Synthesis and evaluation of pseudopeptide analogues of a specific CXCR4 inhibitor, T140: the insertion of an (E)-alkene dipeptide isostere into the betaII'-turn moiety

A 14-residue peptide, T140, strongly inhibits the T-cell line-tropic HIV-1 (X4-HIV-1) infection, since this peptide functions as a specific antagonist against a chemokine receptor, CXCR4. T140 takes an antiparallel beta-sheet structure with a type II' beta-turn. In the present paper, we have designed and synthesized several T140 analogues, in which an (E)-alkene dipeptide isostere was inserted into the type II' beta-turn moiety, as a bridging study to develop nonpeptidic CXCR4 inhibitors. It has been proven that the turn region of T140 can be replaced by the above surrogate with the maintenance of strong anti-HIV activity.     

Flow cytometry analysis of changes in the DNA content of the polychlorinated biphenyl degrader Comamonas testosteroni TK102: effect of metabolites on cell-cell separation

Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four bph genes, bphA1, bphB, bphC, and bphD, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the bphB gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.     

Rapid Assessment of the Physiological Status of the Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102 by Flow Cytometry

The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R² = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = −0.9776 x + 98.36; R² = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (≤1 h) and distinguishes correctly among live, dead, and permeabilized cells.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins II: public release of inaugural version of InGaP database containing gene/protein expression profiles for 127 mouse KIAA genes/proteins.

The inaugural version of the InGaP database (Integrative Gene and Protein expression database; http://www.kazusa.or.jp/ingap/index.html) is a comprehensive database of gene/protein expression profiles of 127 mKIAA genes/proteins related to hypothetical ones obtained in our ongoing cDNA project. Information about each gene/protein consists of cDNA microarray analysis, subcellular localization of the ectopically expressed gene, and experimental data using anti-mKIAA antibody such as Western blotting and immunohistochemical analyses. KIAA cDNAs and their mouse counterparts, mKIAA cDNAs, were mainly isolated from cDNA libraries derived from brain tissues, thus we expect our database to contribute to the field of neuroscience. In fact, cDNA microarray analysis revealed that nearly half of our gene collection is predominantly expressed in brain tissues. Immunohistochemical analysis of the mouse brain provides functional insight into the specific area and/or cell type of the brain. This database will be a resource for the neuroscience community by seamlessly integrating the genomic and proteomic information about the mouse KIAA genes/proteins.     

Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences

Abstract The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of " Sporolactobacillus dextrus " and Bacillus coagulans . Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type of S. inulinus . The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accomodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus .     

Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria

The 16S rRNA or rRNA gene sequences of the type strains of 5 species of Rhodobacter, Rhodopseudomonas blastica and Paracoccus denitrificans were determined. The sequence analysis revealed that Rhodobacter species, whose intracytoplasmic membrane systems were characteristically vesicular, composed a sole cluster. Rhodopseudomonas blastica, whose intracytoplasmic membrane system was lamellar, was included in the cluster of Rhodobacter. The phylogenetic co-clustering of these bacteria conformed to their possessing of the identical types of carotenoids. Paracoccus denitrificans, which is nonphototrophic, is a right member of the Rhodobacter cluster. Rhodobacter species, Rhodopseudomonas blastica and Paracoccus denitrificans are apart from the other phototrophic bacteria and have the common deletions of 21 bases at the positions 1258 to 1278 (Escherichia coli numbering system). It was demonstrated that the morphological character intracyto-plasmic membrane structure, that has been regarded as a generic criterion does not reflect the phylogeny in the phototrophic bacteria. The transfer of Rhodopseudomonas blastica to the genus Rhodobacter is proposed.     

High avidity cytokine autoantibodies in health and disease: Pathogenesis and mechanisms

Numerous reports have documented the presence of autoantibodies working against naturally occurring cytokines in humans in health and disease. In most instances, their physiological and pathophysiological significance remains unknown. However, recent advances in the methodologies for detecting cytokine autoantibodies and their application in research focused on specific disorders have shown that some cytokine autoantibodies play an important role in the pathogenesis of disease. Additionally, levels of cytokine autoantibodies may also correlate with disease severity and progression in certain infectious and autoimmune diseases but not in others. This suggests that cytokine-specific pathogenic differences exist. While multiple lines of evidence support the notion that high avidity cytokine autoantibodies are present and likely to be ubiquitous in healthy individuals, their potential physiological role, if any, is less clear. It is believed that they may function by scavenging pro-inflammatory cytokines and thereby inhibiting deleterious ‘endocrine’ effects, or by serving as carrier proteins, providing a ‘reservoir’ of inactive cytokines and thus modulating cytokine bioactivity. A central hypothesis is that sustained or repeated high-level exposure to cytokines triggers defects in T-cell tolerance, resulting in the expansion of existing cytokine autoantibody-producing B cells.     

Regulation of cell-to-cell communication mediated by astrocytic ATP in the CNS

It has become apparent that glial cells, especially astrocytes, not merely supportive but are integrative, being able to receive inputs, assimilate information and send instructive chemical signals to other neighboring cells including neurons. At first, the excitatory neurotransmitter glutamate was found to be a major extracellular messenger that mediates these communications because it can be released from astrocytes in a Ca(2+)-dependent manner, diffused, and can stimulate extra-synaptic glutamate receptors in adjacent neurons, leading to a dynamic modification of synaptic transmission. However, recently extracellular ATP has come into the limelight as an important extracellular messenger for these communications. Astrocytes express various neurotransmitter receptors including P2 receptors, release ATP in response to various stimuli and respond to extracellular ATP to cause various physiological responses. The intercellular communication "Ca(2+) wave" in astrocytes was found to be mainly mediated by the release of ATP and the activation of P2 receptors, suggesting that ATP is a dominant "gliotransmitter" between astrocytes. Because neurons also express various P2 receptors and synapses are surrounded by astrocytes, astrocytic ATP could affect neuronal activities and even dynamically regulate synaptic transmission in adjacent neurons as if forming a "tripartite synapse". In this review, we summarize the role of astrocytic ATP, as compared with glutamate, in gliotransmission and synaptic transmission in neighboring cells, mainly focusing on the hippocampus. Dynamic communication between astrocytes and neurons mediated by ATP would be a key event in the processing or integration of information in the CNS.     

Formation of high molecular weight caspase-3 complex in neonatal rat brain

Caspase-3 plays an essential role in normal brain development. Recently, a large protein complex known as apoptosome, which catalyzes the activation of caspase-3, has been reported. To investigate structural characteristics of caspase-3 in the developing brain, rat neonatal cortex extract was analysed by gel filtration chromatography. We show here the formation of high molecular complex including procaspase-3 in the extract. When the extract was activated by cytochrome c, caspase-3 recruitment to the apoptosome was not observed, although apoptotic protease activating factor-1 (Apaf-1), caspase-9, and X-linked inhibitor of apoptosis protein (XIAP) existed in the apoptosome. These results indicate that procaspase-3 exists as a high molecular weight complex during brain development.     

Superoxide production from human polymorphonuclear leukocytes by human mannan-binding protein (MBP)

Mannan-binding protein (MBP) is a Ca(2+)-dependent mammalian lectin that plays an important role in innate immunity. In this study, we found that ligand-bound MBP stimulates polymorphonuclear leukocytes (PMN) to induce cell aggregation and superoxide production. The biological response of PMN to ligand-bound MBP was dose- and time-dependent. The PMN aggregation and superoxide production induced by ligand-bound MBP was blocked completely by pertussis toxin, and partially blocked by a platelet activation factor receptor antagonist, TCV-309. These findings suggest that the ligand-bound MBP stimulates PMN through a putative MBP receptor(s) on PMN.