Prolongation of all phospholipid-dependent coagulation tests was found in a patient with macroglobulinemia, despite absence of bleeding manifestations. The purified monoclonal IgM lambda protein and its Fabmu tryptic fragment induced similar changes in normal plasma. Patient IgM and Fabmu completely inhibited Ca++-dependent binding of radiolabeled prothrombin and Factor X to mixed phospholipid micelles. The patient's IgM lambda paraprotein reacted with phosphatidylserine and, to a lesser extent, with phosphatidylinositol and phosphatidic acid, but not with phosphatidylcholine or phosphatidylethanolamine. Prior incubation of phospholipid with patient Fabmu blocked the positive reactions. Substitution of washed platelets for phospholipid led to normalization of patient coagulation tests and corrected all abnormalities produced in normal plasma by patient IgM. Furthermore, binding of 125I-Factor Xa to thrombin-treated platelets was entirely normal in the presence of patient IgM. These studies support the concept that platelets, rather than phospholipid micelles, are the primary locus of prothrombin and Factor X activation in normal hemostasis.
P Thiagarajan, S S Shapiro, L De Marco
We examined the role of prostaglandins and thromboxanes as mediators of plasma-dependent increased polymorphonuclear leukocyte adhesiveness induced by Escherichia coli lipopolysaccharide. The cyclo-oxygenase inhibitors—indomethacin and d,l-6-chloro-α-methyl-carbozole-2-acetic acid (R020-5720)—reduced lipopolysaccharide-induced adherence of polymorphonuclear leukocytes by 74 and 62%, respectively. In addition, inhibitors of thromboxane synthetase—imidazole, 9,11-azoprosta-5,13-dienoic acid, and 1-benzylimidazole—suppressed the stimulation of adherence by 31, 66, and 83%, respectively. Exogenous prostaglandins E1, E2, and F2α did not increase polymorphonuclear leukocyte adherence, nor were they detected in significant quantities in supernates of polymorphonuclear leukocytes exposed to lipopolysaccharide. However, inhibitors of both cyclo-oxygenase and thromboxane synthetase reduced increases in adherence induced by arachidonic acid (10 μg/ml), suggesting that lipopolysaccharide-mediated increases in adherence were due to an arachidonic acid product other than prostaglandin E2 or F2α. 8,11,14-Eicosatrienoic acid, a precursor of monoenoic prostaglandins, did not enhance polymorphonuclear leukocyte adhesiveness.
Philip J. Spagnuolo, Jerrold J. Ellner, Aviv Hassid, Michael J. Dunn
The feeding of cholesterol-rich diets alters the serum lipoproteins of a number of mammalian species. These lipoproteins are characterized by the presence of several classes of particles enriched in cholesteryl esters and apolipoprotein E (apo E). It was the aim of this study to determine whether one or more of these particles arises by de novo hepatic synthesis by characterizing nascent lipoproteins isolated from the hepatic Golgi apparatus of hypercholesterolemic rats. Characterization of these lipoproteins afforded the opportunity to assess morphologic, biochemical, and biophysical properties of newly synthesized lipoproteins before enzymatic alterations and apoprotein transfer known to occur after secretion into the plasma compartment. Golgi very low density lipoproteins (VLDL, d < 1.006 g/ml) from hypercholesterolemic rats contained nearly four times the total cholesterol mass found in control Golgi VLDL. They exhibited electrophoretic mobility intermediate between beta and pre-beta and were devoid of apo C. A second population of hepatic Golgi lipoproteins was isolated from hypercholesterolemic rats at 1.006--1.040 g/ml d. These low density lipoproteins were smaller than VLDL, displayed beta electrophoretic mobility, were enriched in cholesteryl esters, and contained apo E as well as apo B. The fatty acid composition of the core lipids of the nascent lipoproteins was found to reflect that of dietary triglyceride. The liver of the hypercholesterolemic rat thus plays an active role in dietary-induced hypercholesterolemia by synthesizing a modified VLDL and a low density lipoprotein resembling serum low density lipoprotein.
L L Swift, N R Manowitz, G D Dunn, V S LeQuire
Evidence has been sought for a genetically determined predisposition among children with juvenile rheumatoid arthritis (JRA) who are also at particular risk for the development of inflammatory eye disease.
D. Glass, D. Litvin, K. Wallace, L. Chylack, M. Garovoy, C. B. Carpenter, P. H. Schur
In vivo studies demonstrate that the pancreatic enzymes and the ionic environment in the upper gastrointestinal tract are essential determining factors for transport and absorption of cobalamin in man. Jejunal fluid was aspirated from healthy human volunteers after administration of cyano[57Co]cobalamin preparations. Immunochemical analysis of the aspirates demonstrated that all isotopic vitamin was transferred to a protein that is identical to the gastric intrinsic factor in terms of molecular mass (57,500), ionic nature (mean pI, 5.09), and reactivity with anti-intrinsic factor sera. However, in the aspirates from patients with exocrine pancreatic dysfunction the vitamin was found to be coupled > 60% to a protein identical to R proteins in terms of molecular mass (125,000), ionic nature (mean pI, 3.51), and reactivity with anti-R protein and anti-intrinsic factor sera. The preferential transfer of cobalamin to R proteins in the patients and to intrinsic factor in healthy subjects was associated, respectively, with low and normal levels of pancreatic enzymes in the intestine and these in turn were paralleled respectively by impaired and normal ileal absorption of cobalamin. These findings confirm the suggestion that the formation of unabsorbable cobalamin complexes may be the reason of impaired vitamin absorption in exocrine pancreatic insufficiency. Observations made with other selected patients demonstrate: (a) that decreased enzyme activity and nondegradation of R proteins may also be due to nonactivation of pancreatic zymogens in an acidic pH of the intestinal juice the vitamin transported to the jejunum couples to intrinsic factor when pancreatic function is normal, and to intrinsic factor and R protein in exocrine pancreatic insufficiency. The observations made with these selected patients may explain why not all patients with exocrine pancreatic insufficiency develop imparied cobalamin absorption, and also why the malabsorption is corrected by the administration of bicarbonate in certain patients.
G Marcoullis, Y Parmentier, J P Nicolas, M Jimenez, P Gerard
We have studied the interaction between virulent egg yolk-grown Legionella pneumophila Philadelphia 1 and human blood monocytes in vitro. The leukocytes were cultured in antibiotic-free tissue culture medium supplemented with 15% autologous human serum.
Marcus A. Horwitz, Samuel C. Silverstein
Although a normal serum thyrotropin (TSH) concentration is generally considered to be the most important finding to support the clinical impression of euthyroidism in patients with nonthyroidal diseases and decreased serum triiodothyronine (T3), the regulation of TSH secretion in sick patients has not been studied previously. Accordingly, we studied the regulation of TSH secretion in 23 patients with nonthyroidal diseases; 15 of the patients had decreased serum T3. TSH regulation was studied by measuring the TSH response to injected thyrotropin-releasing hormone (TRH) before and after effecting a small decrease in serum thyroxine (T4) and/or T3 concentrations by iodide treatment, 262 mg daily for 10 d. Iodide treatment significantly decreased (> 10%) the free T4 index (FT4-I) and/or free T3 index (FT3-I) in all patients. FT4-I values were correlated (0.611, P < 0.001), with free T4 concentration determined by equilibrium dialysis. Despite decreased FT4-I and/or FT3-I after iodide treatment in all patients, the TSH response to TRH after iodide treatment was augmented in only 8 of 15 patients who had decreased serum T3 (group 1) and in only 5 of 8 patients who had a normal serum T3. Mean base-line TSH concentration was increased significantly (P < 0.05) from 0.9±0.1 to 1.5±0.3 μU/ml in group 1 only. Comparison of the mean TSH response to TRH showed that there was no significant difference between groups 1 and 2. Moreover, no significant difference in thyroidal parameters was observed between patients who had augmented TSH response to TRH after iodides and those who had either similar or decreased TSH response irrespective of the initial serum T3. These studies show that an augmented TSH response to TRH in response to a small reduction in serum T4 and T3 concentration occurred in only 57% of the entire group of patients with nonthyroidal diseases and that the presence or absence of a normal TSH response to this stimulus did not seem to be related to the base-line serum T3 concentration. Because an increase in serum TSH in response to decreased serum T4 and T3 did not occur in about one-half of patients with nonthyroidal diseases, normal serum TSH may not be a reliable index of the euthyroid state in these patients.
Susan J. Maturlo, Robert L. Rosenbaum, Chao Pan, Martin I. Surks
An assay for the detection and quantitation of immune complexes is described. Experimental immune complexes or aggregated human gamma globulin (AHG) were incubated with polymorphonuclear leukocytes (PMN). After challenge of the PMN with opsonized zymosan, chemiluminescence was recorded in a scintillation spectrometer. A quantitative inhibition of chemiluminescence could be demonstrated by the interaction of PMN with immune complexes or AHG. Experimental immune complexes of bovine serum albumin-anti-bovine serum albumin were formed and tested by this assay, and immune complexes formed near antigen excess were best described by this technique. The technique was used to demonstrate immune complexes in the sera from patients with systemic lupus erythematosus, rheumatoid arthritis, and vasculitis. Immune complexes were quantitated by reference to a standard curve using AHG. By this technique, normal human sera had < 10 micrograms of AHG per milliliter of serum. Immune complexes at levels above this were detected in 9/15 patients with systemic lupus erythematosus, 18/30 patients with rheumatoid arthritis, and 2/5 patients with vasculitis. Therefore, this assay is a sensitive, simple method for measurement of circulating immune complexes in the sera of patients with certain connective tissue diseases.
N J Doll, M R Wilson, J E Salvaggio
We studied adherence to human cells by a strain of Escherichia coli. Adherence to erythrocytes was assessed directly by phase-contrast microscopy and indirectly by hemagglutination; adherence to peripheral blood leukocytes, using radiolabeled bacteria and subsequent determination of leukocyte-associated radioactivity; and adherence to renal glomeruli, by microscopy of fluoresceinated bacteria and of Gram-stained nonfluoresceinated bacteria.
Douglas P. Fine, Barbara L. Harper, Edward D. Carpenter, C. Patrick Davis, Tito Cavallo, James C. Guckian
The presence of neutrophils within the lung is a characteristic feature of a variety of lung diseases. To evaluate the potential role of alveolar macrophages in modulating the migration of neutrophils to the lung, normal human alveolar macrophages obtained from volunteers by bronchopulmonary lavage, were exposed for various periods of time in vitro to heat-killed microorganisms, and noninfectious particulates, immune complexes, and the macrophage supernates were evaluated for chemotactic activity. The microorganisms, noninfectious particulates, and immune complexes were chosen as stimuli for alveolar macrophages because these stimuli are representative of a spectrum of pathogenic agents that cause neutrophil accumulation in the lower respiratory tract. After incubation with each of these stimuli, alveolar macrophages released low molecular weight (400-600) chemotactic factor(s) (alveolar macrophage-derived chemotactic factor[s] [AMCF]) with relatively more activity for neutrophils than monocytes or eosinophils. Checker-board analysis of the AMCF revealed that the factor was primarily chemotactic and not chemokinetic for neutrophils. The selectivity for neutrophils vs. monocytes could not be explained by a selective deactivation of monocytes, because the AMCF was more potent in deactivating neutrophils than monocytes. Partial characterization of AMCF demonstrated it was heterogeneous with the following features: (a) stable to heating at 56 and 100°C for 30 min; (b) stable over a pH range of 1.0 to 12.0 for 60 min; (c) stable after exposure to trypsin, papain, chymotrypsin, collagenase, and elastase; (d) partially inhibited by serum chemotactic factor inhibitor(s); (e) two major isoelectric points (pI 7.6 and 5.2); and (f) partially extractable into ethyl acetate, ether, and hexane. Although AMCF was, at least, partially lipid in nature, it did not appear to be similar to previously described lipid chemotactic factors (e.g., hydroxy-derivatives of 5,8,10,14-eicosatetraenoic acid); analysis by gas chromatography-mass spectrophotometry of AMCF extracted into ethyl acetate did not reveal the presence of 5,8,10,14-eicosatetraenoic acid. The macrophage supernates containing the AMCF also stimulated normal human neutrophils to release lysozyme and lactoferrin but not lactate dehydrogenase. These studies suggest that a wide variety of potentially pathogenic stimuli induce normal alveolar macrophages to generate a low molecular weight chemotactic factor(s) that preferentially attracts neutrophils. Because alveolar macrophages are normal residents of alveoli, it is likely that by releasing this factor(s) macrophages play a significant role in amplifying the inflammatory processes seen in many acute and chronic lung diseases.
Gary W. Hunninghake, James E. Gadek, Henry M. Fales, Ronald G. Crystal
Propionic and methylmalonic acidemia are both known to be associated with hyperammonemia. Rats injected with 10 or 20 mmol/kg of propionate or 20 mmol/kg of methylmalonate, along with 1.5 g/kg of a mixture of amino acids, developed severe hyperammonemia, whereas rats administered the same dosages of acetate did not. In vitro, neither propionyl nor methylmalonyl CoA affected the activity of carbamyl phosphate synthetase I, ornithine transcarbamylase, nor the activation constant (KA) of carbamyl phosphate synthetase I for N-acetyl glutamate. Furthermore, rats injected with propionate showed no alteration of liver amino acid concentrations, which could explain impaired ureagenesis. Animals injected with methylmalonate showed an increase in both citrulline and aspartate, suggesting that argininosuccinic acid synthetase may also have been inhibited. Liver ATP levels were unchanged. Citrullinogenesis, measured in intact mitochondria from livers of injected animals, was reduced 20-25% by 20 mmol/kg of propionate or methylmalonate (compared with acetate). This effect was attributable to an impairment in the normal rise of liver N-acetyl glutamate content after amino acid injection. Thus, carbamyl phosphate synthetase I activation was reduced. Liver levels of acetyl CoA and free CoA were reduced. Levels of unidentified acyl CoA derivatives rose, presumably reflecting the accumulation of propionyl and methylmalonyl CoA. Thus, the principal mechanism for hyperammonemia induced by these acids is depletion of liver N-acetyl glutamate, which is in turn attributable to depletion of acetyl CoA and/or competitive inhibition by propionyl and methylmalonyl CoA of N-acetyl glutamate synthetase. Injection of methylmalonate may also have an additional inhibitory effect on argininosuccinic acid synthetase.
Peter M. Stewart, Mackenzie Walser
Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.
B E Seligmann, J I Gallin
The fall in pulmonary compliance in mice with radiation pneumonitis is associated with increased microvascular leakage of plasma proteins into the alveolar spaces and increased surfactant phospholipids in the lung and alveolar fluid. In the present experiments we examined the effect of corticosteroid adminitration on these two effects and on pulmonary mechanics 16 wk after x irradiation of the thorax. Survival in irradiated animals that received corticosteroids was markedly better during the period of corticosteroid administration than that of irradiated animals that received no corticosteroids. The development of abnormalities in pulmonary mechanics and alveolar fluid surface tension appeared to be inhibited in the irradiated animals receiving corticosteroids as compared with irradiated animals not receiving corticosteroids. The increased microvascular protein leakage seen in the lungs of irradiated mice was not significantly different in the corticosteroid-treated group. However, corticosteroid adminstration was associated with a marked increase in the amount of phosphatidyl choline that could be recovered from the alveolar spaces by lavage, over and above the increase resulting from irradiation, and a significant increase in the incorporation of [14C]palmitate into phosphatidyl choline by lung slices. The beneficial effects of steroids in this variety of adult respiratory distress syndrome may be the result of augmented surfactant production which may contribute to the maintenance of relatively normal pulmonary mechanics despite substantial leakage of plasma proteins into the alveolar space.
N J Gross
To investigate the effect of acute elevation of plasma free fatty acids (FFA) on the secretion of splanchnic somatostatin-like immunoreactivity (SLI), the peripheral venous, pancreatic, and gastric venous effluent levels of SLI were measured in normal and chronic alloxan diabetic dogs before and after the infusion of a fat emulsion supplemented with heparin. In normal conscious dogs heparin injected during the infusion of a fat emulsion elevated FFA levels from a mean (±SE) base-line level of 0.7±0.1 meq/liter to a peak value of 1.5±0.1 meq/liter (P < 0.001) and plasma SLI rose from a mean (±SE) base-line value of 145±7 pg/ml to a peak of 253±44 pg/ml (P < 0.05). Neither the infusion of glycerol, of fat emulsion without heparin, of heparin alone nor of saline itself had an effect on either the plasma level of FFA or SLI. In another group of anesthetized dogs with surgically implanted catheters the administration of fat emulsion plus heparin was accompanied by more than a two-fold rise in the concentration of SLI in the venous effluent of the pancreas and of the gastric fundus and antrum in association with an elevation of FFA levels. In a group of conscious diabetic dogs fat emulsion plus heparin raised FFA from a mean base-line level of 1.2±0.2 to 1.6±0.3 meq/liter (P < 0.05) and SLI rose from a mean base-line level of 185±9 pg/ml to a peak value of 310±44 pg/ml (P < 0.01). Although SLI levels were significantly greater than in normal dogs at several time points after the rise in FFA, the magnitude of the increment in diabetic dogs did not differ from normal. These results demonstrate that a rise in FFA levels is a potent stimulus for SLI secretion from the pancreas and stomach and raise the possibility that FFA is an important physiological regulator of SLI secretion.
Taro Wasada, Barbara Howard, Richard E. Dobbs, Roger H. Unger
The complement system was analysed in 14 asymptomatic patients with erythropoietic protoporphyria. In the majority of the sera studied the levels of complement components C1, C4, C2, and C3 were within the normal range. Upon ultraviolet light (330--460 nm) irradiation of the serum samples in vitro, a marked decrease in total hemolytic activity accompanied by reduction of C1, C4, C2, and C3 levels was observed. The loss of total hemolytic activity can be directly correlated with the levels of protoporphyrin (PP) and similar changes can be obtained in normal serum upon addition of PP followedf by ultraviolet light irradiation. It is postulated that after irradiation the excited PP develops the capacity to activate the complement sequence with the production of cleavage products, which may contribute to the skin changes observed in these patients upon sun exposure.
I Gigli, A A Schothorst, N A Soter, M A Pathak
Prostaglandin synthesis and T lymphocyte colony formation have been examined in previously untreated patients with Hodgkin's disease. Mononuclear cells have been isolated from peripheral blood and spleens of these patients. Significant augmentation in prostaglandin E levels were noted in the mononuclear cell cutures from Hodgkin's disease patients compared with controls (1.64 +/- 0.29 vs. 0.39 +/- 0.09 ng/10(6) cells, P < 0.005). Measured prostaglandin E levels increased with advancing stage of disease. Virtually all of the prostaglandins were synthesized by the adherent monocyte cell population. Prostaglandin E was the major product. Clonal expansion of a T lymphocyte precursor cell, which gives rise to colonies > 50 cells, was determined by a layered soft agar method. T colony formation was significantly reduced in patients with stage II, III, and IV disease. There were progressively reduced colony numbers seen with advancing stage of disease (609 +/- 209, 416 +/- 158, 207 +/- 58 compared with normals 2,274 +/- 360 colonies/10(6) cells plated; P < 0.005). The addition of inhibitors of endogenous prostaglandin synthesis resulted in significant augmentation of T colony number. However, a consistent relative decrease in T colony number was seen even when endogenous prostaglandin E synthesis was blocked. It would appear that both the prostaglandin-dependent and independent T colony precursor cells are lost with progressive stage of disease. A causative role of augmented prostaglandin synthesis in this stage-dependent reduction of T colony formation could not be established.
R S Bockman
We used embryonic chick pelvic cartilage as a model to study the mechanism by which cyclic AMP increases RNA synthesis. Isolated nuclei were incubated with [32P]-8-azidoadenosine 3,5'-monophosphate ([32P]N3cAMP) with no resultant specific nuclear binding. However, in the presence of cytosol proteins, nuclear binding of [32P]N3cAMP was demonstrable that was specific, time dependent, and dependent on a heat-labile cytosol factor. The possible biological significance of the nuclear binding of the cyclic AMP-protein complex was identified by incubating isolating nuclei with either cyclic AMP or cytosol cyclic AMP-binding proteins prepared by batch elution DEAE cellulose chromatography (DEAE peak cytosol protein), or both, in the presence of cold nucleotides and [3H]uridine 5'-triphosphate. Poly(A) RNA production occurred only in nuclei incubated with cyclic AMP and the DEAE peak cytosol protein preparation. Actinomycin D inhibited the incorporation of [3H]uridine 5'-monophosphate into poly(A) RNA. The newly synthesized poly(A) RNA had a sedimentation constant of 23S. Characterization of the cytosol cyclic AMP binding proteins using [32P]N3-cAMP with photoaffinity labeling three major cAMP-binding complexes (41,000, 51,000, and 55,000 daltons). The 51,000 and 55,000 dalton cyclic AMP binding proteins were further purified by DNA-cellulose chromatography. In the presence of cyclic AMP they stimulated poly(A) RNA synthesis in isolated nuclei. The 51,000-dalton cyclic AMP-binding protein was the predominant one that bound to the nuclei. While cyclic AMP-dependent protein kinsae activity was present in the cytosol and DEAE peak cytosol proteins, it was not present in the DNA-cellulose-bound, cyclic AMP-binding proteins. We conclude that one possible mechanism by which cyclic AMP increases RNA synthesis is by complexing to a 51,000-dalton cytosol cyclic AMP-binding protein and being subsequently translocated to the nucleus, where it is specifically bound and associated with induction of poly(A) RNA synthesis.
W M Burch Jr, H E Lebovitz
Pokeweed mitogen-induced B lymphocyte differentiation in vitro into antibody secreting plaque-forming cells (PFC) was investigated in nine patients with severe combined immunodeficiency having variable proportions of circulating B lymphocytes. When cultured by themselves, the peripheral blood mononuclear cells did not respond to stimulation with pokeweed mitogen in any patient. In the presence of irradiated allogeneic T cells as helpers, however, PFC responses were elicited in lymphocyte cultures from peripheral blood and/or bone marrow in some patients. In one of these patients, results of allogeneic co-culture experiments were suggestive of genetically restricted suppressor cells. In a single patient with deficiency of the enzyme adenosine deaminase, PFC were generated in bone marrow lymphocyte cultures only when they were supplemented with exogenous adenosine deaminase and allogeneic helper cells. A parallel study of T lymphocyte differentiation in vitro performed in fractionated bone marrow cells was suggestive of arrested differentiation at different steps along the differentiation pathway. In two patients with evidence of functional B cell precursors, deficiencies of helper T cell function could be attributed to differentiation defects at the level of the stem cells in one and the thymus in the other. The findings reported here further substantiate the heterogeneity of the severe combined immunodeficiency disease syndromes.
S G Pahwa, R N Pahwa, R A Good
Conversion of thyroxine (T4) to 3,5,3′-triiodothyronine (T3) in rat brain has recently been shown in in vivo studies. This process contributes a substantial fraction of endogenous nuclear T3 in the rat cerebral cortex and cerebellum. Production of T4 metabolites besides T3 in the brain has also been suggested. To determine the nature of these reactions, we studied metabolism of 0.2-1.0 nM [125I]T4 and 0.1-0.3 nM [131I]T3 in whole homogenates and subcellular fractions of rat cerebral cortex and cerebellum. Dithiothreitol (DTT) was required for detectable metabolic reactions: 100 mM DTT was routinely used. Ethanol extracts of incubation mixtures were analyzed by paper chromatography in t-amyl alcohol:hexane:ammonia and in 1-butanol:acetic acid. Rates of production of iodothyronines from T4 and T3 were greater at pH 7.5 than at 6.4 or 8.6 and greater at 37°C than at 22° or 4°C. Lowering the pH, reducing the protein or DTT concentrations, and preheating homogenates to 100°C all increased excess I− production but reduced iodothyronine production.
Michael M. Kaplan, Kimberlee A. Yaskoski
Using a laser diffraction technique, we have studied factors that influence the deformability of erythrocytes. Variations in suspending medium osmolality and applied shear stress were employed to isolate the individual contributions to whole cell deformability of internal viscosity, surface area-to-volume ratio, and viscoelastic properties of the membrane. An experimental system was devised in which normal cells were modified in vitro to induce specific alterations in each factor. Measurements of deformability as a function of medium osmolality showed characteristic behavior of the modified cells. Reduced surface area-to-volume ratio was detected by an exaggeration of the normal decrease in deformability as medium osmolality was decreased. In contrast, increased internal viscosity was detected by an increase in deformability as osmolality was decreased. Finally, decreased membrane flexibility was detected by reduced deformation at low shear stress. These methods of analysis were applied to cells from patients with hereditary spherocytosis, hereditary pyropoikilocytosis, and hemoglobin CC disease to define the basis of reduced deformability. Hereditary spherocytes showed the combined effects of reduced surface area and increased internal viscosity. Hereditary pyropoikilocytes revealed the effects of severely reduced surface area-to-volume ratio. Hemoglobin CC cells showed only the effects of high internal viscosity. An increase in the membrane shear modulus (decreased membrane deformability) was not evident in these disorders.
N Mohandas, M R Clark, M S Jacobs, S B Shohet
Isolated adipocytes and soleus muscles prepared from mature rats, rendered hypothyroid by a low iodine diet and propylthiouracil, markedly resisted the ability of insulin to increase glucose utilization. In adipocytes, the sum of basal d-(1-14C)-glucose conversion to CO2, glyceride-glycerol, and fatty acid was unaltered by hypothyroidism, although conversion to fatty acid was decreased. The response of each of these metabolic pathways to insulin at all concentrations tested was greatly diminished in hypothyroid rat adipocytes. 3-O-Methylglucose transport rates in the presence of insulin were not significantly different in adipocytes from hypothyroid as compared with euthyroid rats, although basal transport rates were significantly higher in the hypothyroid state. Lipolysis and cyclic AMP accumulation in adipocytes from hypothyroid rats in response to theophylline were markedly diminished compared with euthyroid controls, but insulin was about as effective in inhibiting lipolysis in these cells as in those derived from euthyroid animals. The binding of 125I-insulin to adipocytes at several hormone concentrations was also shown to be unaffected by hypothyroidism.
Michael P. Czech, Craig C. Malbon, Keith Kerman, Wendy Gitomer, Paul F. Pilch
Factor V was isolated from human plasma by barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B chromatography, ammonium sulfate fractionation, and gel chromatography on Ultrogel 22. Degradation of Factor V during purification was largely prevented by ample use of inhibitors of proteolytic enzyme. The purified Factor V was a stable, single-chain molecule with an apparent molecular weight of 330,000. Activation of human Factor V by thrombin resulted in a 10- to 15-fold increase in activity. The activation pattern as monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis was compared with that of bovine Factor V. Differences in the patterns of thrombin activation were noticed between the two species, whereas the final products were similar. The products of human Factor V activation are two closely spaced doublets, one with an apparent molecular weight of approximately 110,000, and the other, approximately 72,000. An antibody was raised against the purified protein. Crossed immunoelectrophoresis showed that the antibody recognized Factor V both before and after activation with thrombin.
Human synovial tissue cells in monolayer can be shown to take up and digest a soluble protein, horseradish peroxidase (HRP). Uptake of HRP was linear with increasing concentrations of substrate and cell protein and with time for up to 4 h. Low temperature (4 degrees C), and sodium fluoride, an inhibitor of glycolysis were the most effective metabolic inhibitors of endocytosis. In addition, colchicine, an inhibitor of microtubule assembly, and yeast mannan, an inhibitor of mannose-specific receptors, reduced HRP uptake. Synovial cells from patients with rheumatoid arthritis (RSC) demonstrated a statistically significantly higher rate of endocytosis (247 +/- 107 ng HRP/100 micrograms cell protein per 2 h.) than cells from control, nonrheumatoid patients (NSC) (100 +/- 80 ng HRP/100 micrograms cell protein per 2 h). Thus, it is possible to discriminate RSC from NSC by their quantitatively different rates of endocytosis. Digestion of HRP by synovial cells is statistically significant by 6 h after uptake. A faster initial rate of digestion was seen in RSC. Over the first 6--8 h of incubation 42% of the endocytosed HRP was still cell-associated in RSC and 67% remained in NSC cultures. However, by 24 h 20--30% of endocytosed HRP was found in both types of cultures. These results indicate that endocytosed molecules may accumulate more rapidly in RSC and persist within their lysosomes for a longer time than in NSC. The quantitative determination of enhanced endocytosis by RSC compared with NSC suggests that this increased activity may have a role in the pathological function of synovial tissue in rheumatoid arthritis.
K A Krakauer, R B Zurier
Chronic granulomatous disease (CGD), an often fatal syndrome of recurrent infections results from the inability of patients' peripheral blood phagocytic leukocytes to generate superoxide despite otherwise normal phagocytic functions such as ingestion and degranulation. Circulating granulocytes and monocytes are the progeny of bone marrow progenitor cells, colony-forming units in culture. We compared the function of cells grown in two different in vitro cuture systems from the bone marrow of a CGD patient with those from normal subjects. The cells of normal colony-forming unit in culture colonies grown in semisolid medium reduced nitroblue tetrazolium dye when stimulated by phorbol myristate acetate; none of the cells from colonies derived from CGD marrow did so. Cells grown in liquid suspension culture from normal marrow generated superoxide nearly as well as normal peripheral blood granulocytes; those from CGD marrow produced no superoxide, similarly cultured cells from both normal and CGD marrow ingested opsonized bacteria at rates equal to peripheral blood granulocytes. CGD marrow-derived cells showed increased exocytic degranulation relative to both normal marrow-derived cells and normal peripheral blood granulocytes. These studies demonstrate that the basic functional characteristics of CGD are embedded in the genetic program of granulocyte progenitors.
P E Newburger, M S Kruskall, J M Rappeport, S H Robinson, M E Chovaniec, H J Cohen
The role of calmodulin in insulin secretion from rat pancreatic islets has been examined by the use of trifluoperazine, an inhibitor of calmodulin-Ca++-directed functions. It was found that 30 microM trifluoperazine caused 50% inhibition, and 100 microM, up to 73% inhibition of 16.7 mM glucose-stimulated insulin release. 100 microM trifluoperazine caused a similar inhibition of 10 mM glyceraldehyde-stimulated release. Therefore, the site of action of trifluoperazine in glucose stimulus-secretion coupling appears to be after the trioses. As trifluoperazine had no effect upon insulin release stimulated by 1 mM 3-isobutyl-1-methylxanthine, the inhibitory effect of trifluoperazine appears to be rather specific. Further, the process of exocytosis per se is not affected. It was also found that although trifluoperazine inhibited the effect of glucose to stimulate insulin release, it did not affect the synergism between glucose and 3-isobutyl-1-methylxanthine to potentiate insulin release. It may be concluded that trifluoperazine selectively inhibits one part of the mechanism by which glucose stimulates insulin release. Calmodulin plays a role in the stimulation of insulin release by glucose at a site between metabolism of trioses and elevation of cytosol Ca++, but is not involved in the final process of exocytosis.
Y Krausz, C B Wollheim, E Siegel, G W Sharp
We have studied the effects of the oral sulfonylurea agent glyburide to modulate insulin receptors on nontransformed human fibroblasts in tissue culture. When glyburide was added to monolayers of human fibroblasts, a dose-dependent increase in the number of cell surface receptors was observed with a maximum effect (19% increase) seen at 1 microgram/ml glyburide. Insulin can induce a loss of insulin receptors in these cells, and when fibroblasts are exposed to 100 ng/ml insulin for 6 h, approximately 60% of the initial complement of cell surface receptors are lost. When the process of insulin-induced receptor loss (or down regulation) was studied in the presence of glyburide, the drug exerted a marked inhibitory effect on this regulatory process. Thus, glyburide inhibited insulin-induced receptor loss in a dose-dependent fashion, and the maximally effective drug concentration (1 microgram/ml) inhibited 34% of the receptor loss. These studies demonstrate a direct in vitro effect of this oral hypoglycemic agent to increase the number of cell surface insulin receptors or prevent their loss, presumably by slowing the rate of receptor internalization. These findings may explain the well known extrapancreatic effect of sulfonylurea agents to improve insulin-mediated tissue glucose metabolism.
M J Prince, J M Olefsky
In vivo androgen kinetics were determined in six young (21--49 yr) and elderly men (62-77 yr) with prostatge hyperplasia (BPH). Steady-state infusions of [14C]testosterone and [3H]androstanediol (3 alpha diol) were given, which allowed determination of the conversions testosterone leads to dihydrotesterone (DHT) in equilibrium or formed from 3 alpha diol. These infusions also yield metabolic clearance data which, together with meaurement of nonisotopic steroid levels, yield estimations of blood production rates. The production rate for testosterone was 6.04 +/- 1.66 vs. 3.69 +/- 0.62 mg/d, whereas the production rate for 3 alpha diol was 319 +/- 57 and 193 +/- 34 micrograms/d (P < 0.05 both groups). The irreversible conversion rate of testosterone to DHT was 3.1 +/- 0.4 and 3.5 +/- 0.9% (NS). The back conversion of 3 alpha diol to dHT was high (68 +/- 25 vs. 81 +/- 17, NS) indicating that 3 alpha diol might cause BPH as a result of conversion to DHT in vivo. The conversion of DHT to 3 alpha diol is reduced in the elderly group (15.8 +/- 2.6 and 6.3 +/- 1.4, P < 0.001). Since DHT formation in the prostate is a key event in the development of BPH and blood DHT appears to be a measure of extrasplanchnic sexual target tissue activity, our in vivo studies suggest that the tissue increase in DHT may result from reduced metabolism and the activity of 3 alpha-oxidoreduction favors the oxidative pathway in elderly men.
I Morimoto, A Edmiston, R Horton