cDNA cloning and characterization of porcine histamine H4 receptor

Fast muscle myosin responds in similar way to F-actin and to phalloidin F-actin. It is activated 7.5 fold at infinite F-actin concentration and 6.8 fold at infinite phalloidin F-actin. The actomyosin dissociation constants are 0.89 +/- 0.34 microM with F-actin and 0.90 +/- 0.71 microM with phalloidin F-actin. Slow muscle myosin responds differently to F-actin and to phalloidin F-actin. It is activated 3.76 fold at infinite F-actin concentration and only 2.27 fold at infinite phalloidin F-actin concentration. The actomyosin dissociation constants are 1.95 +/- 1.27 microM with F-actin and 0.27 +/- 0.16 microM with phalloidin F-actin. At first glance this means that substitution of F-actin with phalloidin F-actin magnifies the difference between fast muscle and slow muscle myosins. Furthermore the change of the dissociation constants may affect the contractile force of the attached crossbridge.     

Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses. Purification and characterization.

A novel trypsin-type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N-terminal 44-amino-acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di-isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high-ionic-strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose-dependent manner, although human mast cell tryptase beta and rat mast cell tryptase (rat MCP-7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.     

Comparative analysis of the structure and chromosomal assignment ofCD1: an evidence for different evolutionary histories between classic CD1 and CD1D class genes

The non-MHC-encoded CD1 family has recently emerged as a novel antigen-presenting system that is distinct from MHC class I and class II molecules. In the present study, we determined the genomic structure of that rat CD1, and compared with those of other previously reported CD1 genes. Rat CD1 was extremely similar to mouse CD1 genes, especially to CD1D1. It is of interest that a tyrosine-based motif for endosomal localization, identified in the human CD1b cytoplasmic tail, was conserved in all CD1 molecules except for CD1a, that was encoded by a single short exon. Comparison of the overall exon-intron organization of CD1 genes revealed that the length of the introns was also characteristic to each of the two classes of CD1 genes; classic (CD1A, CD1B, CD1C and CD1E), and CD1D, which have been categorized by comparison of coding regions. These findings support a hypothesis that the two classes have different evolutionary histories. In contrast to the absence of the classic CD1 genes in rats and mice, the entire region of nonpolymorphic CD1D gene has been conserved through mammalian evolution. Furthermore, we determined chromosomal localization of rat CD1 gene using the fluorescence in situ hybridization method with several probes derived from genomic rat CD1 clones. Similar to human and mouse CD1, rat CD1 mapped outside the MHC loci despite the structural and functional resemblance to MHC. Conserved syntheny of chromosomal segments of RNO2 and MMU3 is implied.     

Glutamate triggers elevation of intracellular Ca2+ concentration in neural precursor cells

Both neurons and glial cells are derived from neuralprecursor cells in the ventricular zone during braindevelopment. The fate of the neural precursor cells isaffected by neurotransmitters such as glutamate. Inthis study, we examined glutamate-triggeredintracellular Ca²⁺ signaling in neural precursorcell lines by the calcium digital imaging method. Whenimmortalized primary-cultured neural precursor cellswere treated with glutamate, a subpopulation of thesecells showed an increase in intracellular Ca²⁺concentration. In an effort to determine the role ofthe glutamate-triggered intracellular Ca²⁺ signalin neural precursor cells, we tried to cultureimmortalized basal ganglial and hippocampal neuralprecursor cell lines in glutamate-free medium. Thehippocampal (MHP-2) cells became adapted to theglutamate-free medium, and when treated with glutamatethe adapted subline (MHP-2-E1) showed an increase inintracellular Ca²⁺ concentration. In contrast,the basal ganglial neural precursor cell lines failedto become adapted to the glutamate-free medium. Theseresults suggest that hippocampal and basal ganglialneural precursor cells differ in their cellularresponse to glutamate as an exogenous stimulus.     

Apoptosis in antibody-dependent monocyte-mediated cytotoxicity with monoclonal antibody 17-1A against human colorectal carcinoma cells: enhancement with interferon γ

 Antibody-dependent cell-mediated cytotoxicity (ADCC) has been considered to be one of the main effector mechanisms by which unconjugated monoclonal antibody (mAb) 17-1A can exert an antitumor effect in vivo. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by monocytes (ADMC) using mAb 17-1A as an antibody and the human colorectal carcinoma cell line, COLO205, as target cells in vitro. The implications of the apoptosis during ADMC was demonstrated by means of both a DNA fragmentation assay and a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, interferon γ (IFNγ) was also found to enhance the induction of apoptosis significantly. The addition of superoxide dismutase did not reduce the level of the apoptosis, although superoxide anion (O₂ ^–) was observed to be produced. However, the release of tumor necrosis factor α (TNFα) was significantly enhanced during ADMC, while, in addition, apoptosis was significantly inhibited by the addition of anti-TNFα antibody. These findings indicated that apoptosis might be implicated in ADMC with mAb 17-1A, which was augmented by IFNγ, while, in addition, TNFα may also be one of the major mediators of apoptosis. Received: 1 August 1996 / Accepted: 27 August 1996     

Multi-stage biofilm reactor for acetic acid production at high concentration

Summary Continuous production of acetic acid by liquid surface culture ofAcetobacter aceti M7 was investigated using a Multi-Stage Biofilm Reactor (MSBFR) composed of ten shallow flow horizontal reactors of laboratory scale. With varying dilution rate in the range from 0.049 to 0.2 h^–1, the maximum exit acetic acid concentration reached was as high as 98.0 g/l at the lowest dilution rate with step feed of ethanol-rich medium to stages 3, 5, and 7. The production rate (4.3 g/l/h) was rather high considering the inhibitory effect of high acetic acid concentration. This may be ascribed to non-homogeneous distribution of acetic acid concentration in the bioreactor and step feed of ethanol-rich medium.     

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C₂₈- and C₂₉-Δ²⁴⁽²⁸⁾-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ²⁴⁽²⁵⁾ intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-²H₃,28-¹³C)-24-methylenecholesterol, followed by ¹³C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ²⁴⁽²⁵⁾ intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.