Changes in Ribosomal Activity of Escherichia coli Cells during Prolonged Culture in Sea Salts Medium

The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the k_max/K_s ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the k_max/K_s value. {k_max is the apparent catalytic rate constant of peptidyl transferase, and K_s is the dissociation constant of the encounter complex between acetyl (Ac)[³H]Phe-tRNA-poly(U)-ribosome and puromycin.} Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.     

Unique features of the hemoglobin system of the Antarctic notothenioid fish Gobionotothen gibberifrons.

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.     

Insect symbionts and applications: The paradigm of cytoplasmic incompatibility-inducing Wolbachia

Wolbachia is a group of Gram‐negative, obligatory intracellular and maternally transmitted alpha‐Proteobacteria. They have been reported to establish symbiotic relationships with a great variety of species of the most diverse animal class, the insects, as well as with several other arthropods and with filarial nematodes. The reproductive alterations Wolbachia causes in its hosts account for its widespread distribution. These alterations include parthenogenesis, feminization, male killing, and cytoplasmic incompatibility (CI). CI is the most frequent and best studied effect Wolbachia has on its hosts. CI is a form of male sterility, ultimately resulting in embryo lethality in diplodiploid host species. As a consequence of CI, Wolbachia infections spread and lead to the replacement of uninfected populations. CI was used nearly four decades ago to control important disease vectors with very encouraging results, and a number of more recent studies have confirmed the effectiveness of CI as a pest population suppression tool as well as a driving mechanism. Furthermore, recent advancements in the field encourage the development of Wolbachia‐based methods for the biological control of insect pests and disease vectors of agricultural, environmental and medical importance.     

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.     

Lipid rafts and T-lymphocyte function: implications for autoimmunity

Experimental evidence indicates that the mammalian cell membrane is compartmentalized. A structural feature that supports membrane segmentation implicates assemblies of selected lipids broadly referred to as lipid rafts. In T-lymphocytes, lipid rafts are implicated in signalling from the T-cell antigen receptor (TCR) and in localization and function of proteins residing proximal to the receptor. This review summarizes the current literature that deals with lipid raft involvement in T-cell activation and places particular emphasis in recent studies investigating lipid rafts in autoimmunity. The potential of lipid rafts as targets for the development of a new class of immune-modulating compounds is discussed.     

Immunolocalization of carbonic anhydrase and phosphoenolpyruvate carboxylase in developing seeds of Medicago sativa.

To investigate the role of carbonic anhydrase (CA; EC 4.2.1.1) and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) during Medicago sativa seed development, the distribution of both proteins was examined using an immunohistological approach. Both enzymes are co-localized in most ovular and embryonic tissues. In early stages of seed development, both proteins were abundant in embryo and integuments, while at subsequent stages both proteins are accumulated in endosperm, nucellus and integuments. At late stages of seed development when both endosperm and nucellus are degraded, significant accumulation of both proteins was observed in the embryo proper. Chlorophyll was found to accumulate in embryos after the heart stage and reached a maximum at mature stage. It is suggested that CA and PEPC play a role in respiratory carbon dioxide refixation while generating malate to support amino acid and/or fatty acids biosynthesis.     

Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco

Polyamines (PAs) exert a protective effect against stress challenges, but their molecular role in this remains speculative. In order to detect the signaling role of apoplastic PA-derived hydrogen peroxide (H2O2) under abiotic stress, we developed a series of tobacco (Nicotiana tabacum cv Xanthi) transgenic plants overexpressing or downregulating apoplastic polyamine oxidase (PAO; S-pao and A-pao plants, respectively) or downregulating S-adenosyl-l-methionine decarboxylase (samdc plants). Upon salt stress, plants secreted spermidine (Spd) into the apoplast, where it was oxidized by the apoplastic PAO, generating H2O2. A-pao plants accumulated less H2O2 and exhibited less programmed cell death (PCD) than did wild-type plants, in contrast with S-pao and samdc downregulating plants. Induction of either stress-responsive genes or PCD was dependent on the level of Spd-derived apoplastic H2O2. Thus, in wild-type and A-pao plants, stress-responsive genes were efficiently induced, although in the latter at a lower rate, while S-pao plants, with higher H2O2 levels, failed to accumulate stress-responsive mRNAs, inducing PCD instead. Furthermore, decreasing intracellular PAs, while keeping normal apoplastic Spd oxidation, as in samdc downregulating transgenic plants, caused enhanced salinity-induced PCD. These results reveal that salinity induces the exodus of Spd into the apoplast, where it is catabolized by PAO, producing H2O2. The accumulated H2O2 results in the induction of either tolerance responses or PCD, depending also on the levels of intracellular PAs.     

Effect of biotic and abiotic factors on reproductive parameters of the synovigenic endoparasitoid Venturia canescens

Abstract.  The main effects and interactions of adult age, access to food and host deprivation, on the egg load of Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae), a larval parasitoid of Ephestia kuehniella (Lepidoptera: Pyralidae) and other pyralids, were studied in the laboratory. Intraspecific variation in the number of ovarioles was also studied. There was a positive and significant correlation between wasp size and both egg load and ovariole number, with the reproductive system of large wasps containing significantly more ovarioles and mature eggs than small wasps. Newly emerged adult wasps contained 27 ± 2.4 ovulated (mature) eggs in their lateral oviducts. Access to food and host deprivation were the only conditions under which egg load increased with parasitoid age. Wasps that had access to hosts immediately after emergence showed a significant decline in their egg complement, irrespective of food presence. Under conditions of both host and food deprivation, there was practically no alteration of egg load with parasitoid age. A three-way analysis of variance revealed that egg load varies significantly with food or host access but not with parasitoid age. All interactions among the three factors were significant. It is confirmed that there is no egg resorption in V. canescens and that egg production stops in the absence of food.     

Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum

The role of phosphoinositide-specific phospholipase C (PI-PLC) signaling in the macrotubule-dependent protoplast volume regulation in plasmolyzed root cells of Triticum turgidum was investigated. At the onset of hyperosmotic stress, PI-PLC activation was documented. Inhibition of PI-PLC activity by U73122 blocked tubulin macrotubule formation in plasmolyzed cells and their protoplast volume regulatory mechanism. In neomycin-treated plasmolyzed cells, macrotubule formation and protoplast volume regulation were not affected. In these cells the PI-PLC pathway is down-regulated as neomycin sequesters the PI-PLC substrate, 4,5-diphosphate-phosphatidyl inositol (PtdInsP(2)). These phenomena were unaffected by R59022, an inhibitor of phosphatidic acic (PA) production via the PLC pathway. Taxol, a microtubule (MT) stabilizer, inhibited the hyperosmotic activation of PI-PLC, but oryzalin, which disorganized MTs, triggered PI-PLC activity. Taxol prevented macrotubule formation and inhibited the mechanism regulating the volume of the plasmolyzed protoplast. Neomycin partly relieved some of the taxol effects. These data suggest that PtdInspP(2) turnover via PI-PLC assists macrotubule formation and activation of the mechanism regulating the plasmolyzed protoplast volume; and the massive disorganization of MTs that is carried out at the onset of hyperosmotic treatment triggers the activation of this mechanism.