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Notes: Abstract Ly-5 (CD45) glycoproteins of the mouse, expressed by all or most hematopoietic cell lineages and specified by a single Ly-5 gene, range in size from isoform T200 of T cells (the smallest), in which exons 4, 5, and 6 are not represented, to isoform B220 of B cells (the largest), in which all three of these optional exons are represented. The main purpose of the present study, utilizing the polymerase chain reaction (PCR), was to ascertain whether known isoforms of intermediate size are generated by single or dual usage of optional exons 4, 5, and 6. Transcripts representing all eight isoforms predictable from varied use of three exons were observed among a diverse panel of nine B-cell tumors in culture, but there was no evident concordance with known contrasting differential features that distinguish members of the B-cell tumor panel. No two B tumors exhibited the same variety of transcripts and the relative quantities of transcripts expressed varied greatly from tumor to tumor. Cloning of B-cell tumors did not alter their distinctive transcript patterns. Separation methods (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE) did not suffice to segregate all corresponding expressed isoforms but did establish that transcripts representing usage of a single optional exon and of two optional exons were actually translated, which supports a provisional inference that all eight isoforms exist. The considerable diversity of B-cell transcript phenotypes was not seen among seven T-cell leukemias, two cytolytic T-cell lines, and three Th1 helper T-cell lines, all of which displayed a uniform phenotype comprising major expression of the T200 transcript (no optional exon) and minor expression of a transcript employing exon 5. However, a panel of five cloned Th2 T-cell lines, which represents a second and functionality different branch of the helper/inducer T-cell category, exhibited a characteristic transcript pattern which distinguished them from a panel of three Th1 T-cell lines. The major transcript in the Th2 lines was also T200, but the Th2 lines showed higher representation of transcript containing optional exons. A single Th2 clone expressed an unusual transcript suggesting a potential isoform not compounded simply by varied inclusion of the three identified optional exons. After activation of the helper T-cell lines with concanavalin A (Con A), expression of transcript containing optional exons appeared to decrease.

Notes: α-Galactosyltransferase which participates in the biosynthesis of blood group B substance was found in urine from group B and AB healthy persons of both secretors and non-secretors.The activity of α-galactosyltransferase in the urine of healthy variant Bm person was lower than that found in a normal group B person. This enzyme in urine of A1Bm persons must be much lower than that in normal AB persons. Its activity was not detected by the method of group O to group B transformation of erythrocytes.

Notes: α-L-Fucosyltransferases were demonstrated in human saliva which catalyze the transfer of L-fucose from GDP-L-[14C]-fucose to oligosaccharides from human milk. An α-(1→4)-L-fucosyltransferase that synthesizes lacto-N-fucopentaose II and lacto-N-difucohexaose I from lacto-N-tetraose and lacto-N-fucopentaose I, respectively, was detected in saliva samples of Le(a-b+) secretors and Le(a + b-) non-secretors in which Lea substance was secreted. This enzyme activity was demonstrable neither in saliva samples of Le(a-b-) secretors nor non-secretors. An α-(1→2)-L-fucosyltransferase, that synthesizes lacto-N-fucopentaose I from lacto-N-tetraose, was detected in saliva samples from Le(a-b+) secretors which secreted H and Leb substances and from Le(a-b-) secretors which secreted only H substance. An α-(1→3)-L-fucosyltransferase was present in all saliva samples of different ABO and Lewis blood groups, irrespective of their ABH secretor status of the donors. The fucosyltransferases in saliva were activated by Mn++ or Mg++ ions, and were inhibited by ATP, GTP and EDTA. They had a broad pH optimun between pH 5.0 and 6.5.

Notes: A β-galactoside α1 → 2 fucosyltransferase (H-enzyme) from human group O plasma which provides H blood group specificity to erythrocyte membranes has been purified approximately 22,000-fold by chromatography on DEAE-Sepharose CL-6B, GDP-hexanolamine-Sepharose 4B and SP-Sephadex C-50. The molecular weight of the H-enzyme was estimated to be 150,000 by gel filtration. Human group O erythrocyte membranes which had lost their H blood group activity by the action of α1 → 2 fucosidase were fucosylated by the transferase, and restored the H activity. Radioactive L-fucose appeared to be incorporated into glycolipid blood group substances of erythrocyte membranes. The activity of the α1 → 2 fucosyltransferase from human plasma, stomach mucosa, erythrocyte membranes and porcine stomach mucosa were specifically inhibited by the rabbit antiserum immunized with the preparation of human plasma H-enzyme. The anti-plasma H-enzyme antiserum did not inhibit the activities of α1 → 3 N-acetygalactosaminyltransferase (A-enzyme), α1 → 3 galactosyltransferase (B-enzyme), and β-N-acetylglucosaminide α1 → 3 and α1 → 4 fucosyltransferases from human plasma and stomach mucosa.

Notes: Abstract The host-vector system for an extreme thermophile, Thermus thermophilus HB27, was developed. The host strain has a mutation in tryptophan synthetase gene (trpB), and the mutation was determined to be a missense mutation by DNA sequence analysis. A Thermus-E. coli shuttle vector pYK109 was constructed. pYK109 consists of Thermus cryptic plasmid pTT8, tryptophan synthetase gene (trpB) of Thermus T2 and E. coli plasmid vector pUC13. pYK109 transformed T. thermophilus HB27 trpB5 to Trp to a frequency of 106 transformants per μg DNA.

Notes: Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4+ CD8−, CD4−CD8+, and CD4−CD8− phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.

Notes: Human B lymphocytes express components of the superoxide generating system of phagocytes, NADPH oxidase. We studied regulation of this ‘B-cell oxidase’ during in vitro blast transformation, using Lucigenin-amplified chemiluminescence (CL) to detect superoxide release. While freshly isolated tonsil B lymphocytes showed no CL responses, culture with phorbol myristate acetate (PMA) and ionomycin induced susceptibility to CL triggering by anti-IgM and anti-HLA-DR. Maximal effects were observed after 3 days of culture with 0.4 ng/ml PMA+1μg/ml ionomycin. Cells from such B lymphoblast cultures showed no CL responses to opsonized zymosan. In contrast, peripheral blood mononuclear cells, where monocytes are the predominant oxidant source, showed CL responses to opsonized zymosan but not to anti-IgM and anti-HLA-DR, either before or afterculture with PMA+ionomycin.Culture of B cells with the surface immunoglobulin cross-linking agent staphylococcus aureus Cowan 1 also led to emergence of a CL response to anti-IgM, which was enhanced by interferon-γ. Interestingly, markedly fewer B blasts than freshly isolated B lymphocytes expressed cytochrome b-558 surface antigen. Thus, the B-cell oxidase is up-regulated during blast transformation and can be triggered via surface IgM and HLA-DR; however, this appears to be restricted to a subset of B lymphoblasts.

Notes: Levels of transcripts initiated at a hut promoter in Bacillus subtilis were analysed. The addition of histidine to the culture medium increased the level of the transcript sixfold. In the presence of histidine and glucose together, the level of the transcript was reduced to the level in the absence of Induction. Furthermore, addition of a mixture of 16 amino acids to cultures of induced cells and of catabolite-repressed cells decreased levels of the transcript 16-fold and 2.6-fold, respectively. Thus, it appears that at least three regulatory mechanisms associated with induction, catabolite repression, and amino acid repression, control the transcriptional activity of the hut promoter. Expression of the hut promoter–lacZ fusions that contained various regions of the hutP gene and deletion analysis of the hutP region revealed a cis-acting sequence associated with catabolite repression that was located between positions +204 and +231 or around position +203.

Notes: [Auszug] It is shown here how mass spectrometry (MS) can be used for on–line data acquisition in fermentation. MS was applied in this work to analyze gas and liquid phases. Gas phase analysis allows fast and accurate measurement of all gases of interest (O2, N2, CO2, Ar, He etc.). Liquid phase ...

Notes: Abstract Two monoclonal antibodies of types IgG2b and IgG2a, anti-spermine-(Spm)-1 (ASPM-1) and anti-Spm-2 (ASPM-2) respectively were found among five clones of murine monoclonal antibodies, which were raised against Spm conjugated with bovine serum albumin via the cross-linker N-(γ-maleimidobutyryloxy) succinimide (GMBS). Antibody specificity was evaluated by a recently developed ELISA binding test, and led to the study of tissue sections by immunocytochemistry (ICC). ASPM-1 showed exclusive immunoreactivity with Spm, with the exception of a negligible cross-reactivity (2.0%) with spermidine (Spd). ASPM-2, on the other hand, reacted almost equally with acetylspermine (Ac-Spm) and N 1-acetylspermidine (N1-Ac-Spd) but with none of the other polyamine-related compounds tested. Complete agreement was obtained with the results of immunoblot analysis. Furthermore, results for antibody specificity obtained with the ELISA inhibition test and ICC model experiments using Sepharose gel beads strongly suggested that ASPM-1 recognizes the Spm molecule possessing at least a free terminal primary amino group, while ASPM-2 recognizes the Spm molecule acylated at both the terminal primary amino groups. An ICC method using ASPM-2 produced strong staining for polyamines (PAs) in the cytoplasm (but very few in the nuclei) of two different tumor cell lines and protein- or peptide-secreting cell systems, including exocrine and endocrine cell types; ASPM-1 showed immunoreactivity only with the tumor cell lines. These results strongly suggest that ASPM-2 may be useful for studies on actively proliferating and neoplastic cells, supporting our previously proposed idea that in immunocytochemistry PAs were converted to a variety of PA derivatives during the fixation process.