To delineate the extent to which bone marrow transplantation provides "enzyme replacement therapy", we have determined metabolite concentrations in two patients with adenosine deaminase (ADA) deficiency treated with bone marrow transplants and rendered immunologically normal. 10 yr after engraftment of lymphoid cells, erythrocyte deoxy ATP was markedly decreased compared to the marked elevations of deoxy ATP observed in untreated patients, but was still significantly elevated (62 and 90 vs. normal of 6.0 +/- 6.0 nmol/ml packed erythrocytes). Similarly, deoxyadenosine and adenosine excretion were both markedly diminished compared to that of untreated patients but deoxyadenosine excretion was still clearly increased (20.1 and 38.6 vs. normal of less than 0.2 nmol/mg creatinine) while adenosine excretion was in the upper range of normal (7.0 and 8.1 vs. normal of 5.6 +/- 3.6 nmol/mg creatinine). Mononuclear cell deoxy ATP content was also elevated compared to normal (5.25 and 14.4 vs. 1.2 +/- 0.3). Separated mononuclear cells of bone marrow transplanted patients contain both donor lymphocytes and recipient monocytes. When mononuclear cells were depleted of the cells enriched for donor lymphocytes (i.e. monocyte depleted) was lower than that of the mixed mononuclear cells (2.2 vs. 5.26). Surprisingly, plasma adenosine was as high as in untreated ADA-deficient patients (3.2 and 1.5 vs. untreated of 0.3-3 microM). Consistent with the elevated plasma adenosine and urinary deoxyadenosine, erythrocyte S-adenosyl homocysteine hydrolase activity was diminished (0.88 and 1.02 vs. normal of 5.64 +/- 0.25). Thus, bone marrow transplantation of ADA-deficient patients not only provides lymphoid stem cells, but also partially, albeit incompletely, clears abnormally increased metabolites from nonlymphoid body compartments.
R Hirschhorn, V Roegner-Maniscalco, L Kuritsky, F S Rosen
Neutrophil-mediated endothelial injury was assessed in vitro using assays of cell lysis and cell detachment. Activation of human peripheral blood neutrophils adherent to human umbilical vein endothelial cell monolayers by serum-treated zymosan produced dose-dependent endothelial cell detachment without concomitant cell lysis. This injury was inhibited by neutral protease inhibitors, but not by catalase or superoxide dismutase. Neutrophils from a patient with chronic granulomatous disease also produced endothelial cell detachment when activated by serum-treated zymosan similar to normal neutrophils. Endothelial detachment was also produced by cell-free postsecretory media from activated neutrophils or by partially purified human neutrophil granule fraction and was inhibitable by tryptic, elastase, and serine protease inhibitors, but not by an acid protease inhibitor. Analysis of iodinated endothelial cell surface proteins that had been exposed to partially purified neutrophil granule fraction showed complete loss of proteins migrating in the region of fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This result was prevented in the presence of neutral protease inhibitors. We conclude that neutrophil-derived neutral proteases mediate endothelial cell detachment in vitro through digestion of endothelial cell surface proteins including fibronectin.
J M Harlan, P D Killen, L A Harker, G E Striker, D G Wright
Activity of the microsomal enzyme, steroid sulfatase, is absent in keratinocytes, fibroblasts, and leukocytes of patients with recessive x-linked ichthyosis. This study was undertaken to determine if cholesterol sulfate, a substrate of this enzyme, accumulates in the pathological scale of these patients. Scales from 8 patients with recessive x-linked ichthyosis, 10 patients with other forms of ichthyosis, and normal human outer stratum corneum were extracted with chloroform/water (1:2:0.8 by vol) and lipids were fractionated by quantitative, sequential thin-layer chromatography. Cholesterol sulfate was identified by cochromatography in several solvent systems, by its staining characteristics, by biochemical analysis, and by mass spectrometry. The mean cholesterol sulfate content of recessive x-linked ichthyotic scale was 12.5 +/- 0.8% of the total lipid, a fivefold increase over normal (P less than 0.0025), whereas the cholesterol sulfate content of other ichthyotic scale was normal. This increase in cholesterol sulfate content was accompanied by a decrease in total neutral lipids (P less than 0.0025) and free sterols (P less than 0.025) but no change in sterol esters or total sterols. These results demonstrate that deficiency of steroid sulfatase in recessive x-linked ichthyosis results in excessive accumulation of a substrate, cholesterol sulfate, in the pathologic scale, which may underly the pathogenesis of the scaling in this disorder. Measurement of cholesterol sulfate content in scale provides an alternative method to enzymatic assay for the diagnosis of this form of ichthyosis.
M L Williams, P M Elias
Experiments were performed on anesthetized opossums to study the influence of vagal efferent stimulation on peristalsis in the esophageal smooth muscle using various stimulus parameters. Current intensity, pulse duration, frequency, and train duration were varied systematically. Electrical and mechanical activities were recorded simultaneously at 5, 3, and 1 cm above the lower esophageal sphincter (LES). Vagal efferent stimulation produced a spike burst and contraction with a latency after the termination of the stimulus. This latency varied at different sites with the same stimulus parameters. For example, a stimulus of 5 mA, 0.5 ms, 10 Hz, and 1-s train produced latencies for the electrical response of 1.48 +/- 0.04, 2.2 +/- 0.12, and 3.5 +/- 0.09 s (+/- SEM) at 5, 3, and 1 cm above LES, respectively. The differences in latency were statistically significant (P less than 0.01). The latency of response at any one site also changed with different stimulus parameters; e.g. at 1 cm above LES, the latency of electrical response at 10 Hz was 3.5 +/- 0.09 s, but at 20 Hz the latency was 2.01 +/- 0.06 s when current intensity, pulse, and train duration remained at 5 mA, 0.5 ms, and 1 s. This decrease in latency with increasing frequency was statistically significant (P less than 0.01). By changing stimulus parameters, antiperistalsis or peristalsis with different speeds of propagation could be induced. Antiperistalsis or simultaneous responses occurred near threshold stimulus parameters. Suprathreshold stimuli produced peristaltic responses. Speed of peristalsis in the distal esophagus was 1.82 +/- 0.08 cm/s with swallowing, which was not different from 1.98 +/- 0.14 cm/s (P greater than 0.05) with vagal stimulation of 5 mA, 0.5 ms, 10 Hz, and 1-s train. These studies suggest that: (a) peristalsis in the smooth muscle part of the esophagus can be explained entirely on the basis of peripheral mechanisms, and (b) the central nervous system may modulate the occurrence, polarity, and speed of propagation by modifying the intensity and frequency of vagal activation.
J S Gidda, B W Cobb, R K Goyal
Factor IXChapel Hill (Factor IXCH), an abnormal Factor IX molecule isolated from the plasma of a patient with mild hemophilia B, has previously been shown to exhibit delayed activation by Factor XIa and calcium. In this study, we have found that Factor IXCH is cleaved upon incubation with human Factor XIa and calcium; however, cleavage of this protein is not observed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the rate of disappearance of the zymogen parallels both the appearance of the heavy chain and the generation of clotting activity. In addition, a protein band that migrates with an apparent molecular weight of 45,000 also increases in parallel with clotting activity. Factor IXCH and normal Factor IX (Factor IXN), after incubation with Factor XIa and calcium, were subjected to amino terminal sequence analysis. Activated Factor IXN is cleaved at an arginine-alanine (Arg-Ala) bond and an arginine-valine (Arg-Val) bond as demonstrated by formation of the three amino terminal sequences corresponding to the amino terminal of the light chain, heavy chain, and activation peptide. However, activated Factor IXCH has only two amino terminal sequences, corresponding to the original amino terminal sequence and the heavy chain (formed by cleavage at the Arg-Val bond). It is concluded that the major defect in Factor IXCH is the inability of Factor XIa to cleave the Arg-Ala bond at a significant rate. The rate of formation of clotting activity of Factor IXCH is approximately 60% of the rate of formation of clotting activity of Factor IXN. The specific clotting activity of activated Factor IXCH is between 20 and 33% of activated Factor IXN.
K M Braunstein, C M Noyes, M J Griffith, R L Lundblad, H R Roberts
Hemodynamic variables (blood pressure, cardiac output, heart rate, plasma volume, splanchnic blood flow, and peripheral subcutaneous blood flow) and plasma concentrations of norepinephrine, epinephrine, and renin were measured in the supine position and after 30 min of quiet standing. This was done in normal subjects (n = 7) and in juvenile-onset diabetic patients without neuropathy (n = 8), with slight neuropathy (decreased beat-to-beat variation in heart rate during hyperventilation) (n = 8), and with severe neuropathy including orthostatic hypotension (n = 7). Blood pressure decreased precipitously in the standing position in the diabetics with orthostatic hypotension, whereas moderate decreases were found in the other three groups. Upon standing, heart rate rose and cardiac output and plasma volume decreased similarly in the four groups. The increases in total peripheral resistance, splanchnic vascular resistance and subcutaneous vascular resistance were all significantly lower (P less than 0.025) in the patients with orthostatic hypotension compared with the other three groups. The increase in plasma norepinephrine concentrations in the patients with orthostatic hypotension was significantly lower (P less than 0.025) than in the patients without neuropathy, whereas plasma renin responses to standing were similar in the four groups. We conclude that in diabetic hypoadrenergic orthostatic hypotension the basic pathophysiological defect is lack of ability to increase vascular resistance, probably due to impaired sympathetic activity in the autonomic nerves innervating resistance vessels; cardiac output and plasma volume responses to standing are similar to those found in normal subjects and in diabetics without neuropathy.
J Hilsted, H H Parving, N J Christensen, J Benn, H Galbo
Serious illness is accompanied by markedly increased susceptibility to colonization of the respiratory tract by gram-negative bacilli and an increase in the number of such organisms which adhere to regional epithelial cells during incubation in vitro. Trypsinization of cells from normal subjects causes a similar increase in bacillary adherence. We studied bacillary adherence to buccal cells in vitro, protease activity of upper respiratory secretions with a fibrin plate technique, and the amount of fibronectin on the surface of buccal cells with a direct radioimmunobinding assay. Among 10 patients seriously ill with acute respiratory failure bacillary adherence to buccal cells and protease activity in secretions were increased compared with controls and cell-surface fibronectin was decreased; all patients were colonized in vivo with gram-negative bacilli. These changes were persistent and 80% of the patients died. Serial determinations were made in eight patients undergoing coronary artery bypass surgery. Following surgery, protease activity and bacillary adherence increased and cell-surface fibronectin decreased; 38% of coronary artery bypass patients became colonized. In these uncomplicated patients the changes observed were transient, largely returning to normal by the third postoperative day. Increased protease activity of secretions and alterations in epithelial cell surfaces as reflected by loss of buccal cell-surface fibronectin occur swiftly after major illness and appear to underlie enhanced cell adherence of bacilli and colonization of the upper respiratory tract. These findings suggest new approaches to the prevention of nosocomial pneumonia.
D E Woods, D C Straus, W G Johanson Jr, J A Bass
The incorporation of labeled compounds into neurophysins of a transplantable human oat cell carcinoma of the lung with ectopic vasopressin production was studied in vitro. Neurophysins in cell extracts and in incubation media were isolated by immunoprecipitation and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When cells were incubated with L-[35S]cysteine for 12 h, SDS-polyacrylamide gel electrophoresis of the immunoprecipitates from cell extract and medium resolved two forms of neurophysins with apparent molecular mass of 10,000 (10K) and 20,000 (20K). Both forms of [35S]-neurophysins were completely displaced from the immunoprecipitates by excess human neurophysin. Incubation of cells with L-[35S]cysteine and D-[3H]-glucosamine hydrochloride revealed that glucosamine was incorporated into the 20K neurophysin region, but not into 10K species. To observe the kinetics of labeling of the two forms of neurophysins, cells were incubated with L[35S]cysteine for varying periods of time. After short labeling periods, most of the radioactivity resided in 20K species, which plateaued after 1 h, whereas 10K neurophysin progressively increased in its height. When cells were chased with unlabeled cysteine after the exposure to a short pulse of labeling, 20K neurophysin peak gradually decreased with an apparent initial half-life of 1 h. In contrast, the label in 10K neurophysin steadily increased, which exceeded the former by 3 h of chase. Analysis of 20K neurophysin in cell extract by isoelectric focusing on polyacrylamide gel demonstrated that it was principally composed of a protein with an apparent isoelectric point (pI) of 5.7. These results suggest that neurophysin is synthesized in ectopic vasopressin-producing tumors by post-translational processing from a glycosylated proneurophysin with an apparent molecular mass of 20,000 daltons and a pI of 5.7.
T Yamaji, M Ishibashi, S Katayama, A Itabashi, N Ohsawa, Y Kondo, Y Mizumoto, K Kosaka
We have investigated alterations in beta adrenergic receptor binding sites of rat reticulocytes occurring in animals rendered hypothyroid by thyroidectomy. Beta adrenergic receptor interactions were assessed by measuring the number of (-)[3H]-dihydroalprenolol binding sites and the ability of an agonist to compete for occupancy of the receptors. The number of receptors was significantly reduced in cells from the hypothyroid animals. In addition, there were significant agonist-specific alterations in binding. Using computer assisted curve fitting techniques, it was found that the ability of (-)isoproterenol to stabilize a high affinity guanine nucleotide sensitive "coupled" form of the receptor was impaired. Reticulocytes from hypothyroid animals have, in addition, a reduction in the concentration of the nucleotide regulatory protein as assessed by the number of 42,000 Mr substrates for cholera toxin catalyzed ADP ribosylation. These alterations are associated with reductions in catecholamine and NaF stimulated adenylate cyclase activity. Diminished coupling of beta adrenergic receptors with other regulatory components of the adenylate cyclase system represents a mechanism by which altered thyroid states modulate beta adrenergic receptor function and beta adrenergic responsiveness of tissues.
G L Stiles, J M Stadel, A De Lean, R J Lefkowitz
The quantitative contributions of pancreatic responsiveness and insulin sensitivity to glucose tolerance were measured using the "minimal modeling technique" in 18 lean and obese subjects (88-206% ideal body wt). The individual contributions of insulin secretion and action were measured by interpreting the dynamics of plasma glucose and insulin during the intravenous glucose tolerance test in terms of two mathematical models. One, the insulin kinetics model, yields parameters of first-phase (phi 1) and second-phase (phi 2) responsivity of the beta-cells to glucose. The other glucose kinetics model yields the insulin sensitivity parameters, SI. Lean and obese subjects were subdivided into good (KG greater than 1.5) and lower (KG less than 1.5) glucose tolerance groups. The etiology of lower glucose tolerance was entirely different in lean and obese subjects. Lean, lower tolerance was related to pancreatic insufficiency (phi 2 77% lower than in good tolerance controls [P less than 0.03]), but insulin sensitivity was normal (P greater than 0.5). In contrast, obese lower tolerance was entirely due to insulin resistance (SI diminished 60% [P less than 0.01]); pancreatic responsiveness was not different from lean, good tolerance controls (phi 1: P greater than 0.06; phi 2: P greater than 0.40). Subjects (regardless of weight) could be segregated into good and lower tolerance by the product of second-phase beta-cell responsivity and insulin sensitivity (phi 2 . SI). Thus, these two factors were primarily responsible for overall determination of glucose tolerance. The effect of phi 1 was to modulate the KG value within those groups whose overall tolerance was determined by phi 2 . SI. This phi 1 modulating influence was more pronounced among insulin sensitive (phi 1 vs. KG, r = 0.79) than insulin resistant (obese, low tolerance; phi 1 vs. KG, r = 0.91) subjects. This study demonstrates the feasibility of the minimal model technique to determine the etiology of impaired glucose tolerance.
R N Bergman, L S Phillips, C Cobelli
The interaction of exercise and insulin on glucose metabolism was examined in 10 healthy volunteers. Four study protocols were used: study 1: plasma insulin was raised by approximately 100 microunits/ml while plasma glucose was maintained at basal levels for 2 h (insulin clamp). Study 2: subjects performed 30 min of bicycle exercise at 40% of VO2 max. Study 3: an insulin clamp was performed as per study 1. Following 60 min of sustained hyperinsulinemia, however, subjects exercised for 30 min as per study 2. Study 4: subjects were studied as per study 3 except that catheters were inserted into the femoral artery and vein to quantitate leg glucose uptake. During the 60-90 min period of hyperinsulinemia (study 1), glucose uptake averaged 8.73 +/- 0.10 mg/kg per min. With exercise alone (study 2), the increment in peripheral glucose uptake was 1.43 +/- 0.30 mg/kg per min. When hyperinsulinemia and exercise were combined (study 3), glucose uptake averaged 15.06 +/- 0.98 mg/kg per min (P less than 0.01) and this was significantly (P less than 0.001) greater than the sum of glucose uptake when exercise and the insulin clamp were performed separately. The magnitude of rise in glucose uptake correlated closely with the increase in leg blood flow (r = 0.935, P less than 0.001), suggesting that the synergism is the result of increased blood flow and increased capillary surface area to exercising muscle. More than 85% of total body glucose metabolism during studies 1 and 3 was accounted for by skeletal muscle uptake. These results demonstrate that (a) insulin and exercise act synergistically to enhance glucose disposal in man, and (b) muscle is the primary tissue responsible for the increase in glucose metabolism following hyperinsulinemia and exercise.
R A DeFronzo, E Ferrannini, Y Sato, P Felig, J Wahren
Recent studies have shown that effective pulmonary ventilation is possible with tidal volumes (VT) less than the anatomic dead-space if the oscillatory frequency (f) is sufficiently large. We systematically studied the effect on pulmonary CO2 elimination (VCO2) of varying f (2-30 Hz) and VT (1-7 ml/kg) as well as lung volume (VL) in 13 anesthetized, paralyzed dogs in order to examine the contribution of those variables that are thought to be important in determining gas exchange by high frequency ventilation. All experiments were performed when the alveolar PCO2 was 40 +/- 1.5 mm Hg. In all studies, VCO2 increased monotonically with f at constant VT. We quantitated the effects of f and VT on VCO2 by using the dimensionless equation VCO2/VOSC = a(VT/VTo)b(f/fo)c where: VOSC = f X VT, VTo = mean VT, fo = mean f and a, b, c, are constants obtained by multiple regression. The mean values of a, b, and c for all dogs were 2.12 X 10(-3), 0.49, and 0.08, respectively. The most important variable in determining VCO2 was VOSC; however, there was considerable variability among dogs in the independent effect of VT and f on VCO2, with a doubling of VT at a constant VOSC causing changes in VCO2 ranging from -13 to +110% (mean = +35%). Increasing VL from functional residual capacity (FRC) to the lung volume at an airway opening minus body surface pressure of 25 cm H2O had no significant effect on VCO2.
A S Slutsky, R D Kamm, T H Rossing, S H Loring, J Lehr, A H Shapiro, R H Ingram Jr, J M Drazen
We have stimulated in a cultured hepatocyte system the synergistic interaction between triiodothyronine (T3) and dietary carbohydrate in the induction of malic enzyme (ME). Kinetic studies revealed that isolated hepatocytes equilibrate with media T3 within 5 min; nuclei equilibrate with media T3 by 45 min; and the half-time of T3 metabolism was 10 h in 10% serum. We demonstrated nuclear T3 receptors in isolated hepatocytes and the induction of ME by T3 in physiological concentrations. However, in the complete absence of T3 glucose could still induce ME. At all concentrations of glucose (100-1,000 mg/dl), T3 (0.3 nM free T3) resulted in a relatively constant (1.4- to 1.7-fold) increase in ME response. The augmentation in ME activity was paralleled by an enhanced rate of enzyme synthesis as determined by [3H]leucine incorporation into immunoprecipitable ME. Cells cultured in serum free media also demonstrated a glucose-dependent increase in ME. Insulin greatly stimulated the glucose induction of ME, whereas dexamethasone had very little influence on ME induction. These studies demonstrate the usefulness of an adult hepatocyte tissue culture model for the study of the effects of T3 on gene expression in cells that are not derived from tumor. They clearly demonstrate that well established effects of T3 can be simulated in such a system at levels of free hormone that approximate those in extracellular body fluids. Our results indicate that an increased concentration of glucose per se can induce the formation of ME in the absence of alterations in extrahepatic hormones or factors. Moreover, our findings confirm inferences from in vivo studies that T3 acts as a multiplier of a glucose-induced signal.
C N Mariash, C R McSwigan, H C Towle, H L Schwartz, J H Oppenheimer
Biotin-responsive multiple carboxylase deficiency is an inherited disorder of organic acid metabolism in man in which there are deficiencies of propionyl-coenzyme A (CoA), 3-methylcrotonyl-CoA, and pyruvate carboxylases that can be corrected with large doses of biotin. It has been proposed that the basic defect in patients with the early infantile form of the disease is in holocarboxylase synthetase, the enzyme that covalently attaches biotin to the inactive apocarboxylases to form active holocarboxylases. We have developed an assay for holocarboxylase synthetase in extracts of human fibroblasts using as substrate apopropionyl-CoA carboxylase partially purified from livers of biotin-deficient rats. Fibroblasts from the initial patient with the infantile form of biotin-responsive multiple carboxylase deficiency were shown to have abnormal holocarboxylase synthetase activity with a maximum velocity about 30-40% of normal, a Km for ATP of 0.3 mM similar to the normal Km of 0.2 mM, and a highly elevated Km for biotin of 126 ng/ml, about 60 times the normal Km of 2 ng/ml. These results show that the primary defect in this patient is a mutation affecting holocarboxylase synthetase activity, and thus a genetic defect of the metabolism of biotin.
B J Burri, L Sweetman, W L Nyhan
We tested the hypothesis that cyclic AMP plays a significant role in modulating the growth of embryonic chick cartilage by determining whether cyclic AMP levels change in growing embryonic cartilage and whether cyclic AMP could stimulate embryonic cartilage growth in a long term in vitro organ culture. Cyclic AMP levels were low (0.1 pmol/mg wet wt) in 8-d chick embryo pelvic cartilage, and increased progressively through the 11th d of embryonic development at which time they reached a maximum (1.8 pmol/mg wet weight) and thereafter remained constant. We developed an in vitro organ culture system to determine whether cyclic AMP, a factor known to stimulate radiolabeled precursor incorporation into macromolecules in short-term studies does, in fact, stimulate growth of cartilage. Individual pelvic cartilages were isolated from 9-d chick embryos, placed in serum-free medium (BGJb-FJ modification) and incubated for 3 to 5 d during which time they increased in size (39 and 60% in length, respectively), wet weight (90 and 141%, respectively), and content of total soluble protein (30 and 48%, respectively). N6-monobutyryl cyclic AMP (BtcAMP) added to the medium caused a dose-dependent (0.05 to 1.0 mM) stimulation of growth. After 3 d of incubation, 1.0 mM BtcAMP increased wet weight (125%), [14C]leucine incorporation into protein (75%), and [3H]thymidine incorporation into DNA (48%) compared with control cartilages incubated in medium alone. 1-methyl-3-isobutyl xanthine, a phosphodiesterase inhibitor, also increased cartilage growth above control while sodium butyrate, AMP, and ATP had no effect. Histological examination of cartilage grown in medium was similar to that of cartilage developing in ovo, whereas, cartilage grown in medium containing BtcAMP showed marked hypercellularity with many immature chondrocytes. Our observations are compatible with the hypothesis that cyclic AMP can significantly modulate the growth of embryonic cartilage.
W M Burch, H E Lebovitz
When gram-negative bacterial lipopolysaccharides (LPS) are injected intravenously into the rabbit or rat, they bind to plasma lipoproteins, particularly high density lipoproteins (HDL). The present studies were performed to examine the mechanisms by which LPS-HDL complexes are removed from the circulation and taken up by various tissues. Our approach was to compare the sites of specific tissue binding and uptake of HDL and of LPS-HDL complexes in the rat and squirrel monkey. In the rat, binding of homologous 125I-HDL was demonstrated principally in the adrenal gland, ovary, liver, and spleen. [3H]LPS-HDL complexes (produced in vitro by incubating Salmonella typhimurium [3H]LPS with rat HDL and lipoprotein-free plasma) bound to the same tissues, but with apparently lower affinities. The specificity of binding of both 125I-HDL and [3H]LPS-HDL to these organs was demonstrated in two ways. First, tissue binding of both radiolabeled preparations was swamped out by raising the circulating levels of HDL-cholesterol from 32 to 140 mg/dl. Second, treatment of the animals with dexamethasone abolished specific binding of both HDL preparations to the adrenal gland while administration of adrenocorticotropin increased the specific adrenal binding of the two preparations. The steady-state plasma clearance rate for 125I-HDL equaled 774 +/- 29 microliters/h and was significantly lower (557 +/- 39 microliters/h) for the LPS-HDL complex, a finding that presumably reflected the lesser ability of the various tissues to bind the LPS-HDL complex. Binding studies were also done in the squirrel monkey, an animal that has the same level of circulating HDL cholesterol as the rat, but nearly three times more cholesterol in low density lipoproteins. Specific binding of homologous 125I-HDL and [3H]LPS-HDL was again found principally in the adrenal gland and liver. The results indicate that the sites of tissue uptake of bacterial LPS are strongly influenced by binding of LPS to HDL. In particular, LPS-HDL binding may be an important determinant of the extent to which LPS are taken up by the adrenal gland during bacterial sepsis.
R S Munford, J M Andersen, J M Dietschy
The possibility that mineralocorticoids have a direct influence on renal Na-K ATPase activity has been the focus of intense research effort and some controversy for a number of years. Early studies were hindered by an inability to differentiate between possible glucocorticoid vs. mineralocorticoid effects on this enzyme within the multitude of cells that comprise the heterogeneous mammalian nephron. This study attempts to circumvent this problem by monitoring Na-K ATPase activity in the rabbit renal cortical collecting tubule (CCT), a proposed target epithelium for mineralocorticoids. Using an ultramicro assay, Na-K ATPase activity was measured in CCT from normal, adrenalectomized (adx), and adx rabbits subjected to one of several corticosteroid treatment protocols. The results indicate that Na-K ATPase activity in the CCT decreased by 86% subsequent to adrenalectomy. Injection of physiological doses of aldosterone (10 micrograms/kg) but not dexamethasone (100 micrograms/kg) restored CCT Na-K ATPase activity in adx rabbits to normal levels within 3 h after injection. An insignificant rise in activity was observed 1.5h after aldosterone treatment. In addition, spirolactone SC 26304, a specific mineralocorticoid antagonist, blocked the action of aldosterone on Na-K ATPase.. Therefore an acute increase in Na-K ATPase activity participates in the action of aldosterone on Na transport in this segment. To differentiate between primary vs. secondary activation of this enzyme, adx animals were treated with amiloride before the injection of aldosterone with the intent of blocking luminal membrane Na entry into CCT. In these animals, pretreatment with amiloride blocked the increase in CCT Na-K ATPase act activity seen with aldosterone alone at 3 h. Thus the increase in activity with aldosterone appears to be a secondary adaptation that is dependent on an aldosterone-enhanced increase in the passive entry of Na across the luminal membrane. The subcellular mechanism by which Na modulates Na-K ATPase activity remains obscure.
K J Petty, J P Kokko, D Marver
To assess the consequences of elevated branched chain amino acid levels on alanine, glutamine, and ammonia metabolism in muscle, L-leucine meals (14.7 g) were consumed by six normal postabsorptive individuals. Bilateral forearm studies were performed, and the dominant arm was subjected to 15 min of light exercise, using a calibrated dynamometer, beginning 45 min after the ingestion of the meal. Large uptakes of leucine were seen across both forearm muscle beds within 30 min of the meal. After exercise, blood flow in the dominant arm increased from 3.1 +/- 0.4 to 5.2 +/- 0.9 ml/100 ml forearm per minute (mean +/- SEM, P less than 0.005). Glutamine flux out of the dominant forearm increased threefold after the ingestion of the leucine meal and increased eightfold over base line after exercise. Less marked changes (significant only at 90 min) in the nonexercised, nondominant arm were also seen. Alanine flux out of the dominant forearm muscle bed increased modestly at 75 and 90 min. No significant change in ammonia flux across either forearm muscle bed was noted. Unexpectedly, large and significant net nitrogen loss from both forearm muscle beds was documented. Thus, following the ingestion of a leucine meal and light exercise, the primary means by which excess nitrogen is routed out of muscle is via glutamine formation and release with alanine and ammonia pathways playing relatively minor roles. More importantly, the ingestion of significant amounts of leucine by normal subjects, presumably in optimal nitrogen balance, results in a net loss of nitrogen from muscle.
T T Aoki, M F Brennan, G F Fitzpatrick, D C Knight
The production of beta-globin messenger RNA (mRNA) in beta-thalassemic erythroblasts was studied during pulse-chase incubations with [3H]uridine. Globin [3H]mRNA was quantitated by molecular hybridization to recombinant DNA probes complementary to globin mRNA and mRNA precursor sequences. Each of six patients with beta +-thalassemia produced normal amounts of globin alpha and beta [3H]mRNA during a 20-min pulse incubation, but the beta/alpha [3H]mRNA ratio declined to steady-state levels during a chase incubation, suggesting posttranscriptional defects in beta-globin mRNA metabolism. beta-globin mRNA precursor production was estimated by measurement of [3H]RNA sequences hybridizing to a pure DNA probe containing only the large intervening sequence (intron) of the beta-mRNA precursor. Four of the patients exhibited abnormal accumulation of 3H-beta-intron sequences (2-10 times normal), indicating abnormal posttranscriptional processing. In the remaining two patients, one of whom is known to carry a mutation in the small intron of the beta-globin gene, accumulation of large 3H beta-intron RNA and beta-globin [3H]mRNA was normal in nuclei, but the ratio of beta/alpha [3H]mRNA in cytoplasm was reduced, suggesting a different posttranscriptional defect in beta-mRNA processing. These findings imply the existence of heterogeneous posttranscriptional abnormalities in beta-globin mRNA metabolism in different patients with beta-thalassemia. The initial rates of gamma- and delta-mRNA synthesis were low in all patients, suggesting that the low level of expression of these genes in adults is mediated at the transcriptional level.
E J Benz Jr, A L Scarpa, B L Tonkonow, H A Pearson, A K Ritchey
The direct effects of steroid hormones on the production of immunoglobulins and DNA synthesis by human T and B lymphocytes was evaluated in cultures of peripheral blood mononuclear cells. As detected by a reverse hemolytic plaque assay, the addition of 0.1 mM to 10 nM hydrocortisone to lymphocytes in culture in the absence of other stimulants or mitogens, resulted in the dramatic induction of immunoglobulin production with responses comparable to those seen in similar cultures stimulated with pokeweed mitogen. Steroid-stimulated immunoglobulin production was first seen after 48 h and peaked at 8-10 d of culture. The production of IgG, IgA, and IgM was induced following incubation with steroid. Glucocorticoids, but not estrogens or androgens, were capable of mediating this effect, and only compounds with affinity for the glucocorticoid receptor were active. The induction of immunoglobulin production was dependent on both T cells and monocytes; cultures depleted of either cell type did not produce immunoglobulin when stimulated with glucocorticoid hormones. Proliferation of B cells or T cells could not be detected by [3H]thymidine incorporation or total cell recovery from steroid-stimulated cultures, even though such cultures demonstrated marked increases in immunoglobulin production. The mechanism responsible for this functional maturation of B cells to become high rate immunoglobulin producing cells is as yet undefined, although it appears to involve more than merely steroid mediated inactivation of suppressor T cells.
J Grayson, N J Dooley, I R Koski, R M Blaese
A highly significant, but unanswered, question in the pathogenesis of psoriasis relates to how normal appearing and diseased skin can coexist, undergo spontaneous flares and remissions, and yet appear to be genetically acquired. A plausible explanation for these disparate observations is that there is a basic defect in epidermal proliferation of skin of subjects with psoriasis and that disease expression is governed by other host factors. To address this question, we compared epidermal proliferation of skin involved and uninvolved with psoriasis with normal skin before and after transplantation to congenitally athymic (nude) mice, a biologic milieu free of humoral factors unique to the donor host. Results demonstrated that (a) before transplant, synthesis of DNA by the epidermal cells from skin uninvolved and involved with psoriasis is significantly higher than normal, 1.6 and 3.6 times, respectively; (b) 6 wk after transplantation, synthesis of DNA by epidermal cells is unchanged for normal skin, increased for uninvolved skin, and decreased for involved skin. These increases and decreases are of such a magnitude that at 6 wk the number of epidermal cells synthesizing DNA per 1,000 basal cells is identical, and is 2.2 times that of normal skin. When removed from the milieu of the afflicted host, skin involved and uninvolved with psoriasis appear equally "diseased." These data support the notion that there is aberrant epidermal proliferation in skin of patients with psoriasis and that host factors appear to play a role both in the expression and nonexpression of this disease.
G G Krueger, D A Chambers, J Shelby
We isolated and partially characterized three Fc-binding macromolecules from human leukocytes. Mononuclear cells from normal individuals and from five patients with chronic lymphocytic leukemia and neutrophils from normal donors were surface radiolabeled by using 125I and lactoperoxidase. After detergent solubilization of the cells, Fc gamma-binding macromolecules were purified by repetitive affinity chromatography under a variety of conditions and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Three radiolabeled macromolecules were isolated that retained specific ability to bind to Fc fragments. A 52,000-64,000-mol wt macromolecule was isolated from normal mononuclear and polymorphonuclear cells. A 43,000-mol wt band was characteristic of mononuclear cells, particularly from patients with chronic lymphocytic leukemia. A 33,000-mol wt molecule could be obtained from normal leukocytes under conditions that suggest it might be a proteolytic fragment.
A Kulczycki Jr, L Solanki, L Cohen
Patients from two families with chronic hemolytic anemia have been studied. The erythrocytes are very fragile and appear microcytic with a great variety of shapes. Clinical evaluation failed to identify traditionally recognized causes of hemolysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed no significant abnormality of the major polypeptide bands. Erythrocytes spectrin-ankyrin and ankyrin-membrane interactions were analyzed with 125I-labeled spectrin, 125I-labeled ankyrin, and inside-out vesicles. Patients' vesicles bound 125I-spectrin normally. Likewise, patients' spectrin and ankyrin competed normally for the binding sites on control membranes. None of the individual components appeared to have abnormal thermal sensitivity. Ankyrin-stripped, inside-out vesicles prepared from the patients bound less 125I-ankyrin than did vesicles prepared from normals (P less than 0.05 for all corresponding points in the high-affinity region). Scatchard analysis showed the most significant abnormality to be a 50% reduction in the high affinity ankyrin binding sites. Similar experiments were performed with blood from patients with spherocytosis and splenectomized controls, but no abnormalities were detected. The water soluble 43,000-dalton fragments of band 3 (the high-affinity ankyrin binding sites) were prepared from one of the patients and competed normally for 125I-ankyrin binding in solution. This suggests that the primary structural defect is a reduction in the number of high affinity membrane binding sites for ankyrin, and is consistent with an abnormal organization of band 3 in the membrane.
P Agre, E P Orringer, D H Chui, V Bennett
Newborn mononuclear cells are known to have increased suppressor activity when compared with adult cells. However, the precise phenotypic description of the suppressor cell subpopulation has not yet been reported. This study was designed to examine the surface markers on human cord blood cells as defined by monoclonal antibodies and to characterize cord blood mononuclear cells that suppress in vitro pokeweed mitogen-driven immunoglobulin production. The pattern of fluorescence with the 9,6-monoclonal antibody suggested either a decreased density or a partial blocking of E-receptors on cord blood lymphocytes. Otherwise, human cord and adult cells had similar proportions of T cell subpopulations. Different subsets of newborn cells isolated by a monoclonal antibody rosetting technique were tested for suppressor activity. Cord blood lymphocytes recognized by the OKT8 monoclonal antibody constituted the major functional suppressor cell subpopulation. These cells were present in both E-rosetting and non-E-rosetting populations. Removal of OKT8+ mononuclear cord blood cells abrogated most of the suppressor activity. Thus the major suppressor cell present in cord blood is an OKT8 positive lymphocyte.
M A Rodriguez, A D Bankhurst, J L Ceuppens, R C Williams Jr
Washed rabbit platelets stimulated with platelet-activating factor, thrombin, or arachidonic acid, released a slow-reacting substance (SRS), whereas platelets aggregated by adenosine diphosphate did not. Production of platelet-derived SRS was neither affected by indomethacin nor aspirin but was reduced by large doses of eicosatetraynoic acid, an inhibitor of the cyclo-oxygenase and lipoxygenase. L-cysteine enhanced markedly the release of SRS from platelets. This SRS activity, which was antagonized by FPL 55712 and inactivated by arylsulfatase, followed the same elution pattern on Amberlite, silicic acid, and reverse phase high-pressure liquid chromatography columns as that described for the SRS from other origins. SRS activity released from platelets preincubated with [14C]arachidonic acid exhibited the same retention time as radioactivity in reverse phase high-pressure liquid chromatography. The release of a SRS from platelets is consistent with their implication in the pathogenesis of asthma and other lung diseases.
J M Mencia-Huerta, L Hadji, J Benveniste
Apolipoprotein (apo)C-III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 microM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 microM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine three- to fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of 5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.
A J Alpert, A L Beaudet
T lymphocyte-mediated bone marrow failure is a recently recognized clinical disorder, but the T lymphocyte subsets responsible for mediating the inhibitory effect have not been identified. We obtained T lymphocytes from the bone marrow of two patients with T lymphocyte-mediated granulopoietic failure, exposed them to monoclonal antibodies (OKT3, OKT4, and OKT8) in cytotoxicity assays, then recombined the treated T cells with autologous T-depleted marrow cells in clonal assays for granulocyte/macrophage progenitors (CFU-GM). Treatment of T cells with OKT3 and OKT8 abrogated their granulopoietic inhibitory effect but treatment with OKT4 did not. Therefore, in these two patients, the lymphocytes that played a role in the inhibition of granulopoiesis were of the cytotoxic/suppressor subset.
G C Bagby Jr
Isolated human T4+ cells proliferate in the autologous mixed lymphocyte reaction (AMLR), whereas isolated T8+ cells do not. However, in the presence of Interleukin 2 or T4+ cells, the T8+ cells demonstrated substantial proliferation. These studies suggest that T8+ cells recognize signals from autologous non-T cells, but require an additional factor for the subsequent proliferative response. Since this stimulus can be provided by T4+ cells, the AMLR appears to constitute an inducer circuit. Different defects in this circuit may be responsible for the common abnormality of the AMLR in different diseases.
J S Smolen, T A Luger, T M Chused, A D Steinberg
Diabetes mellitus in pregnancy is associated with neonatal respiratory distress syndrome due to impaired synthesis of fetal lung surfactant. Pharmacologic agents that promote fetal lung maturity are diabetogenic and have limited use in the management of diabetic pregnancy for prevention of respiratory distress syndrome. Maternal administration of a thyroid analog 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT) results in significant enhancement of fetal lung phospholipid synthesis and accelerated lung maturity. We therefore studied the effects of DIMIT (0.5 mg/kg per d, s.c.) administration to pregnant alloxan-diabetic rabbits on days 25 and 26 of gestation. DIMIT treatment of diabetic maternal rabbits (DD) was associated with reduction of maternal blood glucose (115 +/- 13 vs. 275 +/- 72 mg/dl, P less than 0.05) and fetal glucose (64 +/- 6 vs. 274 +/- 47 mg/dl, P less than 0.001) compared with saline-injected diabetic (D) mothers. Reduction of fetal insulin levels was also associated with maternal DIMIT therapy in diabetic rabbits (56 +/- 5 (D) vs. 24 +/- 4 microunits/ml, P less than 0.001). Maternal diabetes resulted in significant reduction of fetal lung weight (370 +/- 20 vs. 520 +/- 30 mg, P less than 0.005) and lung protein content (6.5 +/- 0.7 vs. 8.7 +/- 0.7 mg/gm, P less than 0.005), which were restored to normal in offspring of DIMIT-treated diabetic rabbits. Maternal DIMIT administration caused significant reduction in fetal lung glycogen content in control (62 +/- 5.8 vs. 25 +/- 5.9 micrograms/mg protein, P less than 0.001) and diabetic (56 +/- 7 vs. 34 +/- 5 micrograms/mg protein, P less than 0.02) offspring. Whereas maternal diabetes was associated with reduction of all major phospholipid species in fetal lung-comprising surfactant, these were restored with DIMIT therapy. The results demonstrate that short-term maternal administration of DIMIT in pregnant diabetic rabbits not only promotes fetal lung phospholipid synthesis, but also appears to ameliorate maternal hyperglycemia. Thus, DIMIT is of potential benefit in the management of diabetic pregnancy.
N Neufeld, S Melmed
A solid-phase radioimmunoassay was developed to detect immunoglobulin (Ig)E antibodies that bound to human IgG. IgE-rheumatoid factor activity was found in the serum of 18 of 20 patients with seropositive rheumatoid arthritis, 1 of 4 patients with seronegative rheumatoid arthritis, 3 of 32 patients with seronegative rheumatoid arthritis, 3 of 32 patients with asthma, and in 1 patient with hypocomplementemic vasculitis and iodide sensitivity. Immunopathologic implications of IgE-rheumatoid factor are discussed.
B L Zuraw, C H O'Hair, J H Vaughan, D A Mathison, J G Curd, D H Katz