Amelioration of vanadium-induced testicular toxicity and adrenocortical hyperactivity by vitamin E acetate in rats

Vanadium toxicity is a challenging problem to the health professionals and a cutting-edge medical problem. Vanadium has been recognized as industrial hazards that adversely affect human and animal reproductive health. Since testicular function is exquisitely susceptible to reactive-oxygen species, the present study elucidates the possible involvement of oxidative stress in vanadium-induced testicular toxicity and the prophylactic effects of vitamin E acetate against such adverse effects of vanadium. The study also characterizes the effects of vanadium on rat adrenal steroidogenesis and determines the underlying mechanisms of testicular and adrenal interactions in response to vanadium exposure. Significantly reduced sperm count associated with decreased serum testosterone and gonadotropins level in the vanadium-injected group of rats compared to control substantially proves the ongoing damaging effects of vanadium-induced ROS on developing germ cells. This is in turn reflected in the appreciable increase in testicular lipid peroxidation level and decline in the activities of steroidogenic and antioxidant enzymes. However, oral administration of vitamin E acetate could protect testes from the toxic effects of vanadium. Vanadium also results in adrenocortical hyperactivity, as evidenced by the elevated secretion of glucocorticoids, adrenal gland hypertrophy and increased activity of adrenal Δ⁵3β-HSD. However, reversibility of these alterations in adrenocortical activities was vividly reflected after vitamin E acetate supplementation. All these studies reveal that oxidative stress is the major mechanism of health deterioration and that vanadium can act as a stressor metal causing chronic stress effects through excitation of hypothalamo-pituitary-adrenal axis. However antioxidant support by vitamin E acetate may provide significant protection.     

Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance

Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.     

ErbB4 isoforms selectively regulate growth factor induced Madin-Darby canine kidney cell tubulogenesis

ErbB4, a member of the epidermal growth factor (EGF) receptor family that can be activated by heregulin beta1 and heparin binding (HB)-EGF, is expressed as alternatively spliced isoforms characterized by variant extracellular juxtamembrane (JM) and intracellular cytoplasmic (CYT) domains. ErbB4 plays a critical role in cardiac and neural development. We demonstrated that ErbB4 is expressed in the ureteric buds and developing tubules of embryonic rat kidney and in collecting ducts in adult. The predominant isoforms expressed in kidney are JM-a and CYT-2. In ErbB4-transfected MDCK II cells, basal cell proliferation and hepatocyte growth factor (HGF)-induced tubule formation were decreased by all four isoforms. Only JM-a/CYT-2 cells formed tubules upon HB-EGF stimulation. ErbB4 was activated by both HRG-beta1 and HB-EGF stimulation; however, compared with HRG-beta1, HB-EGF induced phosphorylation of the 80-kDa cytoplasmic cleavage fragment of the JM-a/CYT-2 isoform. HB-EGF also induced early activation of ERK1/2 in JM-a/CYT-2 cells and promoted nuclear translocation of the JM-a/CYT-2 cytoplasmic tail. In summary, our data indicate that JM-a/CYT-2, the ErbB4 isoform that is proteinase cleavable but does not contain a PI3K-binding domain in its cytoplasmic tail, mediates important functions in renal epithelial cells in response to HB-EGF.     

Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation

An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.     

Phyto-extraction of heavy metals and biochemical changes with Brassica nigra L. grown in rayon grade paper mill effluent irrigated soil

In this study, distribution of metal accumulation and their biological changes of Indian mustard plants (Brassica nigra L.) grown in soil irrigated with different concentration of rayon grade paper effluent (RGPE, 25%, 50%, 75%, 100%, v/v) were studied. A pronounced effect was recorded at 50% (v/v) RGPE on germination of seeds, amylase activity and other growth parameters in Indian mustard plants. An increase in the chlorophyll and protein contents was also recorded at <50% (v/v) RGPE followed by a decrease at higher concentrations of RGPE (>75%). A significant increase lipid peroxidation was recorded, which was evidenced by the increased malondialdehyde (MDA) content in shoot, leaves and seeds in tested plant at all the concentrations of RGPE. This Indian mustard plants (Brassica nigra L.) are well adapted for tolerance of significant amount of heavy metals due to increased level of antioxidants (cysteine and ascorbic acid) in root shoot and leaves of treated plants at all concentration of RGPE. Moreover, it is also important that RGPE should be treated to bring down the metal concentration well within the prescribed limit prior to use in agricultural soil for ferti-irrigation.     

Conformational regulation of the EGFR kinase core by the juxtamembrane and C-terminal tail: a molecular dynamics study

The catalytic domain of epidermal growth factor receptor (EGFR) is activated by dimerization, which requires allosteric coupling between distal dimerization and catalytic sites. Although crystal structures of EGFR kinases, solved in various conformational states, have provided important insights into EGFR activation by dimerization, the atomic details of how dimerization signals are dynamically coupled to catalytic regions of the kinase core are not fully understood. In this study, we have performed unrestrained and targeted molecular dynamics simulations on the active and inactive states of EGFR, followed by principal component analysis on the simulated trajectories, to identify correlated motions in the EGFR kinase domain upon dimerization. Our analysis reveals that the conformational changes associated with the catalytic functions of the kinase core are highly correlated with motions in the juxtamembrane (JM) and C-terminal tail, two flexible structural elements that play an active role in EGFR kinase activation and dimerization. In particular, the opening and closing of the ATP binding lobe relative to the substrate binding lobe is highly correlated with motions in the JM and C-terminal tail, suggesting that ATP and substrate binding can be coordinated with dimerization through conformational changes in the JM and C-terminal tail. Our study pinpoints key residues involved in this conformational coupling, and provides new insights into the role of the JM and C-terminal tail segments in EGFR kinase functions.     

A nonerythropoietic peptide that mimics the 3D structure of erythropoietin reduces organ injury/dysfunction and inflammation in experimental hemorrhagic shock

Recent studies have shown that erythropoietin, critical for the differentiation and survival of erythrocytes, has cytoprotective effects in a wide variety of tissues, including the kidney and lung. However, erythropoietin has been shown to have a serious side effect-an increase in thrombovascular effects. We investigated whether pyroglutamate helix B-surface peptide (pHBSP), a nonerythropoietic tissue-protective peptide mimicking the 3D structure of erythropoietin, protects against the organ injury/ dysfunction and inflammation in rats subjected to severe hemorrhagic shock (HS). Mean arterial blood pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 mL/kg Ringer Lactate for 10 min and 50% of the shed blood for 50 min. Rats were euthanized 4 h after the onset of resuscitation. pHBSP was administered 30 min or 60 min into resuscitation. HS resulted in significant organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung). In rats subjected to HS, pHBSP significantly attenuated (i) organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung), (ii) increased the phosphorylation of Akt, glycogen synthase kinase-3β and endothelial nitric oxide synthase, (iii) attenuated the activation of nuclear factor (NF)-κB and (iv) attenuated the increase in p38 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. pHBSP protects against multiple organ injury/dysfunction and inflammation caused by severe hemorrhagic shock by a mechanism that may involve activation of Akt and endothelial nitric oxide synthase, and inhibition of glycogen synthase kinase-3β and NF-κB.     

The molecular basis for the broad substrate specificity of human sulfotransferase 1A1

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.     

Precise Mapping of the Homothallism Genes HML and HMR in SACCHAROMYCES CEREVISIAE

The HML and HMR loci carry unexpressed copies of MATa and MATα information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).