Antiviral immune responses in the absence of organized lymphoid T cell zones in plt/plt mice

The paucity of lymph node (LN) T cells (plt) mutation in mice results in strongly reduced T cell numbers in LNs and homing defects of both dendritic cells (DCs) and naive T cells. In this study, we investigated the functional significance of the plt phenotype for the generation of antiviral immune responses against cytopathic and noncytopathic viruses. We found that DC-CD8(+) T cell contacts and the initial priming of virus-specific T cells in plt/plt mice occurred mainly in the marginal zone of the spleen and in the superficial cortex of LNs. The magnitude of the initial response and the maintenance of protective memory responses in plt/plt mice was only slightly reduced compared with plt/+ controls. Furthermore, plt/plt mice mounted rapid neutralizing antiviral B cell responses and displayed normal Ig class switch. Our data indicate that the defective homing of DCs and naive T cells resulting from the plt/plt mutation results in a small, but not significant, effect on the induction of protective antiviral T and B cell immunity. Overall, we conclude that the spatial organization of secondary lymphoid T cell zones via the CCR7-CC chemokine ligand 19/CC chemokine ligand 21 pathway is not an absolute requirement for the initial priming and the maintenance of protective antiviral T and B cell responses.     

Evolution of Human Polyomavirus JC: Implications for the Population History of Humans

The polyomavirus JC virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy, is ubiquitous in the human population, infecting children asymptomatically, then persisting in the kidney. The main mode of transmission of JCV is from parents to children through long-term cohabitation. Twelve JCV subtypes that occupy unique domains in Europe, Africa, and Asia have been identified. Here, we attempted to elucidate the evolutionary relationships among JCV strains worldwide using the whole-genome approach with which a highly reliable phylogeny of JCV strains can be reconstructed. Sixty-five complete JCV DNA sequences, derived from various geographical regions and belonging to 11 of the 12 known subtypes, were subjected to phylogenetic analysis using three independent methods: the neighbor-joining, maximum parsimony, and maximum likelihood methods. The trees obtained with these methods consistently indicated that ancestral JCVs were divided into three superclusters, designated as Types A, B, and C. A split in Type A generated two subtypes, EU-a and -b, mainly containing European and Mediterranean strains. The first split in Type B generated Af2 (the major African subtype). Subsequent splits in Type B generated B1-c (a minor European subtype) and all seven Asian subtypes (B1-a, -b, -d, B2, MY, CY, and SC). Type C generated a single subtype (Af1), consisting of strains derived from western Africa. While the present findings provided a basis on which to classify JCV into types or subtypes, they have several implications for the divergence and migration of human populations. Received: 4 April 2001 / Accepted: 31 July 2001     

5-Halogenated thymidine analogues induce a senescence-like phenomenon in HeLa cells

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.     

Histochemical analysis of macrophage migration inhibitory factor in psoriasis vulgaris

Psoriasis is a persistent cutaneous disease characterized by skin inflammation and infiltration of immunocytes such as lymphocytes and monocytes/macrophages, concomitant with abnormal epidermal hyperproliferation. We previously showed that the serum level of macrophage migration inhibitory factor (MIF) and its production by peripheral blood mononuclear cells of patients with psoriasis were closely correlated with the severity of clinical symptoms; however, the precise role of MIF in psoriatic epidermis remains to be clarified. The current study was carried out to elucidate the possible involvement of MIF in psoriasis, using immunohistochemistry and in situ hybridization. In contrast to elevated serum MIF in psoriasis, MIF-positive staining in the lesional psoriatic epidermis was significantly decreased, as demonstrated by immunohistochemical analysis using an anti-MIF antibody. Consistent with this finding, we found, by in situ hybridization, that MIF mRNA concomitantly decreased in the psoriatic lesions. Although the reason for the different MIF levels in the psoriatic epidermis and in the circulation remains unknown, it is hypothesized that MIF, a potential growth factor, might be decreased in psoriatic lesions to counterregulate the abnormal epidermal proliferation caused by dysregulation of cytokines and growth factors.     

Novel carbohydrate specificity of the 16-kDa galectin from Caenorhabditis elegans: binding to blood group precursor oligosaccharides (type 1, type 2, Talpha,and Tbeta) and gangliosides

Galectins, a family of soluble beta-galactosyl-binding lectins, are believed to mediate cell-cell and cell-extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydrate-binding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galbeta1,3GlcNAc, and type 2, Galbeta1,4GlcNAc; Talpha, Galbeta1,3GalNAcalpha; Tbeta, Galbeta1,3GalNAcbeta) and gangliosides containing the Tbeta structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66-69, which enables interactions of Glu(67) with both axial and equatorial -OH at C-3 of GlcNAc (in Galbeta1,4GlcNAc) or at C-4 of GalNAc (in Galbeta1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.     

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.     

Complete deoxygenation from a hemoglobin solution by an electrochemical method and heat treatment for virus inactivation

Hemoglobin (Hb) has been widely studied as a raw material for various types of oxygen carriers. In the purification of Hb from red blood cells including virus inactivation and denaturation of other proteins and the long-term storage of Hb vesicles (HbV), a deoxygenation process is one of the important processes because of the high stability of deoxygenated Hb to heating and metHb formation. Though an oxygenated Hb solution can be deoxygenated with an artificial lung, it is difficult to reduce the oxygen partial pressure of the Hb solution to less than 10 Torr. We developed an electrochemical system for complete deoxygenation of the Hb solution at the cathode compartment using hydrogen containing nitrogen gas at the anode compartment. Oxygen in the Hb solution was reduced to OH(-) at the cathode compartment within several minutes at a potential value of -1.67 V and was finally converted to water by neutralization with H(+) from the anode in the whole system. The resulting completely deoxygenated Hb could tolerate heat treatment at 62 degrees C for 10 h with no denaturation of deoxygenated Hb. The metHb formation rate of reoxygenated Hb at 37 degrees C was not changed after heat treatment. Furthermore, vesicular stomatitis virus (VSV) could be inactivated at an inactivation degree of more than 5.96 log by heat treatment.     

Isolation and characteristics of trypsin from pyloric ceca of the starfish Asterina pectinifera

Trypsin was purified from pyloric ceca of the starfish Asterina Pectinifera by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 28000. Optimum pH and temperature of A. pectinifera trypsin for hydrolysis of N(alpha)-p-Tosyl-L-arginine methyl ester hydrochloride were approximately pH 8.0 and 55 degrees C, respectively. A. pectinifera trypsin was unstable at above 50 degrees C and below pH 5.0, and was not activated by adding Ca(2+). The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was found.