Effect of propionate and pyruvate on citrulline synthesis and ATP content in rat liver mitochondria.

Propionate inhibits citrullinogenesis when succinate (plus rotenone) or glutamate are the oxidizable substrates used. Propionate decreases the intramitochondrial concentration of carbamylphosphate by decreasing the ATP content. When the energy supply for citrullinogenesis is provided by an influx of exogenous ATP, propionate is no longer an inhibitor. Pyruvate inhibits citrullinogenesis with glutamate but not with succinate (plus rotenone) as oxidizable substrates. Propionate and pyruvate deplete mitochondrial ATP but probably by different mechanisms.     

Direct identification of the murine IL-2 receptor p55-p75 heterodimer in the absence of IL-2

The proteins cross-linked to the IL-2R p55 subunit were biochemically compared in distinct cell populations that varied in their capacity to express high affinity IL-2R. We directly cross-linked p75 to p55 in the absence of IL-2 for the cell populations that bear only high affinity IL-2R. Furthermore, although no endogenous IL-2 production was detected, p75 was readily cross-linked to p55 for EL4J-3.4, a p55 transfectant of EL4 that bears high affinity IL-2R. These results strongly suggest that high affinity IL-2R exist as a preformed heterodimer of p55 and p75 which do not require IL-2 for their association. Furthermore, cross-linking of three other proteins of apparent Mr of 100,000, 135,000, and 180,000 to p55 was also seen, raising the possibility of a more complex subunit composition for the IL-2R.     

Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.     

Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.     

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.     

The proline biosynthesis in living organisms

Summary In this article we review recent work on the physiology of proline and ¹-pyrroline-5-carboxylate (P5C) in living organisms and consider recent progress in our understanding of the role of P5C synthetase in collagen metabolism and the regulation of urea cycle in vertebrates. Much of this recent progress has been made possible by advances in our knowledge of the enzymes and genes involved in proline biosynthesis in man. The availability of well characterized P5C synthetase deficiency in man has been an impetus for the cloning of the cDNA encoding for this enzyme from man and facilitated the establishment of the phenotype-genotype relationships in P5C synthetase deficiency in higher vertebrates.Abbreviations GK -glutamyl kinase - GPR -glutamyl phosphate reductase - P5CR ¹-pyrroline-5-carboxylate reductase - GSA glutamic--semialdehyde - P5C ¹-pyrroline-5-carboxylate - P₁ Inorganic phosphate - AMP, ADP, ATP Adenosine 5-mono-, di-, triphosphate - NAD⁺, NADH nicotinamide adenine dinucleotide, and its reduced form - NADP⁺, NADPH nicotinamide adenine dinucleotide phosphate, and its reduced form; DEAF: diethylaminoethyle - OAT ornithine amino transferase; CHO: Chinese hamster ovary - IGF-1 insulin-like growth factor-1 - P5CDH pyrroline 5carboxylate dehydrogenase - IMP inosine 5-monophosphate     

Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid.

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.     

Reconstitution of an active surface CD2 by DNA transfer in CD2-CD3+ Jurkat cells facilitates CD3-T cell receptor-mediated IL-2 production.

To investigate the requirements for CD2 expression in the activation of T lymphocytes via the CD3-TCR complex, we produced and characterized a series of CD2-variants of the IL-2 producing Jurkat leukemia cell line, J32 (surface phenotype, CD2+, CD3+, CD28+). These mutants were derived by radiation and immunoselection, and were cloned under limiting dilution conditions. A total of 3 out of 30 of these mutants selectively lost the expression of both CD2 surface molecules and CD2 mRNA, and retained the expression of the CD3-TCR complex and the CD28 molecule. A mitogenic combination of anti-CD2 antibodies (9.6 + 9-1) failed to stimulate activation of these variants as measured by mobilization of intracellular Ca2+ and by IL-2 production. The CD2- mutants stimulated with anti-CD3 or anti-TCR mAb revealed an 8- to 32-fold decrease in IL-2 production and IL-2 mRNA accumulation as compared with the parental cells. No alteration of CD3-TCR-induced mobilization of intracellular Ca2+ was observed in the CD2- mutants. Reconstitution of CD2 expression by gene transfer in two J32 CD2- mutants restored IL-2 production and IL-2 mRNA accumulation in responses to both anti-CD2 and anti-CD3-TCR mAb. These results are the first direct demonstration of the requirement for CD2 molecules in optimizing IL-2 response in human T cells stimulated via CD3-TCR complex.     

Identification of a Gs-protein coupling domain to the beta-adrenoceptor using site-specific synthetic peptides. Carboxyl terminus of Gs alpha is involved in coupling to beta-adrenoceptors

Competition between Gs-protein and the synthetic peptide, GSA 379-394, derived from the carboxyl-terminal region of the alpha s-subunit, led to complete inhibition of receptor-mediated adenylate cyclase activation in turkey erythrocyte membranes. Related peptides corresponding to the homologous carboxyl-terminal region of alpha t-, alpha il- or alpha o-subunits did not interfere with beta-receptor-Gs coupling. The direct coupling between Gs and adenylate cyclase was not influenced by any of these peptides. These results emphasize the important role of the carboxyl-terminus of G-protein alpha-subunits for the specific recognition of their corresponding receptors and for signal transduction.