Genetics of multidrug resistance.

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Authors

J M Croop, P Gros, D E Housman

Fibronectin degradation products containing the cytoadhesive tetrapeptide stimulate human neutrophil degranulation.

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We investigated whether adhesive glycoproteins, such as fibronectin or fibrinogen, could function to provide a nidus for neutrophil degranulation. Elastase release in recalcified plasma was normal in afibrinogenemic plasma, but 73% less in plasma depleted of fibronectin. Proteolytic digests of fibronectin, but not intact fibronectin (50-1,000 micrograms/ml), induced a concentration-dependent release of neutrophil elastase and lactoferrin. MAbs N293, which recognized the mid-molecule of fibronectin, N294, which was directed toward the 11-kD cell adhesive fragment, and N295, generated against the amino terminal of the 11-kD fragment, inhibited the release of elastase by 7, 24, and 60%, respectively. The cytoadhesive tetrapeptide portion of fibronectin, Arg-Gly-Asp-Ser (250-1,000 micrograms/ml), released 1.94 +/- 0.10 micrograms/ml of elastase from 10(7) neutrophils, in contrast to the lack of release by the control hexapeptide, Arg-Gly-Tyr-Ser-Leu-Gly. Plasmin appeared to be the enzyme responsible for fibronectin cleavage, since neutrophil elastase release in plasma that had been depleted of plasminogen was decreased and reconstitution of plasminogen-deficient plasma with purified plasminogen corrected the abnormal release. Plasmin cleaved fibronectin to multiple degradation products, each less than 200 kD. This fibronectin digest released 1.05 microgram/ml of elastase from 10(7) neutrophils. We suggest that the activation of plasminogen leads to the formation of fibronectin degradation products capable of functioning as agonists for neutrophils.

Authors

Y T Wachtfogel, W Abrams, U Kucich, G Weinbaum, M Schapira, R W Colman

Impaired carbohydrate metabolism of polymorphonuclear leukocytes in glycogen storage disease Ib.

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This study measures hexose monophosphate (HMP) shunt activity, glycolytic rate, and glucose transport in PMN and lymphocytes of patients with glycogen storage disease (GSD) type Ib as compared with controls and with GSD Ia patients. HMP shunt activity and glycolysis were significantly lower in intact PMN cells of GSD Ib patients as compared with GSD Ia patients and with controls. These activities were above normal levels in disrupted GSD Ib PMN. HMP shunt activity and glycolytic rates in lymphocytes were similar in all three groups studied. The rate of 2-deoxyglucose transport into GSD Ib PMN was 30% of that into cells of normal controls. In GSD Ib lymphocytes or in GSD Ia PMN and lymphocytes transport was normal. The striking limitation of glucose transport across the cell membrane of the PMN of GSD Ib patients may account for the impairment of leukocyte function that is characteristic of GSD Ib, but not found in GSD Ia patients.

Authors

N Bashan, Y Hagai, R Potashnik, S W Moses

Adenosine deaminase (ADA) deficiency due to deletion of the ADA gene promoter and first exon by homologous recombination between two Alu elements.

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In 15-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The goal of this study was to determine the precise molecular defect in a patient with ADA-deficient SCID whom we previously have shown to have a total absence of ADA mRNA and a structural alteration of the ADA gene. By detailed Southern analysis, we now have determined that the structural alteration is a deletion of approximately 3.3 kb, which included exon 1 and the promoter region of the ADA gene. DNA sequence analysis demonstrates that the deletion created a novel, complete Alu repeat by homologous recombination between two existing Alu repeats that flanked the deletion. The 26-bp recombination joint in the Alu sequence includes the 10-bp "B" sequence homologous to the RNA polymerase III promoter. This is the first example of homologous recombination involving the B sequence in Alu repeats. Similar recombination events have been identified involving Alu repeats in which the recombination joint was located between the A and B sequences of the polymerase III split promoter. The nonrandom location of these events suggests that these segments may be hot spots for recombination.

Authors

M L Markert, J J Hutton, D A Wiginton, J C States, R E Kaufman

Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet-activating factor.

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We have developed a rat model of ischemic bowel necrosis associated with shock by injection of platelet-activating factor (PAF) or a combination of PAF and endotoxin. Recent investigations have shown that tumor necrosis factor (TNF) also induces shock and necrosis of the gastrointestinal tract. The morphological changes of TNF-induced bowel lesions are indistinguishable from those caused by PAF. The mechanism of TNF-induced bowel necrosis is unclear. In the present study, we have shown that (a) TNF caused PAF production in bowel tissue; (b) the effects of TNF and LPS on PAF production in the intestine are additive; (c) TNF and LPS are synergistic in inducing bowel necrosis; and (d) TNF-induced bowel necrosis is due to PAF release and can be prevented by pretreatment with PAF antagonists.

Authors

X M Sun, W Hsueh

Uptake of cholesterol-rich remnant lipoproteins by human monocyte-derived macrophages is mediated by low density lipoprotein receptors.

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The uptake and degradation of cholesterol-rich remnant lipoproteins, referred to as beta-VLDL, are shown in the present study to be mediated by LDL receptors (apoB,E(LDL) receptors), not by unique beta-VLDL receptors. Human blood monocytes cultured for 5-7 d bound apoB- and/or apoE-containing lipoproteins from different species with affinities equivalent to those demonstrated for the receptors on cultured human fibroblasts. Low density lipoproteins competed effectively and completely with 125I-beta-VLDL for binding to and degradation by monocyte-derived macrophages. Specific polyclonal antibodies to bovine apoB,E(LDL) receptors abolished both LDL and beta-VLDL uptake by normal human monocyte-macrophages. Immunoblots of monocyte-macrophage extracts with these antibodies revealed a single protein in human macrophages with an apparent molecular weight identical to that of the apoB,E(LDL) receptor found on human fibroblasts. Like receptors on cultured human fibroblasts, the apoB,E(LDL) receptors on monocyte-macrophages responsible for 125I-beta-VLDL and 125I-LDL uptake were efficiently down regulated by preincubation of the cells with beta-VLDL or LDL. Finally, monocyte-macrophages from seven homozygous familial hypercholesterolemia subjects were unable to metabolize beta-VLDL or LDL, but demonstrated normal uptake of acetoacetylated LDL. The classic apoB,E(LDL) receptors on human monocyte-macrophages thus mediate the uptake of beta-VLDL by these cells.

Authors

C Koo, M E Wernette-Hammond, Z Garcia, M J Malloy, R Uauy, C East, D W Bilheimer, R W Mahley, T L Innerarity

Physical and genetic mapping of the Salmonella dublin virulence plasmid pSDL2. Relationship to plasmids from other Salmonella strains.

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Plasmids of approximately 80 kb in size are found in nearly all clinical isolates of Salmonella dublin and are believed to be essential for virulence. We have shown previously that the 80-kb plasmid pSDL2 is required for the S. dublin Lane strain to establish a lethal systemic infection in BALB/c mice after oral or intraperitoneal inoculation. We now present a physical and genetic characterization of pSDL2. We have established a complete restriction endonuclease cleavage map of pSDL2 for five enzymes: Xba I, Bam HI, Xho I, Sal I, and Hind III. The region specifying autonomous replication has been localized to a 10.5-kb region of the Sal I A fragment by subcloning on the vector pBR322. Using transposon insertion mutagenesis with Tn5-oriT, a region encoding the virulence phenotype has been mapped within a 6.4-kb portion of the Sal I B fragment. Deletions generated by partial Eco RI restriction digestion demonstrate that at least 50 kb of the plasmid DNA are not required for replication or virulence functions, confirming the map location of these phenotypes. Plasmids of different sizes and restriction patterns were found in mouse virulent strains of S. dublin Vi+, S. enteritidis, and S. choleraesuis. By Southern hybridization, these putative virulence plasmids share a common 4-kb Eco RI fragment with the virulence region of pSDL2, and the plasmids from S. dublin Vi+ and S. enteritidis were shown to express mouse virulence comparable to pSDL2.

Authors

P R Beninger, G Chikami, K Tanabe, C Roudier, J Fierer, D G Guiney

Clearance and early hydrolysis of atrial natriuretic factor in vivo. Structural analysis of cleavage sites and design of an analogue that inhibits hormone cleavage.

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This study examines the clearance and early hydrolysis of atrial natriuretic factor (ANF) in vivo. Radiolabeled ANF was cleared from the circulation of the rat with biphasic kinetics; the majority (90%) of ANF cleared with a t1/2 of 15 s, the remaining peptide was cleared with a t1/2 of 5 min. Microsequence analysis of ANF peptides recovered from the circulation of rats revealed five major degradation products of the intact hormone. The first cleavage occurred between amino acids 12 and 13 of the hormone and would inactivate ANF. Over time, additional fragments of the hormone were generated, including fragments of 6, 7, 21, and 24 amino acids in length. Whole body radioautography of rats injected with [123I]-ANF revealed the kidney as a predominant organ involved in clearance of ANF. Subsequent amino acid sequence analyses of radiolabeled ANF exposed to the kidney in vivo indicated that this organ generated four of the five major hydrolysis products observed in circulation, namely, the 6, 7, 16, and 21 amino acid fragments of the hormone. In an attempt to stabilize ANF in vivo, a synthetic analogue of the hormone was prepared that contained the amino acid analogue, aminoisobutyric acid, substituted at position 13. This analogue completely abolished the in vivo cleavage of ANF at this site. These studies demonstrate the usefulness of a protein chemistry approach in characterizing hormone metabolism in vivo and designing analogues with enhanced in vivo stability to cleavage.

Authors

C L Condra, E A Leidy, P Bunting, C D Colton, R F Nutt, M Rosenblatt, J W Jacobs

Adherence of human basophils to cultured umbilical vein endothelial cells.

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The mechanism by which circulating human basophils adhere to vascular endothelium and migrate to sites of allergic reactions is unknown. Agents have been identified which stimulate the adherence of purified basophils to cultured human umbilical vein vascular endothelial cells (HuVEC). Treatment of HuVEC with interleukin 1, tumor necrosis factor (TNF), bacterial endotoxin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in time and dose-dependent increases of adhesiveness for basophils. Coincubation of basophils and HuVEC for 10 min with C5a, formyl-methionyl-leucyl-phenylalanine, the calcium ionophore A23187, platelet-activating factor, TNF, and TPA also resulted in significant dose-dependent increases in basophil adherence; this effect resulted from activation of the basophil. Adherence of basophils to HuVEC was time and temperature dependent, required divalent cations, and was unaffected by glucocorticoids. Monoclonal antibody 60.3, directed against the beta-subunit of the leukocyte adherence complex CD18, inhibited the binding of basophils to HuVEC. Adherence of basophils to vascular endothelium may be important in initiating basophil infiltrates in vivo.

Authors

B S Bochner, P T Peachell, K E Brown, R P Schleimer

Use of laminar flow and unstirred layer models to predict intestinal absorption in the rat.

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Carbon monoxide (CO) and [14C]warfarin were used to measure the preepithelial diffusion resistance resulting from poor luminal stirring (RL) in the constantly perfused rat jejunum at varying degrees of distension (0.05, 0.1, and 0.2 ml/cm). RL was much greater than epithelial cell resistance, indicating that poor stirring was the limiting factor in absorption and that an appropriate model of stirring should accurately predict absorption. A laminar flow model accurately predicted the absorption rate of both probes at all levels of gut distension, as well as the absorption of glucose when RL was the rate-limiting factor in absorption. In contrast, an unstirred layer model would not have predicted that gut distension would have little influence on absorption, and would have underestimated [14C]warfarin absorption relative to CO. We concluded that in the perfused rat jejunum, laminar flow accurately models luminal stirring and an unstirred layer should be considered to be a unit of resistance in laminar flow, rather than a model of luminal stirring.

Authors

M D Levitt, J M Kneip, D G Levitt

Endocytotic uptake, processing, and retroendocytosis of human biosynthetic proinsulin by rat fibroblasts transfected with the human insulin receptor gene.

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Abstract

The cellular itinerary and processing of insulin and proinsulin were studied to elucidate possible mechanisms for the observed in vivo differences in the biologic half-lives of these two hormones. A rat fibroblast cell line transfected with a normal human insulin receptor gene was used. Due to gene amplification, the cells express large numbers of receptors and are ideal for studying a ligand, such as proinsulin, that has a low affinity for the insulin receptor. Competitive binding at 4 degrees C showed that the concentration of unlabeled insulin and proinsulin that is needed to displace 50% of tracer insulin or proinsulin was 0.85-0.95 nM and 140-150 nM, respectively. Binding to surface receptors and internalization occur at rates that are four to five times faster in cells incubated with insulin compared with proinsulin. Chloroquine led to an increase in cell-associated radioactivity of approximately 1.4-fold in cells incubated with insulin or proinsulin, but inhibited the appearance of degraded insulin by 54% and degraded proinsulin by only 10%. To study the fate of internalized ligand, cells were incubated with insulin and proinsulin until steady state binding occurred. Surface bound ligand was removed by an acid wash and the remaining cell-associated radioactivity represented internalized ligand. Cells were then reincubated in 37 degrees C buffer and the cell-associated radioactivity and radioactivity released into the medium were analyzed by TCA precipitation, Sephadex G-50, and HPLC. The results demonstrated that proinsulin more readily bypasses the intracellular degradative machinery and is therefore released intact from the cell via the retroendocytotic pathway. These results may help to explain the prolonged metabolic clearance rate and biologic responsiveness of proinsulin in vivo.

Authors

J R Levy, A Ullrich, J M Olefsky

Activation of protein breakdown and prostaglandin E2 production in rat skeletal muscle in fever is signaled by a macrophage product distinct from interleukin 1 or other known monokines.

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During sepsis or after injection of endotoxin into rats, there is a large increase in muscle protein breakdown and prostaglandin E2 (PEG2) production. Prior studies showed that partially purified interleukin 1 (IL-1) from human monocytes can stimulate these processes when added to isolated rat muscles. The availability of pure recombinant IL-1 and other monokines has allowed us to investigate the identity of the active agent in this process. Incubation of muscles with recombinant human or murine IL-1 alpha or IL-1 beta or with IL-1 plus a phorbol ester did not stimulate muscle proteolysis or PGE2 production. Homogeneous natural porcine IL-1 ("catabolin") and mouse or human IL-1 beta were also not effective in vitro. In addition, a variety of other human cytokines, including tumor necrosis factor ("cachectin"), epidermal thymocyte-activating factor, eosinophil cytotoxicity-enhancing factor, interferon-alpha, beta, and gamma, platelet-derived growth factor, and transforming growth factor (TGF) beta, which are all released by activated macrophages, TGF-alpha, or mixtures of these polypeptides, also failed to activate proteolysis or PGE2 production. By contrast, a large increase in net protein breakdown could be induced in the rat soleus by polypeptides released from porcine monocytes or by the serum from febrile cattle which had been injected with Pasteurella haemolytica or bovine rhinotracheitis virus. Therefore, a still-unidentified product of activated monocytes appears to be responsible for the negative nitrogen balance that accompanies infectious illness.

Authors

A L Goldberg, I C Kettelhut, K Furuno, J M Fagan, V Baracos

Tumor necrosis factor can induce fever in rats without activating protein breakdown in muscle or lipolysis in adipose tissue.

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Tumor necrosis factor (TNF, cachectin) is a macrophage product that has been suggested to signal the loss of body weight, the decrease in adipose tissue and muscle mass, and anorexia during infections or chronic illness. To test this possibility, young growing rats were injected subcutaneously or intraperitoneally with human or murine recombinant TNF. After 3-4 h, these animals developed a 1-2 degrees fever which lasted approximately 4 h. With repeated daily TNF injections for 5 d, the animals developed fevers similarly each day. In contrast, rats injected with endotoxin show a single febrile episode and then are tolerant to subsequent daily injections of endotoxin (but do not develop tolerance to TNF or interleukin-1). On the first day of TNF treatment, the rats did not grow, but on subsequent days, despite their fevers, they grew at similar rates as controls. Although the TNF-treated rats consumed slightly less food than control animals, the ratio of growth per amount of food intake was identical in the two groups. When rats are administered endotoxin, they develop a fever, and their muscles show increased protein degradation and prostaglandin (PG)E2 production. However, when fevers were induced with TNF, there was no change in muscle proteolysis or PGE2 production, and in adipose tissue no increase in basal or catecholamine-induced lipolysis. Also TNF addition in vitro did not enhance lipolysis in epididymal fat pads or proteolysis in soleus muscles. Thus, TNF treatment can induce fever without producing a catabolic state similar to that induced by endotoxin.

Authors

I C Kettelhut, A L Goldberg

Phenotypic and functional characterization of human cytolytic T cells lacking expression of CD5.

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Although the CD5 (T1) antigen was initially described as a pan-T cell membrane glycoprotein, we report that 14 of 40 normal individuals were found to have 5% or greater of their blood mononuclear cells characterized as CD3 (T3)+ but CD5- by dual immunofluorescence flow cytometry. These cells expressed normal quantities of surface CD3 and CD2 but low levels of CD7, were CD8+ and CD4-, and CD16-. In order to determine whether cells of this phenotype were functional, six CD5- cytolytic T lymphocyte (CTL) clones isolated from normal individuals were studied. The CD5- CTL clones all demonstrated normal cytolytic activity against appropriate target cells. Monoclonal antibodies (MAbs) directed against CD3, CD8, CD2, and lymphocyte function-associated antigen 3, but not against CD5, inhibited cytolytic activity. Changes in intracellular calcium [( Ca2+]i) in response to anti-CD5 and anti-CD3 MAbs were measured. Stimulation by anti-CD5 MAb alone did not give rise to a change in [Ca2+]i. However, under conditions of limiting concentrations of anti-CD3 MAb, preincubation of normal CD5+, but not CD5-, clones with anti-CD5 MAb led to a dramatic enhancement in the ability of anti-CD3 MAb to elicit a rise in [Ca2+]i. We conclude that CD5- T lymphocytes represent a normal lymphoid phenotype. Although CD5 may be involved in T cell activation when present, these CD5- CTL clones appear to express normal cytolytic activity.

Authors

B E Bierer, Y Nishimura, S J Burakoff, B R Smith

Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells.

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The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-binding protein were 58 +/- 17 (mean +/- SD) and 5 +/- 2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182 +/- 34 and 26 +/- 6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2-10 nM 5-methyltetrahydrofolate). Increases in folate-binding protein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing less than 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 microM 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate medium supplemented with reduced folates, the levels of both folate-binding proteins fell linearly towards the levels in KB cells continuously maintained in standard medium. The folate transport protein was identified in and underwent similar changes in human and mouse mammary tumor cells. These studies indicate that the folate transport system is probably regulated by the extracellular folate concentration through changes in intracellular metabolite levels.

Authors

M A Kane, P C Elwood, R M Portillo, A C Antony, V Najfeld, A Finley, S Waxman, J F Kolhouse

Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy.

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We now describe experiments that allow one to determine the consequences of B cell reduction alone vs. those that result from superimposed mild hyperglycemia. Male CD rats underwent a 60% pancreatectomy (Px); controls were sham operated. 1 wk later, either 10% sucrose (SUC) was substituted as fluid supply or tap water was continued (WAT). Plasma glucose and insulin values in Px-WAT remained equal to the sham groups; in Px-SUC the values were euglycemic for 25 d, but then nonfasting plasma glucose rose 15 mg/dl. After 6 wk, B cell mass in Px-WAT was reduced by 45% and non-B cell mass by 57%. In contrast, in Px-SUC both masses were comparable to the sham groups. The insulin response to 27.7 mM glucose was measured using the in vitro perfused pancreas. The responses were reduced in Px-WAT but in proportion to their reduced B cell mass; in contrast, it was 75% less than expected in Px-SUC. Also, the response to arginine given at 16.7 mM glucose was reduced only in Px-SUC. These results show that a lowering of B cell mass that does not result in hyperglycemia has no adverse effect on the remaining B cells. On the other hand, if even mild hyperglycemia develops, B cell function becomes impaired and results in inappropriately reduced insulin stores and insulin responses to marked stimuli.

Authors

J L Leahy, S Bonner-Weir, G C Weir

Interleukin 1 stimulates T lymphocytes to produce granulocyte-monocyte colony-stimulating factor.

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T lymphocytes are thought to cooperatively interact with monocytes to produce colony-stimulating factors (CSF). However, little is known about monocyte-mediated signals leading to CSF-secretion by T lymphocytes, although soluble monocyte products have been implicated. We have employed monoclonal antibody anti-T3B covalently coupled to CnBr-activated Sepharose 4B beads, to show that multimeric ligation of T cell antigen receptor leads to T cell receptiveness to interleukin 1 (IL-1), as indicated by T cell production of CSF, which induces growth of myeloid progenitor cells into neutrophil, eosinophil, and monocyte colonies. To investigate the molecular basis of these findings, total RNA was extracted from T3B Sepharose-primed and IL-1-stimulated T lymphocytes and probed for granulocyte-monocyte-CSF (GM-CSF), granulocyte-CSF (G-CSF), and monocyte-CSF (M-CSF) mRNA. GM-CSF, but not G-CSF or M-CSF, messages were detected. Nuclear "run on" assays revealed that IL-1 action is effective primarily at the level of GM-CSF gene transcription. These results suggest a previously unrecognized role of IL-1 in the regulation of GM-CSF secretion by T cells.

Authors

F Herrmann, W Oster, S C Meuer, A Lindemann, R H Mertelsmann

Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways.

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The serum complement system is a major mediator of inflammation reactions. Two of the complement proteins, the third (C3) and fifth (C5) components, are precursors of potent phlogistic molecules, C3a and C5a. C5a has potent chemotactic activity and plays an active role in pulmonary inflammation. We present evidence suggesting that several complement proteins, including C5, are synthesized locally in the lung in alveolar type II epithelial cells. Lung tissue from normal mice synthesized and secreted C5 protein similar to the C5 protein in mouse serum, whereas lung tissue from C5-deficient mice did not. Lung tissues from both normal and C5-deficient mice synthesized C3. Rat lung tissue synthesized and secreted C5, as well as C2, C4, C3, and factor B. Cultures of type II cells (95% type II cells, 5% macrophages) regularly synthesized all these proteins. In contrast, cultures of macrophages alone synthesized large amounts of C2 and factor B, and in some experiments C3 and C4, but never C5. The C5 synthesized by the rat cells was slightly larger than serum C5 (200 kD compared with 180 kD) and was not processed to the two-chain molecule seen in serum. Rat lung tissue and purified type II cells contained C5 mRNA with the same molecular mass as the C5 mRNA in rat liver and in mouse lung and liver. Human type II cells also synthesized C5, as well as C2, C4, C3, and factor B. Human pulmonary macrophages synthesized only C2, factor B, and, in some experiments, C3. Synthesis of complement proteins in cells that line the alveolar wall may provide a local source of these proteins for inflammatory responses in the lung. Local synthesis of complement proteins could be regulated independently of the synthesis in the liver.

Authors

R C Strunk, D M Eidlen, R J Mason

Requirements for the construction of antibody heterodimers for the direction of lysis of tumors by human T cells.

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We constructed a series of MAb heterodimers consisting of the J5 (anti-common acute lymphoblastic leukemia antigen [CALLA]) antibody and antibodies to a variety of structures present on the surface of activated human T cells, including CD3 antigen (T cell receptor-associated glycoproteins), CD2 antigen (T11/E-rosette receptor), CD25 antigen (IL-2 receptor), and the transferrin receptor. We tested the ability of these heterodimers to direct a CD2 + CD3 + CD8 + CD4 - CD25 + transferrin receptor + MHC-restricted human cytolytic T lymphocyte (CTL) clone to lyse a CALLA + human tumor in vitro. Only heterodimers containing an anti-CD3 antibody or activating antibodies to CD2 could direct the clone to lyse these human tumor targets, even when the clone was additionally activated with anti-CD3 or anti-CD2 antibodies. Our findings may have implications in the design of strategies for the use of such reagents in the treatment of human neoplasia.

Authors

C F Scott Jr, W A Blättler, J M Lambert, R S Kalish, C Morimoto, S F Schlossman

Pathogenesis of Campylobacter fetus infections. Failure of encapsulated Campylobacter fetus to bind C3b explains serum and phagocytosis resistance.

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Campylobacter fetus ssp. fetus strains causing systemic infections in humans are highly resistant to normal and immune serum, which is due to the presence of high molecular weight (100,000, 127,000, or 149,000) surface (S-layer) proteins. Using serum-resistant parental strains (82-40 LP and 23D) containing the 100,000-mol wt protein and serum-sensitive mutants (82-40 HP and 23B) differing only in that they lack the 100,000-mol wt protein capsule, we examined complement binding and activation, and opsono-phagocytosis by polymorphonuclear leukocytes. C3 consumption was similar for all four strains but C3 was not efficiently bound to 82-40 LP or 23D even in the presence of immune serum, and the small amount of C3 bound was predominently the hemolytically inactive iC3b fragment. Consumption and binding of C5 and C9 was significantly greater for the unencapsulated than the encapsulated strains. Opsonization of 82-40 HP with heat-inactivated normal human serum caused greater than 99% killing by human PMN. Similar opsonization of 82-40 LP showed no kill, but use of immune serum restored killing. Findings in a PMN chemiluminescence assay showed parallel results. Association of 32P-labeled 82-40 HP with PMN in the presence of HINHS was 19-fold that for the 82-40 LP, and electron microscopy illustrated that the difference was in uptake rather than in binding. These results indicate that presence of the 100,000-mol wt protein capsule on the surface of C. fetus leads to impaired C3b binding, thus explaining serum resistance and defective opsonization in NHS, mechanisms that explain the capacity of this enteric organism to cause systemic infections.

Authors

M J Blaser, P F Smith, J E Repine, K A Joiner

Acquired deficiencies of protein S. Protein S activity during oral anticoagulation, in liver disease, and in disseminated intravascular coagulation.

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Protein S is a vitamin K-dependent plasma protein which serves as the cofactor for activated protein C. Protein S circulates in both an active, free form and in an inactive complex with C4b-binding protein. To elucidate the role of protein S in disease states and during oral anticoagulation, we developed a functional assay for protein S that permits evaluation of the distribution of protein S between free and bound forms and permits determination of the specific activity of the free protein S. In liver disease, free protein S antigen is moderately reduced and the free protein S has significantly reduced specific activity. In disseminated intravascular coagulation, reduced protein S activity occurs due to a redistribution of protein S to the inactive bound form. During warfarin anticoagulation, reduction of free protein S antigen and the appearance of forms with abnormal electrophoretic mobility significantly decrease protein S activity. After the initiation of warfarin, the apparent half-life of protein S is 42.5 h. In patients with thromboembolic disease, transient protein S deficiency occurs due to redistribution to the complexed form. Caution should be exercised in diagnosing protein S deficiency in such patients by use of functional assays.

Authors

A D'Angelo, S Vigano-D'Angelo, C T Esmon, P C Comp

Bicarbonate absorption stimulates active calcium absorption in the rat proximal tubule.

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Abstract

To evaluate the effect of luminal bicarbonate on calcium reabsorption, rat proximal tubules were perfused in vivo. Perfusion solution contained mannitol to reduce water flux to zero. Total Ca concentration was measured by atomic absorption spectrometry, Ca ion concentration in the tubule lumen (CaL2+) and the peritubular capillary (CaP2+), and luminal pH (pHL) with ion-selective microelectrodes and transepithelial voltage (VTE) with conventional microelectrodes. When tubules were perfused with buffer-free Cl-containing solution, net Ca absorption (JCa) averaged 3.33 pmol/min. Even though VTE was 1.64 mV lumen-positive, CaL2+, 1.05 mM, did not fall below the concentration in the capillary blood, 1.07 mM. When 27 mM of Cl was replaced with HCO3, there was luminal fluid acidification. Despite a decrease in VTE and CaL2+, JCa increased to 7.13 pmol/min, indicating that the enhanced JCa could not be accounted for by the reduced electrochemical gradient, delta CCa. When acetazolamide or an analogue of amiloride was added to the HCO3 solution, JCa was not different from the buffer-free solution, suggesting that HCO3-stimulated JCa may be linked to acidification. To further test this hypothesis, we used 27 mM Hepes as the luminal buffer. With Hepes there was luminal fluid acidification and JCa was not different from the buffer-free solution but delta CCa was significantly reduced, indicating enhanced active calcium transport. We conclude from the results of the present study that HCO3 stimulates active Ca absorption, a process that may be linked to acidification-mediated HCO3 absorption.

Authors

K Bomsztyk, M B Calalb

Role of the hepatic artery in canalicular bile formation by the perfused rat liver. A multiple indicator dilution study.

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The role of the hepatic artery in tracer water exchange and regulation of permeation of small solutes during canalicular bile formation was studied in the rat using a system that permitted perfusion of both hepatic artery and portal vein. Hepatic vein and biliary multiple indicator dilution curves were obtained after injection of indicators into either vessel. The main difference in hepatic venous dilution curves was a 3.1-fold longer t0 (time spent in nonexchanging vessels) and a 5% larger equivalent water space after injection into the hepatic artery. Biliary tracer recovery of water was markedly higher after arterial injection than after portal vein injection. Both taurocholate and taurodehydrocholate stimulated bile flow and increased biliary tracer recovery after injection into either vessel. The biliary recovery of sucrose relative to that of water, which is a measure of biliary sucrose permeation, was much lower when given into the hepatic artery than when given into the portal vein. During taurocholate infusion, it decreased by 33% in the hepatic artery but increased 36% in the portal vein. Taurodehydrocholate, by contrast, did not affect permeation of sucrose given into the portal vein. Our studies demonstrate marked exchange of tracer water in the biliary epithelium. Taurocholate, but not taurodehydrocholate, increases permeation of sucrose into bile in the portal vein bed while both bile salts decrease it in the arterial bed.

Authors

J Reichen

Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.

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Abstract

The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.

Authors

A B Zimrin, R Eisman, G Vilaire, E Schwartz, J S Bennett, M Poncz

Dual mechanisms of regulation of type I iodothyronine 5'-deiodinase in the rat kidney, liver, and thyroid gland. Implications for the treatment of hyperthyroidism with radiographic contrast agents.

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Abstract

Alterations in thyroid hormone status and the administration of radiographic contrast agents can markedly influence iodothyronine metabolism and, in particular, the activity of type I 5'-deiodinase (5'DI). In the present studies, the mechanisms responsible for these effects have been reassessed. As previously reported, the addition of iopanoic acid (IOP) to broken cell preparations resulted in a competitive pattern of 5'DI inhibition. However, the in vivo administration to rats of IOP or 3,3',5'-triiodothyronine (rT3) resulted in a noncompetitive pattern of inhibition of 5'DI in the liver, kidney, and thyroid gland, whereby marked decreases in maximal enzyme velocity (V max) were noted, with no change in the value of the Michaelis-Menten constant. In rats rendered hyperthyroid by the injection of 3,5,3'-triiodothyronine (T3), 5'DI activity was significantly increased in the liver and the kidney. The administration of IOP to these thyrotoxic animals resulted in a rapid loss of enzyme activity characterized by an approximate 80% decrease in 5'DI V max values in both tissues. Furthermore, this inhibitory effect persisted for longer than 60 h after a single IOP injection. IOP administration also decreased 5'DI V max levels in the thyroid gland by 52%. In other experiments, treatment of intact Reuber FAO hepatoma cells with IOP or rT3 induced a rapid decrease in 5'DI V max levels. In cells treated with cycloheximide, these agents enhanced the rate of disappearance of enzyme activity by greater than 12-fold, indicating a predominant effect on accelerating the rate of enzyme inactivation and/or degradation. These studies demonstrate that iodothyronines and other iodinated compounds have complex regulatory effects on 5'DI that entail alterations in the rates of both enzyme activation and inactivation. The previously accepted concept that rT3 and IOP impair thyroxine (T4) to T3 conversion in vivo by acting as competitive inhibitors is an oversimplification. Rather, the clinically beneficial effects of administering these agents to patients with hyperthyroidism may result primarily from the rapid and prolonged inactivation of 5'DI which occurs in the thyroid gland and peripheral tissues.

Authors

D L St Germain

Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease.

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Abstract

The mechanisms regulating activation of the respiratory burst enzyme, NADPH oxidase, of human neutrophils (PMN) are not yet understood, but protein phosphorylation may play a role. We have utilized a defect in a cytosolic factor required for NADPH oxidase activation observed in two patients with the autosomal recessive form of chronic granulomatous disease (CGD) to examine the role of protein phosphorylation in activation of NADPH oxidase in a cell-free system. NADPH oxidase could be activated by SDS in reconstitution mixtures of cytosolic and membrane subcellular fractions from normal PMN, and SDS also enhanced phosphorylation of at least 16 cytosolic and 14 membrane-associated proteins. However, subcellular fractions from CGD PMN plus SDS expressed little NADPH oxidase activity, and phosphorylation of a 48-kD protein(s) was selectively defective. The membrane fraction from CGD cells could be activated for NADPH oxidase when mixed with normal cytosol and phosphorylation of the 48-kD protein(s) was restored. In contrast, the membrane fraction from normal cells expressed almost no NADPH oxidase activity when mixed with CGD cytosol, and phosphorylation of the 48-kD protein(s) was again markedly decreased. Protein kinase C (PKC) activity in PMN from the two patients appeared to be normal, suggesting that a deficiency of PKC is not the cause of the defective 48-kD protein phosphorylation and that the cytosolic factor is not PKC. These results demonstrate that the cytosolic factor required for activation of NADPH oxidase also regulates phosphorylation of a specific protein, or family of proteins, at 48 kD. Although the nature of this protein(s) is still unknown, it may be related to the functional and phosphorylation defects present in CGD PMN and to the activation of NADPH oxidase in the cell-free system.

Authors

S E Caldwell, C E McCall, C L Hendricks, P A Leone, D A Bass, L C McPhail

Activation of T lymphocytes by immobilized monoclonal antibodies to CD3. Regulatory influences of monoclonal antibodies to additional T cell surface determinants.

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Abstract

The effect of soluble or immobilized MAb directed at various additional surface proteins on the proliferation of highly purified T4 cells induced by two immobilized MAb to CD3, OKT3 and 64.1, was examined. High density 64.1 stimulated nearly all T4 cells to enter and progress through the cell cycle. Maximal T4 cell proliferation required stimulation with immobilized 64.1 throughout the length of the incubation and was not effected by any of the additional soluble or immobilized MAb employed. In contrast, low density immobilized 64.1 and all densities of immobilized OKT3 employed stimulated a minority of the cells to enter the cell cycle and proliferate. Immobilized MAb directed at CD2, class I major histocompatibility complex (MHC) encoded gene products or CD11a (LFA-1) dramatically enhanced the response, whereas soluble MAb directed at these determinants did not. Both immobilized and soluble MAb directed at CD5 and CD28 (Tp44) enhanced responses, but they were less effective than immobilized MAb to CD2, LFA-1 or HLA-A,B,C. Soluble anti-CD4 MAb inhibited responses somewhat, whereas immobilized anti-CD4 enhanced responses. Costimulation was observed when MAb to CD3 and class I MHC molecules but not CD2, LFA-1 or CD4 were immobilized to separate surfaces. The data suggest that when anti-CD3 stimulation is suboptimal, responses can be enhanced by MAb to CD5 or CD28 (Tp44) or by immobilized MAb to CD4, CD2, CD11a (LFA-1), or class I MHC encoded gene products. Although crosslinking of CD4, CD2, or CD11a with CD3 may be necessary for costimulation, immobilized MAb to CD3 and class I MHC molecules appear to deliver independent signals that result in enhanced T4 cell activation and proliferation.

Authors

T D Geppert, P E Lipsky

Transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes.

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Abstract

Regulation of tumor necrosis factor (TNF) gene expression was investigated in resting human monocytes and in 12-O-tetradecanoylphorbol-13-acetate (TPA) activated monocytes. TNF transcripts were undetectable in resting monocytes. However, in TPA-activated monocytes, TNF mRNA was first detectable by 3 h and reached maximal levels by 12 h of drug exposure. Using run-on transcription assays, the TNF gene was transcriptionally inactive in resting monocytes, but was rapidly activated after TPA exposure. The protein synthesis inhibitor, cycloheximide (CHX), had no detectable effect on levels of TNF transcripts in resting monocytes, while this agent superinduced the level of TNF mRNA by 50-fold in TPA-activated cells. TPA activated monocytes were also exposed to actinomycin D and/or CHX to determine whether transcriptional or posttranscriptional control of TNF gene expression was responsible for the induction of TNF transcripts. After 1 h of actinomycin D treatment, the amount of TNF transcripts was reduced by 75%. In contrast, no difference in TNF mRNA levels was observed in TPA-activated monocytes exposed to CHX alone or CHX in combination with actinomycin D. These findings indicated that CHX prevented the degradation of TNF mRNA by inhibiting the synthesis of a labile protein. Run-on transcription assays performed on cells exposed to either TPA or the combination of TPA and CHX further indicated that CHX treatment increased transcription of the TNF gene. Thus, TNF gene expression is controlled at the transcriptional level in resting human monocytes, while both transcriptional and posttranscriptional events regulate the level of TNF transcripts in TPA-activated cells.

Authors

E Sariban, K Imamura, R Luebbers, D Kufe

Complete protein sequences of the variable regions of the cloned heavy and light chains of a human anti-cytomegalovirus antibody reveal a striking similarity to human monoclonal rheumatoid factors of the Wa idiotypic family.

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Abstract

The complete amino acid and nucleotide sequences of the variable regions of the heavy and light polypeptide chains of a human neutralizing IgGl anti-cytomegalovirus (CMV) antibody reveal a striking homology to IgM rheumatoid factors (RFs) of the Wa idiotypic family. The anti-CMV antibody and Wa RFs have in common VKIIIb, JKl, and VHIa gene segments but use different DH and JH gene segments. The anti-CMV antibody does not have RF activity and does not express the Wa idiotype. The Wa RFs do not have anti-CMV activity. A subset of Wa RFs, however, and the anti-CMV antibody do share several idiotypes on the VHIa and VKIIIb polypeptides. Since there are major differences in the antigen binding characteristics and some of the other expressed idiotypes, these data suggest that the D and J region amino acids are crucial to such specificities. Although the use of such highly homologous gene segments in different immune responses is well-documented in murine systems, these data represent the first such example in the human.

Authors

M M Newkirk, H Gram, G F Heinrich, L Ostberg, J D Capra, R L Wasserman

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

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Abstract

The possible role of group specific component (Gc) (vitamin D-binding protein) in the clearance of cellular actin entering the circulation was examined with 125I-labeled Gc and actin injected into a rabbit model. Although filamentous F-actin is depolymerized primarily by plasma gelsolin, greater than or equal to 90% 125I-actin injected in either monomeric G- or F-form became complexed eventually with Gc (1:1 molar ratio). Clearance of Gc complexes was much faster (greater than 90% within 5 h) than that of native Gc (t1/2 = 17.2 h). Nephrectomy did not significantly alter the clearance of either Gc or actin. Since Gc complexes are dramatically increased in situations of tissue necrosis such as in fulminant hepatic failure, the current results suggest a crucial role for Gc in sequestration and clearance of released cellular actin.

Authors

P J Goldschmidt-Clermont, H Van Baelen, R Bouillon, T E Shook, M H Williams, A E Nel, R M Galbraith

Role of glucose transporters in the cellular insulin resistance of type II non-insulin-dependent diabetes mellitus.

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Abstract

To examine the role of glucose transport proteins in cellular insulin resistance, we studied subcutaneous adipocytes isolated from lean control, obese control (body mass index [BMI] 33.4 +/- 0.9), and untreated obese non-insulin-dependent diabetes mellitus (NIDDM) patients (BMI 35.2 +/- 2.1; fasting glucose 269 +/- 20 mg/dl). Glucose transporters were measured in plasma membrane (PM), low-density (LDM), and high-density (HDM) microsomal subfractions from basal and maximally insulin-stimulated cells using the cytochalasin B binding assay, and normalized per milligram of membrane protein. In all subgroups, insulin led to an increase in PM glucose transporters and a corresponding depletion of transporters in the LDM. Insulin recruited 20% fewer transporters to the PM in the obese subgroup when compared with lean controls, and this was associated with a decline in LDM transporters with enlarging cell size in the control subjects. In NIDDM, PM, and LDM, transporters were decreased 50% in both basal and stimulated cells when compared with obese controls having similar mean adipocyte size. Cellular depletion of glucose transporters was not the only cause of insulin resistance, because the decrease in rates of [14C]-D-glucose transport (basal and insulin-stimulated) was greater than could be explained by reduced numbers of PM transporters in both NIDDM and obesity. In HDM, the number of transporters was not influenced by insulin and was similar in all subgroups. We conclude that (a) in NIDDM and obesity, both reduced numbers and impaired activity of glucose transporters contribute to cellular insulin resistance, and (b) in NIDDM, more profound cellular insulin resistance is associated primarily with a further depletion of cellular transporters.

Authors

W T Garvey, T P Huecksteadt, S Matthaei, J M Olefsky

Thrombospondin-induced adhesion of human keratinocytes.

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Abstract

Human epidermal keratinocytes obtained from normal skin attached and spread on thrombospondin (TSP)-coated plastic dishes but failed to attach and spread on untreated plastic culture dishes or dishes coated with fibronectin or laminin. These cells produced minimal amounts of immunoreactive TSP. Keratinocytes established in culture on MCDB 153 medium and maintained for one to three passages in an undifferentiated state by continued cultivation in this low Ca2+-containing medium attached and spread on plastic dishes as well as on TSP-coated dishes. These cells also secreted significant amounts of TSP into the culture medium. When the keratinocytes were incubated for one day in MCDB 153 medium supplemented with high Ca2+ or in MEM (which also contains high Ca2+), there was decreased secretion of TSP into the culture medium concomitant with a reduction in attachment and spreading on plastic culture dishes. Proteolytic fragments of TSP were examined for stimulation of keratinocyte attachment and spreading. A 140-kd fragment produced by removal of the 25-kd heparin-binding domain had similar activity to the intact molecule while the 25-kd fragment was without effect. Further proteolytic treatment of the 140-kd fragment gave rise to a fragment consisting of 120 kd and 18-D moieties held together in disulphide linkage. This fragment did not support attachment or spreading. This study reveals that normal epidermal keratinocytes grown under conditions that maintain the undifferentiated state are able to produce TSP and utilize it as an attachment factor. When keratinocytes are grown under conditions that promote differentiation, ability to produce and utilize TSP is diminished. Since TSP is present at the dermal-epidermal junction and because TSP promotes keratinocyte attachment and spreading, this molecule may play an important role in maintaining normal growth of the basal cell layer and may also participate in reepithelialization during wound repair.

Authors

J Varani, B J Nickoloff, B L Riser, R S Mitra, K O'Rourke, V M Dixit

Hormonal control of substrate cycling in humans.

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Abstract

Recent studies have established the existence of substrate cycles in humans, but factors regulating the rate of cycling have not been identified. We have therefore investigated the acute response of glucose/glucose-6P-glucose (glucose) and triglyceride/fatty acid (TG/FA) substrate cycling to the infusion of epinephrine (0.03 microgram/kg.min) and glucagon. The response to a high dose glucagon infusion (2 micrograms/kg.min) was tested, as well as the response to a low dose infusion (5 ng/kg.min), with and without the simultaneous infusion of somatostatin (0.1 microgram/kg.min) and insulin (0.1 mU/kg.min). Additionally, the response to chronic prednisone (50 mg/d) was evaluated, both alone and during glucagon (low dose) and epinephrine infusion. Finally, the response to hyperglycemia, with insulin and glucagon held constant by somatostatin infusion and constant replacement of glucagon and insulin at basal rates, was investigated. Glucose cycling was calculated as the difference between the rate of appearance (Ra) of glucose as determined using 2-d1- and 6,6-d2-glucose as tracers. TG/FA cycling was calculated by first determining the Ra glycerol with d5-glycerol and the Ra FFA with [1-13C]palmitate, then subtracting Ra FFA from three times Ra glycerol. The results indicate that glucagon stimulates glucose cycling, and this stimulatory effect is augmented when the insulin response to glucagon infusion is blocked. Glucagon had minimal effect on TG/FA cycling. In contrast, epinephrine stimulated TG/FA cycling, but affected glucose cycling minimally. Prednisone had no direct effect on either glucose or TG/FA cycling, but blunted the stimulatory effect of glucagon on glucose cycling. Hyperglycemia, per se, had no direct effect on glucose or TG/FA cycling. Calculations revealed that stimulation of TG/FA cycling theoretically amplified the sensitivity of control of fatty acid flux, but no such amplification was evident as a result of the stimulation of glucose cycling by glucagon.

Authors

H Miyoshi, G I Shulman, E J Peters, M H Wolfe, D Elahi, R R Wolfe

Xanthine oxidase-derived hydrogen peroxide contributes to ischemia reperfusion-induced edema in gerbil brains.

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Abstract

The contribution of toxic O2 metabolites to cerebral ischemia reperfusion injury has not been determined. We found that gerbils subjected to temporary unilateral carotid artery occlusion (ischemia) consistently developed neurologic deficits during ischemia with severities that correlated with increasing degrees of brain edema and brain H2O2 levels after reperfusion. In contrast, gerbils treated just before reperfusion (after ischemia) with dimethylthiourea (DMTU), but not urea, had decreased brain edema and brain H2O2 levels. In addition, gerbils fed a tungsten-rich diet for 4, 5, or 6 wk developed progressive decreases in brain xanthine oxidase (XO) and brain XO + xanthine dehydrogenase (XD) activities, brain edema, and brain H2O2 levels after temporary unilateral carotid artery occlusion and reperfusion. In contrast to tungsten-treated gerbils, allopurinol-treated gerbils did not have statistically significant decreases in brain XO or XO + XD levels, and reduced brain edema and brain H2O2 levels occurred only in gerbils developing mild but not severe neurologic deficits during ischemia. Finally, gerbils treated with DMTU or tungsten all survived, while greater than 60% of gerbils treated with urea, allopurinol, or saline died by 48 h after temporary unilateral carotid artery occlusion and reperfusion. Our findings indicate that H2O2 from XO contributes to reperfusion-induced edema in brains subjected to temporary ischemia.

Authors

A Patt, A H Harken, L K Burton, T C Rodell, D Piermattei, W J Schorr, N B Parker, E M Berger, I R Horesh, L S Terada

Skeletal muscle glycolysis, oxidation, and storage of an oral glucose load.

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Abstract

Although muscle is considered to be the most important site for postprandial glucose disposal, the metabolic fate of oral glucose taken up by muscle remains unclear. We, therefore, employed the dual isotope technique (intravenous, [6-3H]-glucose; oral, [1-14C]glucose), indirect calorimetry, and forearm balance measurements of glucose, lactate, alanine, pyruvate, O2, and CO2 in nine normal volunteers to determine the relative importance of muscle glycogenic, glycolytic, and oxidative pathways in disposal of an oral glucose load. During the 5 h after glucose ingestion (1 g/kg), 37 +/- 3% (24.9 +/- 2.3 g) of the load was oxidized and 63 +/- 3% (42.8 +/- 2.7 g) was stored. At least 29% (19.4 +/- 1.3 g) was taken up by splanchnic tissues. Muscle took up 26% (17.9 +/- 2.9 g) of the oral glucose coincident with a 50% reduction in its oxidation of fat. 15% of the oral glucose taken up by muscle (2.5 +/- 0.9 g) was released as lactate, alanine, or pyruvate; 50% (8.9 +/- 1.4 g) was oxidized, and 35% (6.4 +/- 2.3 g) was available for storage. We conclude that muscle and splanchnic tissues take up a comparable percentage of an oral glucose load and that oxidation is the predominant fate of glucose taken up by muscle, with storage in muscle accounting for less than 10% of the oral load. Thus, contrary to the prevailing view, muscle is neither the major site of storage nor the predominant site of disposal of an oral glucose load.

Authors

D Kelley, A Mitrakou, H Marsh, F Schwenk, J Benn, G Sonnenberg, M Arcangeli, T Aoki, J Sorensen, M Berger

Effects of basic fibroblast growth factor on bone formation in vitro.

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Abstract

Basic fibroblast growth factor (bFGF) was studied for its effects on bone formation in cultured rat calvariae. bFGF at 0.1-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to 4.4-fold. bFGF also increased the number of colcemid-induced metaphase arrested cells and the DNA content. Transient (24 h) treatment with bFGF enhanced [3H]-proline incorporation into collagen 24-48 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. The collagen stimulated by bFGF was type I, and this effect was observed primarily in the periosteum-free bone. In contrast, continuous treatment with bFGF for 24-96 h inhibited [3H]proline incorporation into type I collagen. bFGF did not alter collagen degradation. In conclusion, bFGF stimulates calvarial DNA synthesis, which causes an increased number of collagen-synthesizing cells, but bFGF has a direct inhibitory effect on collagen synthesis.

Authors

E Canalis, M Centrella, T McCarthy

Phorbol ester and A23187 have additive but mechanistically separate effects on vasopressin action in rabbit collecting tubule.

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Abstract

Activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration ([Ca++]i) result from phosphatidylinositol biphosphate (PIP2) breakdown. We previously demonstrated that PKC activation inhibits arginine vasopressin (AVP)-induced osmotic water flow in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. To estimate the potential significance of PIP2 turnover as a modulator of water transport in this nephron segment, we examined the effect of Ca on AVP action and explored the mechanisms of action of PKC and increased [Ca++]i. In rabbit CCTs perfused at 37 degrees C, pretreatment with bath A23187 (2 x 10(-8) M, 2 x 10(-6) M), a Ca ionophore, almost totally suppressed AVP (10 microU/ml)-induced peak hydraulic conductivity (Lp). The suppression by 2 x 10(-8) M A23187 was as potent as that by 2 x 10(-6) M A23187, and significant even when it was administered 10 min after AVP. When phorbol myristate acetate (PMA, 10(-9) M), a PKC activator, and A23187 (2 x 10(-8) M) were placed in the bath simultaneously, the combined suppressive effect on peak Lp was greater than that of either inhibitor alone. However, the mechanisms of inhibition by PMA and A23187 were different. While both 10(-7) and 10(-9) M PMA suppression are primarily post-cAMP, A23187 predominantly suppressed a pre-cAMP step: 10(-4) M chlorophenylthio-cAMP-induced peak Lp was not affected by 2 x 10(-8) M A23187, and only partially inhibited by 2 x 10(-6) M A23187. The PMA (10(-7) M) suppression of AVP-induced peak Lp was totally reversed by bath staurosporine (10(-7) M), a PKC inhibitor, but not attenuated by either bath indomethacin (5 x 10(-6) M) or low Ca (1-2 x 10(-6) M) bath medium. In contrast, the A23187 (2 x 10(-8) M) suppression of the peak Lp was not affected by staurosporine, but was significantly reversed by indomethacin or low Ca bath medium. We conclude: (a) Elevation of [Ca++]i, as well as activation of PKC, suppresses the hydroosmotic effect of AVP on CCT at 37 degrees C. (b) When stimulated simultaneously these two intracellular mediators are additive in their antagonism of AVP action. These results suggest that stimulated PIP2 breakdown may be an important modulator of water transport in CCT. (c) Different mechanisms underlie PKC and Ca-mediated suppression of the AVP-induced water transport. The inhibition of AVP action by increased [Ca++]i is primarily pre-cAMP, and involves a cyclooxygenase metabolite(s) of arachidonic acid, while the inhibition by PKC is post-cAMP, and independent of cyclooxygenase products of arachidonic acid.

Authors

Y Ando, H R Jacobson, M D Breyer

Indomethacin secretion in the isolated perfused proximal straight rabbit tubule. Evidence for two parallel transport mechanisms.

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Abstract

We studied indomethacin as a probe of anion transport across the isolated perfused proximal straight tubule of the rabbit and discovered that a substantial component of transport may occur by anion exchange at the basolateral membrane. Various perturbations involving direct or indirect dissipation of the cellular sodium gradient (ouabain, sodium- or potassium-free solutions, cooling to 18 degrees C) resulted in only a 50% inhibition of indomethacin transport, which raised the question of a co-existent alternative pathway for secretion. Similarly, the anion exchange inhibitor, 4,4'-diisothiocyanostilbene (DIDS), diminished indomethacin secretion by only 50%. Cooling followed by DIDS or the reverse sequence resulted in additive inhibition such that the combination abolished active secretion of indomethacin. We conclude that active secretion of indomethacin by the proximal straight tubule appears to be in part sodium gradient dependent; the remainder may be driven by an anion exchanger on the basolateral membrane.

Authors

D de Zeeuw, H R Jacobson, D C Brater

Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in the rat.

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Abstract

Biliary secretion of 3 alpha-sulfated bile acids has been studied in Wistar rats with an autosomal recessive defect in the hepatic transport of bilirubin. Liver function, established by measurement of various enzymes in plasma, by enzyme histochemical methods, and by electron microscopy, appeared to be normal in these rats. Serum levels of unconjugated, monoglucuronidated, and diglucuronidated bilirubin were 0.62, 1.62, and 6.16 mumol/liter, respectively, compared with 0.17, 0.08, and 0.02 mumol/liter in control rats. Biliary bilirubin secretion was strongly reduced in the mutant animals: 0.21 +/- 0.03 vs. 0.39 +/- 0.03 nmol/min per 100 g body wt in control rats. Despite normal biliary bile acid output, bile flow was markedly impaired in the mutant animals, due to a 53% reduction of the bile acid-independent fraction of bile flow. The transport maximum for biliary secretion of dibromosulphthalein (DBSP) was also drastically reduced (-53%). Biliary secretion of intravenously administered trace amounts of the 3 alpha-sulfate esters of 14C-labeled taurocholic acid (-14%), taurochenodeoxycholic acid (-39%), taurolithocholic acid (-73%), and glycolithocholic acid (-91%) was impaired in the jaundiced rats compared with controls, in contrast to the biliary secretion of the unsulfated parent compounds. Hepatic uptake of sulfated glycolithocholic acid was not affected in the jaundiced animals. Preadministration of DBSP (15 mumol/100 g body wt) to normal Wistar rats significantly impaired the biliary secretion of sulfated glycolithocholic acid, but did not affect taurocholic acid secretion. We conclude that separate transport systems in the rat liver exist for biliary secretion of sulfated and unsulfated bile acids; the sulfates probably share secretory pathways with the organic anions bilirubin and DBSP. The described genetic defect in hepatic transport function is associated with a reduced capacity to secrete sulfated bile acids into bile; this becomes more pronounced with a decreasing number of hydroxyl groups on the sulfated bile acid's molecule.

Authors

F Kuipers, M Enserink, R Havinga, A B van der Steen, M J Hardonk, J Fevery, R J Vonk

Identification and isolation of a platelet GPIb-like protein in human umbilical vein endothelial cells and bovine aortic smooth muscle cells.

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Abstract

Glycoprotein Ib (GPIb) is an intrinsic platelet membrane protein that plays a major role in platelet adherence and mediates ristocetin-dependent platelet von Willebrand factor binding. Recent reports that the platelet membrane glycoprotein complex IIb/IIIa is expressed in several cell types prompted us to look for GPIb expression in other vascular cells. Immunoperoxidase staining of human stomach and skin histologic sections with polyclonal as well as monoclonal anti-GPIb antibody revealed the presence of GPIb in the endothelial cell and smooth muscle cell layers. Western blotting using monospecific polyclonal anti-GPIb antibodies confirmed the presence of immunoreactive GPIb in human umbilical vein endothelial and bovine aortic smooth muscle cell cultures. Fab fragments of a monoclonal anti-GPIb antibody were used to immunoprecipitate [3H]leucine labeled GPIb from metabolically labeled cells. The GPIb in these cells was functional as measured by ristocetin-dependent cell agglutination and by vWF binding. Endothelial cells as well as smooth muscle cells bound 125I-labeled vWF in a ristocetin-dependent manner, with a Kd of 7.9 nM.

Authors

A S Asch, B Adelman, M Fujimoto, R L Nachman

Pathogenetic relevance of HLA class II expressing thyroid follicular cells in nontoxic Goiter and in Graves' disease.

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Abstract

HLA class II expressing thyroid follicular cells are found not only in classical thyroid autoimmune diseases, such as Graves' disease, but also in presumably nonautoimmune thyroid disorders such as nontoxic goiter. In this study the immunostimulatory function of the HLA class II expressing thyroid follicular cells derived from patients with nontoxic goiter and with Graves' disease was compared by assessing their capacity to stimulate allogeneic and autologous peripheral blood mononuclear cells, as well as cultured intrathyriodal T lymphocytes. Proliferation of allogeneic peripheral blood mononuclear cells was stimulated by thyroid follicular cells from both nontoxic goiter and Graves' disease thyroids, thus demonstrating that thyroid follicular cells from both disorders are capable of presenting alloantigens. In contrast the proliferation of autologous peripheral blood mononuclear cells was more efficiently stimulated by thyroid follicular cells from Graves' disease than from nontoxic goiter. Cultured intrathyroidal T lymphocytes proliferated specifically in response to autologous HLA class II+ thyroid follicular cells in Graves' disease, but not in nontoxic goiter. The responses were dose dependent and HLA class II restricted. Thyroid autoantigen presentation by HLA class II expressing thyroid follicular cells thus only occurs in Graves' disease, suggesting that HLA class II expression on thyroid follicular cells is an essential feature, but by itself not sufficient for the induction of autoimmunity. Additional factors, the possible nature of which is discussed must also be involved.

Authors

B Grubeck-Loebenstein, M Londei, C Greenall, K Pirich, H Kassal, W Waldhäusl, M Feldmann

Regulation of intestinal and hepatic apoprotein synthesis after chronic fat and cholesterol feeding.

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Abstract

Although diet influences levels of lipoproteins and their corresponding apoproteins, its effects on the molecular regulation of apoprotein synthesis are relatively unknown. Male Sprague-Dawley rats were fed an atherogenic diet containing cholesterol and propylthiouracil (PTU). Intestinal apo AI and AIV mRNA concentrations were decreased by the atherogenic diet, but apo AI and AIV synthesis was increased in vitro (organ explants) and in vivo (polysome runoff), consistent with regulation at the translational level. In contrast, hepatic apo E mRNA concentration and synthesis were increased after the atherogenic diet, consistent with pretranslational regulation. The response to cholesterol feeding for hepatic apo AI and E showed a third pattern of regulation, in which synthesis increased and mRNA content remained stable or fell, again suggesting translational control, but polysome runoff synthesis was unchanged. The apparent importance of translational regulation in the intestine is consistent with the necessity for the tissue to respond rapidly to changes in intraluminal content.

Authors

M F Go, G Schonfeld, B Pfleger, T G Cole, N L Sussman, D H Alpers

Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. An abundant transcript induced by transformation of fibroblasts.

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Abstract

Transfection of an activated rat oncogene into NIH3T3 fibroblasts leads to transformation and induction of a metastatic phenotype. To identify genes whose activation might mediate these processes, we used a differential screening strategy. A 1.5-kb transcript is induced fiftyfold, constitutes 1% of ras transformed cell messenger RNA (mRNA) and is the most abundantly induced message in these cells. Our sequence data shows that it encodes murine cathepsin L, a potent collagenolytic and elastinolytic lysosomal enzyme. The murine clone was used to isolate human cathepsin L complementary DNA (cDNA) clones. The complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L are presented and compared to other papain family cysteine proteinases. Northern analysis shows that both human and murine cathepsin L probes hybridize to a 1.5-kb transcript in several tissues, but also to a 4-kb transcript in human kidney. These clones will facilitate studies of the structure, expression, and function of cathepsin L, including its unexpected upregulation in transformation.

Authors

L J Joseph, L C Chang, D Stamenkovich, V P Sukhatme

Ischemic acute renal failure and antioxidant therapy in the rat. The relation between glomerular and tubular dysfunction.

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Abstract

The effects of antioxidant therapy with probucol were evaluated in rats subjected to 1 h renal ischemia and to 24 h reperfusion. Probucol exerted significant antioxidant effects in renal cortical tubules in vitro when exposed to a catalase-resistant oxidant. At 24 h probucol treatment (IP) improved single nephron glomerular filtration rate (SNGFR) (28.1 +/- 3.3 nl/min) in comparison to untreated ischemic (I) rats (15.2 +/- 3.0), primarily as a result of improving SNGFR in a population of low SNGFR, low flow and/or obstructed nephrons. However, absolute proximal reabsorption remained abnormally low in IP rats at 24 h (5.9 +/- 0.8 nl/min), and cell necrosis was greater than in I rats. Kidney GFR remained low in IP rats due to extensive tubular backleak of inulin measured by microinjection studies. Evaluations after 2 h of reperfusion revealed a higher SNGFR in IP (36 +/- 3.1 nl/min) than I rats (20.8 +/- 2.7 nl/min). Absolute proximal reabsorption was essentially normal (11.6 +/- 1.3 nl/min) in IP rats, which was higher than IP rats at 24 h and the concurrent I rats. Administration of the lipophilic antioxidant, probucol, increased SNGFR and proximal tubular reabsorption within 2 h after ischemic renal failure. Although SNGFR remained higher than I rats at 24 h, absolute reabsorption fell below normal levels and tubular necrosis was more extensive in IP rats. Early improvement in nephron filtration with antioxidants may increase load dependent metabolic demand upon tubules and increase the extent of damage and transport dysfunction.

Authors

J E Bird, K Milhoan, C B Wilson, S G Young, C A Mundy, S Parthasarathy, R C Blantz

Exaggerated triglyceride accretion in human preadipocyte-murine renal line hybrids composed of cells from massively obese subjects.

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Abstract

To learn about adipose differentiation of precursors from postnatal adipose tissue of lean and massively obese subjects, human omental adipocyte precursor-murine renal adenocarcinoma cell (RAG) hybrids were formed by fusion with polyethylene glycol, and cultured selectively with 50 microM ouabain in hypoxanthine aminopterin thymidine (HAT) medium. Under conditions in which the parent cells did not differentiate, a number of hybrids, which were cloned, revealed morphologic and biochemical evidence of differentiation. In addition to activation of human genes within the common nucleus of the hybrids, murine cytoplasmic activators are probably also involved because heterocaryons (fused cells with two interspecific nuclei) revealed the same phenomenon. Hybrids composed of precursors from massively obese subjects disclosed more frequent and prominent differentiation. Since these hybrids, in contrast to those from the lean, recapitulate this phenomenon in subcultures, they provide the potential system for mapping the human gene(s) responsible for adipose differentiation and its exaggeration in massive obesity.

Authors

P E Le Blanc, D A Roncari, D I Hoar, A M Adachi

Proteolytic inactivation of alpha-1-proteinase inhibitor by a neutrophil metalloproteinase.

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Abstract

Human neutrophils triggered with phorbol myristate acetate or opsonized zymosan particles released a metalloproteinase (MP) capable of cleaving and inactivating alpha-1-proteinase inhibitor (alpha-1-PI). Sequence analysis of the amino acids in proteolyzed, native alpha-1-PI revealed a unique single cleavage site between Phe-352 and Leu-353. An analysis of the process regulating the enzyme's activity revealed that the neutrophil MP was released from cells in a latent form whose activation was tightly linked to the generation of hypochlorous acid. These results indicate that human neutrophils use chlorinated oxidants to activate a latent MP that is capable of proteolytically inactivating alpha-1-PI by cleaving the antiproteinase at a unique point in its inhibitory site region.

Authors

P E Desrochers, S J Weiss