Human long-chain acyl-CoA synthetase: structure and chromosomal location.

A complementary DNA clone encoding the entire human long-chain acyl-CoA synthetase was isolated and the total 698-amino acid sequence was deduced. The amino acid sequence of human long-chain acyl-CoA synthetase shows 84.9% identity to that of rat long-chain acyl-CoA synthetase. The nucleotide sequences of the protein coding regions between human and rat long-chain acyl-CoA synthetase mRNAs are highly conserved (85.6%), whereas those of the 3' untranslated regions are less conserved (72%). The location of the human long-chain acyl-CoA synthetase gene was identified on chromosome 4 by spot hybridization of flow-sorted chromosomes. Computer-assisted homology search revealed a significant similarity of the enzyme with the enzymes of the luciferase family. Based on this similarity, the structure of human long-chain acyl-CoA synthetase can be divided into five domains: the N-terminus, two domains similar to those in enzymes of the luciferase family, a long gap region between the similar domains and the C-terminus.     

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor): regulation of its production and possible roles in bone metabolism.

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast-like cells. Local bone-resorbing factors such as IL-1, TNF alpha, and LPS strongly induced expression of LIF/D-factor mRNA in both clonal MC3T3-E1 cells and primary osteoblast-like cells. Neither parathyroid hormone nor 1 alpha,25-dihydroxyvitamin D3 stimulated expression of LIF/D-factor mRNA. LIF/D-factor per se did not stimulate expression of its own mRNA. Appreciable amounts of LIF/D-factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D-factor, IL-1, and IL-6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D-factor produced by osteoblasts is in concert with other bone-resorbing cytokines such as IL-1 and IL-6 involved in the bone resorption seen in the joints of RA patients. LIF/D-factor specifically bound to MC3T3-E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D-factor dose-dependently suppressed incorporation of [3H]thymidine into MC3T3-E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D-factor alone had no effect on enzyme activity. These results suggest that LIF/D-factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation with other osteotropic factors.     

Single crystals of bovine heart cytochrome c oxidase at fully oxidized resting, fully reduced and CO-bound fully reduced states are isomorphous with each other.

Fully reduced and CO-bound fully reduced forms of cytochrome c oxidase from beef heart muscle were crystallized in the presence of sodium ascorbate under N2 or CO atmosphere. Hexagonal bipyramidal and tetragonal crystals were obtained for both forms depending on buffer species. The hexagonal bipyramidal crystals, as large as 0.6 mm in the largest dimension, diffracted X-rays at 7 A resolution, showing an identical space group and cell dimension, P6(2) or P6(4) and a = b = 209 A, c = 283 A, respectively. These parameters coincide with those for crystals of the fully oxidized resting enzyme. This result suggests that a large conformational change, like a subunit arrangement, is not induced by the redox change and/or binding of CO (and possibly O2) to heme a3.     

Immuno-Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.

In order to investigate whether a vasa -like protein is present in germ line cells of Xenopus , antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein.
Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42–69 ± 10³ in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 10³ band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila .     

Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase. Effects on guanylate cyclase activity and ligand-binding specificity.

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130-kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH < 5.6 caused a rapid and spontaneous cleavage of the 130-kDa ANF receptor, yielding a 65-kDa polypeptide that could be detected by photoaffinity labeling by 125I-labeled N alpha 4-azidobenzoyl-ANF(4-28) followed by SDS/PAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130-kDa receptor was converted to a 65-kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDS/PAGE was carried out under non-reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65-kDa ANF-binding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130-kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130-kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130-kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1-28) or ANF(4-28), but not by atriopeptin I [ANF(5-25)] or C-ANF [des-(18-22)-ANF(4-23)-NH2]. On the contrary, the 65-kDa ANF-binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130-kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130-kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand-binding selectivity, may be responsible for the generation of low-molecular-mass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down-regulation of the ANF receptor.     

Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1.

Transmembrane signalling mechanisms of tumor necrosis factor alpha (TNF alpha) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNF alpha stimulated the release of 3H radioactivity from intact cells labeled with [3H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A2. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNF alpha. Pretreatment of the cells with pertussis toxin inhibited the liberation of [3H]arachidonate. GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) reduced the binding affinity of [125I]TNF alpha to beta-escin-permeabilized cells. The addition of TNF alpha together with an unhydrolyzable analog of GTP, GTP gamma S, to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid led to a release of the 3H radioactivity. The production of prostaglandin E2 (PGE2) was markedly stimulated by TNF alpha in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A2, thereby indicating that other process(es) in addition to the activation of phospholipase A2 may be responsible for the enhanced production of PGE2. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH2-PGE2 isomerase) was stimulated by TNF alpha with much the same time and dose requirements as for the production of PGE2, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNF alpha in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A2 and the de novo synthesis of cyclooxygenase.     

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.