Antizyme affects cell proliferation and viability solely through regulating cellular polyamines

Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.     

Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones

Tuberculosis (TB) remains one of the world's leading causes of death from infectious disease. It is caused by infection with Mycobacterium tuberculosis or sometimes, particularly in immune-compromised patients, Mycobacterium avium. The aim of this study was to create a tool that could be used in the search for new anti-TB drugs that inhibit branched-chain amino acid (BCAA) biosynthesis, as these are essential amino acids that are not available to a mycobacterium during growth in an infected organism. To this end, we cloned, overexpressed, purified and characterised for the first time an acetohydroxyacid synthase (AHAS), a key enzyme in the pathway to the biosynthesis of the BCAAs, from the genus Mycobacterium. Nine commercial herbicides of the sulfonylurea and imidazolinone classes were tested for their influence on this enzyme. Four of the sulfonylureas were potent inhibitors of the enzyme. The relative potency of the different inhibitors towards the M. avium enzyme was unlike their potency towards other AHASs whose inhibitor profile has been reported, emphasising the advantage of using a mycobacterial enzyme as a tool in the search for new anti-TB drugs.     

Three forms of GnRH in the brain and pituitary of the turbot, Scophthalmus maximus: immunological characterization and seasonal variation

Three forms of GnRH, chicken (c) GnRH-II, salmon (s) and seabream (sb) GnRH, were immunologically characterized in the brain and pituitary of turbot by ELISA. cGnRH-II and sGnRH were detected in the brain, while sbGnRH and sGnRH (but not cGnRH-II) were detected in the pituitary. In females, the levels of cGnRH-II in the turbot brain extracts increased from May to July, concomitant with an increase in oocyte diameter. In the pituitary, sbGnRH was found to be the dominant form, with levels 100-600-fold those of sGnRH. Both sGnRH and sbGnRH in the pituitary showed variation during the spawning season; sbGnRH increased from May to July and correlated with the increase in oocyte diameter, while sGnRH decreased. The overall patterns were the same for male turbot, although levels were generally lower. These findings suggest that sbGnRH could be controlling reproduction in the turbot. However, the seasonal variation in sGnRH indicates a potential physiological role in turbot reproduction. This study gives the first immunological indications that sbGnRH is present in the pituitary of a pleuronectiform fish, and will provide the basis for further studies on the endocrine regulation of reproduction in flatfish.     

Pairing success of male Gammarus lacustris infected by two acanthocephalans: a comparative study

The costs of parasitism to host reproduction can be best assessedusing field studies to determine overall mating success andexperimental studies to examine how parasites may affect matingbehavior. We compared the influence of two parasites, Polymorphusparadoxus and P. marilis (Acanthocephala), on the pairing successof their intermediate host (Gammarus lacustris, Crustacea) inboth the field and laboratory. Parasitism significantly loweredthe pairing success of male gammarids. In the field, P. paradoxus-infectedmales paired significantly less often than P. marilis-infectedor uninfected males. Those infected by P. marilis were alsofound in precopula significandy less often than uninfected ones.In the laboratory, the pairing success of males infected byeither parasite was significantly reduced in both competitiveand noncompetitive situations. As in the field studies, thepairing success of P. paradoxus-infected males was significantlylower than that of P. marilis-infected and uninfected males.Polymorphus marilis-infected males were also outcompeted byuninfected individuals, however, their pairing success improvedwhen alone with a female (noncompetitive experiments). We relatethe differential influence of the two parasites on the pairingsuccess of male gammarids to their effects on the physiologyand behavior of G. lacustris.     

A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho.     

Daily and yearly burnout symptoms in Israeli shift work residents

Burnout is a syndrome of physical and emotional exhaustion that develops among individuals who are open to public demands. In view of their heavy work load and sleep deprivation, we decided to evaluate the impact of long working hours on burnout and psychological status among a sample of residents during the first 2 years of their residency. Seventy-eight residents participated in the study, all residents completed self-administered questionnaires, and their sleep-wake cycle was monitored by a wrist-worn actigraph for a period of 5-7 days. The questionnaires included a short form suitable for Experience Sampling Method (ESM), and a longer background Questionnaire. The results revealed that sleep duration, Work Load and the interaction between them, explain the Negative Mood the day after the night shift. However, positive mood, and fatigue were not affected by sleep duration or workload. In general, after one year of residency, residents become more stressed, less involved in the job, and had a high level of burnout and psychosomatic symptoms. However, after the second year, the burnout symptoms were almost the same as at the beginning except for the level of stress that remained high. Sleep duration was unrelated to the burnout symptoms.     

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN(-/-)) mice were treated in vitro with H(2)O(2) to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN(-/-) cells but was increased to only 20% in WT cells. In contrast, after 1-8 h of treatment with H(2)O(2), the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN(-/-) cells. Electron microscopy of WT cells treated with H(2)O(2) showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H(2)O(2)-treated OPN(-/-) cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN(-/-) and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN(-/-) cells by approximately 30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN(-/-) cells was not altered. Restoration of OPN expression in OPN(-/-) fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H(2)O(2) treatment. Thus H(2)O(2)-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.     

A monomeric 3(10)-helix is formed in water by a 13-residue peptide representing the neutralizing determinant of HIV-1 on gp41

The peptide gp41(659-671) (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41(659-671) in water. This peptide forms a monomeric 3(10)-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 3(10)-helix is confirmed by its characteristic CD pattern. Studies of the 3(10)-helix have been hampered by the absence of a model peptide adopting this conformation. gp41(659-671) can serve as such a model to investigate the spectral characteristics of the 3(10)-helix, the factors that influence its stability, and the propensity of different amino acids to form a 3(10)-helix. The observation that the 3(10)-helical conformation is highly populated in the peptide gp41(659-671) indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41(659-671) segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41(659-671) is an autonomous folding unit, peptide immunogens consisting of the complete gp41(659-671) sequence are likely to induce antibodies highly cross-reactive with HIV-1.     

Phosphorylation-dependent regulation of Kv2.1 Channel activity at tyrosine 124 by Src and by protein-tyrosine phosphatase epsilon

Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTPepsilon (cyt-PTPepsilon) down-regulates channel activity and counters its phosphorylation and up-regulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTPepsilon. Tyr(124) is phosphorylated by Src in vitro; in whole cells, Y124F Kv2.1 is significantly less phosphorylated by Src and loses most of its ability to bind the D245A substrate-trapping mutant of cyt-PTPepsilon. Phosphorylation of Tyr(124) is critical for Src-mediated up-regulation of Kv2.1 channel activity, since Y124F Kv2.1-mediated K(+) currents are only marginally up-regulated by Src, in contrast with a 3-fold up-regulation of wild-type Kv2.1 channels by the kinase. Other properties of Kv2.1, such as expression levels, subcellular localization, and voltage dependence of channel activation, are unchanged in Y124F Kv2.1, indicating that the effects of the Y124F mutation are specific. Together, these results indicate that Tyr(124) is a significant site at which the mutually antagonistic activities of Src and cyt-PTPepsilon affect Kv2.1 phosphorylation and activity.     

Phytomelatonin in the leaves and fruits of wild perennial plants

Phytomelatonin has been documented in numerous flowering plants, mostly in cultivated species consumed by humans. Although frugivorous animals feed on fruits, the phytomelatonin content of these organs has hardly ever been tested in wild plants. The aim of this study was to determine the levels of phytomelatonin in the leaves and fleshy fruits of 31 wild perennial species known to be eaten by herbivorous and frugivorous mammals and birds. Considerable levels of phytomelatonin were found in the leaves of all the tested species, and some contained melatonin in their fruits as well. The melatonin content was found to vary significantly in different life forms (trees, shrubs, and climbers), with trees possessing the highest levels. The analysis revealed a significant positive correlation between the phytomelatonin levels in the leaves and the fruits of various species. However, the concentration found in the fruits was generally lower than that found in the leaves of the same species. Despite the presence of phytomelatonin in the fleshy fruits of different families, there was no noticeable common attribute among them. Phytomelatonin was exhibited in both the seeds and the pulp, with no obvious preference for either one. Although it was determined that ingested melatonin enters the bloodstream of birds and mammals, its specific role is still not certain. The potential impact of edible phytomelatonin on the circadian rhythm of herbivores and frugivores is discussed on the basis of these findings.