Regulation of prostaglandin generation in carrageenan-induced pleurisy by inducible nitric oxide synthase in knockout mice

In the present study, by comparing the responses in wild-type mice (iNOSWT) and mice lacking (iNOSKO) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the correlation between endogenous nitric oxide (NO) and prostaglandin (PG) generation in carrageenan-induced pleurisy. The inflammatory response in iNOSKO mice was significantly reduced in respect to iNOSWT animals, as demonstrated by the exudate volume (-63%) and numbers of infiltrating cells (-62%). The levels of NOx in the pleural exudate from carrageenan-treated mice were significantly (p < 0.01) decreased in iNOSKO mice (16 +/- 7.6 nmoles/mice) compared to iNOSWT animals (133 +/- 9 nmoles/mice). Similarly, the amounts of PGE2 in the pleural exudates of carrageenan-treated animals were significantly (p < 0.01) lower in iNOSKO compared to iNOSWT mice (120 +/- 20 pg/mice vs. 308 +/- 51 pg/mice). Also the amounts of 6-keto-PGF(1 alpha) produced by lungs from carrageenan-treated iNOSKO mice (1.01 +/- 0.10 ng/tissue mg) were significantly (p < 0.01) reduced compared to iNOSWT carrageenan-treated mice (2.1 +/- 0.09 ng/tissue mg). In conclusion our results confirm, by the use of iNOSKO mice that in carrageenan-induced pleurisy NO positively modulates PG biosynthesis.     

HIV-1-Tat protein activates phosphatidylinositol 3-kinase/ AKT-dependent survival pathways in Kaposi's sarcoma cells

In this study we found that Tat protected vincristine-treated Kaposi's sarcoma cells from apoptosis and from down-regulation of several anti-apoptotic genes such as AKT-1, AKT-2, BCL2, BCL-XL, and insulin-like growth factor I and induced the de novo expression of the interleukin-3 gene. Moreover, we found that Tat enhanced phosphorylation of AKT and BAD proteins. The inhibition of phosphatidylinositol 3-kinase with two unrelated pharmacological inhibitors, wortmannin and LY294002, abrogated both the anti-apoptotic effect and the phosphorylation of AKT induced by Tat. After treatment with Tat, the AKT enzymatic activity showed a biphasic increase: an early activation (15 min), independent from protein synthesis; and a delayed activation (24 h), which was significantly decreased upon blockage of protein synthesis. Experiments with a function blocking anti-vascular endothelial cell growth factor receptor-2 antibody suggested that both the early and delayed AKT activation and the protection from apoptosis were triggered by the interaction of Tat with vascular endothelial cell growth factor receptor-2. Moreover, experiments with function-blocking antibodies directed against insulin-like growth factor I/insulin-like growth factor I receptor or interleukin-3 indicated their involvement in the delayed activation of AKT and their contribution to the anti-apoptotic effect of Tat on vincristine-treated Kaposi's sarcoma cells.     

Ligand binding and structural analysis of a human putative cellular retinol-binding protein

Three cellular retinol-binding protein (CRBP) types (CRBP I, II, and III) with distinct tissue distributions and retinoid binding properties have been structurally characterized thus far. A human binding protein, whose mRNA is expressed primarily in kidney, heart, and transverse colon, is shown here to be a CRBP family member (human CRBP IV), according to amino acid sequence, phylogenetic analysis, gene structure organization, and x-ray structural analysis. Retinol binding to CRBP IV leads to an absorption spectrum distinct from a typical holo-CRBP spectrum and is characterized by an affinity (K(d) = approximately 200 nm) lower than those for CRBP I, II, and III, as established in direct and competitive binding assays. As revealed by mutagenic analysis, the presence in CRBP IV of His(108) in place of Gln(108) is not responsible for the unusual holo-CRBP IV spectrum. The 2-A resolution crystal structure of human apo-CRBP IV is very similar to those of other structurally characterized CRBPs. The side chain of Tyr(60) is present within the binding cavity of the apoprotein and might affect the interaction with the retinol molecule. These results indicate that human CRBP IV belongs to a clearly distinct CRBP subfamily and suggest a relatively different mode of retinol binding for this binding protein.     

Comparison of two microimmunofluorescence tests for detecting antibodies to Chlamydia pneumoniae

Two microimmunofluorescence (MIF) tests were compared for detection of antibodies to Chlamydia pneumoniae: the microimmunofluorescence of Washington University and the microimmunofluorescence of Chlamydia Serofia. Concordant positive results at the same dilution were observed for IgG in 37.33% of sera tested and concordant negative results were found in 44%. Variations of one fold dilution were observed in 36 sera. Extensive variations (2-3 two-fold dilutions) in the numeric titer values were observed in 20 serum samples with titers of antibody generally higher in the Chlamydia Serofia MIF than in the Washington MIF, resulting in a diagnosis of current infection in three patients. IgM were found with both methods only in one patient. The discrepancies observed may be due to several factors including the different TWAR strain used as antigen in the two tests and the dilution of the FITC-labelled conjugated anti-human IgG.We think that MIF serology may also be influenced by the type of response of the host that may depend on the "local strain" of C. pneumoniae that may express different antigens or in different amounts in comparison with the strains used by the commercial kit.     

Influence of active synaptic pools on the single synaptic event

The activity of the single synapse is the base of information processing and transmission in the brain as well as of important phenomena as the Long Term Potentiation which is the main mechanism for learning and memory. Although usually considered as independent events, the single quantum release gives variable postsynaptic responses which not only depend on the properties of the synapses but can be strongly influenced by the activity of other synapses. In the present paper we show the results of a series of computational experiments where pools of active synapses, in a compatible time window, influence the response of a single synapse of the considered pool. Moreover, our results show that the activity of the pool, by influencing the membrane potential, can be a significant factor in the NMDA unblocking from \(Mg^{2+}\) increasing the contribution of this receptor type to the Excitatory Post Synaptic Current. We consequently suggest that phenomena like the LTP, which depend on NMDA activation, can occur also in subthreshold conditions due to the integration of the dendritic synaptic activity.     

Ubiquitin protease Ubp8 is necessary for S. cerevisiae respiration

Healthy mitochondria are required in cell metabolism and deregulation of underlying mechanisms is often involved in human diseases and neurological disorders. Post-translational modifications of mitochondrial proteins regulate their function and activity, accordingly, impairment of ubiquitin proteasome system affects mitochondria homeostasis and organelle dynamics. In the present study we have investigated the role of the ubiquitin protease Ubp8 in S. cerevisiae respiration. We show that Ubp8 is necessary for respiration and its expression is upregulated in glycerol respiratory medium. In addition, we show that the respiratory defects in absence of Ubp8 are efficiently rescued by disruption of the E3 Ub-ligase Psh1, suggesting their epistatic link. Interestingly, we found also that Ubp8 is localized into mitochondria as single protein independently of SAGA complex assembly, thus suggesting an independent function from the nuclear one. We also show evidences on the importance of HAT Gcn5 in sustaining Ubp8 expression and affecting the amount of protein in mitochondria. Collectively, our results have investigated the role of Ubp8 in respiratory metabolism and highlight the role of ubiquitin related pathways in the mitochondrial functions of S. cerevisiae.     

S-100 protein in the testis

Summary S-100, a protein originally believed to be unique to the nervous system, has recently been found in extraneural cell types. We report here on the presence of S-100 in the testis, namely in Leydig cells and in lymphatic endothelial cells, using immunohistochemical and immunochemical methods. We show that the protein in the testis is immunologically identical to brain S-100. The S-100-labelled cells in the testis exhibit morphological similarities with other cell types in different tissues known to contain S-100.     

Polar lipid composition of chloroplast thylakoids isolated from leaves grown under different lighting conditions

The polar acyl lipid composition was determined for samples of chloroplast thylakoids isolated from Pisum sativum plants grown at light intensities of 50 and 300 E·m^-2·s^-1 and from Aesculus hippocastanum leaves taken from shade or sun environments. Lighting conditions had no major effect on lipid class composition except for a small increase in the amount of monogalactosyldiacylglycerol relative to other lipids in low compared with high light and shade compared with sun conditions. The thylakoids from low light and shade environments also had, relative to those from high light and sun conditions, a substantial decrease in the level of trans-hexadecenoic acid in phosphatidyglycerol. In parallel with this there were lower lipid to chlorophyll ratios, higher overall fatty acid unsaturation, lower chlorophyll a to b ratios and increased relative levels of light harvesting chlorophyll a/b polypeptides as expected for an increase in the degree of thylakoid appression. With this in mind, our results on lipid class composition and content of trans-hexadecenoic acid are discussed in the context of the lateral distribution of lipids within the plane of membrane.Abbreviations DGDG digalactosyldiacylglycerol - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LHC light harvesting chlorophyll a/b - MGDG monogalactosyldiacylglycerol - MPL minor phospholipids - PS1 photosystem one - PS2 photosystem two - SDS sodium dodecyl sulphate - SL sulphoquinovosyldiacylglycerol     

Further investigation on the high-conductance ion channel of the inner membrane of mitochondria

By use of the patch-clamp technique, the inner membrane of mouse liver and heart mitochondria is shown to contain a highly conductive (around 100 pS in symmetrical 150 mM KCl) and voltage-dependent ion channel. This channel closely resembles that previously found in cuprizone-treated mouse liver inner mitochondrial membrane. The paper discusses the electrical properties of the channel and its possible physiological function. The reconstitution in giant liposomes of a partially purified ox heart inner membrane fraction containing the channel and the use of various inhibitors are also presented.