Increased lymphocyte adherence to human arterial endothelial cell monolayers in the context of allorecognition.

The interactions of alloreactive T lymphocytes with the vascular endothelium were studied in an in vitro model of lymphocyte adherence to cultured human arterial endothelial cell (HAEC) monolayers. Donor-primed lymphocytes (DPL) were shown to have significantly greater adherence to donor HAEC than were third-party primed lymphocytes. Limiting dilution analysis of adherent DPL showed an enrichment of donor-reactive lymphocytes compared with nonadherent DPL. This study examines the allospecific nature of this increased lymphocyte adherence. HAEC constitutively express class I HLA Ag and can be induced by IFN-gamma to express class II Ag. DPL adherence to class I+ HAEC was inhibited only in the presence of mAb directed against class I Ag. DPL adherence to class I+ and class II+ HAEC was inhibited in the presence of mAb directed against class I and class II Ag. Class I- and class II-specific adherence was also shown to involve CD8 and CD4 molecules, respectively, whereas lymphocyte function-associated Ag do not appear to play a major role in long term alloreactive lymphocyte adherence to HAEC. These findings suggest that alloreactive lymphocyte adherence to HAEC is mediated by two mechanisms. One is based on allorecognition, primarily of HLA Ag, and the other is related to presumably non-Ag-specific interactions between activated lymphocytes and the vascular endothelium. The studies presented provide evidence to suggest that HLA-specific lymphocyte adherence to endothelium may significantly contribute to the development of alloreactive lymphocyte infiltrates within the allograft.     

HIV-gp120 can block CD4-class II MHC-mediated adhesion

A possible component of the immune dysfunction associated with infection by HIV is the inhibition of CD4 function resulting from the avid binding of soluble HIV envelope glycoprotein (gp120) to cell surface CD4. We assessed CD4 function by measuring the ability of CD4+ T cells to form conjugates with cell size lipid vesicles, artificial target cells (ATC), bearing the natural ligand for CD4, MHC class II proteins. Conjugate formation was a transient process with the greatest number of specific cell to ATC conjugates found after approximately 30 min of incubation at 37 degrees C. Addition of gp120 specifically blocked conjugates between CD4+ cells and class II ATC in a concentration-dependent manner. These data indicate that T lymphocyte adhesion mediated by CD4 is a dynamic event and that binding of gp120 to CD4 is able to disrupt the normal progression of the interaction between CD4+ T lymphocytes and class II+ APC.     

Cysteine: Depolarization-Induced Release from Rat Brain In Vitro

Compounds released on depolarization in a Ca2+-dependent manner from rat brain slices were screened to identify candidates for neuroactive substances. Lyophilized superfusates were analyzed by reversed-phase HPLC after derivatization with 9-fluorenyl N-succinimidyl carbonate. One of the compounds that showed an increase of concentration in superfusates in the presence of iodoacetamide was identified as the cysteine (Cys) derivative, S-carboxamidomethylcysteine, by fast atom bombardment mass spectrometry and other methods. This stable Cys derivative originates from endogenous, extracellular Cys. The finding led to a method for quantification of Cys in superfusates by immediate cooling of the superfusates to 0 degrees C and reaction of Cys with N-ethylmaleimide. Depolarization-induced Ca2+-dependent release of Cys was most prominent in the neocortex, followed by the mesodiencephalon, striatum, and cerebellum. This suggests that Cys is released from a neuronal compartment and might be involved in neurotransmission.     

d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

Summary The production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1^–1 per day were reached.Offprint requests to: D. Gygax     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols     

DNA encapsidation by viruslike particles assembled in insect cells from the major capsid protein VP1 of B-lymphotropic papovavirus.

Capsids of polyomaviruses--small, nonenveloped DNA viruses--consist of the major structural protein VP1 and the minor structural proteins VP2 and VP3. The contributions of the individual capsid proteins to functions of the viral particle, such as DNA encapsidation, cell receptor attachment, entry, and uncoating, are still not clear. Here we show that viruslike particles assembled in nuclei of insect cells from VP1 of the monkey B-lymphotropic papovavirus (LPV) are sufficient to unspecifically encapsidate DNA. LPV VP1 expressed in large amounts in insect cells by a baculovirus vector assembled spontaneously in the nuclei to form viruslike particles. After metrizamide equilibrium density gradient purification and nuclease digestion, a fraction of these particles was shown to contain VP1-associated linear, double-stranded DNA with a predominant size of 4.5 kb. The fraction of DNA-containing VP1 particles increased with time and dose of baculovirus vector infection. The DNA-containing particles, further purified by sucrose gradient centrifugation, appeared as "full" particles in negative-staining electron microscopy. As shown by DNA hybridization, the encapsidated DNA consisted of insect cell and baculoviral sequences with no apparent strong homology to LPV sequences. Three non-LPV VP1-derived host proteins with apparent molecular masses of approximately 14, 15, and 16 kDa copurified with the DNA-containing particles and may represent insect cell histones encapsidated together with the DNA. A similar species of host DNA was also found in purified LPV wild-type virions. These data suggest that LPV VP1 alone can be sufficient to encapsidate linear DNA in a sequence-independent manner.