Susceptibility to autoimmune disease is associated with null alleles at one of the two genetic loci encoding complement protein C4. These two genetic loci, C4A and C4B, are highly homologous in primary structure but encode proteins with different functional activities. Expression of C4A and C4B genes is regulated by IFN-gamma in human hepatoma cells and in murine fibroblasts transformed with the respective genes. In these cell lines, IFN-gamma has a significantly greater and longer-lasting effect on expression of C4A than that of C4B. In this study we examined synthesis and regulation of C4A and C4B in peripheral blood monocytes from normal, C4A-null, and C4B-null individuals. Synthesis of C4 in human peripheral blood monocytes decreases during time in culture. IFN-gamma mediates a concentration- and time-dependent increase in steady-state levels of C4 mRNA and a corresponding increase in synthesis of C4 in normal human monocytes. LPS decreases monocyte C4 expression and completely abrogates the effect of IFN-gamma on the expression of this gene. In contrast, LPS and IFN-gamma have a synergistic effect in upregulating expression of another class III MHC gene product, complement protein factor B. The effect of LPS on constitutive and IFN-gamma-regulated C4 synthesis is probably not mediated via release of endogenous monokines IL-1 beta, TNF-alpha, or IL-6. Synthesis of C4, and regulation of its synthesis by IFN-gamma and LPS, are similar in normal, C4A-, and C4B-null individuals. These results demonstrate the synthesis of C4 at extrahepatic sites and tissue-specific regulation of C4 gene expression.
J Kulics, H R Colten, D H Perlmutter
We investigated regulation of the cardiac L-type calcium channel by intracellular ATP and by alpha 1-adrenergic agonism using single adult guinea pig ventricular cells and the whole-cell patch clamp method. Inclusion of 5 mM ATP in the patch clamp pipette prevented calcium current rundown but did not increase the maximal magnitude of the slow inward calcium current (ICa). During beta 1-adrenergic blockade with 10 microM (-)-propranolol, cells preincubated with 1 microgram/ml pertussis toxin for 2-5 h exhibited a rapid twofold increase in ICa after rupture of the membrane patch when 5 mM ATP was present in the patch clamp pipette. In the absence of ATP, the increase in ICa did not occur. In pertussis toxin-treated cells, 100 microM (-)-phenylephrine inhibited the augmentation of ICa. This inhibitory effect was blocked by 100 nM terazosin, a selective alpha 1-antagonist. The inhibitory effect of alpha 1-adrenergic agonism was not mediated by cAMP-dependent phosphodiesterase since incubation with 100 microM (-)-phenylephrine did not augment the activity of this enzyme. We conclude that regulation of the L-type calcium channel in cardiac cells is complex, and is dependent on a pertussis toxin-sensitive substrate, ATP, and an alpha 1-adrenergic receptor. The marked increase in ICa after pertussis toxin treatment in the presence of ATP indicates significant inhibition of ICa by a pertussis toxin substrate, presumably the guanine nucleotide inhibitory protein (Gi) in the basal state. The inhibitory action of (-)-phenylephrine in pertussis toxin-treated cells is consistent with modulation of ICa by an alpha 1-adrenergic receptor not coupled to Gi.
E C Keung, J S Karliner
Interest in human dendritic cells (DC) has been heightened recently by the discovery that this cell type is a primary target of the human immunodeficiency virus, the causative agent of AIDS. DC are bone marrow-derived cells with an extraordinarily potent ability to promote the immunological activity of T lymphocytes. Unfortunately, since DC constitute less than 0.5% of peripheral blood mononuclear cells and die within a few days of their isolation, they are not readily accessible to study. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine with well-recognized effects on granulocyte and macrophage maturation, profoundly affects the morphology and viability of DC isolated from peripheral blood. GM-CSF not only promotes DC survival but also induces DC differentiation to mobile, reversibly adherent cells with long-branched projections. DC cultured in GM-CSF survive for up to 6 wk and retain their ability to stimulate the proliferation of T cells in allogeneic and autologous mixed leukocyte reactions.
S Markowicz, E G Engleman
In the present study we examined mRNA and protein levels for the muscle/adipose tissue glucose transporter (GLUT-4) in various tissues of spontaneously obese mice (C57BL/KsJ, db/db) and their lean littermates (db/+). Obese (db/db) mice were studied at 5 wk of age, when they were rapidly gaining weight and were severely insulin resistant, evidenced by hyperglycemia (plasma glucose 683 +/- 60 vs. 169 +/- 4 mg/dl in db/+, P less than 0.05) and hyperinsulinemia (plasma insulin 14.9 +/- 0.53 vs. 1.52 +/- 0.08 ng/ml in db/+, P less than 0.05). The GLUT-4 mRNA was reduced in quadriceps muscle (67.5 +/- 8.5%, P = 0.02), but unaltered in adipose tissue (120 +/- 19%, NS), heart (95.7 +/- 6.1%, NS), or diaphragm (75.2 +/- 12.1%, NS) in obese (db/db) mice relative to levels in lean littermates. The GLUT-4 protein, measured by quantitative immunoblot analysis using two different GLUT-4 specific antibodies, was not different in five insulin-sensitive tissues including diaphragm, heart, red and white quadriceps muscle, and adipose tissue of obese (db/db) mice compared with tissue levels in lean littermates; these findings were consistent when measured relative to tissue DNA levels as an index of cell number. These data suggest that the marked defect in glucose utilization previously described in skeletal muscle of these young obese mice is not due to a decrease in the level of the major muscle glucose transporter. An alternate step in insulin-dependent activation of the glucose transport process is probably involved.
L Koranyi, D James, M Mueckler, M A Permutt
The biochemical basis underlying the genetic polymorphism of drug N-acetylation was investigated using a combination of in vivo and in vitro assays for arylamine N-acetyltransferase (NAT) activity and content in human liver. The acetylator phenotype of 26 surgical patients was determined using caffeine as an innocuous probe drug by measurement of the 5-acetyl-amino-6-formylamino-3-methyluracil to 1-methylxanthine molar ratio in urine. Liver wedge biopsies from these patients and livers from 24 organ donors were then used for measurement of N-acetyltransferase activity with the substrate sulfamethazine and for quantitation of immunoreactive N-acetyl-transferase protein. In vivo (caffeine metabolites in urine) and in vitro (sulfamethazine acetylation) measures of N-acetyl-transferase activity correlated very highly (r = 0.98). Moreover, in all subjects tested, slow acetylation both in vivo and in vitro was associated with a decrease in the quantity of immunodetectable N-acetyltransferase protein in liver cytosol relative to that seen in cytosols from rapid acetylator livers. Two kinetically distinct enzyme activities, designated NAT-1 and NAT-2, were partially purified from low- and high-activity livers and their relationship to acetylator status was determined. Low acetylation capacity was related to decreases in the liver content of both of these immunologically related proteins. The results demonstrate that genetically defective arylamine N-acetylation is due to a parallel decrease in the quantity of two structurally and functionally similar acetylating enzymes.
D M Grant, K Mörike, M Eichelbaum, U A Meyer
Amylin, a peptide copackaged with insulin in beta-cell granules, was measured in the effluent of the perfused rat pancreases by means of a newly developed specific radioimmunoassay. Its secretion parallels that of insulin in response to 20 mM glucose, 10 mM arginine, or the combination thereof. The relative molar amount of secreted amylin was estimated to be 25-37% that of insulin. Treatment with a borderline diabetogenic dose of streptozotocin reduced amylin response without significantly changing the insulin response. A severely diabetogenic dose of streptozotocin totally abolished amylin release and markedly reduced insulin release. The selective impairment of amylin secretion in streptozotocin-treated rats could represent an early manifestation of beta-cell depletion or injury.
A Ogawa, V Harris, S K McCorkle, R H Unger, K L Luskey
The leukocyte adhesion molecules CD11a/CD18, CD11b/CD18, and CD11c/CD18 (Leu-CAM) are members of the integrin receptor family and mediate crucial adhesion-dependent functions in leukocytes. The molecular basis for their deficient cell surface expression was sought in a patient suffering from severe and recurrent bacterial infections. Previous studies revealed that impaired cell surface expression of Leu-CAM is secondary to heterogeneous structural defects in the common beta subunit (CD18). Cloning and sequencing of complementary DNA encoding for CD18 in this patient revealed two mutant alleles, each representing a point mutation in the coding region of CD18 and resulting in an amino acid substitution. Each mutant allele results in impaired CD18 expression on the cell surface membrane of transfected COS M6 cells. One substitution involves an arginine residue (Arg593----cysteine) that is conserved in the highly homologous fourth cysteine-rich repeats of other mammalian integrin subfamilies. The other substitution involves a lysine residue (Lys196----threonine) located within another highly conserved region in integrins. These data identify crucial residues and regions necessary for normal cell surface expression of CD18 and possibly other integrin beta subunits and define a molecular basis for impaired cell surface expression of CD18 in this patient.
M A Arnaout, N Dana, S K Gupta, D G Tenen, D M Fathallah
D W Crabb
Previous results (Weinberg, J. M., J. A. David, M. Abarzua, and T. Rajan. 1987. J. Clin. Invest. 80:1446-1454) have shown that GSH and glycine (GLY) are cytoprotective during anoxia when added extracellularly. The present studies investigate the role that intracellular GSH plays in this cytoprotection. Proximal renal tubules in suspension prepared with either high (11 +/- 1 nmol/mg protein) or low (6 +/- 1 nmol/mg protein) GSH contents were subjected to 40 min of anoxia and 40 min of reoxygenation. Low GSH tubules were protected from plasma membrane damage during anoxia by exogenous addition of 1 mM GSH or GLY, reducing lactate dehydrogenase (LDH) release from 42 +/- 7 to 14 +/- 1 and 10 +/- 1%, respectively. High GSH tubules were equally protected from anoxic damage without exogenous additions. Since the high GSH content approximates the in vivo values, it may be concluded that GSH may be cytoprotective during anoxia in vivo. However, it is not the intracellular GSH itself that is cytoprotective; rather, this protection resides in the ability to produce GLY, which appears to be the cytoprotective agent. Alanine was also shown to have similar cytoprotective properties, although higher concentrations were required. Sulfhydryl reducing agents such as cysteine and dithiothreitol offered less, but significant protection from anoxic damage. Protection by GSH, GLY, or alanine was not associated with higher ATP levels during anoxia. Tubules that were protected from membrane damage during anoxia recovered oxygen consumption and K and ATP contents significantly better during reoxygenation than unprotected tubules.
L J Mandel, R G Schnellmann, W R Jacobs
Autoantibodies to a polymerase III transcription factor, La (SS-B), are frequently detected in the serum of patients with Sjogren's syndrome and systemic lupus erythematosus. To define the humoral immune response to this protein, we analyzed the patterns of antibody recognition toward 13 recombinant La peptides by immunoblotting and determined the heterogeneity of antibodies reactive with the immunodominant epitopes. The smallest epitopes that were strongly antigenic and recognized by greater than 70% of sera tested (immunodominant) were encoded by the subclones BgX and XA located in the 5' and 3' halves of the La cDNA, respectively. Conformation of the immunodominant La peptides played a major role in antibody recognition. Although greater diversity in antibody binding to carboxyl-terminal La peptides was observed, the overall pattern of peptide recognition by anti-La antibodies was similar in different diseases. The antibody responses to the immunodominant peptides were strongly correlated (r = 0.68, P less than 0.001). One- and two-dimensional isoelectric focusing of affinity purified IgG anti-La peptide antibodies revealed restricted heterogeneity and oligoclonal bands (kappa light chains). These observations suggest that anti-La antibodies are induced and/or maintained by the self antigen and that their diversity is constrained either by mechanisms related to tolerance or by affinity maturation of the humoral immune response.
P Bini, J L Chu, C Okolo, K Elkon
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