Gunn rats are a mutant strain of Wistar rats that have unconjugated hyperbilirubinemia due to absence of hepatic uridine diphosphate-glucuronosyltransferase (UDPGT; EC. 18.104.22.168) activity toward bilirubin. We isolated five UDPGT isoforms from solubilized microsomal fractions from liver of inbred Wistar (RHA) rats and congeneic Gunn rats. UDPGT isoform V (elution pH 7.5) from Wistar (RHA) rats is active toward bilirubin and 4'-hydroxydimethylaminoazobenzene. The corresponding isoform from Gunn rat liver was enzymically inactive but exhibited normal elution pH and mobility on NaDodSO4/polyacrylamide gel electrophoresis (Mr 53,000), and was recognized by a UDPGT-specific antiserum. UDPGT isoform I (elution pH 8.7) from Wistar (RHA) and Gunn rats was active toward 4-nitrophenol. The isoform from Gunn rat liver had only 10% of normal UDPGT activity, however UDPGT activity increased to normal upon addition of 15 mM diethylnitrosamine in vitro. Isoforms II (elution pH 8.4), III (elution pH 8.0), and IV (elution pH 7.8) from Gunn rats had normal UDPGT activities, except that Isoform IV was inactive toward bilirubin.
N Roy Chowdhury, F Gross, A D Moscioni, M Kram, I M Arias, J Roy Chowdhury
We did experiments to determine whether beta-adrenergic agonists increase lung liquid clearance in anesthetized ventilated adult sheep and, if so, whether the increase is mediated by beta receptors and what mechanism is involved. We instilled 100 ml of autologous serum either alone or with a beta-adrenergic agonist (terbutaline, 10(-5) M, or epinephrine, 5.5 X 10(-6) M) into one lower lobe. After 4 h both terbutaline and epinephrine increased lung liquid clearance. The increase in lung liquid clearance was inhibited when propranolol (a beta blocker) or amiloride (a sodium channel blocker) was added to the terbutaline. Increased clearance was not explained by changes in pulmonary hemodynamics, pulmonary blood flow, or lung lymph flow. We conclude that beta-adrenergic agonists increase lung liquid clearance in anesthetized intact adult sheep. This increase is mediated through beta receptors and probably depends on increased active transport of sodium across the alveolar barrier.
Y Berthiaume, N C Staub, M A Matthay
Thrombin, collagen, and Ca2+-ionophore A23187 aggregate platelets in the presence of inhibitors of the first (ADP-mediated) and second (cyclooxygenase-dependent) pathway of platelet activation. This aggregation, via a third pathway, was hypothesized to be mediated by the alkoxyether lipid platelet-activating factor (PAF). We recently demonstrated virtual absence of plasmalogen-type alkoxyether lipids and deficiency in key enzymes of their biosynthesis in Zellweger patients. We hypothesized that PAF synthesis might also be impaired. We report two Zellweger patients with an undetectable A23187-induced PAF synthesis of leukocytes (patients, less than 3 pmol PAF/10(8) granulocytes (PMN); four age-matched controls, 249-2,757 pmol PAF/10(8) PMN; five adult controls, 291-5,433 pmol PAF/10(8) PMN). In a third patient, residual PAF synthesis was detected. However in all patients the thrombin-induced third mechanism of platelet aggregation was present. We therefore conclude that PAF may not be the mediator of the third pathway.
A Sturk, M C Schaap, J W ten Cate, H S Heymans, R B Schutgens, H Przyrembel, P Borst
The developmental origin of the four phenotypically distinct hormone-producing islet cells (insulin, glucagon, somatostatin, pancreatic polypeptide) is unclear. To investigate the potential for phenotypic differentiation of islet cells, we prepared several clonal cell lines from a radiation-induced rat islet tumor and analyzed them for insulin, glucagon, and somatostatin gene expression by cDNA hybridization, immunocytochemistry, and radioimmunoassay. We found expression of all three genes in the tumor and in the parental cell line and mixed variable phenotypes in the clonal lines derived from the parental line. We also observed the ectopic expression of the angiotensinogen gene in the tumor and the cell lines. The relative levels of hormonal gene expression differed among the cell lines but remained fixed during continuous passage. The three islet hormone mRNAs were larger compared to the pancreas owing to longer poly(A) tracts. These observations indicate that neoplastic islet cells retain the potential to differentiate into hormone-specific cellular phenotypes and may mimic developmental pathways of the pancreatic islets.
J Philippe, W L Chick, J F Habener
Free fatty acids are considered to be the major energy source for the myocardium. To investigate the metabolic fate of this substrate in humans, 24 subjects underwent coronary sinus and arterial catheterization. 13 subjects were healthy volunteers and 11 subjects had symptoms of ischemic heart disease. [1-14C]oleate or [1-14C]palmitate bound to albumin was infused at a constant rate of 25 microCi/h. Oxidation was determined by measuring the 14CO2 production. The data demonstrated that a high percentage (84 +/- 17%) of the palmitate and oleate extracted by the myocardium underwent rapid oxidation. A highly significant correlation was present between the arterial level and the amount oxidized (r = 0.82, P less than 0.001 for palmitate; r = 0.77, P less than 0.001 for oleate). The isotope extraction ratio was greater than the chemical extraction ratio. This difference of 6 +/- 2 nmol/ml of blood in the young normal subjects was significantly less than the 12 +/- 4 nmol/ml observed in the ischemic heart disease patients (P less than 0.001).
J A Wisneski, E W Gertz, R A Neese, M Mayr
In view of the similarity of cataracts and neuropathy in galactose-fed and diabetic rats, the present experiments were undertaken to determine whether consumption of galactose-enriched diets (10, 25, or 50% by weight) also increases collagen crosslinking and permeation of vessels by 125I-albumin analogous to that observed in diabetic rats. The observations in these experiments: demonstrate that consumption of galactose-enriched diets for 3 wk selectively increases 125I-albumin permeation of the same vascular beds affected in diabetic rats and by diabetic vascular disease in humans (i.e., the aorta and vessels in the eye, kidney, sciatic nerve, and new tissue formed in the diabetic milieu); demonstrate that the susceptibility of the vasculature to aldose reductase-linked injury (increased permeability) varies greatly in different tissues; indicate that collagen solubility (crosslinking) changes in galactose-fed rats differ sharply from those in diabetic rats; and provide new evidence that consumption of galactose-enriched diets induces a hypogonadal state in male rats.
K Chang, M Tomlinson, J R Jeffrey, R G Tilton, W R Sherman, K E Ackermann, R A Berger, T J Cicero, C Kilo, J R Williamson
Protein S is a vitamin K-dependent glycoprotein cofactor to the serine protease, activated protein C. In this study we demonstrate that 125I-protein S bound to unstimulated platelets in a time- and calcium-dependent saturable reaction. Half-maximal binding occurred at a protein S concentration of 10 nM, with approximately 1,100 binding sites per platelet. The binding of protein S to platelets was followed by rapid cleavage of the protein mediated by a protease confined to the platelet membrane. The membrane protease was Ca++-dependent, inhibited by high concentrations of diisopropyl fluorophosphate, but was resistant to a variety of other protease inhibitors. Functional studies demonstrated that the cleavage of protein S was associated with complete loss of cofactor anticoagulant activity. We conclude that protein S binds to platelets and is inactivated by a novel Ca++-dependent membrane protease. This may represent a physiological reaction that regulates the activity of protein S.
C A Mitchell, H H Salem
The role of membrane transport in the cellular accumulation of 1-beta-D-arabinofuranosylcytosine (ara-C) was studied in freshly isolated human acute leukemia cells. Patient cells had low rates for ara-C transport as compared with human and murine experimental cells and correspondingly low binding capacities for the nucleoside transport inhibitor, nitrobenzylmercaptopurine riboside (NBMPR). At 1 microM ara-C, the rate of net cellular accumulation was close to the membrane transport rate, and NBMPR inhibited transport and accumulation to the same extent. The rate of ara-C accumulation was half maximal at only 3-5 microM, a level much lower than that required for murine cells (67-85 microM). At concentrations below 1 microM the rate of ara-C accumulation was determined primarily by the transport rate, but at higher concentrations above 10 microM, phosphorylation capacity was the principal determinant of the net uptake rate. This difference in the role of transport at high and low ara-C concentrations may explain, in part, the efficacy of high-dose ara-C in patients refractory to standard dose protocols.
J C White, J P Rathmell, R L Capizzi
Two approaches were used to demonstrate that reduction in serum opsonization of Streptococcus pneumoniae via the alternative complement pathway in children with sickle cell disease is related to a deficiency of antibodies to pneumococcal capsular polysaccharide. First, opsonization of S. pneumoniae mediated by the alternative pathway in patients' sera was restored to normal by addition of the purified IgG or IgM fraction of goat antiserum to capsular polysaccharide of the homologous serotype. Secondly, IgG antibody titers to capsular polysaccharide in patients' sera correlated significantly with alternative pathway-mediated opsonization; the correlation between titers of IgM anticapsular antibodies and opsonization approached statistical significance. The sum of the IgG and IgM anticapsular antibody titers correlated most significantly with opsonization. Our results suggest that reduction in alternative pathway-mediated opsonization in sera from children with sickle cell disease is related to low levels of both IgG and IgM anticapsular antibodies.
A B Bjornson, J S Lobel
This paper demonstrates that in the presence of indomethacin, a cyclooxygenase inhibitor, 100% of the mice died when infected with live Listeria, whereas none of the animals died in the absence of the drug. The death of the animals correlated with the numbers of bacteria found extraperitoneally in the spleen and not with the Ia expression of the peritoneal macrophages. Increases in the spleen bacterial numbers between mice treated with either indomethacin or a specific thromboxane synthase inhibitor, OKY1581, and those not receiving either drug, were found as early as 2-4 h after infection. The differences in the initial increased bacterial spleen counts in the presence of indomethacin were reversed by administration of a stable thromboxane A2 analog or another potent vasoconstrictor, phenylephrine. Because thromboxane A2 does not regulate macrophage or T cell functions directly (Tripp, C.S., A. Wyche, E.R. Unanue, and P. Needleman, 1986, J. Immunol., In press; and Ceuppens, J.S., S. Vertessen, H. Deckmyn, and J. Vermylen, 1985, Cell Immunol., 90:458-463), but is probably generated at the site of an infection (Tripp, C.S., K.M. Leahy, and P. Needleman, 1985, J. Clin. Invest., 76:898-901), these data suggest an important role for the vasoconstrictive properties of thromboxane A2 in the regulation of immunity to Listeria infection.
C S Tripp, P Needleman, E R Unanue
Natural killer (NK) cells were assessed in patients with hyperthyroxinemia due to Graves' disease or treatment with thyroxine (T4). Cytolytic activity was measured with 51Cr-labeled K562 tumor cells and NK enumeration was by flow cytometry using NKH-1 monoclonal antibody to identify the relevant surface marker. Activity was uniformly decreased in association with hyperthyroxinemia, regardless of the underlying pathology; however, there was no reduction in the number of NKH-1+ cells. NK activity was enhanced by addition of interleukin 2 (IL-2) in both control and patients' cells although the value in the latter instance failed to reach the basal control level. Production of IL-2 by lymphocytes from hyperthyroxinemic subjects, in response to phytohemagglutinin, was also reduced. Since NK cells are thought to act as a defense against viral infections and some malignancies and may play a role in autoregulation of the immune system, this effect of T4 may have significant biological implications.
M Papic, J Stein-Streilein, M Zakarija, J M McKenzie, J Guffee, M A Fletcher
We have found that canine and rat hepatocytes convert (125I)iodoTyr10-glucagon to a peptide metabolite lacking the NH2-terminal three residues of the hormone. The peptide is released into the cell incubation medium and its formation is unaffected by a variety of lysosomotropic or other agents. Use of specific radioimmunoassays and gel filtration demonstrated in both normal subjects and in chronic renal failure patients a plasma peptide having the properties of the hormone fragment identified by cell studies. Studies of the dog revealed a positive gradient of the fragment across the liver and no differential gradient of the fragment and glucagon across the kidney. We conclude that the glucagon fragment arises from the cell-mediated processing of the hormone on a superficial aspect of the hepatocyte, the glucagon fragment identified during experiments in vitro represents the cognate of a peptide formed during the hepatic metabolism of glucagon in vivo, and measurement of the fragment by COOH-terminal radioimmunoassays could lead to an understimulation of hepatic glucagon extraction.
W A Hagopian, H S Tager
Cholesteryl ester (CE) accumulation in arterial wall macrophages (foam cells) is a prominent feature of atherosclerotic lesions. We have previously shown that murine J774 macrophages, unlike mouse peritoneal macrophages, accumulate large amounts of CE from unmodified low density lipoprotein (LDL). We now report a direct comparison of acyl coenzyme A:cholesterol acyl transferase (ACAT) activity in J774 and mouse peritoneal macrophages. Despite similar chloroquine-inhibitable 125I-LDL degradation in the two macrophages, ACAT activity in LDL-treated J774 macrophages was 10-30-fold higher than that in LDL-treated mouse peritoneal macrophages. In contrast, acetyl-LDL (matched for degradation with LDL) caused marked stimulation of ACAT activity in mouse peritoneal macrophages. From these data we conclude that in the presence of LDL, J774 macrophages have a highly active ACAT cholesterol esterification pathway compared with mouse peritoneal macrophages; and in mouse peritoneal macrophages, there is a marked difference in the ability of acetyl-LDL vs. LDL to stimulate ACAT even when the lipoproteins are matched for degradation.
I Tabas, G C Boykow, A R Tall
Rat Y' bile acid binders (33 kD) have been previously recognized as cytosolic bile acid binding proteins (Sugiyama, Y., T. Yamada, and N. Kaplowitz, 1983, J. Biol. Chem., 258:3602-3607). We have now determined that these Y' binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD), bile acid-metabolizing enzymes. 3 alpha-HSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3 alpha-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3 alpha-HSD-I, the predominant form, was purified and functioned best as a reductase at pH 7.0 with a marked preference for NADPH. Michaelis constant values for mono- and dihydroxy bile acids were 1-2 microM, and cholic acid competitively inhibited the reduction of 3-oxo-cholic acid. Under normal redox conditions, partially purified 3 alpha-HSD-I and freshly isolated hepatocytes catalyzed the rapid reduction of 3-oxo-cholic to cholic acid without formation of isocholic acid, whereas the reverse reaction was negligible. The Y' bile acid binders are therefore 3 alpha-HSD, which preferentially and stereospecifically catalyze the reduction of 3-oxo-bile acids to 3 alpha-hydroxy bile acids.
A Stolz, H Takikawa, Y Sugiyama, J Kuhlenkamp, N Kaplowitz
Five dogs with mucopolysaccharidosis I, a model of human Hurler/Scheie syndrome, were transplanted with marrow from phenotypically normal littermates at 5 mo of age. At 3 and 9 mo posttransplantation, biopsies of cerebral cortex, liver, and cerebrospinal fluid were obtained. The alpha-L-iduronidase levels in these tissues were 0.8-7.4, 26-45, and 6.3-14.9% of the paired donor tissues, respectively. Although iduronidase was present in relatively low levels in the recipients' brains and cerebrospinal fluid at both biopsy times, reduction in brain glycosaminoglycan (GAG) was comparable to that observed in liver. Ultrastructural studies of cells within the transplanted dogs' brains showed less lysosomal distension and storage product than in affected, nontransplanted, littermate controls. The most marked clearing of stored GAG was in cells surrounding blood vessels, but decreased lysosomal storage in neurons and glial cells was also observed. Urinary GAG excretion also decreased to near normal levels by 5 mo posttransplantation.
R M Shull, N E Hastings, R R Selcer, J B Jones, J R Smith, W C Cullen, G Constantopoulos
The cardiotonic agent amrinone inhibits bone resorption in vitro. Milrinone, an amrinone analog, is a more potent cardiotonic agent with lower toxicity. In contrast to amrinone, milrinone stimulated resorption in cultures of neonatal mouse calvaria and fetal rat limb bones. Threshold doses were 0.1 microM in calvaria and 0.1 mM in limb bones; maximal stimulation occurred in calvaria at 0.1 mM. Maximal responses to milrinone and parathyroid hormone were comparable. Milrinone concentrations below 0.1 mM did not affect calvarial cyclic AMP. 0.5 microM indomethacin inhibited milrinone effects in calvaria but usually not in limb bones. 3 nM calcitonin inhibited milrinone-stimulated resorption and there was no escape from this inhibition. Structural homology between milrinone and thyroxine has been reported. We find similarities between milrinone and thyroxine actions on bone, because prostaglandin production was crucial for the effects of both agents in calvaria but not in limb bones, and neither agent exhibited escape from calcitonin inhibition.
N S Krieger, T S Stappenbeck, P H Stern
We have previously shown that insulinlike growth factor I (IGF-I) inhibits growth hormone (GH) secretion and messenger RNA (mRNA) levels in pituitary cells. The effects of IGF-I on new GH mRNA synthesis rates in primary monolayer rat pituitary cells were therefore examined by nuclear runoff transcription assays. IGF-I (1.3 nM) treatment for 1 h inhibited GH gene transcription to 60% of controls. IGF-I (3.25 nM) maximally suppressed GH gene transcription to 30% of control values after 4 h. After 24 h treatment, GH transcription was suppressed to 48% of controls by 3.25 nM IGF-I. IGF-I (3.25 nM) also inhibited the twofold growth hormone-releasing hormone (GHRH) (10 nM)-stimulated GH gene transcription by 30% after 4 h. Transcription of the prolactin (PRL) gene was not suppressed in these cells by IGF-I. Relatively high doses of insulin (200 nM) also suppressed GH gene transcription, but epidermal growth factor and fibroblast growth factor did not change GH mRNA synthesis. The results show that IGF-I exerts a rapid and selective suppression of basal and GHRH-stimulated GH gene transcription. These data indicate a role for IGF-I in negative feedback of GH gene expression and provide evidence for the direct transcriptional regulation of the GH gene by IGF-I in primary rat anterior pituitary cells.
S Yamashita, S Melmed
The insulin receptor contains an alpha subunit with insulin binding properties and a beta subunit with insulin-stimulated tyrosine kinase function. Preparations containing insulin and insulinlike growth factor I (IGF-I) receptors were obtained from solubilized human red cell membranes by affinity chromatography. After separate assays for insulin binding and insulin-stimulated tyrosine kinase activities, a high degree of correlation was found between these activities in preparations from normals and diabetics. Identical studies using IGF-I as the ligand showed a lesser degree of correlation. We compared 24 normal subjects and 14 untreated type II diabetics and found significant diminution in the slope of the line coupling insulin binding and insulin-stimulated kinase activities in the diabetics. This difference was not observed in a similar study of IGF-I-related activities. Compared to normal controls, untreated type II diabetics have reduced tyrosine kinase activity stimulated per unit insulin binding.
R J Comi, G Grunberger, P Gorden
Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.
R W Moreadith, M W Cleeter, C I Ragan, M L Batshaw, A L Lehninger
The effect of intravenous gammaglobulin (IVGG) on the immunoregulatory abnormalities found during acute Kawasaki syndrome (KS) was studied in a randomized trial of IVGG plus aspirin (ASA) versus ASA alone. Before therapy, patients in each treatment group had increased numbers of circulating HLA-DR-bearing Leu 3+ helper T cells, a deficiency of Leu 2+ suppressor/cytotoxic T cells, and increased levels of spontaneous IgG and IgM synthesis by peripheral blood mononuclear cells. There were no significant differences (P greater than 0.1) between immunologic parameters measured on day 1 and day 4 in the ASA-treated group. In contrast, patients treated with ASA plus IVGG had by day 4 a highly significant decrease in HLA-Dr+ Leu 3+ helper T cells (P less than 0.001), an increase in Leu 2+ suppressor/cytotoxic T cells (P less than 0.01), and a decrease in spontaneous IgG (P less than 0.01) and IgM synthesis (P less than 0.001). These changes were associated with a reduction in the secretion of T cell-derived B cell helper factors (P less than 0.001). These findings indicate that treatment with IVGG suppresses the marked T and B cell activation found in patients with acute KS.
D Y Leung, J C Burns, J W Newburger, R S Geha
The benzodiazepine receptor inverse agonist 6,7-dimethoxy-4-ethyl-3-carbomethoxy-beta-carboline (DMCM) (1.5-15 mg/kg) was administered to mice 5 min after a lethal (LD94) injection of pentobarbital. DMCM (1.5-5 mg/kg) increased short-term (1 h) survival in a dose-dependent fashion, with an optimum survival rate more than five times greater than mice receiving pentobarbital alone. Statistically significant increases in long-term (24 h) survival were also observed after both 5 and 10 mg/kg of DMCM (34 and 33%, respectively) compared with animals receiving pentobarbital alone (6%). Two doses of DMCM (5 and 2.5 mg/kg, respectively) administered 55 min apart produced an even greater increase (58%) in 24-h survival rates. Doses of DMCM that increased 1- and 24-h survival were not lethal when administered alone, and were below the dose that produced convulsions in 50% (CD50) of the animals. The protective effects of DMCM were blocked by pretreatment with the benzodiazepine receptor agonist ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo- 4H-imidazo[1,5a][1,4]benzodiazodiazepine-3-carboxylate (Ro 15-1788), which suggests the effects of DMCM are mediated through the benzodiazepine receptor. These findings suggest that DMCM or another benzodiazepine receptor ligand with full inverse agonist qualities could prove effective as an antidote for barbiturate intoxication in man.
H Havoundjian, G F Reed, S M Paul, P Skolnick
Trimetrexate, a highly lipid-soluble quinazoline antifolate now undergoing trials as an anticancer agent, was found to be a potent inhibitor of the dihydrofolate reductase (DHFR) isolated from Toxoplasma gondii. The concentration required for 50% inhibition of protozoal DHFR was 1.4 nM. As an inhibitor of this enzyme, trimetrexate was almost 600-fold (amount of antifolate required to inhibit catalytic reaction by 50%) and 750-fold (inhibition constant) more potent than pyrimethamine, the DHFR inhibitor currently used to treat toxoplasma infection. When the protozoan was incubated with 1 microM trimetrexate, the drug rapidly reached high intracellular concentrations. Since toxoplasma organisms lack a transmembrane transport system for physiologic folates, host toxicity can be prevented by co-administration of the reduced folate, leucovorin, without reversing the antiprotozoal effect. The effectiveness of trimetrexate against toxoplasma was demonstrated both in vitro and vivo. Proliferation of toxoplasma in murine macrophages in vitro was completely inhibited by exposure of these cells to 10(-7) M trimetrexate for 18 h. When used alone, trimetrexate was able to extend the survival of T. gondii-infected mice.
C J Allegra, J A Kovacs, J C Drake, J C Swan, B A Chabner, H Masur
Cells cultured from human giant cell tumors of bone were characterized on the basis of morphological features, proliferative capacity, presence of granulocyte-monocyte antigens, receptors for skeletal hormones, and soluble cell products. Three major cell types were identified. One population consisted of mononuclear cells with fibroblastic morphology, which proliferated in culture and most likely represent the neoplastic element of the tumor. Phenotypically they resembled a connective tissue stromal cell. A second population of mononuclear cells lacked receptors for skeletal hormones and did not persist in culture. These cells were likely of monocyte-macrophage lineage. A third population of cells consisted of large multinucleated giant cells. These cells possessed phenotypic features of osteoclasts including receptors for calcitonin. Human giant cell tumors of bone are most likely a neoplasm of connective tissue stromal cells, which have the capacity to recruit and interact with multinucleated giant cells that exhibit phenotypic features of osteoclasts.
S R Goldring, M S Roelke, K K Petrison, A K Bhan
We have examined membrane protein profiles for alterations during red blood cell aging. To obtain populations of in vivo-aged red cells, we maintained mice in a state of continuous erythropoietic suppression for up to 8 wk using serial hypertransfusion. The circulating t1/2 of red cells from mice which had been erythropoietically suppressed for 8 wk was less than 1 d compared with a t1/2 of 15 d for red cells from normal animals. The most obvious alteration in membrane proteins was an increase in the ratio of the membrane skeletal components 4.1a:4.1b from 0.3 for the normal red cell population to greater than 1 for these old cells. The 4.1a:4.1b ratio thus appears to be a useful index of red cell age. Analyses of the density profile of cells aged in the hypertransfused mice disclosed that these old cells had a density range similar to that of controls, suggesting that cell density does not increase significantly with red cell age in the mouse.
T J Mueller, C W Jackson, M E Dockter, M Morrison
Atrial natriuretic factor (ANF) (1 microM) markedly increased cyclic guanosine monophosphate (cGMP) content in microdissected glomeruli (35-fold) and in microdissected inner medullary collecting ducts (IMCD) (20-fold). ANF caused little or no increase in cGMP content in other nephron segments. The threshold concentration for increased cGMP accumulation by ANF was 0.1-1 nM in IMCD, which is in the range reported for rat plasma. Sodium nitroprusside (1 mM), which selectively stimulates soluble guanylate cyclase, increased cGMP content in glomeruli but not in IMCD. ANF did not alter cAMP accumulation in the absence or presence of vasopressin (AVP) or parathyroid hormone (PTH) in outer and inner medullary tubule suspensions, or in microdissected proximal convoluted tubules (PCT), medullary thick ascending limbs (MAL) or IMCD. These data are compatible with the hypothesis that cGMP is a second messenger for a physiologic action of ANF in the inner medullary collecting duct. ANF apparently activates membrane-bound guanylate cyclase in this segment.
H Nonoguchi, M A Knepper, V C Manganiello
We used microelectrode recordings of muscle sympathetic nerve activity (MSNA) from the peroneal nerve in the leg during arm exercise in conscious humans to test the concept that central command and muscle afferent reflexes produce mass sympathetic discharge at the onset of exercise. Nonischemic rhythmic handgrip and mild arm cycling produced graded increases in heart rate and arterial pressure but did not increase MSNA, whereas ischemic handgrip and moderate arm cycling dramatically increased MSNA. There was a slow onset and offset of the MSNA responses, which suggested metaboreceptor mediation. When forearm ischemia was continued after ischemic handgrip, MSNA remained elevated (muscle chemoreflex stimulation) but heart rate returned to control (elimination of central command). The major new conclusions are that: the onset of dynamic exercise does not produce mass, uniform sympathetic discharge in humans, and muscle chemoreflexes and central command appear to produce differential effects on sympathetic and parasympathetic responses.
R G Victor, D R Seals, A L Mark
Three compounds that share specific antimitochondrial properties are gossypol, rhodamine-123, and lonidamine. We compare the antiproliferative activities of these drugs against six human cell lines derived from breast (T47-D), pancreas (MiaPaCa, RWP-2), prostate (DU-145), colon (HCT-8), and cervix (HeLa) carcinomas. Tumor cells enriched in cathodal LDH isozymes (LDH4 and LDH5) are significantly more sensitive to gossypol and rhodamine-123. When compared for ability to inhibit growth of human marrow in soft agar, 10 microM gossypol shows little effect on colony formation whereas 10 microM rhodamine-123 completely prevents stem cell growth, suggesting that gossypol may have the most favorable therapeutic index. Within 24 h of drug administration, there is a relative increase in intracellular inorganic phosphate pools and a marked decline in soluble high-energy phosphates in sensitive tumor cells, as measured by 31P magnetic resonance spectroscopy. These studies suggest that specific antimitochondrial agents might be selectively administered on the basis of tumor LDH isozyme content and noninvasively monitored for antiproliferative activity by 31P spectroscopy.
C Benz, C Hollander, M Keniry, T L James, M Mitchell
A small but consistent proportion of the von Willebrand factor (vWF) in normal plasma is composed of 189, 176, and 140 kD fragments cleaved from the 225 kD subunit. A monoclonal antibody map of vWF, based on the reactivity of individual antibodies with cyanogen bromide and tryptic fragments of known carboxy and/or amino termini, showed that in normal and IIA von Willebrand disease (vWD) plasmas the 140 kD fragment was derived from the amino-terminal region, whereas the 176 kD fragment was derived from the carboxy-terminal region of the subunit. In type IIA vWD, however, the fragments comprised a greater proportion of circulating vWF. In contrast, plasmin cleaved a 176 kD fragment from the amino terminus and a 145 kD fragment from the carboxy terminus of the subunit. Species similar to these plasmin-cleaved fragments were demonstrated in plasmas from four patients treated with fibrinolytic agents, but not in IIA vWD.
S D Berkowitz, J Dent, J Roberts, Y Fujimura, E F Plow, K Titani, Z M Ruggeri, T S Zimmerman
Deoxycholate (DOC), chenodeoxycholate, 12-O-tetradecanoyl phorbol-13-acetate (TPA), or 1-oleoyl-2-acetyl-glycerol (OAG) activated colonic epithelial protein kinase C as reflected by translocation from the soluble to the particulate cell fraction. Activation of protein kinase C was correlated with stimulation of enhanced proliferative activity of colonic mucosa and reactive oxygen production. TPA and OAG, but not DOC, directly activated soluble protein kinase C in vitro. However, DOC rapidly increased labeled inositol phosphate and diacylglycerol accumulation in colonic epithelial cells. Retinoic acid inhibited protein kinase C activity and suppressed DOC-, TPA-, and OAG-induced increases in reactive oxygen production. The results support a role for protein kinase C in the stimulation of colonic epithelial proliferative activity and reactive oxygen production induced by bile acids, TPA and OAG. In contrast to TPA and OAG, which activate protein kinase C directly, bile acids appear to activate protein kinase C indirectly by increasing the diacylglycerol content of colonic epithelium.
P A Craven, J Pfanstiel, F R DeRubertis
We used a subclone of a rabbit genomic clone for collagenase that cross-hybridizes with human synovial cell messenger RNA (mRNA) to identify a human collagenase complementary DNA (cDNA) clone. The human cDNA clone is 2.1 kilobases (kb) and selects a mRNA transcript of approximately the same size from primary cultures of rheumatoid synovial cells that produce collagenase, but no mRNA is selected from control (nonproducing) synovial fibroblasts. Restriction enzyme analysis and DNA sequence data indicate that our cDNA clone is full length and that it is identical to that recently described for human skin fibroblast collagenase. The cDNA clone identified a single collagenase gene of approximately 17 kb from blots of human genomic DNA. The identity of human skin and synovial cell collagenase and the ubiquity of this enzyme and of its substrates, the interstitial collagens types I, II, and III, imply that common mechanisms controlling collagenolysis throughout the human body may be operative in both normal and disease states.
C E Brinckerhoff, P L Ruby, S D Austin, M E Fini, H D White
The effect of chronic physiologic hyperglucagonemia on basal and insulin-mediated glucose metabolism was evaluated in normal subjects, using the euglycemic insulin clamp technique (+50, +100, and +500 microU/ml). After glucagon infusion fasting glucose increased from 76 +/- 4 to 93 +/- 2 mg/dl and hepatic glucose production (HGP) rose from 1.96 +/- 0.08 to 2.25 +/- 0.08 mg/kg X min (P less than 0.001). Basal glucose oxidation after glucagon increased (P less than 0.05) and correlated inversely with decreased free fatty acid concentrations (r = -0.94; P less than 0.01) and decreased lipid oxidation (r = -0.75; P less than 0.01). Suppression of HGP and stimulation of total glucose disposal were impaired at each insulin step after glucagon (P less than 0.05-0.01). The reduction in insulin-mediated glucose uptake was entirely due to diminished non-oxidative glucose utilization. Glucagon infusion also caused a decrease in basal lipid oxidation and an enhanced ability of insulin to inhibit lipid oxidation and augment lipid synthesis. These results suggest that hyperglucagonemia may contribute to the disturbances in glucose and lipid metabolism in some diabetic patients.
S Del Prato, P Castellino, D C Simonson, R A DeFronzo
To assess the importance of the route of glucose delivery in determining net hepatic glucose balance (NHGB) eight conscious overnight-fasted dogs were given glucose via the portal or a peripheral vein. NHGB was measured using the arteriovenous difference technique during a control and two 90-min glucose infusion periods. The sequence of infusions was randomized. Insulin and glucagon were held at constant basal levels using somatostatin and intraportal insulin and glucagon infusions during the control, portal, and peripheral glucose infusion periods (7 +/- 1, 7 +/- 1, 7 +/- 1 microU/ml; 100 +/- 3, 101 +/- 6, 101 +/- 3 pg/ml, respectively). In the three periods the hepatic blood flow, glucose infusion rate, arterial glucose level, hepatic glucose load, arterial-portal glucose difference and NHGB were 37 +/- 1, 34 +/- 1, 32 +/- 3 ml/kg per min; 0 +/- 0, 4.51 +/- 0.57, 4.23 +/- 0.34 mg/kg per min; 101 +/- 5, 200 +/- 15, 217 +/- 13 mg/dl; 28.5 +/- 3.5, 57.2 +/- 6.7, 54.0 +/- 6.4 mg/kg per min; +2 +/- 1, -22 +/- 3, +4 +/- 1 mg/dl; and 2.22 +/- 0.28, -1.41 +/- 0.31, and 0.08 +/- 0.23 mg/kg per min, respectively. Thus when glucose was delivered via a peripheral vein the liver did not take up glucose but when a similar glucose load was delivered intraportally the liver took up 32% (P less than 0.01) of it. In conclusion portal glucose delivery provides a signal important for the normal hepatic-peripheral distribution of a glucose load.
B A Adkins, S R Myers, G K Hendrick, R W Stevenson, P E Williams, A D Cherrington
During activation the lymphocyte attains functional insulin receptors with precise regulation, a consequence of insulin concentration manipulations. These studies test the hypothesis that insulin receptor (+) monocytes monitor insulin concentrations, so instructing the T lymphocytes. Monocyte-enriched populations were incubated with insulin (0-10(-6) M) followed by co-culture with T lymphocytes and an activating stimulus. A dose-related fall in T lymphocyte insulin receptor binding was observed that was specific for the monocyte as the signalling cell and for insulin as the signal received. Monocytes from normal volunteers during a euglycemic, hyperinsulinemic clamp were cultured with T lymphocytes and an activating stimulus. A decline in specific insulin receptor binding on T lymphocytes was observed, which Scatchard analysis demonstrated to be a consequence of reduction in receptor numbers. These studies demonstrate that the receptor (+) monocyte perceives the concentration of insulin and passes this information to T lymphocytes regulating the number of activation-induced insulin receptors. The interplay between the monocyte and T lymphocyte parallels the interaction of these cell types for recognition of antigen.
J H Helderman, R Ayuso, J Rosenstock, P Raskin
We investigated idiotypic markers of monoclonal antibodies derived from patients with polyclonal B-cell activation. Four monoclonal antibodies with different ligand binding specificities derived from a patient with lepromatous leprosy and three monoclonal anti-DNA antibodies from two patients with SLE were studied. Three new public idiotopes, which were common to monoclonal antibodies from all three patients, were defined by five polyclonal rabbit antiidiotypes, two monoclonal mouse antiidiotopes, and a monoclonal mouse antibody against a synthetic peptide that contains residues of the heavy chain CDR-1 of a monoclonal lupus anti-DNA antibody. The antibody against the synthetic idiotype was found to react with native immunoglobulins in solution. One idiotope was found to be consistently immunogenic in all animals tested. Since the three patients are of different ethnic origins, these shared idiotypes are probably encoded by germline V genes. These genes may be recurrently expressed in states of polyclonal B-cell activation, regardless of etiology. The results suggest that some autoantibodies arise by expansion of a pool of precursors in the normal antibody repertoire.
C Mackworth-Young, J Sabbaga, R S Schwartz
We measured basal and peak acid outputs, food-stimulated acid secretion, and basal and food-stimulated serum gastrin concentrations in a large group of duodenal ulcer patients and normal subjects. Basal and peak acid outputs were significantly higher in ulcer patients. In contrast, acid secretion was similar in the groups when food was infused into the stomach and when sham feeding was combined with meal infusion to simulate normal eating. Meal-stimulated acid secretion, expressed as a percentage of peak acid output to correct for differences in secretory capacity, was lower in ulcer patients (P less than 0.002). Basal serum gastrin concentrations were higher in ulcer patients, which may have contributed to higher basal acid output. However, increases in serum gastrin after food were similar in the groups. Duodenal ulcer patients, as a group, have increased basal and maximal acid secretion, but the amount of acid secreted and gastrin released after eating is normal.
A J Blair 3rd, M Feldman, C Barnett, J H Walsh, C T Richardson
The decrease in plasma lactate during dichloroacetate (DCA) treatment is attributed to stimulation of lactate oxidation. To determine whether DCA also inhibits lactate production, we measured glucose metabolism in muscles of fed and fasted rats incubated with DCA and insulin. DCA increased glucose-6-phosphate, an allosteric modifier of glycogen synthase, approximately 50% and increased muscle glycogen synthesis and glycogen content greater than 25%. Lactate release fell; inhibition of glycolysis accounted for greater than 80% of the decrease. This was associated with a decrease in intracellular AMP, but no change in citrate or ATP. When lactate oxidation was increased by raising extracellular lactate, glycolysis decreased (r = - 0.91), suggesting that lactate oxidation regulates glycolysis. When muscle lactate production was greatly stimulated by thermal injury, DCA increased glycogen synthesis, normalized glycogen content, and inhibited glycolysis, thereby reducing lactate release. The major effect of DCA on lactate metabolism in muscle is to inhibit glycolysis.
A S Clark, W E Mitch, M N Goodman, J M Fagan, M A Goheer, R T Curnow
Previous evaluation of lymphocytes taken from patients with Wiskott-Aldrich syndrome (WAS) and other X-linked immunodeficiencies has revealed deficiencies of a lymphocyte sialoglycoprotein with a relative molecular mass of 115 kD (designated gpL-115) found in normal lymphocytes. The development of monoclonal antibodies to gpL-115 has permitted the detection of molecular heterogeneity in gpL-115 from the lymphocytes of immunodeficient patients. When lymphocytes from normal individuals were analyzed by immunoblotting, gpL-115 with only a single molecular species (115 kD) was detected. Lymphocytes from 17 immunodeficient patients were analyzed after overnight incubation. Two patients had no gpL-115 with an Mr of 115 kD, but gpL-115 with an Mr of either 95 or 135 kD was detected. Nine patients had gpL-115 with Mr equally of 95 and 115 Kd. Other patients exhibited gpL-115 with combinations of 95, 115, and 135 kD. The heterogeneity of the degraded gpL-115 suggests that WAS and other X-linked immunodeficiencies are due to a series of abnormalities, all of which involve gpL-115, and may explain the clinical heterogeneity of the diseases.
D Reisinger, R Parkman
A method for measuring fluorescence in anchored monolayers of human endothelial cells is described and used to demonstrate changes in the cytosolic free-calcium concentration ([Ca2+]c) in these cells exposed to histamine and thrombin; some endothelial responses to both agonists (e.g., mitogenesis) have been suggested to be Ca2+-mediated. Umbilical vein endothelial cells were cultured on microcarriers and loaded with the Ca2+ indicator, indo-1. Enzymatic cell detachment was avoided by monitoring the indo-1 fluorescence ratio (400/480 nm) of a stirred suspension of cell-covered microcarriers. Basal [Ca2+]c was estimated to be 70-80 nM. Thrombin induced a transient dose-dependent increase in [Ca2+]c, which was active-site dependent. Histamine stimulated a dose-dependent increase in [Ca2+]c, which was reversed by removal of histamine and inhibited competitively by the H1-receptor antagonist pyrilamine, but not by the H2-receptor antagonist cimetidine. Furthermore, histamine induced a dose-dependent secretion of von Willebrand factor, which paralleled the rise in [Ca2+]c and was similarly blocked by the H1-receptor antagonist, and which may contribute to platelet deposition at sites of inflammation.
K K Hamilton, P J Sims
We have investigated DNA polymorphism of the class II alpha chain genes in HLA typed patients with insulin dependent diabetes mellitus (IDDM; n = 79), celiac disease (CD; n = 46), dermatitis herpetiformis (DH; n = 53), and controls (n = 86). Preferential allelic associations of HLA genes and gene products have thus been constructed for susceptibility to these diseases. DR alpha and DQ alpha gene polymorphisms indicated heterogeneity of HLA DR3, DRw6, and DR7, and HLA DR2 and DRw6, respectively. In DR7 positive CD patients a 3.8-kilobase (kb) DR alpha fragment, which correlated with DQw3, was found in only 11% of patients compared with 45% of corresponding controls (P less than 0.05). An increased frequency of a DX alpha genotype UU in all three diseases was found (IDDM 59%, DH 45%, CD 48%, compared to 21% in controls, P less than 0.001), which is not explained solely by the increased frequencies of DR3-DX alpha U. We therefore conclude part of the genetic susceptibility for these three conditions is encoded by genes within the DQ-DX subregion.
G A Hitman, M J Niven, H Festenstein, P G Cassell, J Awad, J Walker-Smith, J N Leonard, L Fry, P Ciclitira, P Kumar
Insulin secretion is controlled by a complex set of factors that include not only glucose but amino acids, catecholamines, and intestinal hormones. We report that a novel glucagon-like peptide, co-encoded with glucagon in the glucagon gene is a potent insulinotropic factor. The glucagon gene encodes a proglucagon that contains in its sequence glucagon and additional glucagon-like peptides (GLPs). These GLPs are liberated from proglucagon in both the pancreas and intestines. GLP-I exists in at least two forms: 37 amino acids GLP-I(1-37), and 31 amino acids, GLP-I(7-37). We studied the effects of synthetic GLP-Is on insulin secretion in the isolated perfused rat pancreas. In the presence of 6.6 mM glucose, GLP-I(7-37) is a potent stimulator of insulin secretion at concentrations as low as 5 X 10(-11) M (3- to 10-fold increases over basal). GLP-I(1-37) had no effect on insulin secretion even at concentrations as high as 5 X 10(-7) M. The earlier demonstration of specific liberation of GLP-I(7-37) in the intestine and pancreas, and the magnitude of the insulinotropic effect at such low concentrations, suggest that GLP-I(7-37) participates in the physiological regulation of insulin secretion.
S Mojsov, G C Weir, J F Habener
Monocyte and lymphocyte surface-expressed viral antigens have been demonstrated after exposure of unseparated human mononuclear leukocytes to influenza virus in vitro. The current studies, using [35S]methionine pulse-labeled purified preparations of virus-exposed macrophages, depleted of lymphocytes, demonstrate that the presence of these viral proteins does represent new synthesis. However, purified lymphocytes, depleted of monocytes-macrophages and exposed to influenza virus, showed no detectable viral protein synthesis. In further experiments, unseparated mononuclear leukocytes were exposed to virus and subsequently separated by countercurrent centrifugal elutriation. Both macrophages and lymphocytes were then shown to synthesize influenza proteins. Cell-free control or influenza virus-infected macrophage-derived supernatant fluids did not facilitate influenza virus infection of the lymphocytes. The data suggest that macrophages are required for influenza virus infection of human lymphocytes, and raise the possibility that macrophage facilitation of an abortive infection of lymphocytes plays a role in the generation of effective immunity to viral antigens.
D J Mock, F Domurat, N J Roberts Jr, E E Walsh, M R Licht, P Keng
High density lipoproteins (HDL) stimulated a dose-dependent increase in the release of placental lactogen (hPL) from human placental explants. The stimulation was not prevented by delipidation of HDL but was completely blocked by tryptic digestion. Delipidated apolipoproteins (Apo) AI, AII, and CI also stimulated hPL release but other apolipoproteins were without effect. HDL and Apo CI had no effects on the release of luteinizing hormone and follicle-stimulating hormone from rat pituitary cells or the release of prolactin from human decidual cells. Because placental cells have specific HDL receptors and plasma HDL concentrations increase during pregnancy, these results strongly suggest a role for HDL in the regulation of hPL release during pregnancy possibly independent of their usual role in plasma lipid transport.
S Handwerger, S Quarfordt, J Barrett, I Harman
Since thyroid hormones and mineralocorticoids were observed to stimulate kidney Na-K-ATPase in similar sites and with similar time courses, this study was initiated to evaluate whether aldosterone is involved in the stimulation of Na-K-ATPase observed in collecting tubules 3 h after triiodothyronine (T3) administration to thyroidectomized (TX) rabbits. Results indicate that: Plasma aldosterone level decreased markedly in TX rabbits but was not restored 3 h after T3 injection; Early stimulation of Na-K-ATPase by T3 was abolished when plasma aldosterone level was suppressed by adrenalectomy or when aldosterone effects were blocked by spironolactone; Administration of aldosterone to TX rabbits mimicked the action of T3; Sensitivity of Na-K-ATPase to aldosterone markedly decreased after thyroidectomy. These results demonstrate an interaction between aldosterone and T3 in the control of Na-K-ATPase in the collecting tubule. Triiodothyronine enhances the sensitivity of Na-K-ATPase to aldosterone which, in turn, produces a stimulatory action despite the decreased plasma level observed during hypothyroidism.
C Barlet, A Doucet
In this study, possible paracrine factors in adipose tissue from lean and obese subjects were sought. Conditioned media were prepared by incubation in alpha minimum essential medium of adipocyte precursors derived from lean and massively obese subjects. Adipocyte-precursor-derived conditioned media from the obese stimulated replication of cultured rat perirenal adipocyte precursors by about fourfold over control. The effect of media conditioned by precursors derived from lean subjects was much less evident. The mitogenicity of conditioned media was abolished by trypsin, indicating the protein nature of the mitogenic factor(s). Sephacryl S-200 chromatography of adipocyte-precursor-derived conditioned media from obese subjects revealed one major active fraction with molecular masses in the range of 25,000-40,000. Our results demonstrate that adipocyte precursors derived from massively obese subjects release factors mitogenic on cultured rat adipocyte precursors. These principles may act as paracrine factors contributing to the development of the adipocyte hyperplasia characteristic of massive obesity.
D C Lau, D A Roncari, C H Hollenberg
To investigate the relationship of the lymphoid hyperplasia of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) to supervening malignant lymphoma, we subjected DNA from lymph nodes and peripheral blood mononuclear cells from five AILD patients to Southern blot analysis to detect clonal rearrangements of immunoglobulin and T-cell receptor genes. Lymph nodes and peripheral blood from AILD patients were found to contain clones of lymphoid cells harboring either immunoglobulin or T-cell receptor gene rearrangements that, in some instances, regressed during the course of disease. A lymph node from one patient was involved by immunoblastic lymphoma and manifested an additional gene rearrangement pattern not seen in premalignant specimens from that patient. In contrast, DNA obtained from normal peripheral blood mononuclear cells and 11 examples of other forms of lymphoid hyperplasia showed no gene rearrangements. As a disorder of cellular immunoregulation in which lymphoid cells may escape normal growth controls, AILD provides a natural model to dissect stages of lymphomagenesis in man.
E H Lipford, H R Smith, S Pittaluga, E S Jaffe, A D Steinberg, J Cossman
The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca2+/calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.
E A Tallant, R W Wallace
Factor XIII, the clotting factor essential for covalent stabilization of the fibrin clot, is a heterodimer consisting of a2 and b2 subunits, with catalytic function residing in the a-chain. In order to address questions regarding sites of synthesis and chromosomal localization of the Factor XIII a-chain, cDNA was cloned from a lambda gt11 human placental cDNA library. Nucleotide and amino acid sequences were determined from the cDNA. Amino acid sequencing of purified platelet Factor XIII a-chains confirmed the authenticity of the lambda gt11 clone. The gene for Factor XIII a-chain was mapped uniquely to chromosome 6. Northern blot analysis of human placental and U937 (monocytelike) cell poly (A)+ mRNA showed a single approximately 4.0-kb message for the Factor XIII a-chain. These results provide conclusive evidence that the a-chain is synthesized by placenta and monocyte cell lines.
L J Weisberg, D T Shiu, C S Greenberg, Y W Kan, M A Shuman
Certain aspects of the chronic complications of diabetes suggest that, with time, the abnormal metabolic milieu leads to irreversible changes in some cell populations. Since we have previously observed that high glucose concentrations induce an increase in single strand breaks in the DNA of cultured human endothelial cells, we have investigated whether the same abnormality occurs in cells derived from the in vivo diabetic environment. Peripheral blood lymphocytes obtained from 21 type I diabetic patients and age- and sex-matched controls were tested for rate of unwinding in alkali (a measure of DNA single strand breaks). The patients were subdivided into two groups on the basis of glycohemoglobin values above or below 9%. The group with glycohemoglobin values of 12.9 +/- 2.4% (mean +/- SD), but not the group with glycohemoglobin values of 7.4 +/- 1.5%, showed accelerated unwinding of lymphocyte DNA when compared to controls (P less than 0.01). These studies suggest that poorly controlled diabetes may result in DNA lesions, whose impact on long-term complications deserves to be investigated.
M Lorenzi, D F Montisano, S Toledo, H C Wong
We used the complementary DNA for the human hepatoma Hep G2 glucose transporter to determine the distribution of glucose transporter messenger RNA (mRNA) in rat and human tissues. Under stringent hybridization conditions, a single 2.8-kilobase (kb) transcript is seen in all rat and human tissues examined. The mRNA is most abundant in brain, and is especially enriched in the brain microvascular fraction. The mRNA abundance in rat muscle and fat is 5% that in brain. Rat liver (both adult and fetal) and human liver have very little 2.8-kb mRNA, but it is abundant in cultured human fibroblasts and EB virus-transformed lymphoblasts. The same size mRNA is present in leg muscle of two type II diabetic patients. A very homologous glucose transporter mRNA is expressed in both insulin-sensitive and -insensitive tissues of rat and man. Hepatocytes, which have abundant glucose transport, may express a homologous but nonidentical glucose transporter.
J S Flier, M Mueckler, A L McCall, H F Lodish
Iron and iron compounds--including mammalian hemoglobins--catalyze hydroxyl radical production and lipid peroxidation. To determine whether hemoglobin-mediated lipid peroxidation might be important in hemorrhagic injuries to the central nervous system (CNS), we studied the effects of purified hemoglobin on CNS homogenates and injected hemoglobin into the spinal cords of anesthetized cats. Hemoglobin markedly inhibits Na/K ATPase activity in CNS homogenates and spinal cords of living cats. Hemoglobin also catalyzes substantial peroxidation of CNS lipids. Importantly, the potent iron chelator, desferrioxamine, blocks these adverse effects of hemoglobin, both in vitro and in vivo. Because desferrioxamine is not known to interact with heme iron, these results indicate that free iron, derived from hemoglobin, is the proximate toxic species. Overall, our data suggest that hemoglobin, released from red cells after trauma, can promote tissue injury through iron-dependent mechanisms. Suppression of this damage by desferrioxamine suggests a rational therapeutic approach to management of trauma-induced CNS injury.
S M Sadrzadeh, D K Anderson, S S Panter, P E Hallaway, J W Eaton