Structure of triphosphonoglycosphingolipid containing N-acetylgalactosamine 6-O-2-aminoethylphosphonate in the nervous system of Aplysia kurodai

A phosphonoglycosphingolipid, named F-21, was found in the nervous system of Aplysia kurodai by two-dimensional thin-layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). F-21 was isolated from the nervous tissue of Aplysia in this study, and its chemical structure was characterized as follows, where 2-AEP is 2-aminoethylphosphonate. (Formula; see text) The major aliphatic components of the ceramide portion were palmitic acid (75%), stearic acid (22%), octadeca-4-sphingenine (43%), and anteisononadeca-4-sphingenine (54%). Some information on the steric interactions in the sugar moiety was obtained by NMR spectroscopy. The ring protons of the internal galactose, H1, H3, and H4 and the H3 of the side chain galactose were shifted, as compared to the corresponding protons of dephosphonylated F-21. This may indicate the interactions between the 2-AEP residue of N-acetylgalactosamine and the internal galactose and between the N-acetyl group of N-acetylgalactosamine and the side chain galactose, implying a sterically restricted and unique structure that may relate to some biological functions of F-21.     

Heterogeneity of CD4+ T cells involved in anti-allo-class I H-2 immune responses. Functional discrimination between the major proliferating cells and helper cells assisting cytotoxic T cell responses.

MLR in various combinations with class I H-2 disparity revealed that there are three patterns of MLR in the aspect of responding T subset (CD4 vs CD8) dominance. Irrespective of the CD8 vs CD4 dominance, a single i.v. administration of class I-disparate allogeneic spleen cells resulted in almost complete abrogation of anti-class I proliferative capacity of both CD4+ and CD8+ T cells in six combinations. The suppression of proliferative responses was correlated with the striking reduction in the ability to produce IL-2 upon stimulation with the relevant class I alloantigens. In contrast, i.v. presensitized recipient mice exhibiting only marginal MLR/Il-2 production could generate comparable magnitudes of anti-allo class I CTL as well as graft rejection responses to those induced by normal unpresensitized mice. The administration in vivo of anti-CD4 antibody along with the i.v. presensitization not only suppressed the generation of CTL responses by spleen cells but also induced appreciable prolongation of allo-class I-disparate skin grafts under conditions in which neither alone did it. These results demonstrate that 1) the suppression of graft rejection responses is not necessarily reflected on the reduction of MLR; 2) CD8+ CTL precursors responsible for graft rejection can be activated by either allo-class I-reactive CD8+ or CD4+ Th cells; 3) i.v. presensitization induces functional elimination of CD8+ and CD4+ proliferative/IL-2-producing T cells but not of CD8+ CTL precursors and CD4+ Th whose capacity is expressed by assistance of CTL induction but not by their own proliferation. Thus, this study illustrates the heterogeneity of class I alloantigen-reactive CD4+ T cells in the aspect of their capacity to proliferate themselves vs contribute to CTL induction as well as graft rejection.     

Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.     

Animalizing Effect of A23187 on Sea Urchin Embryos

Pulse treatment of sea urchin embryos with 3 μM A23187 for 2 hr starting at a stage in initial 10 hr period of development at 20°C, followed by a culture in normal sea water up to the pluteus corresponding stage (45 hr after fertilization), yielded many large exogastrulae with thin embryo walls. The pulse treatment starting at a time between 10 and 13 hr after fertilization yielded considerable number of large prisms and gastrulae having thin embryo walls. Probably, the pulse treatment exerts stimulating effects on ectodermal cell determination in whole span of pre-hatching period to produce animalized embryos. On the other hand, pulse treatment with A23187 in pre-hatching period exerts stage-specific effects on gut formation. Embryos, thus treated for 2 hr starting at stages between 3 and 5 hr after fertilization, produced quite small exoguts but those treated at stages between 7 and 8 hr formed well developed and long exoguts. In embryos treated at the other stages than above, guts or exoguts were almost the same in their size to those in normal ones. These effects of A23187 on morphogenesis were canceled by procaine, tetracaine and ruthenium red. Probably, artificial Ca²⁺signal induced by A23187 alters the determination of cell fates, programmed in pre-hatching period.     

A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state.

A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.     

Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase.

With specific monoclonal antibodies, we found that human endometrial stromal cells and decidual cells express two function-related surface antigens. Indirect immunofluorescence staining revealed that both endometrial stromal cells and decidual cells during the first trimester of pregnancy expressed cluster of differentiation (CD) 13 antigen and CD10 antigen, which are identical to aminopeptidase N and neutral endopeptidase, respectively. By flow cytometric analysis, CD13 antigen was detected on 82-93% of the examined cells, and CD10 antigen was detected on 75-93% of the examined cells in endometrial stromal cell-enriched preparations. Furthermore, peptidase activity was detected in these cell preparations by an assay based on the hydrolysis of alanine-p-nitroanilide into p-nitroaniline and alanine.     

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy.

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles have been investigated by nuclear Overhauser effects (NOEs) in 1H nuclear magnetic resonance spectroscopy. Two-dimensional NOE and chemical exchange correlated spectroscopy (NOESY) and rotating frame NOE spectroscopy (ROESY) methods have been applied for obtaining the NOEs. In the NOESY spectra, NOEs between protons within the drug were overwhelmed by spin diffusion even at a short mixing time. This observation reduced the usefulness of the NOESY method on the one hand, however, on the other hand it facilitated remarkably in revealing signals due to the drug, hidden in the broad resonances of the membranes. In the ROESY spectra, the spin diffusion phenomena were less effective; accordingly the conformations of the drugs interacting with membranes were determined by the ROESY method. The observed NOE data showed that dibucaine takes more than two conformations and that both dibucaine and tetracaine are present as a dimer in the membranes. Molecular dynamics calculations supported these findings.     

cDNA Cloning of Indole-3-Acetic Acid-Regulated Genes: Aux22 and SAUR from Mung Bean (Vigna radiata) Hypocotyl Tissue

Five cDNAs of auxin-regulated genes were isolated from mungbean (Vigna radiata) hypocotyl sections by differential hybridizationscreening. They were related to the soybean genes, Aux22 [Ainleyet al. (1988) J. Biol. Chem. 263: 10658] and SAUR [McClure etal. (1989) Plant Cell 1: 229]. Regulation of expression of thesegenes, examined by Northern blot analysis, appeared similarto that reported in soybean hypocotyls. (Received August 10, 1991; Accepted October 14, 1991)     

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.