The comparison of measurements of fibronectin and lactoferrin in ejaculates from vasectomized men, subjects with functional deficiency or aplasia of the seminal vesicles, and reference subjects provided evidence that both the fibronectin and the lactoferrin in human seminal fluid originate from the seminal vesicles and the ampullae. The fibronectin is incorporated in the framework of the seminal gel formed during the immediate postejaculatory phase, whereas the lactoferrin remains in solution. In the seminal gel fibronectin is linked to its predominant structural protein, a high molecular weight seminal vesicle protein (semenogelin). Both the gel-bound fibronectin and semenogelin are progressively fragmented and solubilized by the abundant prostatic kallikrein-like protease (prostate-specific antigen) during and after seminal gel liquefaction. Lactoferrin remains essentially unaffected by the seminal proteases.
H Lilja, J Oldbring, G Rannevik, C B Laurell
In addition to immunologic derangement, hematological abnormalities have been reported in the majority of patients with acquired immunodeficiency syndrome (AIDS). In this study 15 patients with AIDS or AIDS-related complex (ARC) were evaluated for the in vitro growth of hemopoietic progenitor cells. In all patients a significant reduction of growth (mean +/- SEM) of colony-forming unit-granulocyte, erythrocyte, macrophage, (megakaryocyte) (CFU-GEM) (1.2 +/- 0.3), burst-forming unit-erythroid (BFU-E) (17 +/- 10), CFU-megakaryocyte (CFU-Mk) (1.7 +/- 0.6), and CFU-granulocyte-macrophage (CFU-GM) (35 +/- 10) was observed in comparison with normal controls. Depletion of T cells from the bone marrow before culture led to a significant increase in colony growth, which indicated an imbalance of the normally modulating T cell subsets. This increase was reversed by readdition of autologous T cells causing a decrease in colony growth to a degree, dependent on the T4 to T8 ratio. A decreased number of hemopoietic progenitor cells and/or a defective modulation of progenitor cell growth, normally carried out by T lymphocyte subsets, might be the cause of the hematological abnormalities in AIDS patients.
C C Stella, A Ganser, D Hoelzer
Follicle-stimulating hormone (FSH) beta, luteinizing hormone (LH) beta, and alpha subunit messenger RNA (mRNA) levels were examined in rats after castration and sex-steroid replacement. Subunit mRNAs were determined by blot hybridization using rat FSH beta genomic DNA, and alpha and LH beta complementary DNA (cDNA). Rat FSH beta mRNA is 1.7 kilobase in size. After ovariectomy, female FSH beta mRNA levels increased fourfold, whereas those of LH beta and alpha increased twenty- and eightfold, respectively. With estradiol, all subunits returned toward normal levels. Male LH beta and alpha mRNA levels rose eight- and fourfold, respectively, 40 d postcastration, but FSH beta mRNA levels increased minimally. After 7 d of testosterone propionate, LH beta and alpha mRNAs declined to normal levels, whereas FSH beta mRNA increased slightly. We conclude that in female rats FSH beta is negatively regulated by gonadal steroids, but to a lesser extent than LH beta or alpha mRNAs, and there is a differential regulation of FSH beta mRNA levels in males as compared with females at the time points examined.
S D Gharib, M E Wierman, T M Badger, W W Chin
Synthesis and secretion of colonic mucin glycoprotein species were assessed during in vitro culture of colonic mucosal explants. DEAE-cellulose chromatography of endogenously labeled mucin glycoproteins from explant tissue demonstrated the presence of six mucin species (I-VI) similar to those identified earlier in surgical specimens of human colonic tissue. The relative proportions of mucin species I-VI in tissue explants remained constant throughout a 30-h culture period. However, the proportional representation of the various mucin species in media was significantly different from that found in tissue, which suggests that some mucin species (I, II, and III) are differentially secreted, whereas others (IV and V) are retained within intracellular pools. Radiolabeled precursors were incorporated into mucin species I, II, and III at a 2.0-2.6-fold greater rate than their concentration in tissue, supporting the concept that these glycoproteins were both synthesized and secreted at a greater rate than species IV and V. Colonic mucosal explants from patients with ulcerative colitis showed greater than 90% reduction of species IV. However, the amount of species IV recovered from culture media of ulcerative colitis explants was comparable to normal controls. It appears that mucin species IV is differentially secreted rather than retained within intracellular pools in mucosa of patients with ulcerative colitis.
A C Smith, D K Podolsky
Recent studies have shown that the bicarbonate reabsorptive capacity of the proximal tubule is increased in metabolic acidosis. For net bicarbonate reabsorption to be regulated, there may be changes in the rate of apical H+ secretion as well as in the basolateral base exit step. The present studies examined the rate of Na+/H+ exchange (acridine orange method) and Na+/HCO3 cotransport (22Na uptake) in apical and basolateral membranes prepared from the rabbit renal cortex by sucrose density gradient centrifugation. NH4Cl loading was used to produce acidosis (arterial pH, 7.27 +/- 0.03), and Cl-deficient diet with furosemide was used to produce alkalosis (arterial pH, 7.51 +/- 0.02). Maximal transport rate (Vmax) of Na+/H+ antiporter and Na+/HCO3 cotransporter were inversely related with plasma bicarbonate concentration from 6 to 39 mM. Furthermore, the maximal transport rates of both systems varied in parallel; when Vmax for the Na+/HCO3 cotransporter was plotted against Vmax for the Na+/H+ antiporter for each of the 24 groups of rabbits, the regression coefficient (r) was 0.648 (P less than 0.001). There was no effect of acidosis or alkalosis on affinity for Na+ of either transporter. We conclude that both apical and basolateral H+/HCO3 transporters adapt during acid-base disturbances, and that the maximal transport rates of both systems vary in parallel during such acid-base perturbations.
T Akiba, V K Rocco, D G Warnock
The titers and isotypes of antibodies to specific proteins of the human immunodeficiency virus were determined by Western blot analysis of sera from 107 homosexual men. Antibody titers were generally lower in sera from patients with the acquired immunodeficiency syndrome (AIDS) and in sera from men whose condition subsequently progressed to AIDS than in sera from men who had not progressed to AIDS. We found no evidence of isotypic prominence or restriction of the antibody response. In multivariate analysis, lower levels of CD4 helper cells were most highly associated with progression to AIDS. Lower antibody titers to the envelope protein gp110, the core protein p24, and the reverse transcriptase enzyme p51/65 were also predictive of progression to AIDS independent of their association with CD4 cell levels. These data suggest that differences in antibody levels are not simply a consequence of severe immunodeficiency but may be markers for control of infection.
J S McDougal, M S Kennedy, J K Nicholson, T J Spira, H W Jaffe, J E Kaplan, D B Fishbein, P O'Malley, C H Aloisio, C M Black
Differences in the expression of Leu-1 (CD5) define two populations of recovering B cells after human marrow transplantation, Leu-1+ and Leu-1- B cells. The Leu-1+ B cells were polyclonal, of donor origin, and did not express detectable interleukin 2 receptor. Leu-1+ B cells generally appeared 2-4 wk after marrow grafting and often preceded the recovery of Leu-1- B cells. Acute and chronic graft vs. host disease (GvHD) resulted in the recovery of significantly fewer Leu-1+ B cells, whereas Leu-1- B cells were only decreased in acute GvHD. Multivariate analysis showed no significant effect of age, disease, prednisone or azathioprine, or ex vivo treatment of the marrow with anti-Leu-1 and complement on recovery of Leu-1+ and Leu-1- B cells, independent of the effects of GvHD. Leu-1+ B cells are a major lymphocyte population posttransplant. They may reflect a stage of differentiation of normal B cells or a separate B cell lineage.
J H Antin, K A Ault, J M Rappeport, B R Smith
We have investigated the inhibitory potential of prostaglandin E2 (PGE2) with respect to intracellular messengers implicated in the signaling system of T-lymphocyte activation pathway. Using the fluorescent indicator Quin 2, it is demonstrated that PGE2 inhibits the increase in cytosolic-free calcium concentration [Ca2+]i. Reconstitution of calcium mobilization in the presence of PGE2 by the calcium ionophore A23187 results in a partial restoration of both interleukin 2 (IL2) production and cell proliferation and has no effect on the inhibition of transferrin receptor expression. In contrast, the treatment of cell cultures with the tumor promotor 12.0 tetra decanoyl phorbol-13-acetate (TPA) abrogates the suppressor activity of PGE2. When T lymphocyte stimulation is provided by the combination of A23187 and TPA, the PGE2 inhibitory effect does not occur. These data also indicate that the down regulation of transferrin receptor by PGE2 is proximal to protein kinase C activation and is not associated with decreased expression of the functional IL2 receptor.
S Chouaib, R J Robb, K Welte, B Dupont
Lipoprotein lipase (LPL) activity in postheparin plasma of 38 normolipidemic volunteers was related to the magnitude of postprandial lipemia after a fat meal, to triglyceride content of high density lipoprotein2 (HDL2), to hepatic lipase (HL) activity, and to HDL2 levels. LPL activity correlated indirectly with lipemia, triglyceride content of HDL2, HL activity, and levels of HDL2 but not of HDL3. HL activity correlated directly with lipemia and indirectly with HDL2 levels. Triglyceride content of HDL2 correlated directly with lipemia and indirectly with HDL2 levels. In HDL2, abundance of apolipoprotein (apo) A-II and the apoA-I/apoA-II ratio varied widely. The latter correlated positively with LPL activity and HDL2 levels, and, inversely, with HL activity, lipemia, and triglyceride content of HDL2. The study suggests that HDL-cholesterol is not an independent parameter of lipid transport, but is strongly affected by triglyceride metabolism through lipolytic enzymes, as exemplified by postprandial lipemia that affect both composition and plasma levels of HDL2.
J R Patsch, S Prasad, A M Gotto Jr, W Patsch
A dose response curve for glucocorticoid-induced proximal and distal colonic cation transport in vivo was established in adrenalectomized rats. All doses (0.5-50 nmol/100 g body wt) stimulated sodium absorption. Distal sodium absorption did not saturate at dexamethasone levels that saturate the glucocorticoid receptor but also bind to greater than 35% of aldosterone receptors. Saturation of the pure glucocorticoid response occurred in both segments with RU26988, a synthetic glucocorticoid that does not occupy aldosterone receptors. Maximum velocities for pure glucocorticoid-induced sodium absorption were 15 and 16 mu eq/min per g dry tissue, and Michaelis constants (Km) were 4.2 and 4.6 X 10(-9) mol/liter for proximal and distal colon. Kms are similar to the dissociation constant for the colonic glucocorticoid receptor and too low for significant aldosterone receptor occupancy. Dexamethasone increased sodium absorption significantly within 30 min of injection, suggesting the response is not dependent on new protein synthesis. Similar time and dose responses in proximal and distal colon suggest glucocorticoids stimulate the same pathway in both segments.
C P Bastl
Morphological and biochemical studies of human colony-forming units-erythroid (CFU-E) have been hindered by their extreme rarity. Since burst-forming units-erythroid (BFU-E) develop into CFU-E, we used normal human blood BFU-E to generate large numbers of highly purified CFU-E in vitro. Using density centrifugation, sheep erythrocyte rosetting, surface immunoglobulin-positive cell depletion, adherence to plastic, and negative panning with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0.368%, a 22-fold purification with a 43% yield. The panned cells were cultured in methylcellulose with recombinant erythropoietin (rEp) and conditioned medium for 9 d. These cells were then collected and CFU-E were further purified using adherence and density centrifugation. This yielded almost 10(7) erythroid colony forming cells with a purity of 70 +/- 18%. Analysis of these cells by light and electron microscopy showed 94% erythroid cells. The prominent cell was a primitive blast with high nuclear/cytoplasmic ratio, dispersed nuclear chromatin and a distinct large nucleolus. The relation between the number of erythroid colonies and the number of day 9 cells plated in plasma clots was a straight line through the origin with a maximum number of erythroid colonies at 1 U/ml of rEp and no erythroid colonies without rEp. Specific binding with 125I-rEp showed that 60% of the binding was inhibited by excess pure erythropoietin (Ep), but not by albumin, fetal calf serum, and a variety of growth factors or glycoproteins. By days 12-13 of cell culture, when the progenitor cells matured to late erythroblasts, specific binding markedly declined. In this study, human CFU-E have been isolated in sufficient purity to characterize the morphology of these rare cells and in sufficient numbers to measure specific binding of Ep.
K Sawada, S B Krantz, J S Kans, E N Dessypris, S Sawyer, A D Glick, C I Civin
Because of a close association between platelets and macrophages in early fatty streak lesions, the hypothesis was tested that platelets contribute to lesion progression by directly enhancing macrophage cholesteryl ester (CE) accumulation. Both the rate of cholesterol esterification and the accumulation of CE were increased within 24 h of the co-culture of adherent macrophages with platelets. Maximum increases in esterification and CE accumulation were observed within 3 to 4 d of culture and were greater than 10-fold over controls. Optimum accumulation of CE by 5 X 10(5) was obtained with 5 X 10(8) autologous platelets. When similar amounts of free cholesterol were supplied with platelets, red blood cells (RBC), RBC ghosts, or sonicated RBC, only platelets enhanced macrophage CE accumulation, which indicates specificity for platelets. Products released from platelets 30 min after thrombin stimulation were active as well. The results suggest that platelets and/or substances shed by activated platelets are potent mediators of macrophage CE accumulation.
L K Curtiss, A S Black, Y Takagi, E F Plow
Regulatory sequences of the human fetal gamma-globin gene were studied by constructing composite gamma/beta globin promoters and comparing their function to that of intact beta promoters in human K562 cells. The beta-globin gene with either 1,600 or 127 basepairs of beta promoter sequence was not expressed after stable introduction into K562 cells, consistent with the known inactivity of the beta-globin gene in these cells. In contrast, a gamma/beta promoter composed of a gamma fragment spanning positions -408 to -137 joined to the 127-bp beta promoter was able to drive the beta-globin gene. The gene appeared to be inducible with hemin. A zeta-globin 5' flanking fragment also activated the beta promoter. The function of a series of composite gamma/beta promoters was then assessed by their ability to drive directly the neomycin resistance gene, again in stably transformed cells. The -408 to -137 gamma fragment activated the beta promoter in an orientation-specific manner in this assay. Deletion analysis showed that regulatory sequences were present between positions -259 and -137 of the fetal gamma-globin gene flanking region.
H J Lin, N P Anagnou, T R Rutherford, T Shimada, A W Nienhuis
We measured H2O2 release by human alveolar macrophages (AM) from normals and sarcoid patients in suspension immediately after bronchoalveolar lavage in the presence and absence of the triggering agent, phorbol myristate acetate (PMA). AM from 11 sarcoid patients produced a mean (+/- SE) of 21.7 +/- 2.3 and 5.9 +/- 3.4 nmol H2O2/10(6) macrophages in the presence and absence of PMA, respectively. By contrast, AM from normals (n = 6) produced 9.8 +/- 1.7 and 1.6 +/- 0.7 nmol H2O2/10(6) macrophages with and without PMA, respectively. Macrophage activation, as monitored by H2O2 production, did not correlate with the angiotensin-converting enzyme levels, the result of gallium-67 scans, or the percent of lymphocytes in the bronchoalveolar lavage. To determine whether AM from normals could be stimulated to increase their H2O2 production to the level seen in patients with sarcoid, we measured H2O2 released by adherent AM after incubation in each of four potential activating agents: recombinant interferons alpha A, beta, gamma (rIFN alpha A, rIFN beta, and rIFN gamma, respectively), and 1,25-dihydroxyvitamin D3. H2O2 release in the range seen in sarcoid patients could be induced in PMA-triggered AM from normals by rIFN gamma in a time- (t1/2 approximately 1 d) and dose-dependent fashion (threefold increase, EC50 5 antiviral U/ml) and by rIFN alpha A and rIFN beta at higher concentrations, but not by 1,25-dihydroxyvitamin D3.
A O Fels, C F Nathan, Z A Cohn
In order to directly determine the amount of label exchange that occurs in the tricarboxylic cycle from labeled alanine and lactate after the ingestion of a glucose load [1-13C]glucose was administered by continuous intraduodenal infusion to awake catheterized rats to achieve steady state jugular venous glycemia (160 mg/dl) for 180 min. Liver was freeze-clamped at 90 and 180 min, and perchloric acid extracts of the liver were subjected to 13C and 1H nuclear magnetic resonance analysis. Dilution in the oxaloacetate pool was determined by comparing the intrahepatic 13C enrichments of C2, C3 positions of glutamate with the C2, C3 positions of alanine and lactate. In addition steady state flux equations were derived for calculation of relative fluxes through pyruvate dehydrogenase/TCA cycle flux and pyruvate kinase flux/total pyruvate utilization. After glucose ingestion in a 24-h fasted rat direct conversion of glucose was responsible for 34% of glycogen. The intrahepatic dilution factor for labeled pyruvate in the oxaloacetate pool was 2.4. Using this factor, alanine and lactate contributed approximately 55% to glycogen formation. Pyruvate dehydrogenase flux ranged between 24 and 35% of total acetyl-coenzyme A (CoA) production and pyruvate kinase flux relative to total pyruvate utilization was approximately 40%.
G I Shulman, L Rossetti, D L Rothman, J B Blair, D Smith
Human T lymphotropic virus type I (HTLV-I) is an exogenous RNA tumor virus etiologically linked to adult T cell leukemia and related diseases. In this paper, we describe that two 2',3'-dideoxynucleoside analogues, erythro 3'-azido-2',3'-dideoxythymidine (also called azidothymidine) and 2',3'-dideoxycytidine can inhibit the infectivity of HTLV-I against helper/inducer T cells in vitro. Both 2',3'-dideoxynucleoside analogues inhibited the overgrowth of target T cells, which was a consequence of virally mediated transformation, when they were exposed to the virus and cultured with the compounds. A profound decrease in the expression of HTLV-I gag-proteins was also observed. Moreover, we observed that the amount of proviral DNA detected in cellular DNA from the target T cells was substantially reduced when the cells were protected by the compounds against the virus and that at certain concentrations of the compounds the synthesis of viral DNA was completely suppressed. These results may be of value in developing a new pharmacological strategy for preventing the replication and possibly blocking the transmission of HTLV-I and related retroviruses in human beings.
S Matsushita, H Mitsuya, M S Reitz, S Broder
Binding of various 125I-lipoproteins to hepatic receptors was studied on cultured human hepatoma cells (Hep G2). Chylomicrons, isolated from a chylothorax, chylomicron remnants, hypertriglyceridemic very low-density lipoproteins, normotriglyceridemic very low-density lipoproteins (NTG-VLDL), their remnants, low-density lipoproteins (LDL), and HDL-E (an Apo E-rich high-density lipoprotein isolated from the plasma of a patient with primary biliary cirrhosis) were bound by high-affinity receptors. Chylomicron remnants and HDL-E were bound with the highest affinity. The results, obtained from competitive binding experiments, are consistent with the existence of two distinct receptors on Hep G2 cells: (a) a remnant receptor capable of high-affinity binding of triglyceride-rich lipoproteins and HDL-E, but not of Apo E free LDL, and (b) a LDL receptor capable of high-affinity binding of LDL, NTG-VLDL, and HDL-E. Specific binding of Apo E-free LDL was completely abolished in the presence of 3 mM EDTA, indicating that binding to the LDL receptor is calcium dependent. Specific binding of chylomicron remnants was not inhibited by the presence of even 10 mM EDTA. Preincubation of the Hep G2 cells in lipoprotein-containing medium resulted in complete suppression of LDL receptors but did not affect the remnant receptors. Hep G2 cells seem to be a suitable model for the study of hepatic receptors for lipoprotein in man.
F Krempler, G M Kostner, W Friedl, B Paulweber, H Bauer, F Sandhofer
We studied two groups of rats acutely loaded with bicarbonate, control rats on a standard diet and rats kept on a K-free diet for 3 wk. Compared with controls, K-depleted rats had reduced fractional excretion of bicarbonate despite their elevated filtered bicarbonate load. Distal bicarbonate reabsorption increased in K-depleted rats. In the presence of almost identical early distal bicarbonate loads (481 +/- 40 pmol/min in controls and 444 +/- 50 pmol/min in K depletion), distal bicarbonate reabsorption was significantly enhanced in K depletion (247 +/- 17 pmol/min) as compared with controls (179 +/- 18 pmol/min). These values are significantly different from each other, and both are severalfold higher than bicarbonate reabsorption in nonloaded conditions. In conclusion, distal bicarbonate reabsorption is load dependent, and distal bicarbonate reabsorption is stimulated in K depletion.
G Capasso, P Jaeger, G Giebisch, V Guckian, G Malnic
We have compared the capillary density and muscle fiber type of musculus vastus lateralis with in vivo insulin action determined by the euglycemic clamp (M value) in 23 Caucasians and 41 Pima Indian nondiabetic men. M value was significantly correlated with capillary density (r = 0.63; P less than or equal to 0.0001), percent type I fibers (r = 0.29; P less than 0.02), and percent type 2B fibers (r = -0.38; P less than 0.003). Fasting plasma glucose and insulin concentrations were significantly negatively correlated with capillary density (r = -0.46, P less than or equal to 0.0001; r = -0.47, P less than or equal to 0.0001, respectively). Waist circumference/thigh circumference ratio was correlated with percent type 1 fibers (r = -0.39; P less than 0.002). These results suggest that diffusion distance from capillary to muscle cells or some associated biochemical change, and fiber type, could play a role in determining in vivo insulin action. The association of muscle fiber type with body fat distribution may indicate that central obesity is only one aspect of a more generalized metabolic syndrome. The data may provide at least a partial explanation for the insulin resistance associated with obesity and for the altered kinetics of insulin action in the obese.
S Lillioja, A A Young, C L Culter, J L Ivy, W G Abbott, J K Zawadzki, H Yki-Järvinen, L Christin, T W Secomb, C Bogardus
Although 1,25-dihydroxyvitamin D3 stimulates osteoclastic bone resorption in vivo and in organ culture, the mechanism by which it effects this stimulation is unknown. We have recently found that the agent does not stimulate resorption by osteoclasts mechanically disaggregated from bone and incubated on slices of cortical bone. This suggests that the osteoclasts were removed by disaggregation from the influence of some cell type, present in intact bone, that mediates hormone responsiveness. We therefore tested the ability of osteoblastic cells derived from neonatal rat calvariae and of cloned, hormone-responsive osteosarcoma cells (UMR106) to restore hormone responsiveness to unresponsive populations of osteoclasts. We found that osteoblastic cells from both sources induced a two- to fourfold stimulation of osteoclastic bone resorption in the presence of 1,25-dihydroxyvitamin D3. Stimulation was observed at concentrations of 10(-10) M and above. Actinomycin D and cycloheximide did not affect bone resorption by osteoclasts incubated alone, but abolished the capacity of osteoblastic cells to stimulate osteoclastic resorption in the presence of 1,25-dihydroxyvitamin D3. When calvarial cells or osteoblastlike UMR cells were incubated with the hormone, they produced a factor in cell-free supernatants that stimulated bone resorption by disaggregated osteoclasts. These experiments suggest that 1,25-dihydroxyvitamin D3 stimulates bone resorption through a primary action on osteoblastic cells, that are induced by the hormone to produce a factor that stimulates osteoclastic bone resorption.
P M McSheehy, T J Chambers
Interleukin 1 (IL-1) is a family of polypeptides initially found to be produced by activated monocytes and macrophages that mediate a wide variety of cellular responses to injury and infection. Epidermal epithelial cells (keratinocytes) produce "epidermal cell-derived thymocyte activating factor" or ETAF, which has been recently shown to be identical to IL-1. Human epidermis is normally exposed to significant amounts of solar ultraviolet radiation. Certain ultraviolet wavelengths (UVB, 290-320 nm) are thought to be responsible for most of the immediate and long-term pathological consequences of excessive exposure to sunlight. In this study, we asked whether exposure to UVB irradiation induced IL-1 gene expression in cultured human keratinocytes. Cultured human keratinocytes contain detectable amounts of IL-1 alpha and beta mRNA and protein in the absence of apparent stimulation; these levels could be significantly enhanced 6 h after exposure to 10 ng/ml of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Exposure to UVB irradiation with an emission spectrum comparable to that of sunlight (as opposed to that of an unfiltered artificial UV light source) significantly increased the steady state levels IL-1 alpha and beta mRNA in identical populations of human keratinocytes. This was reflected in the production of increased IL-1 activity by these cultures in vitro. In the same cell population, exposures to UVB irradiation did not alter the level of actin mRNA; therefore, the effect of UV irradiation on IL-1 represents a specific enhancement of IL-1 gene expression. Local increases of IL-1 may mediate the inflammation and vasodilation characteristic of acute UVB-injured skin, and systemic release of this epidermal IL-1 may account for fever, leukocytosis, and the acute phase response seen after excessive sun exposure.
T S Kupper, A O Chua, P Flood, J McGuire, U Gubler
The insulin effect following hypoglycemia was studied with the euglycemic clamp technique in seven healthy subjects. Following an initial euglycemic clamp hypoglycemia was induced and after glucose recovery a second clamp was performed. Glucose production (Ra) and utilization (Rd) were studied with [3-3H]glucose. Each subject was studied four times; during infusion of placebo, propranolol, somatostatin, and a control study where hypoglycemia was prevented. Hypoglycemia induced an insulin resistance with a lower steady state glucose infusion rate following the hypoglycemia during placebo as compared to the control study (2.5 +/- 0.5 and 4.8 +/- 1.0 mg/kg min, respectively, P less than 0.05). The insulin resistance was due to an attenuated insulin effect on both inhibition of Ra (impaired by 37%) and stimulation of Rd (impaired by 61%). The insulin-antagonistic effect was completely prevented by propranolol but only partly by somatostatin. Thus, early posthypoglycemic insulin resistance (2.5-3.5 h after hypoglycemia) is a sustained effect mainly due to beta-adrenergic stimulation.
S Attvall, B M Eriksson, J Fowelin, H von Schenck, I Lager, U Smith
This study tested the hypothesis that interactions of endogenous angiotensin II (AII) with the noradrenergic neuroeffector junction are important in renin-dependent hypertension. In the in situ blood-perfused rat mesentery, in normal rats exogenous AII potentiated mesenteric vascular responses to periarterial (sympathetic) nerve stimulation (PNS) more than vascular responses to exogenous norepinephrine (NE). In 2-kidney-1-clip (2K-1C) rats with renovascular hypertension mesenteric vascular responses to PNS and NE were greater than in sham-operated rats, and renovascular hypertension mimicked the effects of exogenous AII with respect to enhancing responses to PNS more than responses to NE. In 2K-1C rats, but not in sham-operated rats, 1-Sar-8-Ile-AII markedly suppressed vascular responses to PNS, without influencing responses to NE. Finally, 1-Sar-8-Ile-AII attenuated sympathetic nerve stimulation-induced neuronal spillover of NE in 2K-1C rats, but not in sham-operated rats. These data indicate that renovascular hypertension enhances noradrenergic neurotransmission, and that this enhancement is mediated in part by AII-induced facilitation of NE release.
J B Zimmerman, D Robertson, E K Jackson
The Lp(a) lipoprotein represents a quantitative genetic trait. It contains two different polypeptide chains, the Lp(a) glycoprotein and apo B-100. We have demonstrated the Lp(a) glycoprotein directly in human sera by sodium dodecyl sulfate-gel electrophoresis under reducing conditions after immunoblotting using anti-Lp(a) serum and have observed inter- and intraindividual size heterogeneity of the glycoprotein with apparent molecular weights ranging from approximately 400,000-700,000 D. According to their relative mobilities compared with apo B-100 Lp(a) patterns were categorized into phenotypes F (faster than apo B-100), B (similar to apo B-100), S1, S2, S3, and S4 (all slower than apo B-100), and into the respective double-band phenotypes. Results from neuraminidase treatment of isolated Lp(a) glycoprotein indicate that the phenotypic differences do not reside in the sialic acid moiety of the glycoprotein. Family studies are compatible with the concept that Lp(a) glycoprotein phenotypes are controlled by a series of autosomal alleles (Lp[a]F, Lp[a]B, Lp[a]S1, Lp[a]S2, Lp[a]S3, Lp[a]S4, and Lp[a]0) at a single locus. Comparison of Lp(a) plasma concentrations in different phenotypes revealed a highly significant association of phenotype with concentration. Phenotypes B, S1, and S2 are associated with high and phenotypes S3 and S4 with low Lp(a) concentrations. This suggests that the same gene locus is involved in determining Lp(a) glycoprotein phenotypes and Lp(a) lipoprotein concentrations in plasma and is the first indication for structural differences underlying the quantitative genetic Lp(a)-trait.
G Utermann, H J Menzel, H G Kraft, H C Duba, H G Kemmler, C Seitz
Clearance of immune complexes made of antiinsulin antibodies and 123I-insulin was studied with scintillation scanning in anesthetized rats. Complexes made with purified guinea pig antiinsulin IgG2 (cytophilic isotype) were rapidly cleared by the liver whereas those made with IgG1 remained in the plasma, as did 123I-labeled IgG1 or IgG2 of control animals. Hepatic clearance of insulin-antiinsulin IgG complexes was not inhibited by either an excess of insulin or decomplementation, thereby ruling out interaction with insulin and C3b receptors. Insulin and guinea pig antiinsulin serum or its purified IgG isotypes formed large aggregates exceeding 5 IgG. Antiinsulin antibodies of diabetics, mostly IgG1 and IgG3 (cytophilic isotypes), formed complexes that either remained in plasma (small aggregates) or were cleared by the liver (large aggregates). In conclusion, clearance of insulin-antiinsulin IgG complexes is probably mediated by Fc gamma receptors on macrophages and requires cytophilic subclass composition and formation of large IgG aggregates.
F Sodoyez-Goffaux, J C Sodoyez, M Koch, N Dozio, E R Arquilla, B McDougall, C J De Vos, R von Frenckell
Three preparations of purified von Willebrand factor (vWF), obtained from unrelated patients affected by type IIB von Willebrand disease, were found to have normal sialic acid content (between 129 and 170 nmol/mg of vWF, as compared with 158 +/- 17 nmol/mg in four normal preparations) and to induce platelet aggregation in the presence of physiologic levels of divalent cations and without addition of ristocetin. A monoclonal antibody that blocks the vWF binding domain of the platelet glycoprotein (GP)Ib caused complete inhibition of IIB vWF-induced aggregation. In contrast, a monoclonal antibody that blocks the receptor for adhesive proteins on the platelet GPIIb/IIIa complex failed to inhibit the initial response of platelets to high concentrations of IIB vWF. Moreover, IIB vWF caused agglutination of formalin-fixed platelets that was blocked only by the anti-GPIb antibody, suggesting that the binding of vWF to GPIb, even in the absence of ristocetin, results in platelet-platelet interaction that is followed by exposure of the GPIIb/IIIa receptors for adhesive proteins. Endogenous ADP, normally active platelet metabolism and fibrinogen binding to GPIIb/IIIa were necessary for maximal and irreversible platelet aggregation. In the absence of fibrinogen, however, aggregation was mediated by vWF binding to GPIIb/IIIa. A 52/48-kD tryptic fragment containing the GPIb binding domain of normal vWF completely blocked the aggregation induced by all three IIB vWF preparations. The present study defines in detail the mechanisms involved in IIB vWF-induced platelet aggregation. Moreover, it establishes that the GPIb binding domain of normal and IIB vWF are closely related and that desialylation is not required for the direct interaction of IIB vWF with GPIb.
L De Marco, M Mazzuccato, M Grazia Del Ben, U Budde, A B Federici, A Girolami, Z M Ruggeri
The primary structure of apolipoprotein E (apo E) was investigated in seven type III hyperlipoproteinemic patients with the apo E-2/2 phenotype. Six of the patients had identical two-dimensional tryptic peptide maps. These differed from the normal apo E3 map by the altered mobility of a single peptide. Amino acid analysis and sequencing showed that apo E2 in these patients had a substitution of 158 Arg----Cys. The presence of this mutation in six of the seven type III patients confirms that this is the most common form of apo E2. The seventh type III patient had a unique map with a new peptide resulting from a substitution of 136 Arg----Ser. He was heterozygous for this and for the more common apo E2 (158 Arg----Cys) variant. His very low-density lipoprotein contained approximately five times more apo E2 (136 Arg----Ser) than apo E2 (158 Arg----Cys), as determined by cysteamine treatment and peptide mapping. This new apo E2 mutant thus appears to contribute significantly to the patient's hyperlipidemia.
M R Wardell, S O Brennan, E D Janus, R Fraser, R W Carrell
Hyperthyroidism caused by nodular goiters is a common disease of aging cats. Growth and iodine metabolism were studied by autoradiography in normal and hyperfunctioning thyroid tissue obtained from cats injected with 125I before surgery, and in xenografts, grown in nude mice, after double-labeling with 131I and [3H]thymidine. Hyperthyroid cat goiters contain single or multiple hyperplastic nodules, consisting of highly cellular tissue with an iodine metabolism exceeding that of the surrounding normal tissue. Xenografts of hyperplastic hot tissue in thyroxine-treated nude mice retain their original histologic pattern and continue to accumulate radioiodine intensely. Autoradiographs assessed for [3H]thymidine incorporation reveal autonomously proliferating follicular cells within the hyperplastic foci but not within the normal tissue. Administration of sera from donor cats into host mice fails to stimulate the xenografts. Neither hyperfunction nor growth of toxic cat goiters depends on extrathyroidal stimulators. The basic lesion appears to be an excessive intrinsic growth capacity of some thyroid cells.
H J Peter, H Gerber, H Studer, D V Becker, M E Peterson
Chronic inflammatory myositis similar to human polymyositis occurs in mice after infection with a strain of Coxsackievirus B 1 (CVB 1). To investigate the role of T cells in the pathogenesis of this disorder, we compared disease expression in T cell-deficient athymic nude (nu/nu) mice and heterozygotes (nu/+) with normal T cell function. Acute infectious myositis occurred in nu/nu and nu/+ mice. Chronic (greater than 21 d postinfection) weakness and myositis, however, developed only in nu/+. Resistance to disease in nu/nu mice was not explained by insusceptibility to infection; the amount of virus lethal for 50% of mice and virus replication were comparable in both groups. Additionally, anti-CVB 1 antibody production was similar in both groups. Reconstitution of infected nu/nu mice with spleen cells from normal mice resulted in disease. These results demonstrate that chronic weakness after infection with this virus is not simply a sequela of acute myonecrosis and suggest that T cells play a pivotal role in the pathogenesis of chronic myositis.
S R Ytterberg, M L Mahowald, R P Messner
The kinetics of apolipoprotein (apo) B-100 in particles containing apo E (B,E particles) or lacking apo E (B particles) were studied in Watanabe heritable hyperlipidemic (WHHL) rabbits deficient in low density lipoprotein (LDL) receptors, and compared with those of normal rabbits after injection of radioiodinated very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and LDL. In both groups results of kinetic modeling were consistent with the hypothesis that all apo B enters the plasma in VLDL, mainly as B,E particles, followed by delipidation and partial conversion to IDL and LDL, with concomitant conversion of some B,E particles to B particles. In WHHL rabbits, production of VLDL apo B was reduced by 40%, but LDL production was increased threefold. Defective removal of B,E and B particles in all three lipoprotein classes, coupled with preserved processes of delipidation, can account for the observed increases in the concentration of apo B (threefold in VLDL, fivefold in IDL, and twenty-twofold in LDL) in WHHL rabbits.
N Yamada, D M Shames, R J Havel
Hageman factor (HF, Factor XII) is activated by glass, collagen, and ellagic acid, and initiates blood coagulation via the intrinsic pathway. C1q inhibits collagen-induced platelet aggregation and adherence of platelets to glass, effects attributable to the collagen-like region of C1q. We examined the actions of C1q on HF activation. Incubation of C1q with HF before addition of HF-deficient plasma extended the activated partial thromboplastin time. Similarly, when glass tubes were coated with C1q before testing, the partial thromboplastin time of normal plasma was increased. C1q reduced the activation of HF by ellagic acid, as measured by the release of p-nitroaniline from the synthetic substrate H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride, an effect inhibited by monoclonal anti-human C1q murine IgG and by digestion of C1q by collagenase. Thus, C1q inhibits activation of HF in vitro in clot-promoting and amidolytic assays and suggests a regulatory mechanism for the inhibition of coagulation.
E H Rehmus, B M Greene, B A Everson, O D Ratnoff
To investigate the role of thyroxine-binding globulin (TBG) and albumin in the availability of thyroid hormones to peripheral tissues, comprehensive kinetic studies of thyroxine (T4) and triiodothyronine (T3) were carried out in eight subjects with familial dysalbuminemic hyperthyroxinemia (FDH), in four subjects with inherited TBG excess, and in 15 normals. In high-TBG subjects, the reduction of T4 and T3 plasma clearance rates (by 51% and 54%, respectively) was associated with normal daily productions; T4 and T3 distribution volumes were significantly reduced. In FDH subjects T4 clearance was less reduced (by 31%) than in high TBG; consequently T4 production rate was significantly increased (by 42%); T4 and T3 distribution volumes and T3 clearance rate were unchanged. Increased T3 peripheral production in FDH (by 24%) indicates that T4 bound to abnormal albumin is more available to tissues than T4 carried by TBG, thus suggesting an important role of albumin in T4 availability to the periphery.
R Bianchi, G Iervasi, A Pilo, F Vitek, M Ferdeghini, F Cazzuola, G Giraudi
Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min. Human alveolar and murine peritoneal macrophages, cells that had previously diapedised, could not be induced to upregulate Mac-1 or p150,95. Detergent permeabilization studies in monocytes indicated that these proteins were stored in internal latent pools, which were reduced upon stimulation. Electron microscopy utilizing rabbit antiserum against p150,95 revealed these proteins on the plasma membrane, and in intracellular vesicles and peroxidase negative granules. Together with other functional studies, these findings suggest that the mobilization of Mac-1 and p150,95 from an intracellular compartment to the plasma membrane regulates the monocyte's ability to adhere and diapedese.
L J Miller, D F Bainton, N Borregaard, T A Springer
We examined alpha-, beta-, and gamma-T cell receptor (TCR) gene activation within acute lymphoblastic leukemias (ALLs) that represent early stages of B and T cell development. We wished to determine if TCR rearrangement and expression was lineage restricted, showed any developmental hierarchy, or could identify new subsets of T cells. Rearrangement of gamma and beta TCR genes occurred early in development but in no set order, and most T-ALLs (22/26) were of sufficient maturity to have rearranged both genes. T-ALLs preferentially rearranged C gamma 2 versus the C gamma 1 complex; no preference within the beta locus was apparent. Once rearranged, the beta TCR continued to be expressed (11/13), whereas the gamma TCR was rarely expressed (3/14). The alpha TCR was expressed only in more mature T-ALLs (8/14) that usually displayed T3. The 3A-1 T cell associated antigen appeared earliest in development followed by T11 and T3. Within pre-B cell ALL a higher incidence of lineage spillover was noted for gamma TCR rearrangements (8/17) than for beta rearrangements (3/17). This also contrasts with the only occasional rearrangement of immunoglobulin (Ig) heavy chains (3/25) in T-ALL. However, in pre-B ALL the pattern of gamma TCR usage was distinct from that of T cells, with the C gamma 1 complex utilized more frequently. Almost all ALLs could be classified as pre-B or T cell in type by combining Ig and TCR genes with monoclonal antibodies recognizing surface antigens, although examples of lineage duality were noted. Unique subpopulations of cells were discovered including two genetically uncommitted ALLs that failed to rearrange either Ig or TCR loci. Moreover, two T lymphoblasts were identified that possessed the T3 molecule but failed to express alpha plus beta TCR genes. These T-ALLs may represent a fortuitous transformation of T cell subsets with alternative T3-Ti complexes.
C A Felix, J J Wright, D G Poplack, G H Reaman, D Cole, P Goldman, S J Korsmeyer
Hereditary spherocytosis (HS) is an inherited disorder of erythrocyte shape associated with spectrin deficiency and hemolytic anemia. In a subset of patients with the autosomal dominant form of HS, spectrin displays a reduced capacity to bind protein 4.1 and, therefore, actin; both functions that are critical to the membrane skeleton. A specific structural defect has not been identified in the spectrin from these patients. Chymotryptic digestion of the isolated spectrin chains shows impaired cleavage of the distal peptide of the beta subunit, the beta IV domain. In previous work, we have shown that mild oxidation markedly diminishes the binding capacity of normal spectrin for protein 4.1. Here we observe that chemical reduction of freshly isolated, untreated HS spectrin dramatically improves its function. Thus, a primary structural defect in the beta subunit of spectrin in this subtype of HS may lead to oxidant sensitivity, and secondarily, to a functional defect in the binding of spectrin to protein 4.1 and actin.
P S Becker, J S Morrow, S E Lux
Weight-maintaining fat-rich, "prudent," carbohydrate-rich, as well as energy-restricted diets (300 kcal/d) were fed in succession for 7 d to 12 healthy males of ideal body weight under metabolic ward conditions. At the end of each period isolated fat cells were prepared from subcutaneous abdominal adipose tissue and incubated in vitro in the absence or presence of adenosine deaminase, either alone or in combination with various lipolytic or antilipolytic hormones and agents. Variations in total energy intake and dietary composition had characteristic and specific effects on fat cell lipolysis in vitro. High carbohydrate and prudent diets resulted in low rates of nonstimulated glycerol release and impaired insulin action in the presence of adenosine deaminase (320 mU/ml). High-fat and energy restricted diets were characterized by high rates of nonstimulated glycerol release. Sensitivity of antilipolysis to insulin and prostaglandin E2 was 10 to 200 times lower respectively on energy-restricted than on fat-rich diets. The effects of alpha 2- and beta-adrenergic catecholamines and of N6-phenylisopropyladenosine were not affected by the preceding diets.
H Kather, E Wieland, A Scheurer, G Vogel, U Wildenberg, C Joost
The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on hematopoietic reconstitution after autologous bone marrow transplantation was evaluated in a primate model. Animals were given a continuous intravenous infusion of recombinant human GM-CSF for several days both before and after transplantation or only after the transplant procedure. Marrow ablation was accomplished by total body irradiation. In both groups of animals, the neutrophil count reached 1,000/mm3 by 8-9 d posttransplant compared with an interval of 17 and 24 d for two concurrent controls. After withdrawal of GM-CSF, neutrophil counts fell to values comparable to those observed in untreated controls. Accelerated recovery of platelet production was also observed in four of the five animals. Two additional animals were initially given GM-CSF several weeks posttransplantation because of inadequate engraftment. Prompt and sustained increases in neutrophil and platelet counts were observed. We conclude that GM-CSF may be useful in accelerating bone marrow reconstitution.
A W Nienhuis, R E Donahue, S Karlsson, S C Clark, B Agricola, N Antinoff, J E Pierce, P Turner, W F Anderson, D G Nathan
Relationship between the efficiency of cholesterol absorption and apolipoprotein E (apoE) phenotype was studied in a random sample of middle-aged Finnish men. Subjects that were either heterozygous or homozygous for the allele epsilon 2 absorbed less and synthesized more cholesterol than those with the phenotype E4/3 and E4/4, the values for the individuals with the most frequent phenotype E3/3 (56% of the population sample) falling in between. Among the whole study group, the sum of the subscripts of apoE phenotype (e.g., E2/3 = 5) was correlated positively with the fractional absorption of cholesterol (r = 0.40; P less than 0.05) and negatively with the serum level of lathosterol, a cholesterol precursor sterol reflecting the activity of cholesterol synthesis (r = -0.48; P less than 0.01). Thus, apoE polymorphism appears to affect the efficiency of cholesterol absorption and may by this mechanism contribute to the variation in plasma total and low density lipoprotein cholesterol concentration.
Y A Kesäniemi, C Ehnholm, T A Miettinen
The specificity of serpin superfamily protease inhibitors such as alpha 1-antitrypsin or C1 inhibitor is determined by the amino acid residues of the inhibitor reactive center. To obtain an inhibitor that would be specific for the plasma kallikrein-kinin system enzymes, we have constructed an antitrypsin mutant having Arg at the reactive center P1 residue (position 358) and Ala at residue P2 (position 357). These modifications were made because C1 inhibitor, the major natural inhibitor of kallikrein and Factor XIIa, contains Arg at P1 and Ala at P2. In vitro, the novel inhibitor, alpha 1-antitrypsin Ala357 Arg358, was more efficient than C1 inhibitor for inhibiting kallikrein. Furthermore, Wistar rats pretreated with alpha 1-antitrypsin Ala357 Arg358 were partially protected from the circulatory collapse caused by the administration of beta-Factor XIIa.
M Schapira, M A Ramus, B Waeber, H R Brunner, S Jallat, D Carvallo, C Roitsch, M Courtney
The mechanism of action of erythropoietin is thought to require specific interaction with the target cell surface and involve alteration of cellular calcium metabolism. Using the rabbit reticulocyte membrane as a model of the immature red cell membrane, we investigated the effects of human recombinant erythropoietin on membrane Ca2+-ATPase (calcium pump) activity in vitro. Erythropoietin in a concentration range of 0.025 to 3.0 U/ml progressively decreased membrane Ca2+-ATPase activity by up to 64% (P less than 0.01). These concentrations have been shown by others to stimulate in vitro erythroid growth. The action of erythropoietin on reticulocyte Ca2+-ATPase required an incubation time of 1 h before enzyme assay for maximum effect and was neutralized by antierythropoietin antiserum. Other nonhemopoietic growth factors (epidermal growth factor, insulin) had no effect in this assay. Ca2+-ATPase activity of membranes prepared from rabbit mature red blood cells was not inhibited by erythropoietin. The novel effect of erythropoietin on reticulocyte membrane Ca2+-ATPase activity is a mechanism by which erythropoietin can influence cellular Ca2+ metabolism.
W D Lawrence, P J Davis, S D Blas
We explored the role for protein kinase C (PKC) in modulating vasopressin (AVP)-stimulated hydraulic conductivity (Lp) in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. In control studies, 10 microU/ml AVP increased Lp (mean +/- SE, X 10(-7) centimeters/atmosphere per second) from 4.4 +/- 0.9 to 166.0 +/- 10.4. Pretreatment with dioctanoylglycerol (DiC8) suppressed AVP stimulated peak Lp (peak Lp, 21.9 +/- 3.1). Pretreatment with 10(-9) and 10(-7) M 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) also blocked the increase in Lp in a dose-dependent fashion (peak Lp, 59.3 +/- 7.5 and 18.6 +/- 4.8, respectively). Inactive phorbol ester, 4 alpha-phorbol 12 beta,13 alpha-didecanoate (10(-7) M), had no effect. PMA also suppressed the increase in Lp induced by 10(-4) M 8-p-chlorophenylthio-cyclic AMP (CcAMP): peak Lp was 169.4 +/- 14.9 in control, 79.2 +/- 5.5 with 10(-9) M PMA, and 25.7 +/- 2.9 with 10(-7) M PMA. Furthermore, when 10(-7) M PMA was added to the bath 10 min after exposure to AVP, the Lp response to AVP was blocked. Peak Lp was 52.4 +/- 9.6 with PMA vs. 165.1 +/- 10.0 in control. Phosphatidic acid (PA), which is thought to stimulate phosphatidylinositol (PI) turnover, produced similar inhibitory effects on AVP as well as CcAMP-stimulated Lp: PA suppressed 10-microU/ml AVP-induced peak Lp from a control value of 159.6 +/- 7.9 to 88.9 +/- 15.8, and 10(-4) M CcAMP induced peak Lp from 169.4 +/- 14.9 to 95.5 +/- 7.7. We conclude that PMA, at concentrations known to specifically activate PKC, suppresses the hydroosmotic effect of AVP on CCT; This suppression is primarily a post-cAMP event; Inhibition of AVP-stimulated Lp by DiC8 and PA also suggests an inhibitory role for the PKC system; The ability of pre- and post-AVP administration of PMA to blunt the AVP response suggests that agents that act through modulation of PI turnover in CCT may regulate the hydroosmotic effect of AVP.
Y Ando, H R Jacobson, M D Breyer