Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs

The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H(2)/CO(2) is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.     

Identification of a novel transporter for dicarboxylates and tricarboxylates in plant mitochondria. Bacterial expression, reconstitution, functional characterization, and tissue distribution

A cDNA from Arabidopsis thaliana and four related cDNAs from Nicotiana tabacum that we have isolated encode hitherto unidentified members of the mitochondrial carrier family. These proteins have been overexpressed in bacteria and reconstituted into phospholipid vesicles. Their transport properties demonstrate that they are orthologs/isoforms of a novel mitochondrial carrier capable of transporting both dicarboxylates (such as malate, oxaloacetate, oxoglutarate, and maleate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate). The newly identified dicarboxylate-tricarboxylate carrier accepts only the single protonated form of citrate (H-citrate2-) and the unprotonated form of malate (malate2-) and catalyzes obligatory, electroneutral exchanges. Oxoglutarate, citrate, and malate are mutually competitive inhibitors, showing K(i) close to the respective K(m). The carrier is expressed in all plant tissues examined and is largely spread in the plant kingdom. Furthermore, nitrate supply to nitrogen-starved tobacco plants leads to an increase in its mRNA in roots and leaves. The dicarboxylate-tricarboxylate carrier may play a role in important plant metabolic functions requiring organic acid flux to or from the mitochondria, such as nitrogen assimilation, export of reducing equivalents from the mitochondria, and fatty acid elongation.     

HIV-1 envelope gp41 peptides promote migration of human Fc epsilon RI+ cells and inhibit IL-13 synthesis through interaction with formyl peptide receptors

We evaluated the effects of synthetic peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the structure of HIV-1(MN) envelope gp41 on both chemotaxis of human basophils and the release of preformed mediators (histamine) and of cytokines (IL-13). Peptides 2019 and 2021 were potent basophil chemoattractants, whereas the other peptides examined were ineffective. Preincubation of basophils with FMLP or gp41 2019 resulted in complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with low concentration (5 x 10(-7) M) of FMLP, which binds with high affinity to N-formyl peptide receptor (FPR), but not to FPR-like 1, did not affect the chemotactic response to a heterologous stimulus (gp41 2019). In contrast, a high concentration (10(-4) M) of FMLP, which binds also to FPR-like 1, significantly reduced the chemotactic response to gp41 2019. The FPR antagonist cyclosporin H inhibited chemotaxis induced by FMLP, but not by gp41 2019. None of these peptides singly induced the release of histamine or cytokines (IL-4 and IL-13) from basophils. However, low concentrations of peptides 2019 and 2021 (10(-8)-10(-6) M) inhibited histamine release from basophils challenged with FMLP but not the secretion caused by anti-IgE and gp120. Preincubation of basophils with peptides 2019 and 2021 inhibited the expression of both IL-13 mRNA, and the FMLP-induced release of IL-13 from basophils. These data highlight the complexity of the interactions between viral and bacterial peptides with FPR subtypes on human basophils.     

Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.     

Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin

In contrast to canonical mRNAs, translation of leaderless mRNA has been previously reported to continue in the presence of the antibiotic kasugamycin. Here, we have studied the effect of the antibiotic on determinants known to affect translation of leadered and leaderless mRNAs. Kasugamycin did not affect the Shine-Dalgarno (SD)-anti-SD (aSD) interaction or the function of translation initiation factor 3 (IF3). Thus, the preferential translation of leaderless mRNA in the presence of kasugamycin can neither be attributed to an expanding pool of 30S subunits with a "blocked" aSD nor to a lack of action of IF3, which has been shown to discriminate against translation initiation at 5'-terminal start codons. Using toeprinting, we observed that on leaderless mRNA 70S in contrast to 30S translation initiation complexes are comparatively resistant to the antibiotic. These results taken together with the known preference of 70S ribosomes for 5'-terminal AUGs lend support to the hypothesis that translation of leaderless mRNAs may as well proceed via an alternative initiation pathway accomplished by intact 70S ribosomes.     

Soil micromycete diversity in the hypersaline Dead Sea coastal area,Israel

In the present study, a soil microfungal community was examined over a one-year period (1999–2000) at the western shore of the Dead Sea. A total of 78 species from 40 genera were isolated. The most prominent features of mycobiota of the territory studied were: (i) the prevailing number of melanin-containing micromycetes (46 species, 65.5 % of the total isolate number); (ii) a large share of teleomorphic Ascomyceta (26 species, 18.5 % of isolates); (iii) combination of true soil and plant surface inhabiting species; (iv) spatial and temporal variation of the mycobiota composition; (v) very low fungal density (nearly 500-fold lower than in the Judean Desert soil). These features are formed under the extremely stressful xeric and oligotrophic conditions in which the Dead Sea coastal micromycete community exists. Nine species (Alternaria alternata, A. raphani, Aspergillus niger, Aureobasidium pullulans, Chaetomium globosum, Ch. murorum, Cladosporium cladosporioides, Penicillium aurantiogriseum, and Stachybotrys chartarum) were considered a characteristic micromycete complex for the Dead Sea coastal habitat based on the spatial and temporal occurrence of these species. Many of the micromycetes isolated, including almost all the species listed above, are known to be distributed worldwide occurring in different soil types. This confirms the conclusion of many mycologists working in areas with saline and arid soils that there is no halo-and thermophilous mycobiota characteristic for those soils.     

Elevated CO2 concentrations and stomatal density: observations from 17 plant species growing in a CO2 spring in central Italy

Stomatal density (SD) and stomatal conductance ( g _s) can be affected by an increase of atmospheric CO₂ concentration. This study was conducted on 17 species growing in a naturally enriched CO₂ spring and belonging to three plant communities. Stomatal conductance, stomatal density and stomatal index (SI) of plants from the spring, which were assumed to have been exposed for generations to elevated [CO₂], and of plants of the same species collected in a nearby control site, were compared. Stomatal conductance was significantly lower in most of the species collected in the CO₂ spring and this indicated that CO₂ effects on g _s are not of a transitory nature but persist in the long term and through plant generations. Such a decrease was, however, not associated with changes in the anatomy of leaves: SD was unaffected in the majority of species (the decrease was only significant in three out of the 17 species examined), and also SI values did not vary between the two sites with the exception of two species that showed increased SI in plants grown in the CO₂-enriched area. These results did not support the hypothesis that long-term exposure to elevated [CO₂] may cause adaptive modification in stomatal number and in their distribution.