Neutrophil kinetics of recombinant human granulocyte colony-stimulating factor-induced neutropenia in rats.

Single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately induced a decrease in the number of circulating neutrophils in rats. This neutropenia occurred 10 minutes after the injection but disappeared 40 minutes after injection. This transient neutropenia was dose-dependently induced by rhG-CSF and also induced by repeated injections. We studied the kinetics of circulating neutrophils in transient neutropenia. rhG-CSF markedly decreased the number of 3H-diisopropylfluorophosphate (3H-DFP) labeled neutrophils in the circulation 10 minutes after injection but the labeled neutrophils recovered to near the control level 40 minutes after the injection. These results indicate that the neutrophil margination accounts for the neutropenia and the marginated neutrophils return to the circulation.     

Efficient gene delivery into dendritic cells by fiber-mutant adenovirus vectors

Recent studies have demonstrated the usefulness of dendritic cells (DCs) genetically modified by adenovirus vectors (Ad) to immunotherapy, while sufficient gene transduction into DCs is required for high doses of Ad. The RT-PCR analysis revealed that the relative resistance of DCs to Ad-mediated gene transfer is due to the absence of Coxsackie-adenovirus receptor expression, and that DCs expressed adequate alpha(v)-integrins. Therefore, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob can efficiently transduce and express high levels of the LacZ gene into DCs. The gene delivery by fiber-mutant Ad was more efficient than that by conventional Ad in both murine DC lines and normal human DCs (NHDC). Furthermore, NHDC transduced with fiber-mutant Ad and conventional Ad at 8000-vector particles/cell resulted in a 70-fold difference in beta-galactosidase activity. We propose that alpha(v)-integrin-targeted Ad is a very powerful tool with which to implement DC-based vaccination strategies.     

The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force

Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. The energy source of its flagellar motor was investigated. We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar flagellar motor of V. cholerae is driven by an Na+ motive force.     

Seasonal and daily flight timing of oviposition in several stonefly species (plecoptera) in the field

Peak preovipositional flight season and peak preovipositional flight time of day were compared among 12 species of Nemouridae, Chloroperlidae, Perlodidae and Perlidae. Species with a later peak date of preovipositional flight were found to have a later peak preovipositional flight time of day than species with an earlier peak in preovipositional flight season. A later peak preovipositional flight season correlated with a lower light intensity. Similarly, a later peak preovipositional flight time of day correlated with a lower light intensity and a later sunset. Individuals of one species (Sweltsa sp.), whose preovipositional flight date was later, flew over the stream at a later time of day. Species differences in peak preovipositional flight season and peak preovipositional flight time of day may be driven by species specific sensitivity for different light intensities.     

Imaging analysis of subcellular correlation of androgen receptor and estrogen receptor alpha in single living cells using green fluorescent protein color variants

Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically. In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we investigated the subcellular dynamics of AR and ERalpha fused with green fluorescent protein color variants in single living cells using time-lapse microscopy and the technique of fluorescence recovery after photobleaching. AR and ERalpha showed punctate colocalization in the nucleus with estrogen, but not androgen. N-terminal AR deletion mutant did not form a nuclear punctate pattern with either androgen or estrogen. In the presence of AR, but not ERalpha, N-terminal AR deletion mutant formed a punctate nuclear pattern with androgen. AR had different mobility depending on the ligand and the presence of ERalpha. On the other hand, AR had little effect on the stability of ERalpha. ERalpha mutant that does not bind coactivators did not alter the mobility of AR. Taken together, using an imaging technique, we clarified that possible homo/hetero dimerization between AR and ERalpha could be attributed to androgen-estrogen interaction in living cells.     

First isolation of Bartonella henselae type I from a cat-scratch disease patient in Japan and its molecular analysis

We isolated Bartonella henselae from an inguinal lymph node of a 36-year-old male patient with cat-scratch disease. The patient had many areas of erythema on his body, swelling of the left inguinal lymph nodes with pain and slight fever. The diagnosis was made on the basis of polymerase chain reaction for B. henselae DNA from the lymph node biopsies and blood sample, and isolation of the organism, histology of the lymph node and serology with an indirect immunofluorescent antibody test. We also analyzed the genome profiles for five strains of 90 isolates from the lymph node by pulsed-field gel electrophoresis after Not I endonuclease digestion. We found two different genomic profiles. These results suggest that the patient had been either co-infected or re-infected with two genetically different strains of B. henselae.     

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

Ether-linked analogue of 2-arachidonoylglycerol (noladin ether) was not detected in the brains of various mammalian species

2-Eicosa-5',8',11',14'-tetraenylglycerol (2-AG ether, HU310, noladin ether) is a metabolically stable ether-linked analogue of 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand. 2-AG ether has been used as a valuable experimental tool by a number of investigators. Recently, several groups reported that 2-AG ether is present in mammalian brains. We examined in detail whether 2-AG ether actually exists in the brains of various mammalian species. We found that 2-AG ether is not present, at least in an appreciable amount, in the rat brain by gas chromatography-mass spectrometry analysis and fluorometric high performance liquid chromatography analysis. The level of 2-AG ether in the rat brain was below 0.2 pmol/g brain, if at all present. Similar results were obtained for the mouse brain, hamster brain, guinea-pig brain and pig brain. The fact that 2-AG ether was not detected in the brains of various mammalian species is consistent with the fact that an ether bond is formed through enzymatic replacement of the fatty acyl moiety of 1-acyl dihydroxyacetone phosphate by a fatty alcohol, the resultant 1-O-alkyl dihydroxyacetone phosphate being a common intermediate of the biosynthesis of ether-linked lipids in mammalian tissues. It is rather questionable whether 2-AG ether is present in appreciable amounts in the brain and acts as an 'endogenous' cannabinoid receptor ligand.