Further characterization of the autologous mixed lymphocyte reaction: induction of double negative gamma delta T lymphocytes.

To investigate the specific nature of the autologous mixed lymphocyte reaction (AMLR), we applied a method in which mixtures of NY-nonadherent responder cells and NY-adherent stimulator cells were treated with neuraminidase before culture and then cultured to assay the AMLR. This method produced a marked enhancement of DNA replication in the responder cells and the results were reproducible, regardless of the individuals tested. Using this method, we were able to make the following observations regarding the specific nature of the AMLR. (i) The AMLR is an IL-2-independent reaction, as revealed by bioassay to detect the presence of IL-2 by a blocking test using anti-IL-2R sera and as shown by the absence of mRNA for IL-2 in Northern hybridization. (ii) It is also HLA-DR dependent as proven by the fact that anti-DR sera almost completely inhibited the reaction. (iii) The AMLR was also found to induce the generation of activated CD4+ helper T cells in direct response to stimulation by NY-adherent cells, in which HLA-DR antigens were involved. (iv) Also, it induced the generation of CD4-CD8- double-negative (DN) lymphocytes, including gamma delta T cells with a cytotoxic activity against NK-resistant target cells and with a variety of lymphocyte activation markers (CD56, HLA-DR, CD25, transferrin receptors, CD38, and LFA-1). However, the AMLR did not induce the generation of NK cell markers CD16 and CD57. (v) The DN lymphocytes and gamma delta T cells appeared to be generated from the precursors of CD4-CD8- DN cells, in direct response to the stimulator cells. These results strongly suggest that the AMLR may be a phenomenon which induces the proliferative response of gamma delta T cells and their precursors, in addition to that of alpha beta T cells, particularly of CD4+ helper T cells.     

Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis , were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.     

Construction of shuttle vectors derived fromAcetobacter xylinum for cellulose-producing bacteriumAcetobacter xylinum

Summary Shuttle vector pUF106 was constructed by ligation ofAcetobacter xylinum plasmid pFF6 toEscherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. pUF106 transformed cellulose-producingA. xylinum ATCC10245 as well asE. coli JM109.     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

Budding-specific lectin induced in epithelial cells is an extracellular matrix component for stem cell aggregation in tunicates.

We have examined immunocytochemically the expression, localization and in vivo function of a calcium-dependent and galactose-binding 14 x 10(3) Mr lectin purified from the budding tunicate, Polyandrocarpa misakiensis. Lectin granules first appeared in the inner epithelium of a double-walled bud vesicle. Soon after the bud entered the developmental phase, the granules were secreted into the mesenchymal space, where the lectin-positive extracellular matrix (ECM) developed. The lectin was also produced and secreted by granular leucocytes during budding. Hemoblasts, pluripotent stem cells in the blood, were often found in association with the ECM and they aggregated with epithelial cells to form organ rudiments. The lectin showed a high binding affinity for hemoblast precursors. The blockage of epithelial transformation of stem cells by galactose in in vivo bioassy was ineffective in the presence of the lectin. Polyclonal anti-lectin antibody prevented the hemoblasts spreading on the ECM and moving toward the epithelium, but it did not block the cell-cell adhesion of hemoblasts. By three days of bud development, lectin granules and ECM have almost disappeared from the developing bud together with a cessation of hemoblast aggregation. These results show that Polyandrocarpa lectin is a component of the ECM induced specifically in budding and suggest strongly that it plays a role in bud morphogenesis by directing the migration of pluripotent stem cells to the epithelium.     

Cloning of genes responsible for acetic acid resistance in Acetobacter aceti.

Five acetic acid-sensitive mutants of Acetobacter aceti subsp. aceti no. 1023 were isolated by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Three recombinant plasmids that complemented the mutations were isolated from a gene bank of the chromosome DNA of the parental strain constructed in Escherichia coli by using cosmid vector pMVC1. One of these plasmids (pAR1611), carrying about a 30-kilobase-pair (kb) fragment that conferred acetic acid resistance to all five mutants, was further analyzed. Subcloning experiments indicated that a 8.3-kb fragment was sufficient to complement all five mutations. To identify the mutation loci and genes involved in acetic acid resistance, insertional inactivation was performed by insertion of the kanamycin resistance gene derived from E. coli plasmid pACYC177 into the cloned 8.3-kb fragment and successive integration into the chromosome of the parental strain. The results suggested that three genes, designated aarA, aarB, and aarC, were responsible for expression of acetic acid resistance. Gene products of these genes were detected by means of overproduction in E. coli by use of the lac promoter. The amino acid sequence of the aarA gene product deduced from the nucleotide sequence was significantly similar to those of the citrate synthases (CSs) of E. coli and other bacteria. The A. aceti mutants defective in the aarA gene were found to lack CS activity, which was restored by introduction of a plasmid containing the aarA gene. A mutation in the CS gene of E. coli was also complemented by the aarA gene. These results indicate that aarA is the CS gene.     

Occurrence of D-rhamnan as the common antigen reactive against monoclonal antibody E87 in Pseudomonas aeruginosa IFO 3080 and other strains.

S. Sawada and co-workers reported that a monoclonal antibody (MAb), E87, interacted with about 80% of Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P. aeruginosa IFO 3080 (S. Sawada, T. Kawamura, Y. Masuho, and K. Tomibe, J. Infec. Dis. 152:1290-1299, 1985). In the present study, the rhamnose-rich polysaccharide was shown to be structurally and immunologically identical to the D-rhamnan of P. aeruginosa IID 1008 (S. Yokota, S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito, Eur. J. Biochem. 167:203-209, 1987). Furthermore, a set of enzymes responsible for the formation of GDP-rhamnose (probably in a D-form) from GDP-D-mannose was found in the 100,000 x g supernatant fractions obtained from all of nine P. aeruginosa strains reactive against MAb E87. The result strongly supports a possibility that lipopolysaccharides having a D-rhamnan chain widely occur as the common antigen among various P. aeruginosa isolates.     

Comparison of the intestinal bacteria in specific pathogen free mice from different breeders

Specific pathogen free BALB/c mice from 3 commercial laboratory animal breeders in Japan were compared on the composition of caecal flora revealed by selective and nonselective cultivation as well as direct microscopical observation on smears, and relative caecal weight. Large differences were detected in viable counts of total bacteria and almost all bacterial groups, while direct microscopical counts which consisted mainly of fusiform bacteria were almost equal, resulting in diverse recovery rates among 3 breeders. Eubacterium and spiral shaped organisms were not detected from all breeders. There also existed variations within breeders, especially those in the number of Enterobacteriaceae. Relative caecal weight also differed among breeders, suggesting the participation of variety of caecal bacteria in determining this parameter. As these variations in bacteriological status of animals can influence experimental results, standardization of intestinal microbial flora is required.