Developmental changes in humoral suppressor activity released from concanavalin A-stimulated human lymphocytes on B cell differentiation

Cell-free culture supernatants (Con A-activated supernatants) were obtained by incubating peripheral blood lymphocytes (PBL) from cord blood, healthy children of various ages, and healthy adults with mitogenic doses of concanavalin A (Con A) for 48 hr. It is well known that human T lymphocytes are activated by Con A to manifest suppressor function in vitro. One mechanism whereby these suppressor cells act has been shown to be by the secretion of a soluble suppressor factor. The present study has investigated the Con A-inducible suppressor cell function in cord blood, children of various ages, and adults by comparing the ability of each Con A-activated supernatant to inhibit the generation of immunoglobulin-producing cells (Ig-PC) in pokeweed mitogen- (PWM) stimulated cultures of adult PBL. Con A-activated supernatants from adults could markedly suppress the generation of Ig-PC by allogeneic as well as autologous PBL in response to PWM. Such suppression appeared to be equally effective on the generation of IG-PC of 3 major classes, IgG, IgM, and IgA. On the contrary, Con A-activated supernatants from cord blood and newborn infants showed only a negligible suppression on PWM-induced adult B cell differentiation. But the suppressor activity found in Con A-activated supernatants gradually increased with advancing age, and reached approximately to the adult level at 4 yr of age or later. The results suggest that human T lymphocytes may be relatively deficient in their Con A-induced suppressor cell function in the early period of life.     

Participation of poly(ADP-ribosyl)ation in the depression of RNA synthesis caused by treatment of mouse lymphoma cells with methylnitrosourea

When mouse lymphoma cells (L-1210) are treated with methylnitrosourea, a DNA-damaging agent, polyadenosine diphosphoribose (poly(ADP-ribose)) synthetase activity increases 5-8-fold in 2-3 h, while RNA polymerase activity remains constant for an initial 2 h and then gradually decreases to 25-30% of the control level in 5 h. Both alpha-amanitin-sensitive and -resistant RNA polymerase activities are depressed to the same degree by the treatment with methylnitrosourea. The depression in RNA synthesis is virtually prevented when the treated cells are cultured in the presence of 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) synthetase. Analyses of the RNA extracted from the cells labeled with [3H]uridine by agarose gel electrophoresis and by poly(U)-Sepharose column chromatography show that the contents of ribosomal precursor RNA and poly(A)-containing RNA are both low in the methylnitrosourea-treated cells as compared with those in the untreated cells and that the reduction in the contents of these kinds of RNA is almost completely prevented by the addition of 3-aminobenzamide to the culture medium. These results suggest that the enhancement of poly(ADP-ribosyl)ation causes the decrease in both synthesis of ribosomal RNA and messenger RNA.     

Mechanisms of in vivo generation of cytotoxic activity against allograft: 1. Local differentiation of mature cytotoxic T lymphocytes in rejection of allogeneic lymphocytes

Although cytotoxic activity was not detected within the spleen and regional lymph nodes from mice immunized sc with allogeneic lymphocytes, such activity was detected consistently in glass-nonadherent and anti-θ-sensitive peritoneal exudate cells (PE cells) from Day 5 after immunization and reached a maximum by Day 7. Immunized spleen cells developed cytotoxic T lymphocytes (CTLs) earlier and more effectively than normal spleen cells when transferred ip into X-irradiated syngeneic normal mice together with immunizing antigen, while they did not become cytotoxic when transferred without antigen. These results suggest that spleen and lymph node cells which may have differentiated into some transitional state by in vivo immunization may differentiate into mature CTLs, following direct contact with antigen at the site of graft. CTLs generated there appear to be responsible for the rejection of allogeneic lymphocytes. Cytotoxicity of PE cells was also generated in X-irradiated mice and augmented cytotoxicity was generated by treatment with cyclophosphamide.     

Molecular phylogeny of Neocalanus copepods in the subarctic Pacific Ocean, with notes on non-geographical genetic variations for Neocalanus cristatus

Monophylies of Neocalanus cristatus, Neocalanus plumchrus andNeocalanus flemingeri were revealed by nucleotide sequencesof mitochondrial 16S rRNA gene (410 bp) but not of nuclear 18SrDNA gene (1802 bp). Intraspecific variations in mitochondrial16S rRNA of N. cristatus collected from geographically distantregions were very low (<0.5%).     

Farnesol-Induced Generation of Reactive Oxygen Species via Indirect Inhibition of the Mitochondrial Electron Transport Chain in the Yeast Saccharomyces cerevisiae

The mechanism of farnesol (FOH)-induced growth inhibition of Saccharomyces cerevisiae was studied in terms of its promotive effect on generation of reactive oxygen species (ROS). The level of ROS generation in FOH-treated cells increased five- to eightfold upon the initial 30-min incubation, while cells treated with other isoprenoid compounds, like geraniol, geranylgeraniol, and squalene, showed no ROS-generating response. The dependence of FOH-induced growth inhibition on such an oxidative stress was confirmed by the protection against such growth inhibition in the presence of an antioxidant such as α-tocopherol, probucol, or N-acetylcysteine. FOH could accelerate ROS generation only in cells of the wild-type grande strain, not in those of the respiration-deficient petite mutant ([rho⁰]), which illustrates the role of the mitochondrial electron transport chain as its origin. Among the respiratory chain inhibitors, ROS generation could be effectively eliminated with myxothiazol, which inhibits oxidation of ubiquinol to the ubisemiquinone radical by the Rieske iron-sulfur center of complex III, but not with antimycin A, an inhibitor of electron transport that is functional in further oxidation of the ubisemiquinone radical to ubiquinone in the Q cycle of complex III. Cellular oxygen consumption was inhibited immediately upon extracellular addition of FOH, whereas FOH and its possible metabolites failed to directly inhibit any oxidase activities detected with the isolated mitochondrial preparation. A protein kinase C (PKC)-dependent mechanism was suggested to exist in the inhibition of mitochondrial electron transport since FOH-induced ROS generation could be effectively eliminated with a membrane-permeable diacylglycerol analog which can activate PKC. The present study supports the idea that FOH inhibits the ability of the electron transport chain to accelerate ROS production via interference with a phosphatidylinositol type of signal.     

Filamentous Bacteriophages of Vibrio parahaemolyticus as a Possible Clue to Genetic Transmission

We have previously reported the isolation and characterization of two filamentous bacteriophages of Vibrio parahaemolyticus, designated Vf12 and Vf33. In this study, to understand the potential of these phages as tools for genetic transmission, we investigated the gene structures of replicative-form (RF) DNAs of their genomes and the distribution of these DNAs on chromosomal and extrachromosomal DNAs. The 7,965-bp nucleotide sequences of Vf12 and Vf33 were determined. An analysis of the overall gene structures revealed that Vf12 and Vf33 had conserved regions and distinctive regions. The gene organization of their conserved regions was similar to that of CTX phage of Vibrio cholerae and coliphage Ff of Escherichia coli, while their distinctive regions were characteristic of Vf12 and Vf33 phage genomes. Southern blot hybridization testing revealed that the filamentous phage genomes integrated into chromosomal DNA of V. parahaemolyticus at the distinctive region of the phage genome and were also distributed on some plasmids of V. parahaemolyticus and total cellular DNAs of one Vibrio damsela and one nonagglutinable Vibrio strain tested. These results strongly suggest the possibilities of genetic interaction among the bacteriophage Vf12 and Vf33 genomes and chromosomal and plasmid-borne DNAs of V. parahaemolyticus strains and of genetic transmission among strains through these filamentous phages.     

Molecular definition of a region of chromosome 21 that causes features of the Down syndrome phenotype

Down syndrome (DS) is a major cause of mental retardation and heart disease. Although it is usually caused by the presence of an extra chromosome 21, a subset of the diagnostic features may be caused by the presence of only band 21q22. We now present evidence that significantly narrows the chromosomal region responsible for several of the phenotypic features of DS. We report a molecular and cytogenetic analysis of a three-generation family containing four individuals with clinical DS as manifested by the characteristic facial appearance, endocardial cushion defect, mental retardation, and probably dermatoglyphic changes. Autoradiograms of quantitative Southern blots of DNAs from two affected sisters, their carrier father, and a normal control were analyzed after hybridization with two to six unique DNA sequences regionally mapped on chromosome 21. These include cDNA probes for the genes for CuZn-superoxide dismutase (SOD1) mapping in 21q22.1 and for the amyloid precursor protein (APP) mapping in 21q11.2-21.05, in addition to six probes for single-copy sequences: D21S46 in 21q11.2-21.05, D21S47 and SF57 in 21q22.1-22.3, and D21S39, D21S42, and D21S43 in 21q22.3. All sequences located in 21q22.3 were present in three copies in the affected individuals, whereas those located proximal to this region were present in only two copies. In the carrier father, all DNA sequences were present in only two copies. Cytogenetic analysis of affected individuals employing R and G banding of prometaphase preparations combined with in situ hybridization revealed a translocation of the region from very distal 21q22.1 to 21qter to chromosome 4q. Except for a possible phenotypic contribution from the deletion of chromosome band 4q35, these data provide a molecular definition of the minimal region of chromosome 21 which, when duplicated, generates the facial features, heart defect, a component of the mental retardation, and probably several of the dermatoglyphic changes of DS. This region may include parts of bands 21q22.2 and 21q22.3, but it must exclude the genes S0D1 and APP and most of band 21q22.1, specifically the region defined by S0D1, SF57 and D21S47.     

Purification, cDNA cloning, and expression of UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III from rat kidney.

UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) catalyzes the addition of N-acetylglucosamine in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-linked sugar chains. The enzyme has been purified over 153,000-fold in 1.5% yield from a Triton X-100 extract of rat kidney by fractionation procedures utilizing QAE-Sepharose, Cu(2+)-chelating Sepharose, and affinity chromatography on UDP-hexanolamine and substrate-conjugated Sepharose. The purified protein migrates as one major and one minor band with apparent molecular masses of 62 kDa and 52 kDa, respectively. The purified enzyme was digested with trypsin, and the amino acid sequences of four peptides were determined. Oligonucleotide primers were designed according to those amino acid sequences and used in the polymerase chain reaction. Screening for the cDNA for GnT-III was carried out by plaque hybridization using a rat kidney cDNA library (lambda gt10) and a polymerase chain reaction product as the probe. Rat kidney GnT-III has 536 amino acids and three putative N-glycosylation sites. There is no sequence homology to other previously cloned glycosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases. The GnT-III activity in transiently transfected COS-1 cells was found to be about 500-3600-fold as compared to that in non- or mock-transfected cells.     

Expansion of murine T cells bearing a unique T cell receptor beta-chain in Friend virus-induced tumor in situ.

Heterogeneity of V alpha 1+ and V beta 10+ TCR alpha beta-chains, which are predominantly used in anti-FBL-3 CTL clones established in vitro, was investigated at a nucleotide level in FBL-3 tumor-infiltrating lymphocytes (TIL) in vivo. The majority (90%) of V beta 10+ beta-chains dominated in TIL used homogeneous V beta 10D beta 2.1 sequences identical to that used in the T cell clones with cytotoxic functions. The homogeneous TCR beta-chain expression was dominant and found to be about 10% of the total TCR beta-chains in the TIL population, which was a greater than 300-to 900-fold increase than in the regional lymph nodes. This is in good agreement with the in vitro data showing that about 11% CTL clones used the homogeneous V beta 10D beta 2.1+ beta-chain. However, the J beta segment does not seem to contribute greatly to the recognition and selection of this TCR because some of homogeneous VD+ beta-chains were associated with J beta segments other than J beta 2.7 of the CTL clones. The frequency of the V alpha 1J alpha 112-2+ alpha-chain expression of the CTL type was much less (3- to 80-fold increase compared to that of lymph node) and also varied in sample materials, indicating the lower contribution of the alpha-chain for the oligoclonality of the TCR. The results were also confirmed by quantitative PCR and RNase protection assays. This suggests that the dominant expression of the homogeneous TCR beta-chain is due to the expansion of the particular anti-FBL-3 CTL in the tumor in situ. Also, the TCR beta-chain, especially the V beta D beta region, rather than alpha-chain is more important for the recognition and selection of the anti-FBL-3 TIL with cytotoxic functions.     

Immunocytochemical and morphometric studies on the development of renin-immunoreactive cells in the rat kidney]

The development of renin-immunoreactive cells was immunocytochemically studied in Wistar-Imamichi rats. The renin-immunoreactivity was localized in the walls of the abdominal aorta, renal artery and arterioles in the kidney. The renin-immunoreactive cells converged with the progress of development from the renal artery via several arterioles to the afferent arteriole in the kidney. In the afferent arteriole, renin-immunoreactive cells appeared only in those of matured glomeruli. The matured glomerulus first appeared at 17 days of gestation followed by the initial appearance of renin-immunoreactive cells at 18 days. The juxtaglomerular index showing the relation between matured glomeruli and renin-immunoreactive cells increased rapidly until 20 days of gestation, shifted thereafter at a level similar to that at 20 days until 7 days after birth, and increased again until 20 days after birth. Although the area of the transverse section at the hilus of the kidney increased all through the period examined, the number of matured glomeruli increased rapidly from 17 days of gestation to 7 days after birth and maintained a fixed level thereafter. This led to the decrease in the number of matured glomeruli per unit area after 7 days after birth. The fine structure of renin-immunoreactive cells showed no difference according to their sites of localization.