M L Nibert, D B Furlong, B N Fields
Previous data suggested an active Cl- conductance in the renal proximal convoluted tubule, although single channel conductance and regulation were not found. We have investigated the presence and regulation of the Cl- channel in proximal convoluted tubules by patch clamp analysis. The current-voltage relationship of whole cells with 130 mM NaCl in the pipette was nonlinear. The addition of 1-34 PTH (10(-8) M), forskolin, or cAMP significantly increased whole cell Cl- conductance. We found a single Cl- channel in excised apical membranes possessing conductance of 33 picosiemens (pS) at positive and 22.5 pS at negative potential, which was blocked by 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid (10(-4) M) and was selective to Cl- (Cl/Na = 10). The channel was activated by prolonged membrane depolarization, by a catalytic subunit of protein kinase A (PKA), or by purified kinase C (PKC), but not by Ca2+ (1 microM) inside the membrane. During cell-attached patch clamping, the channel was similarly activated by PTH, phorbol ester, or dibutyryl cAMP in a dose-dependent manner. To investigate second messenger contributions to the PTH-action, the PTH-evoked channels were modified further by the subsequent addition of several blockers of the second messengers. This suggested that PKA and PKC were involved in Cl- channel activation. We therefore conclude that renal proximal convoluted tubule cells possess an apical Cl- channel activated by PTH via the PKA and PKC pathways.
M Suzuki, T Morita, K Hanaoka, Y Kawaguchi, O Sakai
Hereditary elliptocytosis (HE) Sp alpha I/74 is a disorder associated with defective spectrin (Sp) heterodimer self-association and an abnormal tryptic cleavage of the 80-kD alpha I domain of Sp resulting in increased amounts of a 74-kD peptide. The molecular basis of this disorder is heterogeneous and mutations in codons 28, 46, 48, and 49 (codons 22, 40, 42, and 43 in the previous nomenclature which did not include the six NH2-terminal amino acids) have been reported. In this study we present data on seven unrelated HE Sp alpha I/74 kindred from diverse racial backgrounds in whom we identified four different mutations all occurring in exon 2 of alpha Sp at codon 28. Utilizing the polymerase chain reaction we established a CGT----CTT; Arg----Leu 28 mutation in one kindred of Arab/Druze origin. In two unrelated white kindred of English/European origin the substitution is CGT----AGT; Arg----Ser 28 and in two apparently unrelated white kindred from New Zealand, the mutation is CGT----TGT; Arg----Cys 28. Finally, in one American black kindred and in a black kindred from Ghana the mutation involves CGT----CAT; Arg----His 28. Allele specific oligonucleotide hybridization confirmed that the probands are heterozygous for the respective mutant alleles. All four point mutations abolished an Aha II restriction enzyme site which allowed verification of linkage of the mutation with HE Sp alpha I/74. Our results imply that codon 28 of alpha Sp is a "hot spot" for mutations and also indicate that Arg 28 is critical for the conformational stability and functional self association of Sp heterodimers.
T L Coetzer, K Sahr, J Prchal, H Blacklock, L Peterson, R Koler, J Doyle, J Manaster, J Palek
Myeloperoxidase (MPO), H2O2, and chloride comprise a potent antimicrobial system believed to contribute to the antimicrobial functions of neutrophils and monocytes. The mechanisms of microbicidal action are complex and not fully defined. This report describes the MPO-mediated inactivation, in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, of a class of cytoplasmic membrane enzymes (penicillin-binding proteins, PBPs) found in all eubacteria, that covalently bind beta-lactam antibiotics to their active sites with loss of enzymatic activity. Inactivation of "essential" PBPs, including PBP1-PBP3 of E. coli, leads to unbalanced bacterial growth and cell death. MPO treatment of bacteria was associated with loss of penicillin binding by PBPs, strongly suggesting PBP inactivation. In E. coli, PBP inactivation was most rapid with PBP3, where the rate of decline in binding activity approximated but did not equal loss of viability. Changes in E. coli morphology (elongation), observed just before bacteriolysis, were consistent with early predominant inactivation of PBP3. We conclude that inactivation of essential PBPs is sufficient to account for an important fraction of MPO-mediated bacterial action. This feature of MPO action interestingly recapitulates an antibacterial strategy evolved by beta-lactam-producing molds that must compete with bacteria for limited ecologic niches.
R M Rakita, H Rosen
Members of two unrelated families with type I hereditary angioneurotic edema (HANE) were found to have elevated levels of C1 inhibitor (C1INH) mRNA. DNA sequence analysis of PCR-amplified monocyte C1INH mRNA revealed normal and mutant transcripts, as expected in this disorder that occurs in heterozygous individuals. Single base mutations near the 3' end of the coding sequence were identified in affected members of each family. One mutation consisted of insertion of an adenosine at position 1304 which created a premature termination codon (TAA), whereas the second consisted of deletion of the thymidine at position 1298 which created a premature termination codon (TGA) 23 nucleotides downstream. These mutations are approximately 250 nucleotides upstream of the natural termination codon. Nuclear run-off experiments in one kindred revealed no difference in transcription rates of the C1INH gene between the patients and normals. C1INH mRNA half-life experiments were not technically feasible because of the prolonged half-life of the normal transcript. Dideoxynucleotide primer extension experiments allowed the differentiation of the normal and mutant transcripts. These studies showed that the mutant transcript was not decreased relative to the normal, and this therefore was at least partially responsible for the C1INH mRNA elevation. This elevation may be due to the decreased catabolism of the mutant transcript.
D Frangi, M Cicardi, A Sica, F Colotta, A Agostoni, A E Davis 3rd
Concanavalin A (Con A) is a tetrameric plant lectin that disrupts plasma membrane-cytoskeletal interactions and alters plasma membrane fluidity. We used Con A as a probe to explore beta-adrenergic and muscarinic cholinergic receptor-mediated regulation of cAMP in intact neonatal rat ventricular myocytes. Preincubation with Con A, 0.5 micrograms/ml, attenuated 1 microM (-)-norepinephrine (NE)-induced downregulation of beta-adrenergic receptors and resulted in a 50% augmentation of cAMP accumulation stimulated by 1 microM NE. Con A also augmented forskolin (1-10 microM)-stimulated cAMP accumulation by an average of 37% (P less than 0.05); however, Con A preincubation had no effect on basal or cholera toxin-stimulated cAMP content. The muscarinic cholinergic agonist carbachol (1-100 microM) decreased 1 microM NE-stimulated cAMP generation by an average of 32% (n = 7, P less than 0.05); preincubation with Con A further enhanced the inhibitory effect of carbachol by 18% (n = 7, P less than 0.05). Carbachol (1 microM) for 2 h decreased muscarinic cholinergic receptor density in whole cells by 33%; preincubation with Con A prevented this receptor downregulation. Con A pretreatment did not affect (-)-isoproterenol- or forskolin-stimulated adenylate cyclase activity in cell homogenates, suggesting that an intact cytoarchitecture is necessary for Con A to augment cAMP formation. We conclude that Con A, through its modulation of beta-adrenergic and muscarinic cholinergic receptor signaling, amplifies both stimulatory and inhibitory adenylate cyclase-linked pathways in intact neonatal ventricular myocytes. These data suggest the possibility that plasma membrane-cytoskeletal interaction is an important regulator of transmembrane signaling because interference with this interaction results in alterations in cAMP accumulation mediated by both beta-adrenergic- and muscarinic cholinergic-adenylate cyclase pathways.
K J Rocha-Singh, D K Hines, N Y Honbo, J S Karliner
Inhibition of pancreatic glucagon secretion during hyperglycemia could be mediated by (a) glucose, (b) insulin, (c) somatostatin, or (d) glucose in conjunction with insulin. To determine the role of these factors in the mediation of glucagon suppression, we injected alloxan while clamping the arterial supply of the pancreatic splenic lobe of dogs, thus inducing insulin deficiency localized to the ventral lobe and avoiding hyperglycemia. Ventral lobe insulin, glucagon, and somatostatin outputs were then measured in response to a stepped IV glucose infusion. In control dogs glucagon suppression occurred at a glucose level of 150 mg/dl and somatostatin output increased at glucose greater than 250 mg/dl. In alloxan-treated dogs glucagon output was not suppressed nor did somatostatin output increase. We concluded that insulin was required in the mediation of glucagon suppression and somatostatin stimulation. Subsequently, we infused insulin at high rates directly into the artery that supplied the beta cell-deficient lobe in six alloxan-treated dogs. Insulin infusion alone did not cause suppression of glucagon or stimulation of somatostatin; however, insulin repletion during glucose infusions did restore the ability of hyperglycemia to suppress glucagon and stimulate somatostatin. We conclude that intra-islet insulin permits glucose to suppress glucagon secretion and stimulate somatostatin during hyperglycemia.
C J Greenbaum, P J Havel, G J Taborsky Jr, L J Klaff
In this report we demonstrate that proteolytic cleavage of the constituent subunit is one of the causes determining the heterogeneous size distribution of plasma von Willebrand factor (vWf) multimers. As shown by two-dimensional nonreduced/reduced agarose/polyacrylamide gel electrophoresis, the structure of circulating vWf molecules may deviate from that represented by assemblage of a variable number of identical subunits. Indeed, even though the largest multimers in normal plasma appear to be composed predominantly of intact 225-kD subunits, those of intermediate and smaller size contain also 189-, 176-, and 140-kD proteolytic fragments. Different subunit composition patterns are repeated regularly in multimers of increasing molecular mass, yielding series of bands with similar structure. One of these series consists of molecules without evidence of proteolytic fragmentation, and its smallest member appears to be a dimer of 225-kD subunits. Type IIA von Willebrand disease, characterized by absence of the largest multimers, displays a pattern wherein the fragments of 176 and 140 kD are relatively increased, that of 189 kD is markedly decreased or absent, but the composition of individual multimers is otherwise similar to that of species seen also in normal plasma. In contrast to those in the circulation, all normal platelet vWf multimers contain only intact subunit. These results suggest that proteolytic cleavage of plasma vWf subunits occurs after release from cellular sites, whereas platelet vWf stored in alpha-granules is protected from proteolysis. These findings provide information that may be relevant for understanding the normal processing of vWf multimers and for elucidating the pathogenesis of some of the congenital and acquired structural abnormalities of this molecule.
J A Dent, M Galbusera, Z M Ruggeri
Renal cortical tubules consist of polarized epithelial cells where Na/H antiport activity has been demonstrated on the apical and/or basolateral membrane. Apical Na/H antiport activity plays an important role in transcellular bicarbonate (HCO3-) reabsorption, whereas basolateral Na/H antiport activity could be involved in transcellular HCO3- secretion as well as cell volume and pH control. To determine whether this heterogeneity in both localization and function is due to the existence of more than one Na/H antiporter, we studied the tissue distribution of Na/H antiporter mRNA by use of reverse transcription (RT) and polymerase chain reaction (PCR) in isolated nephron segments from rat renal cortex. The primers used were directed against the rat renal cortical Na/H antiporter cDNA which is homologous to the human growth factor-activatable Na/H antiporter. RT/PCR of beta-actin mRNA were performed as positive controls. Na/H antiporter mRNA expression in the proximal tubule was not detectable in S1 and S2 segments from superficial and most midcortical nephrons, which exhibit exclusively luminal Na/H antiport activity. It was expressed in S1 and S2 segments from juxtamedullary nephrons which have also basolateral Na/H antiport activity. Beta-actin mRNA was expressed uniformly in all segments of the proximal tubule. Na/H antiporter mRNA was also expressed in cortical thick ascending limb and cortical collecting duct, segments with basolateral Na/H antiport activity as well as in the glomeruli. In conclusion, at least two different Na/H antiporters exist in the renal cortex, i.e., the proximal tubule. The close correlation between functional localization of basolateral Na/H antiport activity and mRNA expression suggests that the rat kidney Na/H antiporter DNA homologous to the human growth factor activatable Na/H antiporter encodes a basolateral exchanger. The observed expression in a minority of midcortical proximal tubules could reflect a certain heterogeneity in these nephron segments.
R Krapf, M Solioz
Autonomous colony formation is a frequent event in erythroleukemia. In 13 cases of early erythroid leukemias, we investigated whether erythropoietin (Epo) autocrine stimulation was responsible for the growth factor autonomy. Epo transcripts were detected by Northern blotting in cells from one patient. These cells also expressed an Epo receptor (1,000 receptors per cell) with a 420-pM affinity and Epo was detected in the supernatant of cultured cells. In 8 of the 13 cases, Epo transcripts were revealed by the polymerase chain reaction ranging from 0.5 to 500 copies per cell. In situ hybridization proved that these Epo transcripts were present in the blast cells. No Epo gene abnormalities were detected by Southern blotting. In two cases, leukemic cells were grown in the presence of Epo-neutralizing antibodies or Epo antisense oligomers. In one case, the antibody significantly reduced autonomous growth. In contrast, the antibody had no effect in the second case in which blast cells transcribed the Epo gene at a low level. However, Epo antisense oligomers partially inhibited autonomous growth. This inhibition was reversed by addition of exogenous Epo. Overall, these results suggest that an extracellular or intracellular autocrine Epo stimulation occurs in some cases of erythroid malignancies.
M T Mitjavila, J P Le Couedic, N Casadevall, S Navarro, J L Villeval, A Dubart, W Vainchenker
Eosinophil granule major basic protein (MBP), a potent toxin for helminths and mammalian cells in vitro, is a single polypeptide chain rich in arginine. MBP has been localized on damaged helminths and tissues in hypersensitivity diseases including bronchial asthma. The MBP cDNA indicates that MBP is translated as a slightly acidic preproprotein with an acidic propart. To test the hypothesis that the acidic pro-part of proMBP inhibits the toxicity of mature MBP, acidic polyamino acids (aa) were used as antagonists of MBP toxicity to K562 cells and guinea pig tracheal epithelium and used as antagonists of MBP airway hyperresponsiveness in primates. The acidic poly aa inhibited MBP toxicity and MBP airway hyperresposiveness. The acidic poly aa inhibited MBP toxicity in a charge-dependent manner similar to that proposed for proMBP, suggesting that the acidic pro-part of proMBP functions to mask mature MBP toxicity. This inhibition was not limited to MBP, but also applied to polyarginine and eosinophil cationic protein. These acidic poly aa may be useful to inhibit the actions of a number of cationic toxins released by the eosinophil in numerous hypersensitivity diseases.
R L Barker, R H Gundel, G J Gleich, J L Checkel, D A Loegering, L R Pease, K J Hamann
Types A and B Niemann-Pick disease both result from the deficient activity of the lysosomal hydrolase, acid sphingomyelinase (E.C. 220.127.116.11). Type A Niemann-Pick disease is a severe neurodegenerative disorder of infancy which leads to death by three years of age, whereas Type B disease has a later age at onset, little or no neurologic involvement, and most patients survive into adulthood. To investigate the molecular basis for the remarkable phenotypic heterogeneity, the nature of the mutations causing Type B Niemann-Pick disease in Ashkenazi Jewish patients was determined. The entire acid sphingomyelinase coding region from an Ashkenazi Jewish Type B patient was polymerase chain reaction-amplified, subcloned, and completely sequenced. A three-base deletion was identified of nucleotides 1821-1823 in the cDNA which predicted the removal of an arginine residue from position 608 of the acid sphingomyelinase polypeptide (delta R608). The other cDNA clones from this patient had the R496L mutation previously identified in Type A Niemann-Pick disease patients. Both Ashkenazi Jewish Type B patients were heteroallelic for the delta R608 mutation, whereas this allele was not present in 15 unrelated non-Jewish Type B patients, with the notable exception of one mildly affected patient of Arabic descent who was homoallelic for the delta R608 mutation. These results indicate that the delta R608 mutation predicts the Type B Niemann-Pick disease phenotype, even in the presence of the R496L Type A allele, thereby providing the first genotype/phenotype correlation for this lysosomal storage disease. Although only two patients have been studied, it appears that the delta R608 mutation occurs frequently in Type B Niemann-Pick disease patients of Ashkenazi Jewish descent.
O Levran, R J Desnick, E H Schuchman
The molecular mechanism involved in altered regulation of the rate-limiting enzyme in hepatic gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), during endotoxemia is not completely understood. We examined, therefore, the effect of a nonlethal dose of Escherichia coli endotoxin on PEPCK gene expression in fasted rats. 5 h after endotoxin treatment, the PEPCK transcription rate and the amount of mRNA(PEPCK) were significantly decreased at a time when the insulin/glucagon (I/G) molar ratio and plasma corticosterone levels were significantly increased. Similar results were observed in a time course study, in which altered cAMP induction of PEPCK gene expression paralleled changes in the I/G molar ratio. In diabetic rats treated with endotoxin, PEPCK gene expression was decreased in the absence, however, of an increased I/G molar ratio. This finding indicates that other factors, such as inflammatory mediators or cytokines, alter PEPCK gene transcription during endotoxemia. IL-6, a putative mediator of endotoxin action in the liver, had no effect on PEPCK gene expression in fasted rats, but did decrease cAMP induction of PEPCK gene expression. These results indicate that, during endotoxemia, regulation of PEPCK gene expression is influenced by inflammatory mediators in addition to the classical endocrine hormones. IL-6, however, does not appear to be involved directly in the altered regulation of the PEPCK gene during endotoxemia.
M Hill, R McCallum
Human peripheral blood mononuclear cells are analyzed for preproenkephalin gene expression and peptide processing. Met-enkephalin immunoreactivity as detected with a specific antiserum is found in the cytoplasm of monocytes but not in T lymphocytes. Secretion of met-enkephalin was analyzed with an RIA that is specific for the met-enkephalin pentapeptide. Unfractionated PBMC spontaneously released 40 pg/ml met-enkephalin and this increased two- to fourfold after stimulation with PHA. Lower levels (less than 100 pg/ml) of met-enkephalin were detected in supernatants from purified T cells that were activated with PHA and IL-2. In contrast, stimulation of purified monocytes with LPS or PMA resulted in the release of up to 600 pg/ml of the processed peptide. To examine whether T cells can produce met-enkephalin precursor peptides, T cell conditioned media were treated with trypsin and carboxypeptidase-B, which is known to release met-enkephalin from the propeptide. This increased levels of met-enkephalin to 400 pg/ml, indicating that lymphocytes secrete the propeptide but do not process it to met-enkephalin. The 1.4-kb preproenkephalin mRNA is detected in activated blood mononuclear cells and in purified monocytes and T cells. To determine whether monocytes or lymphocytes express met-enkephalin in vivo, lymphoid tissues were analyzed by immunohistochemistry. In human spleen tissue, positive cells were found in the red pulp but not in the follicles, which is also consistent with met-enkephalin expression in monocytes. In summary, these results show that human peripheral blood mononuclear cells express preproenkephalin mRNA and that monocytes, but not T cells, process the propeptide to metenkephalin.
W Kuis, P M Villiger, H G Leser, M Lotz
Because T cell-derived cytokines may affect the functioning of eosinophils, we have investigated the capacity of human eosinophils to respond to IL-2. IL-2 was a potent chemoattractant with ED50 of 10(-12) M with eosinophils from all normal and eosinophilic donors tested. The monoclonal antibodies anti-Tac and TU27 against p55 (Tac/CD25) and p75 receptor subunits, respectively, each inhibited IL-2-dependent eosinophil migration. The molar potency of IL-2 and the inhibitory activity of each of the above antibodies suggest that high affinity heterodimeric IL-2 receptor complexes mediated the migration responses of eosinophils to IL-2. Binding of monoclonal antibody against p75 was not detectable by flow cytometry, and high affinity binding sites for 125I-IL-2 were below the limits of quantitation on eosinophils from individuals with eosinophilia. Expression of p55 (Tac/CD25) by eosinophils, without requirement for in vitro activation, was demonstrable by flow cytometry, radioimmunoprecipitation, and Northern blotting for mRNA. Surface expression of p55 on eosinophils from normal or eosinophilic individuals increased during culture for 24-48 h with a biologic activity purified from stimulated U937 cells and to a lesser extent with granulocyte-macrophage CSF or lymphocyte chemoattractant factor but not with nine other cytokines. These studies indicate that blood eosinophils respond to IL-2 and identify one mechanism whereby activation of T lymphocytes may influence the function of eosinophils.
T H Rand, D S Silberstein, H Kornfeld, P F Weller
Insulin deficiency decreases tissue protein synthesis, albumin mRNA concentration, and albumin synthesis in rats. In contrast, insulin deficiency does not change, or, paradoxically, increases estimates of whole body protein synthesis in humans. To determine if such estimates of whole body protein synthesis could obscure potential differential effects of insulin on the synthetic rates of individual proteins, we determined whole body protein synthesis and albumin and fibrinogen fractional synthetic rates using 5-h simultaneous infusions of [14C]leucine and [13C]bicarbonate, in six type 1 diabetics during a continuous i.v. insulin infusion (to maintain euglycemia) and after short-term insulin withdrawal (12 +/- 2 h). Insulin withdrawal increased (P less than 0.03) whole body proteolysis by approximately 35% and leucine oxidation by approximately 100%, but did not change 13CO2 recovery from NaH13CO3 or estimates of whole body protein synthesis (P = 0.21). Insulin deficiency was associated with a 29% decrease (P less than 0.03) in the albumin fractional synthetic rate but a 50% increase (P less than 0.03) in that of fibrinogen. These data provide strong evidence that albumin synthesis in humans is an insulin-sensitive process, a conclusion consistent with observations in rats. The increase in fibrinogen synthesis during insulin deficiency most likely reflects an acute phase protein response due to metabolic stress. These data suggest that the absence of changes in whole body protein synthesis after insulin withdrawal is the result of the summation of differential effects of insulin deficiency on the synthesis of specific body proteins.
P De Feo, M G Gaisano, M W Haymond
Immune complex-induced injury is an important pathogenic factor in antibody-mediated nephritis, systemic lupus erythematosus, rheumatoid arthritis, and other diseases. In this study we investigated the role mast cells in immune complex-mediated injury in mouse skin. Reverse Arthus reaction was induced in mast cell-deficient WBB6F1-W/Wv mice and their congenic controls (WBB6F1(-)+/+). Serial skin sections were evaluated for neutrophil infiltration, edema, and hemorrhage. In WBB6F1-W/Wv mice the neutrophil influx was only 40% and edema 60% of that in congenic controls. Hemorrhage was also significantly reduced in the mast cell-deficient mice. After mast cell reconstitution, the magnitude of the reaction in WBB6F1-W/Wv was equivalent to that in WBB6F1(-)+/+ mice. Mast cell release in reverse Arthus reaction was evaluated by measuring fluorescence intensity after avidin-FITC staining of mast cell granules. There was a 70% decrease in fluorescence intensity. The 5-lipoxygenase inhibitor A-63162 significantly decreased neutrophil accumulation (40%), edema (60%), and hemorrhage in WBB6F1(-)+/+, but not in mast cell-deficient mice. Mast cell reconstitution of WBB6F1-W/Wv mice restored the effect of A-63162. The results indicate that mast cells and their mediators, including leukotrienes, make an important contribution to reverse Arthus reaction.
Y Zhang, B F Ramos, B A Jakschik
The object of this study was to further localize autoantigenic structures on IIb-IIIa and, if possible, to precisely identify the epitopes recognized by human autoantibodies. In this paper, we identify a 50-kD chymotryptic fragment of IIIa that is recognized by a high percentage of human autoantibodies, typified by the prototype IgG autoantibody RA, which binds to IIIa on intact platelets as well as in an immunoblot assay under nonreduced conditions. Using an immunoblot assay, a carboxy-terminal region of this fragment (33 kD) that contains the cysteine-rich domains of IIIa was found to carry the epitope(s) recognized by the prototype autoantibody RA. The amino-terminal amino acid sequence of the reduced 33-kD fragment, the smallest fragment that retains the RA epitope, is XPSQQDEXSP, and that of the reduced 50-kD fragment is IVQVTFD. This indicates that the 33-kD fragment consists of approximately 175 amino acids beginning at residue 479 and extending at least through residues 636-654, while the 50-kD fragment spans the same region but begins at residue 427. It is apparent that the 33-kD fragment is generated from the 50-kD fragment by additional chymotryptic hydrolysis but remains associated because of the multiple disulfide bonds that are characteristic of this cysteine-rich domain. Sera from 48% of patients with chronic ITP and 2 of 8 patients with acute ITP contain antibodies that bind to the 50-kD fragment in an ELISA. Antibodies of the same specificity are also found in one-third of patients with either secondary immune thrombocytopenia or apparent non-immune thrombocytopenia. We conclude that the 50-kD cysteine-rich region of IIIa is a frequent target of autoantibodies in ITP, but that such antibodies may also be present in cases of thrombocytopenia that cannot be linked to an apparent autoimmune process.
R Kekomaki, B Dawson, J McFarland, T J Kunicki
Protein kinase C is involved in mediating the effects of elevated Ca2+ in ileal villus Na+ absorbing cells to inhibit NaCl absorption. The present studies were undertaken to understand the mechanism by which this occurs. The effects of carbachol and the calcium ionophore A23187, agents which elevate intracellular Ca2+ and inhibit NaCl absorption in ileal villus cells, were studied. Carbachol treatment of villus cells caused a rapid decrease in protein kinase C activity in cytosol, with an accompanying increase in microvillus membrane C kinase. Exposure of the villus cells to calcium ionophore also caused a quantitatively similar decrease in cytosol C kinase and increase in C kinase activity in the microvillus membrane. This increase caused by carbachol and Ca2+ ionophore was specific for the microvillus membrane. In fact, 30 s and 10 min after exposure of the cells to carbachol, basolateral membrane protein kinase C decreased, in a time-dependent manner; whereas 10 min of Ca2+ ionophore exposure did not alter basolateral C kinase. Exposure of villus cells to Ca2+ ionophore or carbachol caused similar increases in microvillus membrane diacylglycerol content. As judged by the ability to inhibit Na+/H+ exchange measured in ileal villus cell brush border membrane vesicles, the protein kinase C which translocated to the microvillus membrane was functionally significant. Inhibition of Na+/H+ exchange required ATP and was reversed by the protein kinase C antagonist H-7. In conclusion, the effect of carbachol and Ca2+ ionophore in regulation of ileal NaCl absorption is associated with an increase in microvillus membrane diacylglycerol content and functionally active protein kinase C. The effects of both carbachol and Ca2+ ionophore are different on brush border and basolateral membrane distribution of protein kinase C.
M E Cohen, J Wesolek, J McCullen, K Rys-Sikora, S Pandol, R P Rood, G W Sharp, M Donowitz
Although intravenous Escherichia coli endotoxin has been used extensively in experimental studies to increase lung endothelial permeability, the effect of E. coli endotoxin on lung epithelial permeability has not been well studied. To examine this issue in sheep, bidirectional movement of protein across the lung epithelial barrier was studied by labeling the vascular space with 131I-albumin and by instilling 3 ml/kg of an isosmolar protein solution with 125I-albumin into the alveoli. E. coli endotoxin was administered according to one of three protocols: intravenous alone (5-500 micrograms/kg), intravenous (5 micrograms/kg) plus low-dose alveolar endotoxin (10 micrograms/kg), and high-dose alveolar endotoxin alone (50-100 micrograms/kg). Alveolar liquid clearance was estimated based on the concentration of the instilled native protein. Sheep were studied for either 4 or 24 h. Although intravenous E. coli endotoxin produced a marked increase in transvascular protein flux and interstitial pulmonary edema, there was no effect on the clearance of either the vascular (131I-albumin) or the alveolar (125I-albumin) protein tracer across the epithelial barrier. High-dose alveolar E. coli endotoxin caused a 10-fold increase in the number of leukocytes, particularly neutrophils, that accumulated in the air spaces. In spite of the marked chemotactic effect of alveolar endotoxin, there was no change in the permeability of the epithelial barrier to the vascular or alveolar protein tracers. Also, alveolar epithelial liquid clearance was normal. Morphologic studies confirmed that the alveolar epithelial barrier was not injured by either intravenous or alveolar E. coli endotoxin. Thus, the alveolar epithelium in sheep is significantly more resistant than the lung endothelium to the injurious effects of E. coli endotoxin.
J P Wiener-Kronish, K H Albertine, M A Matthay
To make synthetic peptide vaccines effective in a broad population of outbred humans, one would have to incorporate enough antigenic determinants to elicit recognition by T cells of most HLA types. We have previously defined multideterminant regions of the human immunodeficiency virus (HIV) envelope that include overlapping determinants seen by proliferating T cells of three or four haplotypes of mice. We have now tested the hypothesis that synthetic peptides encompassing such multideterminant regions will be recognized by T cells of multiple murine MHC types as well as by human T cells representing multiple HLA types. Six such peptides of 20-33 residues in length were prepared, and tested for their ability to stimulate T cells from mice of four distinct MHC types immunized with recombinant envelope protein rgp 160, as well as from 42 HIV-infected humans of different HLA types. Results identify several such peptides that are broadly recognized by mice of four H-2 types and by 52-73% of infected humans who still retain IL-2 productive responses to control recall antigens such as influenza A virus or tetanus toxoid. 86% of such infected donors tested against at least three peptides respond to at least one of the six peptides, and 77% of an additional group of seropositives respond to a mixture of the peptides. Moreover, the peptides can be used to immunize mice to elicit T cells reactive with the intact HIV envelope protein. These peptides therefore may be useful for both vaccine development in the broad human population, and diagnostic or prognostic use.
J A Berzofsky, C D Pendleton, M Clerici, J Ahlers, D R Lucey, S D Putney, G M Shearer
Monoclonal antibodies (MAb) directed against bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF) provide partial protection in experimental models of septic shock. To determine if additional benefit accrues from a combination of anti-TNF and anti-LPS MAb in the treatment of septic shock, a neutropenic rat model was developed to study active infection with Pseudomonas aeruginosa 12.4.4. Animals were treated intravenously with an irrelevant MAb (group 1); anti-TNF MAb (group 2); MAb directed against P. aeruginosa 12.4.4 LPS (group 3); or a combination of anti-TNF and anti-LPS MAb (group 4). None of the control animals in group 1 survived the 7-d period of neutropenia (0/16). In contrast, the survival rate was 44% in group 2 (P less than 0.02); 37% in group 3 (P less than 0.05); and 75% in group 4 (P less than 0.0002). The combination of monoclonal antibodies provided greater protection than either MAb given alone (P less than 0.05). Serum TNF levels during infection were significantly greater in groups 1 and 3 (20.1 +/- 3.3 U, mean +/- SE) than in groups 2 and 4 (0.9 +/- 0.8 U, P less than 0.0001). These results indicate that a combination of monoclonal antibodies to LPS and TNF have additive benefit in experimental Pseudomonas aeruginosa sepsis. This immunotherapeutic approach may be of potential utility in the management of serious, gram-negative bacterial infection in neutropenic patients.
S M Opal, A S Cross, J C Sadoff, H H Collins, N M Kelly, G H Victor, J E Palardy, M W Bodmer
The emphysema of alpha 1-antitrypsin (alpha 1AT) deficiency is conceptualized to result from insufficient alpha 1AT allowing neutrophil elastase to destroy lung parenchyma. In addition to the deficiency of alpha 1AT in these individuals resulting from mutations in the alpha 1AT gene, it is recognized that, for unknown reasons, there are also increased numbers of neutrophils in their lungs compared with normal individuals. With the knowledge that alveolar macrophages have surface receptors for neutrophil elastase, we hypothesized that the neutrophil accumulation in the lower respiratory tract in alpha 1AT deficiency may result, in part, from release of neutrophil chemotactic activity by alveolar macrophages as they bind uninhibited neutrophil elastase. Consistent with this hypothesis, alpha 1AT-deficient alveolar macrophages spontaneously released nearly threefold more neutrophil chemotactic activity than normal alveolar macrophages. Analysis of alpha 1AT-deficient macrophage supernates by reverse-phase HPLC, molecular sieve chromatography, radioimmunoassay, and absorption with anti-LTB4 antibody revealed that the majority of the chemotactic activity was leukotriene B4 (LTB4), a mediator absent from normal macrophage supernates. Consistent with this hypothesis, incubation of normal macrophages with human neutrophil elastase resulted in the release of the same neutrophil chemotactic mediator. Furthermore, purified human alpha 1AT was able to prevent the neutrophil elastase from stimulating the macrophages to release the chemotactic factor. Together, these findings suggest that the absence of a normal antineutrophil elastase screen in the lower respiratory tract permits free neutrophil elastase to bind to alveolar macrophages, resulting in the release of LTB4, a process which attracts neutrophils to the alveoli of alpha 1AT deficient individuals, thus accelerating the lung destruction that characterizes this disorder.
R C Hubbard, G Fells, J Gadek, S Pacholok, J Humes, R G Crystal
TSH secretion is decreased by both T4 and T3. This negative feedback control of TSH secretion has been correlated with an increase in pituitary nuclear T3 content, and it is not clear whether T4 exerts its effect directly on the thyrotroph or after its deiodination to T3. However, levels of the pituitary enzyme catalyzing T4 to T3 conversion, 5'D-II, are decreased in the presence of an increased amount of T4. Thus, it is unclear why the thyrotroph would have a mechanism for modulating the production of T3, if T3 is, in fact, the sole bioactive signal providing negative feedback inhibition. To examine this apparent paradox, we administered EMD 21388, a compound which inhibits the binding of T4 to transthyretin resulting in a rapid increase in circulating free T4 levels, to rats pretreated with radiolabeled T4 and T3. We observed increases in pituitary and liver T4 content of greater than 150%, without increases in the respective tissue T3 contents. The EMD 21388-treated rats also exhibited a 25% decrease in pituitary 5'D-II activity (103.8 +/- 15.8 fmol 125I released.mg protein-1.h-1, vs. control, 137.4 +/- 15.9, mean +/- SE), as did rats treated with sodium salicylate, another compound that inhibits T4-TTR binding (100.8 +/- 7.1). TSH levels significantly decreased 2 h after the administration of EMD 21388. These data demonstrate that despite a T4-mediated decrease in pituitary 5'D-II activity, an increase in T4 independently decreases TSH secretion.
S L Abend, S L Fang, S Alex, L E Braverman, J L Leonard
Repair of arterial injury produced by balloon angioplasty leads to the formation of a neointima and a narrowing of the vascular lumen. In this study, we examined the possibility that smooth muscle cells (SMC) in injured rat carotid arteries are stimulated to produce type-1 transforming growth factor-beta (TGF-beta 1) during neointima formation in vivo. Levels of TGF-beta 1 transcripts (2.4 kb) were significantly increased within 6 h after carotid injury and reached a maximum (five to sevenfold) by 24 h. Regenerating left carotids had sustained increases in TGF-beta 1 mRNA levels (about fivefold) over the next 2 wk, during which time a substantial neointimal thickening was formed. No changes in basal TGF-beta 1 mRNA levels were found in contralateral uninjured carotids at any of the times examined. Immunohistochemical studies showed that a large majority of neointimal SMC were stained for TGF-beta 1 protein in an intracellular pattern, consistent with active TGF-beta 1 synthesis in this tissue. Neointima formation and TGF-beta 1 immunoreactivity were correlated with increases in fibronectin, collagen alpha 2(I), and collagen alpha 1(III) gene expression. Infusion of purified, recombinant TGF-beta 1 into rats with a preexisting neointima produced a significant stimulation of carotid neointimal SMC DNA synthesis. These results suggest that TGF-beta 1 plays an important role as an endogenous growth regulatory factor produced by neointimal SMC themselves during progressive neointimal thickening after balloon angioplasty.
M W Majesky, V Lindner, D R Twardzik, S M Schwartz, M A Reidy
Antibodies against capsular polysaccharides are important in the defense against many pathogenic bacteria. To determine the mechanism for the variability in responses to polysaccharides, a panel of well characterized serologic reagents that identify diagnostic primary amino acid sequences in the framework and hypervariable regions of heavy (H) and light (L) chains were created to characterize the variable region diversity in circulating human antibodies. 10 normal adult volunteers were immunized with the type b capsular polysaccharide of Haemophilus influenzae (Hib PS). By immunoblot analyses each individual was found to use at least three different variable L (VL) families, but all had preferential usage of VH3-derived H chains. Four individuals had lesser populations of VH1-derived H chains and three had populations of VH4-derived H chains, but anti-Hib PS antibodies derived from the VH2, VH5, and VH6 families were not detected. The anti-Hib PS antibodies from all subjects were also identified by serologic markers for two specific types of VH3 H chains. These H chains are structurally related to the 20P1 and 30P1 VH genes that are preferentially rearranged in the early human repertoire. These findings document the VH restriction of physiologic responses to Hib PS immunization, and demonstrate a technique to directly assess the structural and genetic diversity of specific serum antibodies.
G J Silverman, A H Lucas
Angiotensin II is known primarily for its effects on blood pressure and electrolyte homeostasis, but recent studies suggest that angiotensin II may play a role in the regulation of cellular growth. This study was undertaken to identify the angiotensin II receptor subtypes expressed during fetal and neonatal development and to characterize their cellular localization. Using an in situ receptor binding assay on sagittal frozen sections of fetal and neonatal rats, bound 125I-[Sar1,Ile8]-angiotensin II was visualized by film and emulsion autoradiography. Bound radioligand was detected by E11 (embryonic day 11) and maximal binding occurred by E19-21. Radioligand binding remained unaltered 30 min after birth, whereas a noticeable and stable decrease was observed 12 h postparturition. The highly abundant angiotensin II receptors were shown to be AT2 by the marked reduction in radioligand binding achieved with PD123177 (10(-7)M), a specific AT2 receptor antagonist, whereas DuP 753 (10(-5)M), an AT1 receptor antagonist, had little effect. Emulsion autoradiography showed radioligand binding in the undifferentiated mesenchyme of the submucosal layers of the intestine and stomach, connective tissue and choroid surrounding the retina, subdermal mesenchyme adjacent to developing cartilage, diaphragm, and tongue. Residual AT2 receptors were found on the dorsal subdermal region of the tongue 72 h after birth. AT1 receptors were detected in the placenta at E13 and in the aorta, kidney, lung, liver, and adrenal gland at E19-21, consistent with an adult distribution. The transient expression of AT2 receptors in the mesenchyme of the fetus suggests a role of angiotensin II in fetal development.
E F Grady, L A Sechi, C A Griffin, M Schambelan, J E Kalinyak
To define the roles of circadian rhythmicity (intrinsic effects of time of day independent of the sleep or wake condition) and sleep (intrinsic effects of the sleep condition, irrespective of the time of day) on the 24-h variation in glucose tolerance, eight normal men were studied during constant glucose infusion for a total of 53 h. The period of study included 8 h of nocturnal sleep, 28 h of continuous wakefulness, and 8 h of daytime sleep. Blood samples for the measurement of glucose, insulin, C-peptide, cortisol, and growth hormone were collected at 20-min intervals throughout the entire study. Insulin secretion rates were derived from C-peptide levels by deconvolution. Sleep was polygraphically monitored. During nocturnal sleep, levels of glucose and insulin secretion increased by 31 +/- 5% and 60 +/- 11%, respectively, and returned to baseline in the morning. During sleep deprivation, glucose levels and insulin secretion rose again to reach a maximum at a time corresponding to the beginning of the habitual sleep period. The magnitude of the rise above morning levels averaged 17 +/- 5% for glucose and 49 +/- 8% for calculated insulin secretion. Serum insulin levels did not parallel the circadian variation in insulin secretion, indicating the existence of an approximate 40% increase in insulin clearance during the night. Daytime sleep was associated with a 16 +/- 3% rise in glucose levels, a 55 +/- 7% rise in insulin secretion, and a 39 +/- 5% rise in serum insulin. The diurnal variation in insulin secretion was inversely related to the cortisol rhythm, with a significant correlation of the magnitudes of their morning to evening excursions. Sleep-associated rises in glucose correlated with the amount of concomitant growth hormone secreted. These studies demonstrate previously underappreciated effects of circadian rhythmicity and sleep on glucose levels, insulin secretion, and insulin clearance, and suggest that these effects could be partially mediated by cortisol and growth hormone.
E Van Cauter, J D Blackman, D Roland, J P Spire, S Refetoff, K S Polonsky
Hepatic cholesterol metabolism was studied in rats fed purified diets supplemented (9% wt/wt) with either fish oil (FO) (n-3 fatty acids) or corn oil (CO) (n-6 fatty acids) for 4 wk. Rats were equipped with permanent catheters in heart, bile duct, and duodenum to allow studies under normal feeding conditions. [3H]-cholesteryl oleate-labeled small unilamellar liposomes, which are rapidly endocytosed by hepatocytes, were intravenously injected to label intrahepatic cholesterol pools, and plasma and bile were collected. FO as compared to CO induced a lowering of plasma cholesterol levels by 38% and of triglyceride levels by 69%. This reduction in plasma lipids in FO rats was accompanied by: (a) an increased bile acid pool size (28%); (b) a fourfold increase in the ratio cholic acid/chenodeoxycholic acid in bile; (c) increased biliary excretion of cholesterol (51%); (d) accelerated excretion of endocytosed free cholesterol into bile; (e) accelerated incorporation of endocytosed cholesterol in bile acids; (f) a significant increase in the bile acid-independent fraction of bile flow; and (g) a threefold increase in hepatic alkaline phosphatase activity. The results show that FO induces changes in transport and metabolic pathways of cholesterol in the rat liver, which result in a more rapid disposition of plasma-derived cholesterol into the bile.
M J Smit, A M Temmerman, H Wolters, F Kuipers, A C Beynen, R J Vonk
We explored the action of luminal AVP in rabbit CCD perfused in vitro at 37 degrees C. Nanomolar concentrations of luminal AVP induced a sustained hyperpolarization of transepithelial voltage (Vt) in contrast to a transient hyperpolarization caused by basolateral AVP. 10 microM basolateral ouabain abolished the latter but not the former change in Vt. Despite a sustained hyperpolarization (from -20.7 +/- 2.9 to -34.1 +/- 4.7 mV; P less than 0.01), 10 nM luminal AVP only slightly altered net Na+ and K+ fluxes (7.6% stimulation and no significant change, respectively). Instead, luminal AVP appeared to modulate an acetazolamide-sensitive electrogenic ion transport because 200 microM basolateral acetazolamide suppressed the luminal AVP-induced hyperpolarization (percentage of Vt from -50.4 +/- 10.8 to -5.1 +/- 1.4; P less than 0.005). In terms of water transport, 10 nM luminal AVP did not change hydraulic conductivity (Lp, x 10(-7) cm/atm per s) (from 3.9 +/- 0.8 to 5.0 +/- 1.2), but suppressed the increase in Lp induced by 20 pM basolateral AVP (134.9 +/- 19.2 vs. 204.3 +/- 21.1 in control; P less than 0.05). These findings demonstrate distinct luminal action of AVP, suggesting amphilateral regulation of epithelial transport by AVP in the CCD.
Y Ando, K Tabei, Y Asano
We have examined the onset and duration of the inhibitory effect of an intravenous infusion of lipid/heparin on total body carbohydrate and fat oxidation (by indirect calorimetry) and on glucose disappearance (with 6,6 D2-glucose and gas chromatography-mass spectrometry) in healthy men during euglycemic hyperinsulinemia. Glycogen synthase activity and concentrations of acetyl-CoA, free CoA-SH, citrate, and glucose-6-phosphate were measured in muscle biopsies obtained before and after insulin/lipid and insulin/saline infusions. Lipid increased insulin-inhibited fat oxidation (+40%) and decreased insulin-stimulated carbohydrate oxidation (-63%) within 1 h. These changes were associated with an increase (+489%) in the muscle acetyl-CoA/free CoA-SH ratio. Glucose disappearance did not decrease until 2-4 h later (-55%). This decrease was associated with a decrease in muscle glycogen synthase fractional velocity (-82%). The muscle content of citrate and glucose-6-phosphate did not change. We concluded that, during hyperinsulinemia, lipid promptly replaced carbohydrate as fuel for oxidation in muscle and hours later inhibited glucose uptake, presumably by interfering with muscle glycogen formation.
G Boden, F Jadali, J White, Y Liang, M Mozzoli, X Chen, E Coleman, C Smith
In contrast to adult lymphocytes, neonatal lymphocytes secrete minimal amounts of Ig in response to stimulation with immobilized MAb to CD3. This deficiency could be overcome by the addition of supplemental IL-2, IL-4, or IL-6, resulting in the secretion of all Ig isotypes. There were no major differences in the distribution of Ig isotypes secreted in response to the cytokines alone or in combination. The Ig secreted in response to IL-4 or IL-6 was inhibited by MAb to CD25, suggesting that the effects of IL-4 and IL-6 were dependent on IL-2. Stimulation of neonatal lymphocytes with anti-CD3 was sufficient to induce expression of IL-2 receptors (CD25) on both T and B cells. IL-4 exerted direct effects on neonatal T cells by increasing IL-2 production and promoting IL-6 production by anti-CD3-stimulated neonatal lymphocytes. Antibody to IL-4 or IL-6 did not inhibit Ig secretion in response to IL-2 and antibody to IL-6 did not consistently inhibit Ig secretion in response to IL-4. Finally, in the presence of cyclosporin, anti-CD3-stimulated neonatal lymphocytes secreted Ig only with the combination of IL-2 and IL-4. These results have delineated unique, but not Ig isotype-specific, effects of cytokines in supporting Ig secretion by anti-CD3-stimulated neonatal lymphocytes. Deficient production of these cytokines is likely to contribute to the decreased capacity of neonatal lymphocytes to generate an Ig response.
J B Splawski, P E Lipsky
We have recently shown in vitro that the peroxisomal fraction of a rat liver homogenate has the highest capacity to beta-oxidize prostaglandins. In order to evaluate the relative importance of peroxisomes for this conversion also in vivo, we administered [3H]prostaglandin F2 alpha to an infant suffering from Zellweger syndrome, a congenital disorder characterized by the absence of intact peroxisomes. As a control, labeled compound was administered to two healthy volunteers. Urine was collected, fractionated on a SEP-PAK C18 cartridge, and subjected to reversed-phase high-performance liquid chromatography. The Zellweger patient was found to excrete prostaglandin metabolites considerably less polar than those of the control subjects. The major urinary metabolite in the control subjects was practically absent in the urine from the Zellweger patient. The major urinary prostaglandin F2 alpha metabolite from the Zellweger patient was identified as an omega-oxidized C20-prostaglandin, 9,11-dihydroxy-15-oxoprost-5-ene-1,20-dioic acid. The major urinary prostaglandin F2 alpha metabolite from the control subjects had chromatographic properties of a tetranor (C16) prostaglandin, in accordance with earlier published data. The present results, in combination with our previous in vitro data, indicate that peroxisomal beta-oxidation is of major importance for in vivo chain shortening of prostaglandins.
U Diczfalusy, B F Kase, S E Alexson, I Björkhem
Our primary aim was to determine the extent to which intraplasmic retinyl palmitate (RP) transfers to other lipoprotein particles when chylomicron remnants are not produced and/or the plasma RP residence time is increased. The study was conducted on three familial type I hyperlipoproteinemic patients, four lipoprotein lipase (LpL)-deficient heterozygotes, and three controls on a metabolic research unit. To each subject, a fat load was administered containing 16% of total daily calories in type I patients, 40% in heterozygotes and controls, plus 60,000 U/m2 vitamin A. Triglyceride (TG) and RP levels were evaluated in chylomicron and nonchylomicron fractions. Delay in the clearance of chylomicron fraction RP and the marked deficiency in nonchylomicron-RP (presumed lack of remnant production) in all three type I patients suggests that RP does not demonstrate significant intraplasmic transfer from chylomicrons to existent apolipoprotein B100 particles. In contrast to noncoincident TG and RP peaking in the normal subject, heterozygotes were found to demonstrate coincident plasma TG and RP curves, which is consistent with a common catabolic pathway for both TG and RP and inconsistent with intraplasmic RP transfer. This corroborates reports on compromised chylomicron clearance in heterozygotes. We conclude that RP is an appropriate representative marker for intestinally derived particles in LpL-deficient or partially deficient individuals.
D L Sprecher, S L Knauer, D M Black, L A Kaplan, A A Akeson, M Dusing, D Lattier, E A Stein, M Rymaszewski, D A Wiginton
Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment.
D H Ryan, B L Nuccie, C N Abboud, J M Winslow
MUC-2, the first described intestinal mucin gene, has become important as a prototype for secreted mucins in several organ systems. However, little is known about its protein backbone structure and hence its role in diseases such as colon cancer, ulcerative colitis, and cystic fibrosis, which are known to have mucin abnormalities. Studies in this manuscript show that MUC-2 contains two distinct regions with a high degree of internal homology, but the two regions bear no significant homology to each other. Region 1 consists mostly of 48-bp repeats which are interrupted in places by 21-24-bp segments. Several of these interrupting sequences show similarity to each other, creating larger composite repeat units. Region 1 has no length polymorphisms. Region 2 is composed of 69-bp tandem repeats arranged in an uninterrupted array of up to 115 individual units. Southern analysis of genomic DNA samples using TaqI and HinfI reveals both length and sequence polymorphisms which occur within region 2. The sequence polymorphisms have different ethnic distributions, while the length polymorphisms are due to variable numbers of tandem repeats.
N W Toribara, J R Gum Jr, P J Culhane, R E Lagace, J W Hicks, G M Petersen, Y S Kim
Free radicals have been implicated in the pathogenesis of reperfusion injury, but it is unclear how they exert their deleterious effects on cellular metabolism. Several lines of indirect evidence suggest that free radicals elevate intracellular Ca2+ concentration ([Ca2+]i) and inhibit glycolysis as part of their mechanism of injury. We tested these ideas directly in hearts subjected to hydroxyl radicals produced by the Fenton and Haber-Weiss reactions. Nuclear magnetic resonance spectra were obtained from Langendorff-perfused rabbit hearts before, during, and after 4 min of perfusion with H2O2 (0.75 mM) and Fe(3+)-chelate (0.1 mM). Isovolumic left ventricular pressure exhibited progressive functional deterioration and contracture after exposure to H2O2 + Fe3+. Phosphorus nuclear magnetic resonance (NMR) spectra revealed partial ATP depletion and sugar phosphate accumulation indicative of glycolytic inhibition. To measure [Ca2+]i, fluorine NMR spectra were acquired in a separate group of hearts loaded with the Ca2+ indicator 5F-BAPTA [5,5'-difluoro derivative of 1,2-bis-(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid]. Mean time-averaged [Ca2+]i increased from 347 +/- 14 nM in control to 1,026 +/- 295 nM 4 min after free radical generation (means +/- SEM, n = 7), and remained elevated thereafter. We conclude that free radicals induce clear-cut, specific derangements of cellular metabolism in the form of glycolytic inhibition and calcium overload. The observed increase in [Ca2+]i suggests that the deleterious effects of free radicals are at least partially mediated by secondary changes in cellular calcium homeostasis.
M C Corretti, Y Koretsune, H Kusuoka, V P Chacko, J L Zweier, E Marban
The role of CD4+ and CD8+ T lymphocytes in terminating respiratory syncytial virus (RSV) replication, causing disease, and protecting from reinfection was investigated using a BALB/c mouse model in which CD4+ or CD8+ lymphocytes or both were depleted by injections of Mab directed against the respective mouse lymphocyte determinants. Kinetics of RSV replication, illness, and pathology were assessed after primary infection and rechallenge. Both CD4+ and CD8+ lymphocyte subsets were involved in terminating RSV replication after primary infection. When both T lymphocyte subsets were depleted RSV replication was markedly prolonged, yet no illness was evident, suggesting that host immune response rather than viral cytocidal effect was the primary determinant of disease in mice. Both CD4+ and CD8+ lymphocytes contributed to illness, although CD8+ lymphocytes appeared to play the dominant role in this particular system. Analysis of histological responses suggested that CD4+ lymphocytes were required for the appearance of peribronchovascular lymphocytic aggregates seen in normal mice after rechallenge, and that the presence of alveolar lymphocytes was correlated with illness. It is postulated that antibody is an illness-sparing mechanism for protecting mice from RSV infection, and that T lymphocytes are an important determinant of illness. Further delineation of RSV-induced immunopathogenesis in primary infection and reinfection will provide important information for the development of vaccine strategies.
B S Graham, L A Bunton, P F Wright, D T Karzon
Maple Syrup Urine Disease (MSUD) in Mennonites is associated with homozygosity for a T to A transversion in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex. This causes a tyrosine to asparagine substitution at position 393 (Y393N). To assess the functional significance of this missense mutation, we have carried out transfection studies using E1 alpha-deficient MSUD lymphoblasts (Lo) as a host. The level of E1 beta subunit is also greatly reduced in Lo cells. Efficient episomal expression in lymphoblasts was achieved using the EBO vector. The inserts employed were chimeric bovine-human cDNAs which encode mitochondrial import competent E1 alpha subunit precursors. Transfection with normal E1 alpha cDNA into Lo cells restored decarboxylation activity of intact cells. Western blotting showed that both E1 alpha and E1 beta subunits were markedly increased. Introduction of Y393N mutant E1 alpha cDNA failed to produce any measurable decarboxylation activity. Mutant E1 alpha subunit was expressed at a normal level, however, the E1 beta subunit was undetectable. These results provide the first evidence that Y393N mutation is the cause of MSUD. Moreover, this mutation impedes the assembly of E1 alpha with E1 beta into a stable alpha 2 beta 2 structure, resulting in the degradation of the free E1 beta subunit.
C R Fisher, J L Chuang, R P Cox, C W Fisher, R A Star, D T Chuang
The Tax oncoprotein of the type I human T cell leukemia virus (HTLV-I) activates transcription of cellular and viral genes through at least two different transcription factor pathways. Tax activates transcription of the c-fos proto-oncogene by a mechanism that appears to involve members of the cAMP response element binding protein (CREB) and activating transcription factor (ATF) family of DNA-binding proteins. Tax also induces the nuclear expression of the NF-kappa B family of rel oncogene-related enhancer-binding proteins. We have investigated the potential role of these CREB/ATF and NF-kappa B/Rel transcription factors in Tax-mediated transformation by analyzing the oncogenic potential of Tax mutants that functionally segregate these two pathways of transactivation. Rat fibroblasts (Rat2) stably expressing either the wild-type Tax protein or a Tax mutant selectively deficient in the ability to induce NF-kappa B/Rel demonstrated marked changes in morphology and growth characteristics including the ability to form tumors in athymic mice. In contrast, Rat2 cells stably expressing a Tax mutant selectively deficient in the ability to activate transcription through CREB/ATF demonstrated no detectable changes in morphology or growth characteristics. These results suggest that transcriptional activation through the CREB/ATF pathway may play an important role in Tax-mediated cellular transformation.
M R Smith, W C Greene
We have established a recombinant HIV gene transfer system based on transient expression of the HIV packaging functions and a recombinant vector genome in monkey kidney Cos cells. The recombinant HIV retroviral vector introduced the neoR gene into CD4+ cells with high efficiency, comparable to that achieved with the highest titer amphotropic murine recombinant retrovirus. Vector preparations were devoid of replication competent, infectious HIV. Gene transfer was dependent on CD4 expression, as shown by expression of the CD4 gene in HeLa cells, and could be inhibited by soluble CD4. This specific and efficient gene transfer system may be useful for development of gene therapy for which T cells are the desired targets.
T Shimada, H Fujii, H Mitsuya, A W Nienhuis
Airway inflammation is thought to play an important role in the pathogenesis of asthma. We have used in situ hybridization and an immunoassay to determine whether granulocyte macrophage colony-stimulating factor (GM-CSF) (a cytokine capable of eosinophil activation) is present in the airway of asthmatics (n = 6) who have 37.0 +/- 15.1% airway eosinophilia after endobronchial allergen challenge. Levels of immunoreactive GM-CSF (less than 4 pg/ml pre-allergen versus 180.5 +/- 46.9 pg/ml post-allergen) increased significantly 24 h after endobronchial allergen stimulation. The cellular source of bronchoalveolar lavage (BAL) GM-CSF, as determined by in situ hybridization and immunoperoxidase staining, was derived predominantly from UCHL-1 positive BAL lymphocytes, as well as from a smaller population of alveolar macrophages. Before local endobronchial allergen challenge, less than 1% of lymphocytes and alveolar macrophages recovered by BAL expressed GM-CSF mRNA, whereas after allergen stimulation 92.6 +/- 3.4% of lymphocytes and 17.5 +/- 22.7% of alveolar macrophages expressed GM-CSF mRNA. This study provides evidence that in an experimental model of allergen-induced asthma, activation of the immune and inflammatory response (BAL lymphocyte and alveolar macrophage production of GM-CSF) is temporally associated with an inflammatory cell influx of eosinophils into the airway.
D H Broide, G S Firestein
Hypoxia in vivo is associated with constriction of the distal vasculature in the lung. Uniquely situated at the interface between blood and the vessel wall proper, the vascular endothelium may release vasoactive mediators in the setting of hypoxia. Endothelin-1 is a potent vasoconstrictor released by endothelial cells that could function as a paracrine regulator of vascular tone. We found that physiologic low oxygen tension (PO2 = 30 Torr) increased endothelin secretion from cultured human endothelial cells four to eightfold above the secretion rate at ambient oxygen tension. This increase in secretion was accompanied by a corresponding increase in the transcriptional rate of the preproendothelin gene resulting in increased steady-state mRNA levels of preproendothelin. In contrast, the transcription of a number of other growth-factor-encoding genes, including transforming growth factor-beta, was unaffected by hypoxia. Endothelin transcript production increased within 1 h of hypoxia and persisted for at least 48 h. In addition, the stimulatory effects of low oxygen tension on endothelin mRNA levels were reversible upon reexposure to 21% oxygen environments. These findings suggest a role for endothelin in the control of regional blood flow in the vasculature in response to changes in oxygen tension.
S Kourembanas, P A Marsden, L P McQuillan, D V Faller
The hepatitis C virus is a positive stranded hepatotropic RNA virus. We describe a method of detecting positive and negative strands of hepatitis C viral RNA using the polymerase chain reaction. We tested serum and liver tissue from nine patients with chronic hepatitis C. The positive RNA strand of HCV was detected in the sera and livers of all nine, the negative strand was detected in the livers of eight (89%), and in the sera of five (55%). Titers of both strands of HCV RNA were determined by serial endpoint dilutions. The amount of the negative strand in the serum and liver was usually 10-100 times less than the positive strand. Predigestion of serum with ribonucleases did not alter the detection of the negative strand. This suggests that the negative strand found in the serum may be protected from digestion by being associated with virions.
T L Fong, M Shindo, S M Feinstone, J H Hoofnagle, A M Di Bisceglie