Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Cell Sorting and Chondrogenic Aggregate Formation in Limb Bud Recombinants and in Culture

The cartilage pattern of the developing chick limb changes along the proximal-distal (PD) axis. It is assumed that these spatial changes are brought about by differences in the cellular properties of distal mesoderm, the progress zone (PZ). To examine whether these differences are actually maintained in the individual cells composing the PZ, we dissociated early (stage 20) and late (stage 25) PZ tissues into single cells, then mixed and recombined them with ectodermal jackets. The recombinants were grafted to limb bud stumps and allowed to develop into limb-like structures. Early PZ cells were distributed within whole cartilage elements along the PD axis of the limb-like structures, while cells from late PZ participated only in the formation of distal cartilage elements.
A difference in distribution pattern between the cells of early and late PZ in mixed culture was also observed. Cells of early PZ aggregated rapidly in patches and formed cartilage nodules, while the cells of late PZ distributed in regions surrounding these cell aggregates and gradually differentiated to cartilage cells. These results suggest that the cellular properties in the PZ concerning the rate of chondrogenic aggregate formation change during limb bud development, and that this change may relate to the cartilage pattern formation along the PD axis.     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

Characteristics of specific125I-ω-conotoxin GVIA binding in rat whole brain

Characteristics of specific¹²⁵I-omega-conotoxin (-CgTX) binding were systematically investigated in crude membranes from rat whole brain. Kd and Bmax Values for the binding were 49.7 pM and 181.5 fmol/mg of protein, respectively. The effects of various types of Ca channel antagonists on the binding were investigated. Dynorphin A (1–13), in particular, specifically inhibited¹²⁵I--CgTX binding, but not that of [³H](+)PN200-110. Spider venom fromPlectreurys tristes did not specifically inhibit specific binding of¹²⁵I--CgTX, because the venom also inhibited the binding of [³H](+)PN200-110 to a similar degree. The amount of specific binding of¹²⁵I--CgTX was less in the cerebellum than that in any other area of whole brain. The cross-linker disuccinimidyl suberate did not label with¹²⁵I--CgTX and its binding sites in rat whole brain, although it did in chick whole brain, which was used as a positive control. These findings suggested that dynorphine A (1–13) was a selective blocker of -CgTX-sensitive Ca channels in crude membranes from rat whole brain and that -CgTX-sensitive Ca channels were mainly present a rat brain except cerebellum.     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN, IFN treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

Biosynthesis of brassinolide from castasterone in cultured cells of Catharanthus roseus

Feeding experiments with tritium- and deuterium-labeled castasterone (CS) were conducted with three cell lines of Catharanthus roseus, including crown gall cells and nontransformed cells. In all three cell lines, the conversion of CS to brassinolide (BL) was observed and unequivocally confirmed by gas chromatography/mass spectrometry (GC/MS). This is the first conclusive evidence that CS is the biosynthetic precursor of BL.Biosynthesis of brassinosteroids in Catharanthus roseus. Part II. Part I of this series: Yokota et al. (1990a).     

Long-term follow up of patients with common variable immunodeficiency treated with intravenous immunoglobulin: Reevaluation of intravenous immunoglobulin replacement therapy

Five patients with common variable immunodeficiency treated in our hospital between December 1979 and December 1990 were given six kinds of intravenous immunoglobulin preparations (pepsin treated, S-sulfonated, polyethylene glycol treated, pH4 treated, alkylated, and pH4.25 formulation preparation) for replacement therapy. Duration of the therapy ranged from 7.6 to 11 years. Incidences of fever and acute infections were variable among patients, but no significant differences were seen in the incidences among periods given each preparation. Three cases revealed abnormal pulmonary functions in tests. Adverse reactions were rarely seen in our study periods, and no severe reactions were observed. No significant differences were seen in incidences of adverse reactions. Postinfusion levels of serum complement slightly decreased from preinfusion levels. However, the decrease in complement was not related to any adverse reaction. No long-term complications such as transmission of hepatitis have been observed. Our data suggest that no obvious differences exist between the efficacy and safety of each IVIG preparation. Differences of efficacy of IVIG replacement therapy may be due to the variable pathophysiology of each patient.Abbreviations CVID common variable immunodeficiency - IVIG intravenous immunoglobulin     

First finding of urease-positive thermophilic strains of Campylobacter in river water in the Far East, namely, in Japan and their phenotypic and genotypic characterization

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter , respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

Compilation of All Genes Encoding Bacterial Two-component Signal Transducers in the Genome of the Cyanobacterium, Synechocystis sp. Strain PCC 6803

Bacteria have devised sophisticated signaling systems for elicitinga variety of adaptive responses to their environment, whichare generally referred to as the "two-component regulatory system."The widespread occurrence of the two-component systems in bothprokaryotes and eukaryotes implies that it is a powerful devicefor a wide variety of adaptive responses of cells to their environment.The two-component signal transducers contain one or more ofthree conserved and characteristic phosphotransfer signalingdomains, named the "transmitter, receiver, and alternative transmitter."The recently determined entire genomic sequence of Synechocystissp. strain PCC 6803 allowed us to compile systematically a completelist of genes encoding such two-component signal transductionproteins. The results of such an effort, made in this study,revealed that at least 80 ORFs were identified as members ofthe two-component signal transducers in this single speciesof cyanobacteria.