Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae

The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon. Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis. Recently, putative roles for hsp60 and hsp70-like proteins have been proposed in Saccharomyces cerevisiae. hsp70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix. In this paper, we report that a brief temperature shock (44 degrees C) impairs coupling of oxidative phosphorylation in S. cerevisiae as measured indirectly by the Cl-CCP/oligomycin assay. Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions. Pretreatment of cells for a short period of time at 37 degrees C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport. Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity. We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Silver Enhancement of Nickel-Diaminobenzidine as Applied to Single and Double Immunoperoxidase Staining

A reliable and practical method is proposed for increasing sensitivity and detection efficiency of immunocytochemical techniques, based on silver enhancement of the nickel-diaminobenzidine product of the peroxidase reaction. The procedure produces a strong signal at the site of the end product of the peroxidase reaction which is visible as black grains at the light microscopic level. The method has been used to detect peroxidase labeled probes in immunocytochemical tissue preparations and blotting assays and is ideal for the purposes of double staining and photographic documentation.     

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4⁺ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4⁺ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4⁺ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4⁺ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4⁺ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4⁺ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4⁺ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.     

Development of ectopic roots from abortive nodule primordia

The symbiotic phenotype of five Tn5-induced mutants of Rhizobium etli affected in different anabolic pathways (namely, gluconeogenesis and biosynthesis of lysine, purine, or pyrimidine) was analyzed. These mutants induced, on the root of Phaseolus vulgaris, a normal early sequence of morphogenetics events, including root hair deformation and development of nodule primordia. Later on, however, from the resulting root outgrowths, instead of nodules, one or more ectopic roots (spaced closely related and agravitropic) emerged. Therefore, this group of mutant was collectively called "root inducer" (RIND). It was observed that the RIND-induced infection threads aborted early inside the invaded root hair, and that the resulting abortive nodules lack induction of late nodulin genes. Moreover, experiments performed using a conditional mutant (a methionine-requiring invader) revealed that bacterial invasion plays a key role in the maintenance of the program of nodule development and, in particular, in the differentiation of the most specific symbiotic tissue of globose nodules, the central tissue. These data indicate that, in P. vulgaris, the nodule primordium is a root-specified pro-meristematic tissue.     

The Italian Twin Project: from the personal identification number to a national twin registry.

The unique opportunity given by the "fiscal code", an alphanumeric identification with demographic information on any single person residing in Italy, introduced in 1976 by the Ministry of Finance, allowed a database of all potential Italian twins to be created. This database contains up to now name, surname, date and place of birth and home address of about 1,300,000 "possible twins". Even though we estimated an excess of 40% of pseudo-twins, this still is the world's largest twin population ever collected. The database of possible twins is currently used in population-based studies on multiple sclerosis, Alzheimer's disease, celiac disease, and type 1 diabetes. A system is currently being developed for linking the database with data from mortality and cancer registries. In 2001, the Italian Government, through the Ministry of Health, financed a broad national research program on twin studies, including the establishment of a national twin registry. Among all the possible twins, a sample of 500,000 individuals are going to be contacted and we expect to enrol around 120,000 real twin pairs in a formal Twin Registry. According to available financial resources, a sub sample of the enrolled population will be asked to donate DNA. A biological bank from twins will be then implemented, guaranteeing information on future etiological questions regarding genetic and modifiable factors for physical impairment and disability, cancers, cardiovascular diseases and other age related chronic illnesses.