Histidine decarboxylase activities in mutant mice deficient in mast cells

The histamine contents were very low in the whole bodies of various types of mutant mice (W^v/W^v, W^v/W, W/W), in which the number of mast cells was decreased, but the L-histidine decarboxylase activities in these mutant mice were not much lower than in control wild type mice. These findings suggest the presence of high histidine decarboxylase activity in cells other than mast cells. Histidine decarboxylase in the whole body of mice was difficult to assay, because the enzyme was rapidly destroyed by proteases, but inclusion of a protease inhibitor, such as Leupeptin, Antipain, Chymostatin, or Pepstatin in the assay mixture permitted the accurate assay of histidine decarboxylase in crude extracts.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

Selective involvement of superoxide anion, but not downstream compounds hydrogen peroxide and peroxynitrite, in tumor necrosis factor-alpha-induced apoptosis of rat mesangial cells

Tumor necrosis factor-alpha (TNF-alpha) induces reactive oxygen species (ROS) that serve as second messengers for intracellular signaling. Currently, precise roles of individual ROS in the actions of TNF-alpha remain to be elucidated. In this report, we investigated the roles of superoxide anion (O-(2)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) in TNF-alpha-triggered apoptosis of mesangial cells. Mesangial cells stimulated by TNF-alpha produced O-(2) and underwent apoptosis. The apoptosis was inhibited by transfection with manganese superoxide dismutase or treatment with a pharmacological scavenger of O-(2), Tiron. In contrast, although exogenous H(2)O(2) induced apoptosis, TNF-alpha-triggered apoptosis was not affected either by transfection with catalase cDNA or by treatment with catalase protein or glutathione ethyl ester. Similarly, although ONOO(-) precursor SIN-1 induced apoptosis, treatment with a scavenger of ONOO(-), uric acid, or an inhibitor of nitric oxide synthesis, N(G)-nitro-L-argininemethyl ester hydrochloride, did not affect the TNF-alpha-triggered apoptosis. Like TNF-alpha-induced apoptosis, treatment with a O-(2)-releasing agent, pyrogallol, induced typical apoptosis even in the concurrent presence of scavengers for H(2)O(2) and ONOO(-). These results suggested that, in mesangial cells, TNF-alpha induces apoptosis through selective ROS. O-(2), but not H(2)O(2) or ONOO(-), was identified as the crucial mediator for the TNF-alpha-initiated, apoptotic pathway.     

5′-O-Dephosphorylated 2′,5′-oligoadenylate (2-5A) with 8-methyladenosine at the 2′-terminus activates human RNase L

Human ribonuclease L (RNase L), an interferon-induced endoribonuclease, becomes enzymatically active after binding to 2-5A. The 5′-phosphoryl group of 2-5A is reportedly necessary for the conformational change leading to RNase L activation. However, we found that 5′-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2′-terminus were more effective as an activator of RNase L than the parent 2-5A tetramer. Introduction of 8-methyladenosine is thought to induce a dramatic shift of 2-5A in the binding site of RNase L.     

Dicer Is Required for Maintaining Adult Pancreas

Dicer1, an essential component of RNA interference and the microRNA pathway, has many important roles in the morphogenesis of developing tissues. Dicer1 null mice have been reported to die at E7.5; therefore it is impossible to study its function in adult tissues. We previously reported that Dicer1-hypomorphic mice, whose Dicer1 expression was reduced to 20% in all tissues, were unexpectedly viable. Here we analyzed these mice to ascertain whether the down-regulation of Dicer1 expression has any influence on adult tissues. Interestingly, all tissues of adult (8–10 week old) Dicer1-hypomorphic mice were histologically normal except for the pancreas, whose development was normal at the fetal and neonatal stages; however, morphologic abnormalities in Dicer1-hypomorphic mice were detected after 4 weeks of age. This suggested that Dicer1 is important for maintaining the adult pancreas.     

Suppression for the proliferation of fibroblasts by external DNA

Phase-contrast and fluorescence microscopic observation showed that DNA added in the cell-culture medium for fibroblasts localized just on the surface of fibroblasts. The DNA bound to fibroblasts was found to be eluted by treating with collagenase. The suppression for the proliferation of fibroblasts by external DNA was confirmed with microscopic observation for the cells cultured in the presence and absence of DNA. Proliferation of the cells decreased from 412 to 155% by the addition of DNA. These results indicate that DNA has an affinity for collagen, the most major extracellular-matrix produced by fibroblasts, and suppresses the growth of fibroblasts.     

A 3-Mb Sequence-Ready Contig Map Encompassing the Multiple Disease Gene Cluster on Chromosome 11q13.1-q13.3

Despite the presence of several human disease genes on chromosome11q13, few of them have been molecularly cloned. Here, we reportthe construction of a contig map encompassing 11q13.1–q13.3using bacteriophage P1 (P1), bacterial artificial chromosome(BAC), and P1-derived artificial chromosome (PAC). The contigmap comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and1 YAC clone and spans a 3-Mb region from D11S480 to D11S913.The map encompasses all the candidate loci of Bardet-Biedlesyndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5),one-third of the distal region for hereditary paraganglioma2 (PGL2), and one-third of the central region for insulin-dependentdiabetes mellitus 4 (IDDM4). In the process of map construction,61 new sequence-tagged site (STS) markers were developed fromthe Not I linking clones and the termini of clone inserts. Wehave also mapped 30 ESTs on this map. This contig map will facilitatethe isolation of polymorphic markers for a more re.ned analysisof the disease gene region and identi.cation of candidate genesby direct cDNA selection, as well as prediction of gene functionfrom sequence information of these bacterial clones.     

Membrane fluidity change in erythrocytes induced by complement system.

The structural change in erythrocyte membranes induced by antibody and complement was studied using phospholipid spin-labels. Sheep erythrocytes were labeled with phosphatidylcholine spin-label and various intermediate cells (erythrocyte-antibody complex (EA), EA bound with complement components from C1 to C7 (EAC1-7), EAC1-8, and EAC1-9) were prepared. Electron spin resonance spectra of EA, EAC1-7, and EAC1-8 were very similar to that of the erythrocytes, while that of EAC1-9 was markedly different. The overall splitting value for the lysed EAC1-9 (53 G) was much smaller than that for the erythrocytes (57 G), indicating a marked fluidization around the phosphatidylcholine label. The unlysed EAC1-9 membranes contained a limited fraction of the fluidized area. When EA was reacted with complement in the presence of 36% bovine serum albumin, the membranes were fluidized similarly to the lysed EAC1-9, although the hemolysis was largely blocked. The membranes of unlysed EAC1-9 prepared in isotonic (ethylenedinitrilo)tetraacetic acid were also fluidized, but to somewhat smaller extent. The role of C9 in the modification of erythrocyte membranes was also demonstrated using Mg2+ ghosts, which were prepared by hypotonic hemolysis in the presence of Mg2+. The membranes of Mg2+ ghost of EAC1-7 were markedly fluidized when bound with C8 and C9, but not affected by binding of C8 only. The component C8 was found to give a latent effect on the membranes that caused irreversible fluidization upon osmotic shock. The terminal component thus creates a fluidized area in the erythrocyte membranes through which small ions and molecules may diffuse more easily and the resulting osmotic unbalance may finally cause hemolysis.