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).

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.

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.

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).

Vasomodulatory effect of novel peroxovanadate compounds on rat aorta: Role of rho kinase and nitric oxide/cGMP pathway

The present study was undertaken to assess the role of reactive oxygen species (ROS) in rat aortic ring vasoreactivity and integrity by using various peroxovanadate (pV) compounds. All the pV compounds (1 nM-300 mu M) used in the present study exerted concentration-dependent contractions on endothelium intact rat aortic rings. All compounds with an exception of DPV-asparagine (DPV-asn) significantly altered vascular integrity as shown by diminished KCl responses. Phenylephrine (PE)-mediated contractions (3 nM-300 mu M) were unaltered in the presence of these compounds. Acetylcholine (Ach)-mediated relaxation in PE (1 mu M) pre-contracted rings was significantly reduced in presence of diperoxovanadate (DPV), poly (sodium styrene sulfonate-co-maleate)-pV (PSS-CoM-pV) and poly (sodium styrene 4-sulfonate)-pV (PSS-pV). However, no significant change in Ach-mediated responses was observed in the presence of poly (acrylate)-pV (PM-pV) and DPV-asn. DPV-asn was thus chosen to further elucidate mechanism involved in peroxide mediated modulation of vasoreactivity. DPV-asn (30 nM-300 mu M) exerted significantly more stable contractions, that was found to be catalase (100 U/ml) resistant in comparison with H(2)O(2) (30 nM-300 mu M) in endothelium intact aortic rings. These contractile responses were found to be dependent on extracellular Ca(2+) and were significantly inhibited in presence of ROS scavenger N-acetylcysteine (100 mu M). Intracellular calcium chelation by BAPTA-AM (10 mu M) had no significant effect on DPV-asn (30 nM-300 mu M) mediated contraction. Pretreatment of aortic rings by rho-kinase inhibitor Y-27632 (10 mu M) significantly inhibited DPV-asn-mediated vasoconstriction indicating role of voltage-dependent Ca(2+) influx and downstream activation of rho-kinase. The small initial relaxant effect obtained on addition of DPV-asn (30 nM-1 mu M) in PE (1 mu M) pre-contracted endothelium intact rings, was prevented in the presence of guanylate cyclase inhibitor, methylene blue (10 mu M) and/or nitric oxide synthase (NOS) inhibitor, L-NAME (100 mu M) suggesting involvement of nitric oxide and cGMP. DPV-asn, like H(2)O(2), exerted a response of vasoconstriction in normal arteries and vasodilation at low concentrations (30 nM-1 mu M) in PE-pre contracted rings with overlapping mechanisms. These findings suggest usefulness of DPV-asn having low toxicity, in exploring the peroxide-mediated effects on various vascular beds. The present study also convincingly demonstrates role of H(2)O(2) in the modulation of vasoreactivity by using stable peroxide DPV-asn and warrants future studies on peroxide mediated signaling from a newer perspective. (C) 2011 Published by Elsevier Ltd.