Penicilloyl groups, which have been connected to penicillin allergy, are derived from penicillin by cleavage of the beta lactam ring and bind covalently to proteins. Fixation of penicilloyl groups was studied in seven patients given large amounts of penicillin. Penicilloyl groups were found essentially on the albumin molecule at sites not accessible to anti-penicilloyl antibodies, except after pronase digestion. The amount of penicilloyl groups was proportional to the cumulated doses of penicillin. The decline of penicilloyl groups with time after treatment interruption was exponential. The half-life of penicilloylated albumin was lower than or equal to that of normal albumin. The presence of anti-penicilloyl antibodies was demonstrated in 19 out of 34 penicillin-treated patients (including the seven mentioned above). The relative scarcity of penicillin allergy as compared with the frequent occurrence of anti-penicilloyl antibodies may be partly related to unavailable sites of penicilloyl groups within the albumin molecule.
P Lafaye, C Lapresle
1.0 micrograms/kg body wt human corticotropin-releasing factor (hCRF) and 0.005 IU/kg body wt lysine vasopressin (LVP) were administered in a bolus dose to patients receiving daily or alternate-day glucocorticoid therapy. In normal subjects with this hCRF-LVP test, the plasma ACTH increment was significantly greater (approximately 2.5-fold) 15 min after injection than under the CRF test. In patients receiving daily glucocorticoid therapy (greater than 15 mg prednisolone or an equivalent daily dose), the plasma ACTH and cortisol responses to hCRF-LVP were suppressed 2 wk to 1 mo after the beginning of glucocorticoid administration but partially improved at 2-10 mo, and was markedly suppressed several years later. On the other hand, in patients receiving alternate-day glucocorticoid therapy, the plasma ACTH response was normal at 2 wk, normal or higher at 1-3 mo, and normal after 4 mo. A normal plasma cortisol response was observed throughout the test period in patients receiving alternate-day therapy after pulse therapy, whereas plasma cortisol response was gradually improved in patients receiving alternate-day therapy after several months of daily therapy.
K Hashimoto, S Suemaru, T Takao, M Sugawara, T Hattori, J Kageyama, K Takahashi, Z Ota
Histamine-releasing factors (HRFs) have been shown to be released from a variety of human cells, including T lymphocytes and alveolar macrophages. We considered the possibility that known cytokines might possess such activity on human basophils and/or mast cells and therefore tested preparations of human recombinant IL 3, IL 4, IL 5, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) upon a panel of basophil donors. IL 3 and GM-CSF possessed significant histamine-releasing activity in 8 of 10 and 12 of 14 subjects, respectively. In each instance, a dose response could be demonstrated. IL 4 and G-CSF had no such activity, whereas IL 5 had activity in only 2 of 14 donors tested. We conclude that IL 3 and GM-CSF represent two effective HRFs, and suggest that HRF, as isolated based upon histamine-releasing activity, is likely to be heterogeneous in terms of molecular identity. Whether previously described HRFs relate specifically to IL 3 or GM-CSF must await primary sequence analysis of HRF and/or studies with monospecific antisera.
M Haak-Frendscho, N Arai, K Arai, M L Baeza, A Finn, A P Kaplan
The changes in hepatic glucose production (Ra), tissue glucose disposal (Rd), and plasma glucose and insulin concentration that took place over a 16-h period from 10 to 2 p.m. were documented in 14 individuals; 8 with non-insulin-dependent diabetes mellitus (NIDDM) and 6 with normal glucose tolerance. Values for Ra were higher than normal in patients with NIDDM at 10 p.m. (4.73 +/- 0.41 vs. 3.51 +/- 0.36 mg/kg per min, P less than 0.001), but fell at a much faster rate throughout the night than that seen in normal subjects. As a consequence, the difference between Ra in normal individuals and patients with NIDDM progressively narrowed, and by 2 p.m., had ceased to exist (1.75 +/- 0.61 vs. 1.67 +/- 0.47 mg/kg per min, P = NS). Plasma glucose concentration also declined in patients with NIDDM over the same period of time, but they remained quite hyperglycemic, and the value of 245 +/- 27 mg/dl at 2 p.m. was about three times greater than in normal individuals. Plasma insulin concentrations also fell progressively from 10 to 2 p.m., and were similar in both groups throughout most of the 16-h study period. Thus, the progressive decline in Ra in patients with NIDDM occurred despite concomitant falls in both plasma glucose and insulin concentration. Glucose disposal rates also fell progressively in both groups, but the magnitude of the fall was greater in patients with NIDDM. Consequently, Rd in patients with NIDDM was higher at 10 p.m. (3.97 +/- 0.48 vs. 3.25 +/- 0.13 mg/kg per min, P less than 0.001) and lower the following day at 2 p.m. (1.64 +/- 0.21 vs. 1.97 +/- 0.35 mg/kg per min, P less than 0.01). These results indicate that a greatly expanded pool size can exist in patients with NIDDM at a time when values for Ra are identical to those in normal subjects studied under comparable conditions, which suggests that fasting hyperglycemia in NIDDM is not simply a function of an increase in Ra.
Y D Chen, C Y Jeng, C B Hollenbeck, M S Wu, G M Reaven
Hepatocytes are considered to be the predominant source of alpha 1-antitrypsin (AAT), the major antiprotease in human plasma. The development of emphysema in the hereditary PiZ AAT deficiency state suggests that inhibition of leukocyte elastase in the lung is a major function of this protein. In addition, patients with AAT deficiency are at increased risk for developing cholestasis in infancy and chronic liver disease as adults. The mechanism for hepatic cell injury, however, is not understood. Transgenic mice that express the normal human AAT gene demonstrate abundant AAT in hepatocytes and specific cell types of numerous nonhepatic tissues. Immunoperoxidase techniques have previously disclosed AAT in many of the cell types seen in transgenic mice; however, the issue of local synthesis vs. endocytosis in these cell types has remained unresolved. In this study, AAT mRNA was seen in a variety of tissues in the transgenic mouse. Immunoelectron microscopy of renal tubular and small intestinal epithelial cells in the transgenic mice demonstrated AAT within the cisternae of the rough endoplasmic reticulum, as in hepatocytes. These findings support the possibility of local synthesis in the various cell types. The results suggest that in addition to maintaining tissue integrity in the lung, the protease/antiprotease balance may have physiological functions in other organs as well.
J A Carlson, B B Rogers, R N Sifers, H K Hawkins, M J Finegold, S L Woo
The effects of hypoinsulinemic nonketotic streptozotocin diabetes on hepatic apo B synthesis and secretion was studied in primary cultures of rat hepatocytes. Diabetic rats were characterized by their significantly elevated serum glucose, apo B, and triglyceride levels, while serum insulin levels were less than a third of normal. Serum transminase activities of diabetic rats were significantly elevated when compared with control rats, which was attributed to an increase in liver transaminase activity in diabetic rats. The pattern of enzyme activities of hepatocytes reflected that observed in livers of donor rats and the pattern was retained by primary cultures of hepatocytes over the culture period. Hepatocytes from diabetic rats secreted only one third of the apo B secreted by hepatocytes from control rats, which was determined by monoclonal immunoassay of rat total apo B. Decreases in secretion were confirmed by measurement of secretory [35S]methionine-labeled lipoprotein apo B radioactivity. The decreased apo B content of media of hepatocytes from diabetic rats was not due to increased apo B catabolism since hepatocytes from diabetic rats were shown to degrade less lipoprotein-apo B than hepatocytes from normal rats in control experiments. In addition, the apo B content of detergent-solubilized hepatocytes from diabetic rats was significantly less than that of hepatocytes from control rats. These results suggest that insulin is necessary for normal hepatic apo B synthesis and secretion and that the hyperlipidemia associated with hypoinsulinemia in vivo is primarily of intestinal origin.
J D Sparks, C E Sparks, M Bolognino, A M Roncone, T K Jackson, J M Amatruda
Familial dysautonomia is a hereditary disorder that affects autonomic and sensory neurons. Nerve growth factor (NGF) is required for the normal development of sympathetic and sensory neurons and it has been postulated that an abnormality involving NGF may be responsible for familial dysautonomia. Previous studies have shown that the beta-NGF gene is not linked to the disease. However, NGF appears to be abnormal by immunochemical assays; the putative altered form of NGF could result from a disturbance in the processing pathway. To study the processing of the 35-kD glycosylated NGF precursor and the secretion of NGF in familial dysautonomia, we have employed a recombinant vaccinia virus vector to express high levels of NGF mRNA in primary fibroblast cultures from patients with the disorder; the processing pathway was then studied directly. Cells from several unrelated patients all produce the same 35-kD NGF precursor, process this normally to NGF within the cell, and release NGF into the medium. There are no differences in the ability of cells from patients and from unaffected relatives to process and secrete NGF. The use of similar recombinant vaccinia virus vectors to express proteins at high level in primary cell lines should facilitate the detection of posttranslational processing defects in a variety of human disorders.
R H Edwards, W J Rutter
To examine putative relationships between adrenergic receptors on accessible circulating cells and relatively inaccessible extravascular catecholamine target tissues, we measured mononuclear leukocyte (MNL) and lung beta-adrenergic receptors and platelet and lung alpha-adrenergic receptors in tissues obtained from 15 patients undergoing pulmonary resection. Plasma catecholamine concentrations were measured concurrently to explore potential regulatory relationships between the activity of the sympathochromaffin system and both intravascular and extravascular adrenergic receptors. MNL and lung membrane beta-adrenergic receptor densities were correlated highly (r = 0.845, P less than 0.001). Platelet alpha 2-adrenergic receptor and lung alpha 1-adrenergic receptor densities were not. Lung alpha 1-adrenergic receptor densities were positively related to plasma norepinephrine (r = 0.840, P less than 0.01) and epinephrine (r = 0.860, P less than 0.01) concentrations; in contrast, lung beta-adrenergic receptor densities were not positively related to plasma catecholamine concentrations (they tended to be inversely related to plasma norepinephrine and epinephrine [r = -0.698, P less than 0.05] levels). This apparent reciprocal regulation of alpha- and beta-adrenergic receptors by the sympathochromaffin system was only demonstrable with adrenergic receptor measurements in the extravascular catecholamine target tissue. Neither MNL beta-adrenergic receptor nor platelet alpha-adrenergic receptor densities were correlated with plasma catecholamine levels. Thus, although measurements of beta-adrenergic receptors on circulating mononuclear leukocytes can be used as indices of extravascular target tissue beta-adrenergic receptor densities (at least in lung and heart), it would appear that extravascular tissues should be used to study adrenergic receptor regulation by endogenous catecholamines in humans. These data provide further support for the concept of up regulation, as well as down regulation, of some adrenergic receptor populations during short-term activation of the sympathochromaffin system in humans.
S B Liggett, J C Marker, S D Shah, C L Roper, P E Cryer
Cl self-exchange by the rabbit cortical collecting tubule (CCT) occurs via an apical anion exchanger in series with a basolateral Cl conductance. We studied the effects of organic acids on CCT Cl self-exchange. We found no evidence for transport of acid anions by the self-exchange system. Rather, Cl self-exchange was inhibited by a variety of organic acids. The degree of inhibition correlated with the chloroform/water partition coefficient and was enhanced by lowering pH, indicating inhibition by the lipid-soluble, protonated species. Inhibition by the representative acid iso-butyrate was dose-dependent and showed sidedness (basolateral greater than apical). Iso-butyrate also reversibly reduced transepithelial conductance without altering K permeability, suggesting inhibition of the principal cell basolateral Cl conductance. Because small organic compounds with similar lipid solubilities but no carboxyl group had no effect, both the carboxyl group and the lipid-solubility of organic acids appear to be important. The results are consistent with blockade of chloride channels by organic acids.
K Matsuzaki, J B Stokes, V L Schuster
We have studied the binding of von Willebrand factor to extracellular matrices of endothelial cells and to the vessel wall of human umbilical arteries in relation to its function in supporting platelet adhesion. CLB-RAg 201, an MAb against von Willebrand factor, completely inhibits the binding of von Willebrand factor to collagen type I and type III. CLB-RAg 201 does not inhibit the binding of 125I-von Willebrand factor to extracellular matrices of endothelial cells, to smooth muscle cells, or to the subendothelium. CLB-RAg 201 partly inhibits platelet adhesion to these surfaces, but this directly affects the interaction between von Willebrand factor and platelets and is not due to inhibition of binding of von Willebrand factor to these surfaces. Another MAb, CLB-RAg 38, does not inhibit the binding of von Willebrand factor to collagen. CLB-RAg 38 completely inhibits the binding of von Willebrand factor to extracellular matrices. CLB-RAg 38 inhibits platelet adhesion to cellular matrices completely insofar as it is dependent on plasma von Willebrand factor. CLB-RAg 38 does not inhibit the total binding of von Willebrand factor to subendothelium, as there are too many different binding sites, but it completely inhibits the functional binding sites for von Willebrand factor that support platelet adhesion. The epitopes for CLB-RAg 38 and 201 on the von Willebrand factor molecule are located on different fragments of the molecule. These results indicate that von Willebrand factor binds to subendothelium and matrices of cultured cells by a mechanism that is different from that by which it binds to collagen.
P G de Groot, M Ottenhof-Rovers, J A van Mourik, J J Sixma
The nonionic detergent, Pluronic L-81 (L-81) has been shown to block the transport of intestinal mucosal triacylglycerol (TG) in chylomicrons. This results in large lipid masses within the enterocyte that are greater in diameter than chylomicrons. On removal of L-81, mucosal TG is rapidly mobilized and appears in the lymph. We questioned whether the blocked TG requires partial or complete hydrolysis before its transport. Rats were infused intraduodenally with [3H]glyceryl, [14C]oleoyl trioleate (TO) and 0.5 mg L-81/h for 8 h, followed by 120 mumol/h linoleate for 18 h. Mesenteric lymph was collected and analyzed for TG content and radioactivity. An HPLC method was developed to separate TG on the basis of its acyl group species. The assumed acyl group composition was confirmed by gas liquid chromatography analysis. TG lymphatic output was low for the first 8 h but increased to 52 mumol/h at the 11th h of infusion (3 h after stopping L-81). 38% of the infused TO was retained in the mucosa after the 8-h infusion. 95% of mucosal TG was TO, 92% of the radioactivity was in TG, and 2.4% of the 14C disintegrations per minute was in fatty acid. HPLC analysis of lymph at 6, 10, 12, and 14.5 h of infusion showed a progressive rise in TG composed of one linoleate and two oleates, to 39%; and in TG composed of two linoleates and one oleate to 20% at 14.5 h of infusion. On a mass basis, however, 80% of the TG acyl groups were oleate. 3H/14C ratios in the various TG acyl group species reflected the decrease in oleate. We conclude that first, unlike liver, most mucosal TG is not hydrolyzed before transport. The mechanism of how the large lipid masses present in mucosal cells after L-81 infusion are converted to the much smaller chylomicrons is unknown. Second, the concomitant infusion of linoleate did not impair lymph TG delivery after L-81 blockade.
J Halpern, P Tso, C M Mansbach 2nd
During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side-chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. We have previously shown in the rat that the contribution of the 25-hydroxylation pathway can be quantitated in vivo by measuring production of [14C]acetone from [14C]26-cholesterol. In the present study, we adapted this method to human subjects. 4 d after oral administration of 100 microCi of [14C]26-cholesterol and 1 d after beginning a constant infusion of 16.6 mumol/min unlabeled acetone, three men and two women underwent breath collections. Expired acetone was trapped and purified as the 2,4 dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied by the acetone infusion rate to calculate production of [14C]acetone. [14C]Acetone production averaged 4.9% of total release of 14C from [14C]26-cholesterol, estimated by 14CO2 output. The method was validated by showing that [14C]acetone production from [14C]isopropanol averaged 86.9% of the [14C]-isopropanol infusion rate. We conclude that in man, as in the rat, the 25-hydroxylation pathway accounts for less than 5% of bile acid synthesis.
W C Duane, P A Pooler, J N Hamilton
The relationships between contractile function, myocardial oxygen consumption, and tissue high energy phosphate and lactate content were investigated during partial coronary flow disruption. The experimental preparation was an isolated, isovolumic retrograde blood-perfused rabbit heart. Both developed pressure (r = 0.94) and dp/dt (r = 0.95) exhibited strong linear correlations with myocardial oxygen consumption that were stable for up to 45 min after blood flow reduction. In contrast, tissue high energy phosphate content exhibited nonlinear relationships with both developed pressure and oxygen consumption such that systolic mechanical function and oxidative metabolism declined to 20 and 30% of control values, respectively, before significant abnormalities in myocardial high energy phosphate stores were observed. Similarly, developed pressure and oxygen consumption decreased to 36 and 48% of control, respectively, before abnormal tissue lactate content was detected. The results of this study indicate that: (a) mechanical function is closely related to the rate of oxidative energy production during partial coronary flow disruption, and (b) despite the development of significant contractile dysfunction, tissue high energy phosphate content remains at normal levels except under the most severely flow-deprived conditions. The preservation of tissue energy stores can be explained by the apparent coupling of contractile performance to oxidative energy production, which could function to maintain myocardial energy balance during partial coronary flow restriction.
R C Marshall
The Ro/SS-A antigen was purified from an Epstein-Barr virus-transformed human B lymphoblastoid cell line. The amino terminal amino acid sequence of the 60-kD polypeptide bearing this antigenic epitope was determined to be: (formula; see text) A peptide composed of residue 6-19 was synthesized by the solid-phase method. Immunodiffusion-defined monospecific autoimmune sera to Ro/SS-A reacted with this synthetic peptide in ELISA, whereas autoantibodies with other specificities such as anti-La/SS-B and anti-Sm, as well as normal human sera, were not reactive. In addition, rabbit anti-peptide 6-19 antisera reacted specifically with native human Ro/SS-A antigen in ELISA. Furthermore, this synthetic peptide inhibited the binding of rabbit anti-peptide antiserum to native human Ro/SS-A. An additional synthetic peptide corresponding to residues 7-24 partially inhibited the binding of a patient anti-Ro/SS-A serum to native Ro/SS-A. These results suggest that the amino terminal portion of the molecule represents a major epitope of Ro/SS-A. The determination of the amino acid sequence of Ro/SS-A and the availability of synthetic peptide(s) bearing this antigen should provide additional approaches to further characterize the autoimmune response to this antigen.
T S Lieu, M M Newkirk, J D Capra, R D Sontheimer
The influence of leukocytes and Haemophilus influenzae type b (Hib) capsule on blood brain barrier permeability (BBBP) to circulating 125I-albumin in normal and leukopenic rats was assessed after intracisternal inoculation of encapsulated (Rd-/b+/02) or unencapsulated (Rd-/b-/02) isogenic strains of Hib. Both normal and leukopenic animals had increased BBBP 18 h after inoculation, with normal rats demonstrating significantly increased BBBP after challenge with the encapsulated strain. Despite cerebrospinal fluid (CSF) pleocytosis in normal rats, CSF bacterial concentrations were not lower. Normal rats cleared unencapsulated Rd-/b-/02 more effectively than leukopenic rats, with BBBP correlating with CSF bacterial density and not leukocyte concentrations. Challenge with heat-killed Rd-/b+/02 resulted in increased BBBP in both normal and leukopenic rats, with greater BBBP at higher bacterial concentrations. The data suggest: (a) significant increases in BBBP occur in the near absence of CSF leukocytes; (b) CSF leukocytes can augment changes in BBBP; (c) type b capsule inhibits host clearance mechanisms within the CSF; and (d) BBBP appears to correlate with bacterial concentrations within the CSF.
A J Lesse, E R Moxon, A Zwahlen, W M Scheld
The effects of corticotropin-releasing hormone (CRH) and dexamethasone on proopiomelanocortin (POMC) mRNA levels in cultured pituitary adenoma cells were studied in 10 patients with Cushing's disease. As a control, POMC mRNA levels in cells from nonadenomatous tissues were examined in four patients. Human POMC mRNA in the cells was analyzed by Northern blot hybridization. Human POMC DNA probe hybridized with only a single size class of RNA (approximately 1,200 nucleotides) from the adenoma and nonadenoma cells of each patient. The size of POMC mRNA did not change through the culture or after incubation with CRH or dexamethasone. CRH increased POMC mRNA levels in these cells in a dose- and time-dependent manner. The minimum concentration of CRH required to elevate POMC mRNA levels in these cells exposed for 15 h was 0.1 nM. The minimum duration of 1 nM CRH treatment required to increase these levels was 3 h under our conditions. Inhibitory effects of 1 and 10 micrograms/dl dexamethasone on ACTH release and POMC mRNA levels in nonadenoma cells were greater than those in adenoma cells. These results suggest the following: (a) that the mRNA in cultured pituitary adenoma cells is qualitatively the same as that in vivo; (b) that responses of mRNA levels to CRH are time- and dose-dependent; and (c) that adenoma cells resist the inhibitory effect of dexamethasone on POMC mRNA levels and ACTH release.
T Suda, F Tozawa, M Yamada, T Ushiyama, N Tomori, T Sumitomo, Y Nakagami, H Demura, K Shizume
Glomerular mesangial cell (MC)--derived IL-1 may be an important factor in the development of the hypercellularity and sclerosis characteristic of many forms of glomerulonephritis. To define the regulation of IL-1 synthesis by human MC, Northern blot analyses were performed using specific probes for monocytic IL-1 alpha and beta mRNA. Proliferating MC expressed mRNA for both IL-1 alpha and beta, whereas nonproliferating MC contained no detectable IL-1 mRNA. Synchronized MC expressed IL-1 alpha and beta mRNA within 2 h of stimulation with serum. This serum effect could be reproduced with platelet-derived growth factor and epidermal growth factor. Immune precipitations of 35S-methionine-labeled cells indicate that the mesangial IL-1 is synthesized as a 33-kD precursor protein with a pI of 7.2. Extracellular mesangial IL-1 has a pI of 7.0 and molecular weight of 17 kD, consistent with its identification as IL-1 beta. Cellular proliferation in glomerular disease may be driven in part by peptide growth factor-mediated induction of mesangial IL-1 gene expression and protein synthesis.
D H Lovett, A Larsen
We have localized the binding region of a previously described monoclonal anti-factor VIII (FVIII) inhibitory antibody (C5) to amino acid residues Thr351-Ser365 of the thrombin-generated 54-kD fragment of the heavy chain of FVIII. Synthetic FVIII peptides were examined for the ability to competitively inhibit the binding of C5 to FVIII in an ELISA system. The synthetic FVIII peptide Thr351-Ser365 blocked C5 binding to FVIII in a dose-dependent manner in this system. Two other synthetic FVIII peptides, Asn340-Glu354 and Glu342-Asp356, which partially overlapped Thr351-Ser365, also blocked C5 binding to FVIII. Blocking of C5 binding with these peptides, however, required much greater concentrations (greater than 100 times stronger) than that required for Thr351-Ser365. The Thr351-Ser365 peptide also neutralized the FVIII inhibitory activity of C5 in plasma. A human FVIII inhibitor (anti-FVIII heavy chain alloantibody) was also partially neutralized by Thr351-Ser365. Thr351-Ser365 lies between a thrombin cleavage site (Arg372) and an activated protein C cleavage site (Arg336) and may be at or near a region of functional importance in the expression of FVIII procoagulant activity.
P A Foster, C A Fulcher, R A Houghten, S de Graaf Mahoney, T S Zimmerman
The cytotoxic effect of a lymphocyte-specific immunotoxin formed by disulfide conjugation of an anti-T11 monoclonal antibody with the ribosome-inactivating protein gelonin was assessed in vitro on peripheral blood T cells and in vivo on splenic and lymph node T cells of macaque monkeys. This immunotoxin was cytotoxic to proliferating peripheral blood T cells in vitro as measured by both direct and indirect assays. Two sequential intravenous infusions into macaque monkeys achieved plasma concentrations of immunotoxin far in excess of those shown to be cytotoxic for cultured T cells and coated all T cells in lymph nodes and spleen with intact immunotoxin for four days. However, the cytotoxic effect of the immunotoxin on T cells in vivo was considerably less than that predicted by the in vitro studies. Further experiments suggested that the state of activation of the targeted T cell population in vivo, or the appearance of anti-immunotoxin antibodies, which occurred in all infused monkeys, might attenuate immunotoxin-mediated cell killing in vivo. These studies illustrate the significant differences between the action of immunotoxin conjugates in vitro, and those seen when these conjugates are utilized as therapeutic agents in vivo.
K A Reimann, V S Goldmacher, J M Lambert, L V Chalifoux, S B Cook, S F Schlossman, N L Letvin
We determined the sequence of a mutant CYP21B gene isolated from a patient with the severe, "salt-wasting" form of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Codon 318 in this gene is changed from CAG, encoding glutamine, to TAG, a nonsense codon. This is predicted to result in a completely nonfunctional enzyme due to premature termination of translation. In addition, when the cloned mutant gene was transfected into mouse Y1 adrenal cells, the resulting mRNA levels were decreased compared with transfected normal CYP21B genes. This mutation was carried by 3 of 20 unrelated patients with 21-hydroxylase deficiency alleles as determined by hybridization with a specific oligonucleotide probe. This mutation is also seen in the normal CYP21A pseudogene, so that its presence in the abnormal CYP21B gene may be the result of a gene conversion event.
H Globerman, M Amor, K L Parker, M I New, P C White
The presence of serum antibodies to the diol-epoxide DNA adducts of representative polycyclic aromatic hydrocarbons (PAH), chrysene, benz[a]anthracene and benzo[a]pyrene, was determined by ELISA using serum samples obtained from normal healthy individuals. Antibodies that reacted against PAH adducted-DNA, but not against PAH-adducted protein, were found in the serum of approximately 40% of the test individuals. Specificity analysis of the antibodies demonstrated that serological cross-reactions between the benzo[a]pyrene and the chrysene diol-epoxide adducts were present. Similar cross-reactivity between the benz[a]anthracene and the chrysene adducts was observed. Sera containing antibodies that were apparently specific for each of the three PAH-DNA adducts were also identified. The presence of antibodies to PAH-DNA adducts indicates both past exposure to these carcinogenic PAH and their metabolic activation to the DNA damaging metabolites. These antibodies may prove to be useful in both retrospective and prospective epidemiological studies of various diseases associated with PAH exposure.
M J Newman, B A Light, A Weston, D Tollurud, J L Clark, D L Mann, J P Blackmon, C C Harris
Erythropoietin (EP) mRNA was measured in normal and anemic mice during fetal and postnatal development. Normal fetal livers at 14 d of gestation contained a low level of EP mRNA. By day 19 of gestation, no EP mRNA was detected in normal or anemic fetal livers or normal fetal kidneys, but anemic fetal kidneys had low levels of EP mRNA. Newborn through adult stage mice responded to anemia by accumulating renal and hepatic EP mRNA. However, total liver EP mRNA was considerably less than that of the kidneys. Juvenile animals, 1-4 wk old, were hyperresponsive to anemia in that they produced more EP mRNA than adults. Moreover, nonanemic juveniles had readily measured renal EP mRNA, whereas the adult level was at the lower limit of detection. Because of the very low level of fetal EP mRNA, placental transfer of EP was evaluated. When administered to the pregnant mouse, 125I-EP was transferred in significant amounts to the fetuses. These results indicate that in mice the kidney is the main organ of EP production at all stages of postnatal development and that adult kidney may also play some role in providing EP for fetal erythropoiesis via placental transfer of maternal hormone.
M J Koury, M C Bondurant, S E Graber, S T Sawyer
Expression of Fc receptors for IgE (FcER) or IgA (FcAR) on purified natural killer (NK) cells was investigated. No FcER+ and a few FcAR+ NK cells were detectable on freshly separated NK (NKH-1+) cells from normal donors. Incubation of NK cells with IgE-anti-IgE immune complexes or IgA-anti-IgA immune complexes induced up to 10 and 20% FcER+ or FcAR+ cells, respectively. These FcR were induced on CD3- but not on CD3+ NKH-1+ cells. In contrast, NK cells from patients with various dysgammaglobulinemias could not be induced to express FcER or FcAR corresponding to their abnormal circulating IgE and/or IgA levels. Enriched FcER+ or FcAR+ induced NK cell supernatants from normals enhanced IgE or IgA synthesis from Ig secreting B cell lines in an isotype-specific fashion without increasing proliferation. Thus NK cells, after interaction with specific Ig isotypes in complexes, express FcR and produce differentiation factors for that isotype.
H Kimata, A Saxon
Calcium has been implicated as an important factor in prostaglandin production. Phospholipase A2, the enzyme believed to be rate limiting for prostaglandin synthesis, is stimulated by Ca2+; however, the levels of Ca2+ necessary to stimulate phospholipase A2 in cell-free systems are higher than levels achieved in intact cells in response to agonists that stimulate prostaglandin synthesis. We examined the calcium dependency of prostaglandin E2 (PGE2) synthesis in the glomerular mesangial cell. Vasopressin enhanced PGE2 synthesis by mechanisms independent of extracellular Ca2+ concentration. The Ca2+ concentration dependency of PGE2 production was established by rendering cells permeable with digitonin and clamping Ca2+ concentration at various levels. When cytosolic free Ca2+ concentration ([Ca2+]f) was set at levels equal to those measured after stimulation with vasopressin in the intact cell, the PGE2 production by the Ca2+-clamped permeabilized cells was approximately one-half of that obtained in nonpermeabilized cells stimulated with vasopressin. Since stimulation of mesangial cells with vasopressin increases protein kinase C activation as well as [Ca2+]f the effects on PGE2 production of protein kinase C activation with phorbol myristate acetate (PMA) were examined. When permeabilized cells were exposed to Ca2+ concentrations in the range of [Ca2+]f measured in cells treated with vasopressin the addition of PMA approximately doubled PGE2 production. No increase in PGE2 production was observed with PMA when Ca2+ concentration was fixed at basal levels of less than 100 nM. Ca2+-dependent acylhydrolase activity and PGE2 production were inhibited by calmodulin inhibitors, W-7 and compound 48/80. Thus, vasopressin-induced PGE2 production could be explained by a synergistic effect of protein kinase C activation together with an increase in [Ca2+]f. A synergistic action of Ca2+ and PMA on acylhydrolase activity could also be observed in nonpermeabilized cells where A23187 was used to increase [Ca2+]f. The effect of PMA was mimicked by another stimulant of protein kinase C, 1-oleoyl 2-acetylglycerol, albeit with lower potency. Neither PMA nor 1-oleoyl 2-acetylglycerol alone had any effect on acylhydrolase activity. Vasopressin, in the presence of GTP gamma S, stimulated phospholipase C in permeabilized cells when [Ca2+]f was fixed at less than 100 nM, without an associated increase in acylhydrolase activity. This evidence, together with inhibition of acylhydrolase activity with phospholipase A2 inhibitors, dibucaine and mepacrine, indicates that the primary acylhydrolase activity was due to phospholipase A2. The enhanced phospholipase A2 activity observed with protein kinase C activation when [Ca2+]f is increased may be related to phosphorylation of phospholipase A2 itself or phospholipase A2 modulatory proteins. These experiments demonstrate that both Ca2+ and protein kinase C play important roles in the regulation of phospholipase A2 and PGE2 synthesis.
J V Bonventre, M Swidler
Immunocytochemical analyses revealed the presence and distribution of apolipoprotein E (apo E) in normal human brain tissue as well as in 77 human intracranial neoplasms. In normal brain tissues, the perikarya of astrocytes exhibited a strong positive reaction, whereas the Bergmann glia were stained to a moderate degree. However, no immunoreactivity was observed with neurons, oligodendrocytes, ependymal cells, and choroidal epithelium. Among the intracranial neoplasms, oligodendroglioma, choroid plexus papilloma, hemangioblastoma, primary malignant lymphoma, neurinoma, meningioma, pituitary adenoma, and craniopharyngioma were all negative. Immunoreactivity in the peripheral neuroblastoma was nil. However, the perikarya of astrocytomas and glioblastomas showed a positive reaction. Analyses on the degree of anaplasia and the amount of apo-E as an intensity of immunostaining showed a negative correlation. The astrocytic elements were stained in mixed oligoastrocytomas and medulloblastomas with glial differentiation. A few cases of ependymomas showed weak perikaryal immunostaining. Western blot analyses with anti-apo E antibody of a freshly prepared surgical specimen with astrocytomas revealed a single band with a molecular weight of approximately 37,000. The well differentiated cultured human astrocytoma cells secreted apo E into the medium. These lines of evidence suggest that apo E may serve as a potential marker specific for astrocytomas and glioblastomas, as well as an indicator of astrocytic tumor cell differentiation. The apo E localization in human brain tumors could be clinically relevant and diagnostically useful.
M Murakami, Y Ushio, Y Morino, T Ohta, Y Matsukado
Human heart failure is associated with a diminished contractile response to beta-adrenergic agonists. We hypothesized that alterations in the activity of a guanine nucleotide-binding regulatory protein (G protein) might be partially responsible for this abnormality. We therefore measured the activity of G proteins in failing human myocardium utilizing bacterial toxin-catalyzed ADP ribosylation. The activity of a 40,000-mol wt pertussis toxin substrate (alpha G40) was increased by 36% in failing human hearts when compared with nonfailing controls. In contrast, there was no change in the level of the stimulatory regulatory subunit (Gs). The increased activity in alpha G40 was associated with a 30% decrease in basal as well as 5'-guanylyl imidodiphosphate-stimulated adenylate cyclase activity. These data suggest that increased alpha G40 activity is a new marker for failing myocardium and may account at least in part for the diminished responsiveness to beta 1-adrenergic agonists in the failing human heart.
A M Feldman, A E Cates, W B Veazey, R E Hershberger, M R Bristow, K L Baughman, W A Baumgartner, C Van Dop
Because intravenous infusion of beta-hydroxybutyrate (beta-OHB) has been reported to decrease urinary nitrogen excretion, we investigated in vivo metabolism of leucine, an essential amino acid, using L-[1-13C]leucine as a tracer during beta-OHB infusion. Leucine flux during beta-OHB infusion did not differ from leucine flux during normal saline infusion in nine normal subjects, whereas leucine oxidation decreased 18-41% (mean = 30%) from 18.1 +/- 1.1 mumol.kg-1.h-1 (P less than 0.01), and incorporation of leucine into skeletal muscle protein increased 5-17% (mean = 10%) from 0.048 + 0.003%/h (P less than 0.02). Since blood pH during beta-OHB infusion was higher than the pH during saline infusion, we performed separate experiments to study the effect of increased blood pH on leucine kinetics by infusing sodium bicarbonate intravenously. Blood pH during sodium bicarbonate infusion was similar to that observed during the beta-OHB infusion, but bicarbonate infusion had no effect on leucine flux or leucine oxidation. We conclude that beta-OHB decreases leucine oxidation and promotes protein synthesis in human beings.
K S Nair, S L Welle, D Halliday, R G Campbell
The bleeding disorder of hemophilia A currently treated by replacement therapy of the missing coagulation factor, factor VIII, is frequently complicated by the development of neutralizing antibodies. The therapeutic potential of attenuated forms of the lipid-associated glycoprotein tissue factor, a known initiator of coagulation, was investigated as a factor VIII-by-passing activity. The protein moiety of tissue factor (Apo-TF) was partially purified and exhibited minimal procoagulant activity before relipidation in vitro. In pilot studies, Apo-TF injection into rabbits previously anticoagulated with an antibody to factor VIII was found to have a procoagulant effect. The efficacy of the material was further demonstrated when injection of Apo-TF in hemophilic dogs resulted in a normalization of the cuticle bleeding time. Little or no change in the blood parameters associated with disseminated intravascular coagulation was observed at lower doses, although mild to moderate effects were seen at higher doses. These data suggest a novel role for Apo-TF preparations as a potential therapeutic agent for hemophiliacs with antibodies to factor VIII once the potential thrombogenicity of such materials is evaluated.
D P O'Brien, A R Giles, K M Tate, G A Vehar
Osteoinduction is the formation of ectopic bone that follows implantation of demineralized allogeneic bone matrix (DABM) and is believed to be secondary to the release of associated inductive factors from bone matrix. To clarify the role of vitamin D in osteoinduction, we implanted DABM from vitamin D-deficient rats (-D rats) into normal rats (+D rats). Because mitogens and osteocalcin might be involved in osteoinduction, these were measured. Mitogenic activity in extracts from mineralized allogeneic bone matrix (ABM) and DABM from both +D and -D rats was determined with an assay that utilizes monolayer cultures of embryonic chick calvarial cells. Osteocalcin in serum and DABM was measured by radioimmunoassay. DABM from -D rats did not promote osteoinduction as effectively as DABM from +D rats. Resorption of implant matrix from -D rats was diminished compared with resorption of matrix from +D rats (P less than 0.01), and the decrease was attributed to a corresponding decrease in the number of osteoclasts in the implants (P less than 0.02). Bone formation (P less than 0.01) and total implant mineralization (P less than 0.001) were significantly reduced in implants from -D rats, and the reductions corresponded with a decline in the number of osteoblasts (P less than 0.05). Mitogenic activity in DABM from +D rats was only slightly decreased as compared with activity in ABM, but DABM from -D rats contained significantly less activity (P less than 0.001). No mitogenic activity was identified in implants of DABM from either +D or -D rats 3 wk after implantation. Serum osteocalcin was significantly higher in -D as compared with +D animals. In contrast, the concentrations of osteocalcin in DABM from the two groups of animals were not significantly different from each other. These findings indicate that the diminished osteoinductive activity of DABM from -D rats results from deficiency of one or more mitogenic factors that are essential for inducing the proliferation and differentiation of bone cells at the implant site and that osteocalcin does not play a role in this regard.
R T Turner, J Farley, J J Vandersteenhoven, S Epstein, N H Bell, D J Baylink
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] increases intestinal calcium absorption through events that include binding of 1,25(OH)2D3 to the intracellular vitamin D receptor. In vitro studies using mammalian cell cultures reveal an increase in vitamin D receptor content after exposure to 1,25(OH)2D3. To test the hypothesis that 1,25(OH)2D3 increases enterocyte vitamin D receptor content in vivo, male rats were fed either a normal calcium diet (NCD, 1.2% Ca) or low calcium diet (LCD, 0.002% Ca). After 21 d LCD increased serum 1,25(OH)2D3 levels (27 +/- 3 vs. 181 +/- 17 pg/ml, P less than 0.001) and increased transepithelial mucosal to serosal calcium fluxes (Jms) across duodenum (65 +/- 21 vs. 204 +/- 47 nmol/cm2.h, NCD vs. LCD, P less than 0.01) and jejunum (23 +/- 3 vs. 46 +/- 4, P less than 0.007). No change in serosal to mucosal calcium fluxes (Jsm) were observed. LCD increased 1,25(OH)2D3 receptor number threefold in duodenum (32.9 +/- 6.7 vs. 98.7 +/- 13.7 fmol 1,25(OH)2D3/mg protein) and jejunum (34.1 +/- 9.5 vs. 84.9 +/- 7.7) without a change in the receptor affinity for 1,25(OH)2D3 (Kd is 0.17 +/- 0.06 vs. 0.21 +/- 0.02 nM for NCD and LCD duodenum, respectively). Duodenal polyadenylated vitamin D receptor mRNA determined by Northern blot analysis did not increase appreciably during LCD, suggesting that upregulation in vivo may not be due primarily to increased receptor synthesis. The results of this study indicate that under physiologic conditions as during chronic dietary calcium restriction, increased intestinal vitamin D receptor content accompanies increased calcium active transport. Upregulation of the vitamin D receptor by 1,25(OH)2D3 may result primarily from posttranslational processes that decrease degradation of the receptor with increased receptor synthesis responsible for a negligible portion of the accumulation.
M J Favus, D J Mangelsdorf, V Tembe, B J Coe, M R Haussler
Standard and ion-sensitive microelectrodes were used to identify the basis of electrophysiologic changes that occur in canine cardiac Purkinje fibers superfused with "ischemic" solution (40 min) and then returned to standard Tyrode's solution. Maximum diastolic potential (EMDP) decreased (-92.6 +/- 2.4 to -86.0 +/- 4.0 mV; n = 19; P less than 0.001) during exposure to "ischemia," and after reperfusion, rapidly hyperpolarized to -90.0 +/- 4.7 (2 min) and then depolarized to -47.0 +/- 7.5 mV (10 min; P less than 0.001). No significant change in intracellular K activity (alpha ik) was noted throughout. Extracellular K activity (alpha ek) changed only during reperfusion, reaching a nadir at 5 min (3.5 +/- 0.4 to 2.6 +/- 0.5 mM, P less than 0.03), and thus can not account for the decrease in EMDP during reperfusion. Mean alpha iNa increased (8.7 +/- 1.3 to 10.9 +/- 1.9 mM; n = 10; P less than 0.01) during ischemia, but rapidly declined during reperfusion to 5.1 +/- 2.2 mM (10 min; P less than 0.01). Exposure to acetylstrophanthidin (4-5 x 10(-7) M) during the final 10 min of ischemia increased alpha iNa to 19.9 +/- 3.8 mM (n = 5), which was unchanged at 5 min of reperfusion. This suggests that Na-K pump inhibition during ischemia was minimal and that the pump was stimulated early during reperfusion, accounting for the initial transient hyperpolarization. Resting tension did not change significantly during exposure to ischemia; however, return to control Tyrode's solution caused a marked rise to 11.3 +/- 9.9 mg/mm2 (n = 13, P less than 0.001). This is consistent with a calcium overload state during reperfusion. The depolarization seen during reperfusion may result from activation of a Ca-activated, nonselective cation channel or enhanced electrogenic Na/Ca exchange.
R Yee, K K Brown, D E Bolster, H C Strauss
Mechanisms involved in basolateral H/OH/HCO3 transport in the in vitro microperfused rat cortical thick ascending limb were examined by the microfluorometric determination of cell pH using (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein. The mean cell pH in this segment perfused with 147 mM sodium and 25 mM HCO3 at pH 7.4 was 7.13 +/- 0.02 (n = 30). Lowering bath HCO3 from 25 to 5 mM (constant PCO2 of 40 mmHg) acidified the cells by 0.31 +/- 0.02 pH units at a rate of 0.56 +/- 0.08 pH units/min. Removal of bath sodium acidified the cells by 0.28 +/- 0.03 pH units at a rate of 0.33 +/- 0.04 pH units/min. The cell acidification was stilbene inhibitable and independent of chloride. There was no effect of bath sodium removal on cell pH in the absence of CO2/HCO3. Depolarization of the basolateral membrane (step increase in bath potassium) independent of the presence of chloride. Cell acidification induced by bath sodium removal persisted when the basolateral membrane was voltage clamped by high potassium/valinomycin. Although these results are consistent with a Na/(HCO3)n greater than 1 cotransporter, a Na/H antiporter was also suggested: 1 mM bath amiloride inhibited the cell pH defense against an acid load (rapid ammonia washout), both in the presence and absence of CO2/HCO3, and inhibited the cell acidification induced by bath sodium reduction from 50 to 0 mM. In conclusion, an electrogenic Na/(HCO3)n greater than 1 cotransporter in parallel with a Na/H antiporter exist on the basolateral membrane of the rat cortical thick ascending limb.
The effects of chronic caffeine on the A1 adenosine receptor-adenylate cyclase system of rat cerebral cortical membranes were studied. Caffeine treatment significantly increased the number of A1 adenosine receptors as determined with the A1 adenosine receptor antagonist radioligand [3H]xanthine amine congener (XAC). R-PIA (agonist) competition curves constructed with [3H]XAC were most appropriately described by a two affinity state model in control membranes with a KH of 2.1 +/- 0.8 and a KL of 404 +/- 330 nM with 50 +/- 4% of receptors in the high affinity state (%RH). In contrast, in membranes from treated animals, there was a marked shift towards the high affinity state. In three of seven animals all of the receptors were shifted to a unique high affinity state which was indistinguishable from the KH observed in membranes from control animals. In four of seven animals the %RH increased from 50 to 69% with KH and KL indistinguishable from the control values. Thus, the agonist specific high affinity form of the receptor was enhanced following caffeine treatment. Maximal inhibition of adenylate cyclase activity in cerebral cortical membranes by R-PIA (1 microM) was significantly increased by 28% following caffeine treatment, consistent with an increased coupling of receptor-Gi protein with adenylate cyclase. Importantly, the quantity of Gi (alpha i) in rat cerebral cortex, determined by pertussis toxin-mediated labeling, was also increased to 133% of control values by this treatment. Thus, multiple components and interactions of the A1 adenosine receptor-adenylate cyclase complex are regulated by caffeine. These changes are likely compensatory measures to offset blockade of A1 receptors in vivo by caffeine and lead to a sensitization of this inhibitory receptor system.
V Ramkumar, J R Bumgarner, K A Jacobson, G L Stiles
We have characterized cell-to-cell communication (coupling) within intact human skin by microinjecting single keratinocytes with a gap junction-permeant tracer (Lucifer Yellow). 25-50 keratinocytes from different layers of the epidermis were seen to be coupled after most injections (n = 31). A few noncommunicating cells were also microinjected (n = 3) or observed within large territories of coupled keratinocytes. Microinjections of dermal fibroblasts demonstrated an extensive coupling (greater than 100 fibroblasts); however, none of the keratinocyte (n = 34) or fibroblast (n = 3) injections revealed coupling between the epidermal and dermal compartments. Cell coupling was found to be more extensive in epidermal ridges than in suprapapillary plates and, in both regions, was less extensive after injection of the basal layer of the epidermis than after that of the suprabasal layers. This study shows that junctional cell-to-cell communications take place in normal and fully differentiated human tissue. The quantitative data gathered also indicate a regional heterogeneity of keratinocyte-to-keratinocyte communication within intact adult skin and the lack of effect of retinoids on this pattern.
D Salomon, J H Saurat, P Meda
Plasma samples were obtained from 34 bone marrow transplant (BMT) recipients before and after administration of the preparative regimen and tested for their ability to promote and/or support growth of hemopoietic colonies. The ability of plasma samples to promote colony formation on their own was tested on normal nonadherent target cells without addition of exogenous growth factors. The growth-supporting activity was examined in the presence of medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) and/or erythropoietin (EPO). A series of kinetic changes was routinely observed. Pretransplant samples rarely gave rise to colonies without addition of exogenous growth factors. Plasma samples obtained after completion of the preparative regimen demonstrated increments of growth-promoting activities for megakaryocyte and granulocyte-macrophage progenitors (CFU-Meg and CFU-GM), respectively, that peaked between 7 and 21 d after transplantation. By day 30, activity levels of some patients had returned to pretransplant values, whereas in other patients, activities remained elevated. Persisting activity levels were associated with delayed engraftment. In contrast, activities for progenitors committed to erythropoiesis (BFU-E) and pluripotent precursors (CFU-GEMM) were only rarely observed. The activities were independent of febrile episodes. Their growth-promoting influence on CFU-GM could be neutralized completely by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies. These data suggest that at least some of the observed activities in post-BMT plasma are related to GM-CSF. The growth-supporting activities of pretransplant plasma samples are lower than normal plasma when tested on CFU-Meg and CFU-GM. The growth-supporting activities improved transiently within the first month after BMT. A decline during the second and third month was followed by a gradual return to activity levels that were comparable to normal plasma. The effects of these plasma samples on BFU-E and CFU-GEMM were assessed with PHA-LCM and EPO. Similar to CFU-Meg- and CFU-GM-supporting capabilities, they improved transiently after BMT with a return of normal support function after 5-6 mo. The observed endogenous production of growth-promoting and growth-supporting activities for hemopoietic progenitors may serve as a background to design clinical trials for the timely administration of recombinant hemopoietic growth factors to BMT recipients.
K Yamasaki, L A Solberg Jr, N Jamal, G Lockwood, D Tritchler, J E Curtis, M M Minden, K G Mann, H A Messner
The isolation of recombinant cDNA clones expressing antigens found in Onchocerca volvulus infective larvae is described. To isolate such clones, an expression cDNA library constructed from adult O. volvulus RNA was screened with antiserum raised against infective larvae. One clone, designated lambda RAL-1 was characterized further. The recombinant antigen produced by lambda RAL-1 stimulates proliferation of peripheral blood mononuclear cells from O. volvulus infected humans. Lambda RAL-1 is derived from a 1450 bases message that encodes a protein with an apparent molecular weight of 42,000 in adult O. volvulus. The inserted DNA of lambda RAL-1 contains an open reading frame of 1008 bp. The amino acid sequence predicted by this open reading frame contains three repeats of the sequence KKPEDWD. The identification of clones such as lambda RAL-1 will provide quantities of purified antigens sufficient to begin to study the immune response to and explore the development of immunity against the infectious form of the parasite.
T R Unnasch, M Y Gallin, P T Soboslay, K D Erttmann, B M Greene
Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.
N E Cooke, J Ray, M A Watson, P A Estes, B A Kuo, S A Liebhaber
An apparent increase of calmodulin (CaM) activity was previously observed in the heart and kidney but not in the liver of spontaneously-hypertensive rats (SHR) and mice compared with their corresponding normotensive controls. As this change was due to an elevated recovery of CaM in the organs of the hypertensive animals, the present study was designed to evaluate its activity in hypertension. A CaM activator, detected in heart and kidney supernatants from hypertensive animals, was found to be responsible for this enhanced recovery. Similar results were obtained with passaged, cultured aortic smooth muscle cells from SHR, indicating that the anomaly was not a mere consequence of elevated blood pressure but rather a genetic expression of cells of hypertensive origin. The activator was heat stable, nondialyzable, and recovered in the fraction precipitated with 30-50% ammonium sulfate. Preliminary extraction studies suggest that the activator is contained in a glycolipid fraction. The stimulation of phosphodiesterase by this activator was calcium and CaM dependent. The activator appears to affect the affinity of the phosphodiesterase for CaM rather than the maximal stimulation. The activator was also present at a low concentration in the heart and kidney of normotensive animals. These findings indicate that at least some of the calcium abnormalities implicated in the pathogenesis of hypertension could be the result of interactions between CaM, calcium, and this activator.
S L Huang, Y I Wen, D B Kupranycz, S C Pang, G Schlager, P Hamet, J Tremblay
The present study was designed to test whether there are diurnal changes in the firing rate of sympathetic nerves to brown adipose tissue and whether these diurnal rhythms influenced the response to insulin injected into the suprachiasmatic nucleus or ventromedial hypothalamic nucleus (VMH). Food intake was highest at the beginning of the dark period (1800-2200 hours) and lowest during the daylight hours (0600-1000 and 1200-1600 hours). The basal sympathetic firing rate was highest at noon (1000-1200 hours) when food intake was lowest. At midnight, when food intake was highest, sympathetic firing rate was lowest. Injection of insulin (77, 144, and 288 pmol) into the VMH produced a dose-dependent depression of sympathetic firing rate at each of the four measurement periods (0400-0600 hours, 1000-1200 hours, 1600-1800 hours, and 2200-2400 hours), but the magnitude of the effect was greater at noon than at night. In contrast, insulin injections into the suprachiasmatic nucleus decreased the sympathetic firing rate at noon but produced a significant increase in the sympathetic firing rate at night. These data show that a diurnal rhythm exists for the sympathetic firing rate. The decrease in firing rate in response to insulin when injected into the VMH is in the same direction but varies in magnitude throughout the day, whereas the responsiveness of the suprachiasmatic nucleus to injections of insulin shows a reversal of response in relation to day/night cycles. The highly significant inverse relationship between basal sympathetic firing rate and food intake suggests that sympathetic activity may be part of an important control system for energy balance.
T Sakaguchi, M Takahashi, G A Bray
Corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide involved in the regulation of ACTH secretion, has been detected by RIA in extracts of human placenta. We wished to determine whether this immunoreactive substance is a product of CRH gene expression in the placenta. We have found authentic human CRH (hCRH) mRNA in human placental tissue that is similar in size to hypothalamic CRH mRNA. Furthermore, the transcriptional initiation site for placental hCRH mRNA is identical to that previously predicted for hypothalamic hCRH mRNA, 23-26 nucleotides downstream from a canonical promoter element. Placental hCRH mRNA increases more than 20-fold in the 5 wk preceding parturition, in parallel with a rise in placental hCRH peptide content. These data strongly suggest that the hCRH gene is expressed in the placenta and that this expression changes dramatically during gestation.
D M Frim, R L Emanuel, B G Robinson, C M Smas, G K Adler, J A Majzoub
The pathophysiological significance of the glucose-fatty acid cycle in skeletal muscle in vivo is uncertain. We have examined the short term effects of increased availability of nonesterified FFA on tissue-specific glucose uptake and storage in rat tissues in vivo basally and during a hyperinsulinemic (150 mU/liter) euglycemic clamp. Circulating FFA were elevated to 2 mmol/liter (FFA 1) or 4 mmol/liter (FFA 2). Elevated FFA produced a dose-dependent inhibition of myocardial glucose utilization in both basal (FFA1, 42%; FFA2, 68%; P less than 0.001, by analysis of variance) and clamp groups (FFA1, 39%; FFA2, 49%; P less than 0.001) and also suppressed brown adipose tissue glucose utilization during the clamp (-42%, P less than 0.001). In contrast to heart, glucose utilization in skeletal muscle was suppressed by FFA only in the FFA1 basal group (-36%, P less than 0.001); in other groups (e.g., FFA2 clamp) elevated FFA produced increased skeletal muscle glucose utilization (+68%, P less than 0.001) that was directed toward glycogen (+175%, P less than 0.05) and lipid deposition (+125%, P less than 0.005). FFA stimulated basal glucose utilization in white (e.g., FFA2, +220%, P less than 0.005) and brown adipose tissue (e.g., FFA2, +200%, P less than 0.005). Thus elevated FFA can acutely inhibit glucose utilization in skeletal muscle in addition to cardiac muscle in vivo supporting a possible role for the glucose-fatty acid cycle in skeletal muscle in acute insulin resistance. However, at high levels or with elevated insulin, FFA stimulates glucose utilization and storage in skeletal muscle. By promoting accumulation of glucose storage products, chronic elevation of FFA may lead to skeletal muscle (and therefore whole body) insulin resistance.
A B Jenkins, L H Storlien, D J Chisholm, E W Kraegen
We previously reported that intestinal apo B48 synthesis in the rat was unaltered by dietary triglyceride intake but demonstrated regulation in response to biliary lipid availability. Studies are now presented in which the mechanisms underlying biliary lipid dependent expression of intestinal apo B48 synthesis have been investigated further. Bile salt replacement was effective in a dose- and structure-dependent manner in reexpressing intestinal apo B48 synthesis after prolonged bile diversion. Further experiments suggested that this effect of bile salt may be related to facilitated uptake of fatty acid. A role for mucosal phospholipid flux was suggested by studies in which infusion of lysolecithin, with or without Na taurocholate, produced complete reexpression of apo B48 synthesis in jejunal enterocytes. Over a four- to sixfold range of apo B48 synthesis rates in both jejunum and ileum, there was no change in apo B mRNA size or abundance as determined by RNA blot hybridization. Analysis of both intestinal mucosa and microsome lipid content in a variety of settings revealed that apo B48 synthesis rates were correlated with microsome triglyceride fatty acid content (r = 0.65, P less than 0.005) but not free fatty acid or phospholipid content. These studies demonstrate a physiologic role for elements of biliary lipid flux in the regulation of apo B gene expression. The data suggest that an integrated mechanism may exist whereby apo B48 synthesis is related to microsome triglyceride flux, particularly at low levels of lumenal substrate availability.
N O Davidson, M J Drewek, J I Gordon, J Elovson
Erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the differentiation and proliferation of erythroid cells. To determine the cellular mechanism of action of these growth factors, we measured changes in intracellular free calcium concentration [( Cac]) in single human erythroid precursors in response to recombinant erythropoietin and GM-CSF. [Cac] in immature erythroblasts derived from cultured human cord blood erythroid progenitors was measured with fluorescence microscopy digital video imaging. When stimulated with erythropoietin, [Cac] in the majority of erythroblasts increased within 3 min, peaked at 5 min, and returned toward baseline at 10 min. The percentage of cells that responded to erythropoietin stimulation increased in a dose-dependent manner. Additional stimulation with GM-CSF in cells previously exposed to erythropoietin resulted in a second [Cac] increase. Immature erythroblasts treated with GM-CSF followed by erythropoietin responded similarly to each factor with a rise in [Cac]. The source of transient calcium is intracellular since erythroblasts were incubated in medium devoid of extracellular calcium. Our observations suggest that changes in [Cac] may be an intracellular signal that mediates the proliferative/differentiating effect of hematopoietic growth factors.
B A Miller, R C Scaduto Jr, D L Tillotson, J J Botti, J Y Cheung
Expressions and functional roles of novel IL-2 binding molecules (p70, 75) in the differentiation of B cells into Ig secreting cells were explored by using human several B cell lines and tonsillar B cells. Affinity-crosslinking studies revealed that five of nine B cell lines expressed p70 and p75 without detectable Tac antigen (p55) expression and the expression was associated with B cell maturation. In tonsillar B cells, small high-density B cells did not express p70 and p75, whereas large low-density B cells, which were thought to be activated in vivo, expressed them. Binding assays of radiolabeled IL-2 showed that the affinity of these molecules was intermediate (kD = 1-3 nM, 700-3,000 sites/cell). Furthermore, high concentrations of IL-2 (greater than 100 U/ml) induced Ig productions in large B cells and two of five cell lines. These results taken together suggest that B cells may express novel IL-2 binding molecules, associated with B cell differentiation and differentiate into Ig secreting cells by IL-2 through novel IL-2 binding molecules.
T Tanaka, O Saiki, S Doi, M Suemura, S Negoro, S Kishimoto
We have recently developed a micropuncture technique to assess repeatedly function of the same nephrons in chronic renal disease and subsequently examine the morphology of their glomeruli by serial thin-section histological analysis. Using this approach, a potential causal linkage between early functional patterns and late structural abnormalities was examined in glomeruli of two established rat models of glomerular sclerosis. The models are (a) puromycin aminonucleoside (PAN) administration in unilaterally nephrectomized Munich-Wistar rats and (b) adriamycin (ADM) treatment in nonnephrectomized Munich-Wistar rats. Single nephron GFR (SNGFR) and glomerular capillary hydraulic pressure (PGC) were measured repeatedly for 8 (PAN rats) or 31 wk (ADM rats). In all animals studied, values for PGC remained at, or slightly below, levels measured before PAN or ADM administration. SNGFR values declined progressively in all glomeruli in PAN rats. Although some glomeruli in ADM rats had an increase in SNGFR above levels observed in nonnephrectomized control rats, these hyperfiltering glomeruli did not have abnormally high PGC nor did they exhibit glomerular sclerosis at the completion of the study. Histological analysis revealed the existence of a significant inverse correlation between the degree of sclerosis and SNGFR assessed at the time of sacrifice in both PAN and ADM groups. Chronic administration of captopril, an angiotensin I converting enzyme inhibitor, in PAN rats substantially attenuated development of glomerular sclerosis without affecting PGC in earlier stages. The observations in these models indicate that glomerular hyperfiltration and hypertension are not required for the development of glomerular sclerosis in renal diseases, and angiotensin I converting enzyme inhibitor can exert its protective effect independently of its effect on glomerular capillary pressure.
A Fogo, Y Yoshida, A D Glick, T Homma, I Ichikawa
Since transferrin is required for cellular proliferation, we investigated transferrin synthesis by a small cell lung cancer line (NCI-H510) that survives in serum-free media without added transferrin. Immunoassays for human transferrin demonstrated that these cells contained immunoreactive human transferrin. Immunofluorescence studies showed that the protein is expressed on the surface of cells, presumably bound to transferrin receptor. Media conditioned by NCI-H510 cells support proliferation of human leukemic cells that would not survive in media lacking transferrin. [35S]Methionine incorporation documented transferrin synthesis by NCI-H510 cells as well as three other small cell lines. Transferrin synthesis by NCI-H510 cells increased more than 10-fold when cells entered active phases of the cell cycle, and this increase was seen before large increases in transferrin-receptor expression. Further experiments examining the effects of agents that affect iron metabolism show that the addition of transferrin-iron or hemin to the media is associated with a more rapid initial rate of proliferation and lower rates of transferrin synthesis than control cells. Gallium salts, which inhibit iron uptake, inhibited proliferation of these cells. If the cells recovered from this effect, transferrin synthesis remained greatly increased compared to control. We conclude that transferrin synthesis by these malignant cells is ultimately related to an iron requirement for cellular proliferation. It appears that this synthesized transferrin acts as part of an important autocrine mechanism permitting proliferation of these cells, and perhaps permitting tumor cell growth in vivo in areas not well vascularized.
M Vostrejs, P L Moran, P A Seligman
The first adult case of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) deficiency is described. The impaired conversion of cortisol to cortisone (indicated by urinary cortisol and cortisone metabolites and failure to metabolize 11 alpha-[3H]cortisol to [3H]H2O), was associated with hypertension, hypokalemia, and suppression of the renin-angiotensin-aldosterone system. When established on a fixed Na+/K+ intake, dexamethasone, given orally, produced a natriuresis and potassium retention. Plasma renin activity became detectable. When hydrocortisone (10 mg daily s.c. for 4 d) was added, there was marked Na+ retention, a kaliuresis (urinary Na+/K+ falling from 1.2 to 0.15), with suppression of plasma renin activity and an increase in blood pressure. These changes were also seen with the subject on no treatment. Conversion of cortisone to cortisol was not affected. These results suggest that cortisol acts as a potent mineralocorticoid in 11 beta-OHSD deficiency. The major site for the oxidation of cortisol to cortisone is the kidney. In this patient congenital deficiency of 11 beta-OHSD results in high intrarenal cortisol levels which then act on renal type I mineralocorticoid receptors. This condition can be treated with dexamethasone, which suppresses cortisol secretion and binds to the type II glucocorticoid receptor. We suggest that 11 beta-OHSD exerts a critical paracrine role in determining the specificity of the type I receptor. In the normal state cortisol is converted by 11 beta-OHSD to cortisone which thus allows aldosterone to bind preferentially to the type I receptors in the kidney and gut. In this patient deficiency of 11 beta-OHSD results in high intrarenal cortisol concentrations that then bind to the type I receptor.
P M Stewart, J E Corrie, C H Shackleton, C R Edwards
In order to determine whether microvascular blood flow is important in the regulation of intra-islet cellular interactions, rat pancreata were isolated and perfused in vitro, both anterogradely or retrogradely, with and without anti-insulin or anti-somatostatin gamma-globulin. Expressed as percent change, anterograde infusion of insulin antibody increased efflux concentrations of glucagon (110 +/- 20%, P less than 0.0005) and somatostatin (2,112 +/- 73%, P less than 0.0005) above their respective control. Retrograde infusion of insulin antibody did not affect efflux concentrations of glucagon (P less than 0.50) or somatostatin (P less than 0.50). The anterograde infusion of anti-somatostatin antibody had no effect upon insulin (P less than 0.50) or glucagon (P less than 0.50) efflux concentrations, whereas retrograde anti-somatostatin antibody infusion produced immediate increases in efflux concentrations of both insulin (115 +/- 33%, P less than 0.0005) and glucagon (77 +/- 8%, P less than 0.0005). These results strongly suggest that (a) the vascular compartment is important in the regulation of intra-islet cellular interactions and further suggest that (b) the order of islet cellular perfusion and interaction is from the B cell core outward to the mantle, and (c) the mantle is further subordered with the majority of D cells downstream or distal to the majority of A cells. Thus, in the vascular compartment, B cells inhibit A-cell secretion and A cells stimulate D-cell secretion.
E Samols, J I Stagner, R B Ewart, V Marks
The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using 125I-labeled IGF-I demonstrated two high affinity specific binding sites (Kd 1.3 and 4.0 nM with maximal rate (Bmax) 200 and 500 fmol/mg protein, respectively). The exogenous addition of IGF-I, IGF-II, or insulin resulted in marked proliferation of human SCLC cells as evaluated using an in vitro growth assay. These peptides stimulated the growth of SCLC cell lines NCI-H82, NCI-H209, NCI-H345, and NCI-N417. The concentration of IGF-I producing maximal SCLC cell growth was 10-100-fold less than that of insulin or IGF-II, whereas the maximal growth stimulated by the optimal concentration of these peptides were similar. An MAb that specifically binds to the IGF-I receptor (but not to the insulin receptor) mediates a dose-dependent inhibition of cell growth in basal media as well as IGF-I, IGF-II, or insulin-supplemented media. The IGF-I receptor thus appears to be the common pathway for the mitogenic activity by IGF-I, IGF-II, and insulin for human SCLC cell lines. The demonstration of an IGF-I precursor molecule, specific IGF-I receptor binding, IGF-I-mediated growth stimulation, and inhibition of basal cell growth by an MAb to the IGF-I receptor suggests that an IGF-I-like molecule can function in vitro as an autocrine growth factor for human SCLC cell lines.
Y Nakanishi, J L Mulshine, P G Kasprzyk, R B Natale, R Maneckjee, I Avis, A M Treston, A F Gazdar, J D Minna, F Cuttitta
The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. The cochemotaxin attaches to sialic acid residues within the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the nature of the cochemotaxin and found that vitamin D-binding protein is the putative cochemotaxin. Vitamin D-binding protein enhanced the chemotactic activity of native C5a des Arg, but had no effect on the chemotactic activity of either native C5a or FMLP. Sialic acid prevented both enhancement by vitamin D-binding protein of the chemotactic activity of native C5a des Arg and formation of C5a des Arg-vitamin D-binding protein complexes, detected by molecular sieve chromatography. Furthermore, vitamin D-binding protein and cochemotaxin exhibited identical molecular weights, isoelectric points, antigenic reactivity, and amino acid composition.
H D Perez, E Kelly, D Chenoweth, F Elfman
Several serum proteins have been shown to be important in modulating leukocyte chemotaxis and inflammation. We investigated the possibility that the multifunctional serum protein Gc-globulin (vitamin D-binding protein) may also enhance the neutrophil chemotactic activity of complement-derived peptides. Purified Gc-globulin by itself did not induce chemotaxis of human neutrophils. However, as little as 0.01 nM Gc-globulin greatly enhanced the neutrophil chemotactic activity of C5a and its derivative, C5a des Arg over a wide concentration range. The effect was most pronounced at nonchemotactic doses of C5a (0.01 nM) and C5a des Arg (1 nM). Gc-globulin was unable to augment the neutrophil chemotactic activity of FMLP and leukotriene B4. This enhancing activity was not due to a nonspecific effect of anionic proteins since other purified serum proteins, of similar size and charge as Gc-globulin (alpha 1 acid glycoprotein, alpha 2 HS glycoprotein, alpha 2 histidine-rich glycoprotein), could not increase the chemotactic activity of C5a des Arg. Serum depleted of Gc-globulin by immunoaffinity chromatography totally lacked chemotactic enhancing activity for C5a des Arg. Gc-globulin-depleted serum activated with zymosan also had significantly less chemotactic activity than control- (sham-depleted) activated serum. Finally, radioiodinated C5a or C5a des Arg formed a 1:1 complex with purified Gc-globulin when analyzed by gel filtration chromatography. These results indicate that Gc-globulin is the major chemotactic enhancing factor in serum and may function as an up-regulator of the chemotactic activity of C5-derived peptides.
R R Kew, R O Webster
The clinical utility for establishing the immune phenotype in patients with non-Hodgkin's lymphoma is controversial. To help resolve this dilemma, we studied 104 consecutive patients with diffuse large cell lymphoma, the most common subtype of potentially curable non-Hodgkin's lymphomas. The presence or absence of the human class II histocompatibility antigen was determined using the monoclonal antibody anti-HLA-DR (Ia), and the results correlated with pretreatment clinical features and survival. We found that eight HLA-DR negative patients had similar pretreatment clinical characteristics compared with 96 HLA-DR positive patients, but HLA-DR negative patients had a significantly shorter survival duration compared with HLA-DR positive patients (P = 0.003 log-rank). The median survival of the HLA-DR negative patients was 0.5 years compared to 2.8 yr for the HLA-DR positive patients. No HLA-DR negative patient survived beyond 1.5 yr. A multi-variate analysis, adjusting for prognostic factors of known clinical significance, confirmed the importance of HLA-DR as a prognostic factor (P = 0.016). We conclude that determining the presence of HLA-DR is a relatively simple pretreatment study that identifies a small but important group of patients who are not curable using currently available combination chemotherapy.
T P Miller, S M Lippman, C M Spier, D J Slymen, T M Grogan