an 8-kDa peptide involved in testosterone production by luteinizing hormone in rat Leydig cells

Results of Western blot analysis carried out with an interstitial cell extract from male guinea pig and ovarian extract from immature female rats administered equine chorionic gonadotropin (eCG) provide supportive evidence to our earlier suggestion that an 8-kDa peptide is involved in acquisition of steroidogenic capacity by the rat Leydig cells. It was found that though the signal was observed in other tissues such as liver, kidney and lung which do not produce gonadal hormones, the peptide was modulated only by lutenizing hormone (LH) in the rat Leydig cells.

Synergistic effect of UVB radiation and age on HMPS enzymes in rat lens homogenate

The behaviour of rat lenticular enzymes, glucose-6-phosphate dehydrogena.se (G6PD, EC: 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGD, EC: 1.1.1.44) as a function of age and UVB irradiation (in vitro) was investigated by irradiating the lens homogenate from 3-and 12-month-old rats at 300 nm (100 μW cm 2). In the 3-month-old group the specific activities of G6PD and 6PGD were reduced by 26% and 42%, respectively, after 24 h of irradiation, whereas in the 12-month-old group the decrease was 38% and 49% respectively, which suggests that the susceptibility of HMPS enzymes to UVB damage is higher in older lenses. The decrease in specitic activity was associated with a change in apparent Km and Vmax (marginal in 3 months and significant in 12 months) of these enzymes due to UVB irradiation. UVB irradiation also decreased the levels of NADPH and NADPH/NADP ratio. These changes, altered activities of G6PD and 6PGD and altered levels of NADPH. may in turn have a bearing on lens transparency.

Diminished chaos of heart rate time series in patients with major depression

Background: Depression and anxiety have been linked to serious cardiovascular events in patients with preexisting cardiac illness. A decrease in cardiac vagal function as suggested by a decrease in heart rate (HR) variability has been linked to sudden death. Methods: We compared LLE and nonlinearity scores of the unfiltered (UF) and filtered time series (very low, low, and high frequency; VLF, LF and HF) of HR between patients with depression (n = 14) and healthy control subjects (n = 18). Results: We found significantly lower LLE of the unfiltered series in either posture, and HF series in patients with major depression in supine posture (p < .002). LLE (LF/UF), which may indicate relative sympathetic activity was also significantly higher in supine and standing postures in patients (p < .05); LF/HF (LLE) was also higher in patients (p < .05) in either posture. Conclusions: These findings suggest that major depression is associated with decreased cardiac vagal function and a relative increase in sympathetic function, which may be related to the higher risk of cardiovascular mortality, in this group and illustrates the usefulness of nonlinear measures of chaos such as LLE in addition to the commonly used spectral measures.

Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: A potential role for a novel signaling pathway in the epididymis

Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.

Major depression with ischemic heart disease: Effects of paroxetine and nortriptyline on measures of nonlinearity and chaos of heart rate

Depression is associated with increased cardiovascular mortality in patients with preexisting cardiac illness. A decrease in cardiac vagal function as suggested by a decrease in heart rate variability (HRV) or heart period variability has been linked to sudden death in patients with cardiac disease as well as in normal controls. Recent studies have shown decreased vagal function in cardiac patients with depression as well as in depressed patients without cardiac illness. In this study, we compared 20 h awake and sleep heart period nonlinear measures using quantification of nonlinearity and chaos in two groups of patients with major depression and ischemic heart disease (mean age 59-60 years) before and after 6 weeks of treatment with paroxetine or nortriptyline. Patients received paroxetine, 20-30 mg/day or nortriptyline targeted to 190-570 nmol/l for 6 weeks. For HRV analysis, 24 patients were included in the paroxetine treatment study and 20 patients in the nortriptyline study who had at least 20,000 s of awake data. The ages of these groups were 60.4 +/- 10.5 years for paroxetine and 60.8 +/- 13.4 years for nortriptyline. There was a significant decrease in the largest Lyapunov exponent (LLE) after treatment with nortriptyline but not paroxetine. There were also significant decreases in nonlinearity scores on S-netPR and S-netGS after nortriptyline, which may be due to a decrease in cardiac vagal modulation of HRV. S-netGS and awake LLE were the most significant variables that contributed to the discrimination of postparoxetine and postnortriptyline groups even with the inclusion of time and frequency domain measures. These findings suggest that nortriptyline decreases the measures of chaos probably through its stronger vagolytic effects on cardiac autonomic function compared with paroxetine, which is in agreement with previous clinical and preclinical reports. Nortriptyline was also associated with a significant decrease in nonlinearity scores, which may be due to anticholinergic and/or sympatholytic effects. As depression is associated with a strong risk factor for cardiovascular mortality, one should be careful about using any drug that adversely affects cardiac vagal function. Copyright (C) 2002 S. Karger AG, Basel.

Phenobarbitone-mediated translocation of the cytosolic proteins interacting with the 5 '-proximal region of rat liver CYP2B1/B2 gene into the nucleus

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M-r of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation. (C) 2002 Elsevier Science (USA).