p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin.     

Lysophospholipids and lysophospholipase D in rabbit aqueous humor following corneal injury

We previously found that lysophosphatidic acid (LPA)-like activity eliciting Cl(-) currents in Xenopus oocytes is increased in rabbit aqueous humor (AH) following corneal freeze wounds. The purpose of this study was to examine whether actual levels of LPA in AH from wounded eyes are higher than those from control eyes, and to determine the sources and enzymatic pathways of AH LPA in control and wounded conditions. Lysophospholipase D (lysoPLD) activity was measured by the enzymatic determination of choline following incubation of AH samples with exogenous lysophosphatidylcholines (LPCs). The molecular species compositions of LPA and LPC in fresh and incubated AH were determined by liquid chromatography-tandem mass spectrometry. A high, but similar activity of lysoPLD in the samples from both control and freeze-wounded eyes was detected. Its enzymatic properties resemble those of plasma lysoPLD, identified as autotaxin. Levels of LPCs, predominant substrates of lysoPLD in AH, were several times higher in the AH samples from injured eyes than those from the control eyes. Our results suggest that lysoPLD is constitutively released from corneal tissues and/or ciliary body into the AH, with no injury-induced increase in release following freeze-wounding. They also suggest that wound-induced increases in LPA-like biological activity are due to linoleoyl species-rich molecular composition in AH from wounded eyes. A possible mechanism of the altered molecular composition is an increase in the AH concentrations of LPCs, linoleoyl species of which are preferentially converted to corresponding unsaturated LPA by the constitutively active lysoPLD.     

Wnt5a regulates distinct signalling pathways by binding to Frizzled2

Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the β‐catenin‐independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin‐mediated route in response to Wnt5a, and inhibition of clathrin‐dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a‐dependent lipoprotein receptor‐related protein 6 (LRP6) phosphorylation and β‐catenin accumulation. Wnt3a‐dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a‐dependent accumulation of β‐catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the β‐catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin‐dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization‐dependent and ‐independent mechanisms.     

Microglia and macrophages as innate producers of interferon-gamma in the brain following infection with Toxoplasma gondii

We previously reported the requirement of interferon-gamma (IFN-gamma) expression by cells other than T and natural killer (NK) cells in the brain, in addition to T cells, for prevention of toxoplasmic encephalitis following infection with Toxoplasma gondii. In the present study, we analysed the identity of the IFN-gamma-producing non-T, non-NK cells in the brain using infected athymic nude and SCID mice that lack T cells but express IFN-gamma in their brains. Intracellular staining for IFN-gamma followed by flow cytometry revealed that approximately 45-60% of the cells expressing IFN-gamma in their brains were positive for CD11b or F4/80 on their surfaces. Smaller portions of the cells were positive for pan-NK marker. Further smaller portions were positive for CD11c, and these cells were less than 5% of the IFN-gamma-expressing cells in brains of infected SCID mice. In addition to IFN-gamma proteins, large amounts of mRNA for IFN-gamma were detected in CD11b+ cells purified from brains of infected mice, but it was not the case in the cells obtained from uninfected animals. In infected SCID mice depleted of NK cells by treatment with anti-asialo-GM1 antibody, cells expressing IFN-gamma in their brains were all positive for CD11b, and the IFN-gamma-producing cells were detected in both CD45low and CD45high populations. These results suggest that CD11b+ CD45low microglia and CD11b+ CD45high blood-derived macrophages are the major non-T, non-NK cells which express IFN-gamma in the brain of mice infected with T. gondii.     

Monophyletic origin of naked barley inferred from molecular analyses of a marker closely linked to the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>)

To elucidate the origin of naked barley, molecular variation of the marker sKT7 tightly linked to the nud locus was examined. A total of 259 (53 wild, 106 hulled domesticated, and 100 naked domesticated) barley accessions were studied. Restriction analysis of the sKT7 PCR-amplified product revealed the alleles I, II, III, and IV. All four alleles were found in wild barley, but allele IV was found only in a single accession from southwestern Iran. Hulled domesticated accessions showed alleles I, II, or III, but all naked domesticated accessions had allele IV. The distribution of allele IV in wild barley and its pervasive presence in naked domesticated lines support the conclusion that naked barley has a monophyletic origin, probably in southwestern Iran. The available results suggest two scenarios for the origin of naked barley: either directly from a wild barley with allele IV or from a hulled domesticated line with allele IV that later became extinct. Naked domesticated accessions from different regions of the world have extremely homogeneous DNA sequences at the sKT7 locus, supporting the monophyletic origin of naked barley. For allele IV, four haplotypes (IVb to IVe) were found in 30 naked accessions: IVb was predominant (66.7%) and widely distributed, while the other three haplotypes, differing by only one nucleotide at different positions relative to IVb, showed a localized distribution. The geographical distribution of the haplotypes of sKT7 allele IV suggests migration routes of naked domesticated barley in central and eastern Asia.Communicated by F. Salamini     

Crystal structures of the apo- and holomutant human lysozymes with an introduced Ca2+ binding site

The three-dimensional structures of apo- and holomutant human lysozymes (D86/92 lysozyme), in which a calcium binding site was designed and created for enhancing molecular stability by replacing both Gln86 and Ala92 with aspartic acids, were refined at 1.8-A resolution by x-ray crystallography. The overall structures and crystallographic thermal factors of all three proteins, the apo-, holo-D86/92, and the wild-type human lysozymes, were essentially identical; these results showed that the introduction of the calcium binding site did not affect either the overall structure or molecular rigidity of the proteins. However, structure analyses of the apo-D86/92 lysozyme revealed that the mutations affected the side chain conformation of residue 86 and hydrogen networks between the protein and the internal solvent molecules. In the structure of the holo-D86/92 lysozyme, seven oxygen ligands formed a slightly distorted pentagonal bipyramid around the calcium ion, indicating that the coordination around the calcium ion was quite similar to that in baboon alpha-lactalbumin. The pentagonal bipyramid coordination could be one of the most widely found and appropriate calcium binding schemes in proteins.     

A bivalent single-chain Fv fragment against CD47 induces apoptosis for leukemic cells

We constructed a single-chain antibody fragment (scFv) of murine monoclonal antibody, MABL, which specifically bound to human CD47 (hCD47) and induced apoptosis of the leukemic cells. The scFv of MABL antibody with a 15-residue linker (MABL scFv-15) formed both dimer (Mr 50 kDa) and monomer (Mr 25 kDa). Both MABL scFv-15 dimer and monomer had binding activity for hCD47. MABL scFv-15 dimer strongly induced apoptosis of hCD47-introduced mouse leukemic cells in vitro and exhibited anti-tumor effect in a myeloma transplanted mice model. However, MABL scFv-15 monomer scarcely exhibited these activities. These results strongly demonstrate that the ligation of CD47 antigen by two antigen-binding sites of MABL dimer is needed for inducing apoptosis. The parent MABL antibody caused hemagglutination due to the CD47 expressed on erythrocytes. Interestingly, MABL scFv-15 dimer did not cause hemagglutination. This apoptosis-inducing dimer appears to be a lead candidate for novel leukemic therapy.     

Isolation and characterization of a novel 2-<Emphasis Type=Italic>sec</Emphasis>-butylphenol-degrading bacterium <Emphasis Type=Italic>Pseudomonas</Emphasis> sp. strain MS-1

A novel bacterium capable of utilizing 2-sec-butylphenol as the sole carbon and energy source, Pseudomonas sp. strain MS-1, was isolated from freshwater sediment. Within 30 h, strain MS-1 completely degraded 1.5 mM 2-sec-butylphenol in basal salt medium, with concomitant cell growth. A pathway for the metabolism of 2-sec-butylphenol by strain MS-1 was proposed on the basis of the identification of 3 internal metabolites—3-sec-butylcatechol, 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid, and 2-methylbutyric acid—by gas chromatography-mass spectrometry analysis. Strain MS-1 degraded 2-sec-butylphenol through 3-sec-butylcatechol along a meta-cleavage pathway. Degradation experiments with various alkylphenols showed that the degradability of alkylphenols by strain MS-1 depended strongly on the position (ortho ≫ meta = para) of the alkyl substitute, and that strain MS-1 could degrade 2-alkylphenols with various sized and branched alkyl chain (o-cresol, 2-ethylphenol, 2-n-propylphenol, 2-isopropylphenol, 2-sec-butylphenol, and 2-tert-butylphenol), as well as a dialkylphenol (namely, 6-tert-butyl-m-cresol).     

Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast.

SMT3 of Saccharomyces cerevisiae is an essential gene encoding a ubiquitin-like protein similar to mammalian SUMO-1. When a tagged Smt3 or human SUMO-1 was expressed from GAL1 promoter, either gene rescued the lethality of the smt3 disruptant. By indirect-immunofluorescent microscopy, the HA-tagged Smt3 was detected mostly in nuclei and also at the mother-bud neck just like septin fibers. Indeed immunoprecipitation experiments revealed that Cdc3, one of septin components, was modified with Smt3. Furthermore, the protein level of the Cdc3-Smt3 conjugate was reduced and the septin rings disappeared in a ubc9-1 mutant at a restrictive temperature, where the Smt3 conjugation system should be defective. Thus, we conclude that Smt3 was conjugated to Cdc3 in septin rings localized at the mother-bud neck. Around the time of cytokinesis the Cdc3-Smt3 conjugate disappeared. We discuss the biological significance of this Smt3 conjugation to a septin component.     

Effect of absolute and relative changes of hematocrit on erythropoiesis in mice

The serial changes of peripheral reticulocytes and marrow erythroid progenitor cells (CFU-e) in mice were monitored under the conditions of absolute or relative changes in red cell mass to study the regulatory mechanism of erythropoiesis. A decreased number of marrow CFU-e and peripheral reticulocytes was observed in the mice with relative polycythemia induced by dehydration as well as in the mice with absolute polycythemia induced by hypertransfusion. On the other hand, a transient increase in the number of marrow CFU-e followed by a gradual increase in the number of peripheral reticulocytes was seen after a considerable amount of exsanguination. Similar stimulatory effects on marrow CFU-e were also observed either by rehydrating the dehydrated mice or by overhydrating the untreated mice to relatively decrease the level of hematocrit. The results suggested that in addition to factors relating to the balance between oxygen supply and requirement, which has been well known, erythropoiesis is greatly affected by hematocrit.