M B Burg, P F Kador
A flow cytometric immunofluorescence procedure utilizing a specific antibody to rat adipose tissue lipoprotein lipase (LPL) was developed to quantify differentiated and undifferentiated preadipocytes present in the adipose tissue vascular stroma. This method is highly sensitive and specific for cells capable of synthesizing LPL in significant quantities. Pubescence in female rats was associated with an increase in differentiated preadipocytes and in fat cell number with enlargement of the fat depots in the perirenal, parametrial, and the subcutaneous dorsal and femoral regions. A concomitant decline in the percentage of undifferentiated preadipocytes occurred in all but the femoral depot. Ovariectomy reduced pubertal adipose growth in the femoral and parametrial but not the dorsal or perirenal regions. Furthermore, the femoral undifferentiated preadipocyte pool was not preserved in the ovariectomized animals. Thus, ovarian factors influence the pubescence-associated regional preadipocyte differentiation and conversion to adipocytes. The femoral depot contains an ovarian-dependent infinite pool of fat cell precursors. These features could account for the association between ovarian hormones and body fat topography.
G R Krakower, R G James, C Arnaud, J Etienne, R H Keller, A H Kissebah
Experimental aneurysmatic dilatation of the rabbit common carotid artery was induced by a single, periarterial application of calcium chloride in vivo. Vessels were fixed in situ after 3 d, 1 wk, 3 wk, 6 wk, and 12 wk by intracardiac perfusion of glutaraldehyde and tissues prepared for light, scanning, and transmission electron microscopy. Progressive focal aneurysmal dilatation was seen limited to the site of calcium application with endothelial damage and thrombus formation in areas of irregular luminal contour. Disruption of the elastic network of the intima and media was seen with varying degrees of intimal fibromuscular hyperplasia and medial disorganization. The calcium-elastic tissue complex was the focus of the inflammatory, arteriosclerotic reaction and subsequent aneurysm formation. The inflammatory cell infiltration initially included primarily neutrophils followed by lymphocytes, plasma cells, monocytes, and multinucleated giant cells. These studies support the hypothesis that disruption of the elastic tissue network of the vascular wall represents an important pathogenetic factor in the initiation of aneurysmal dilatation. In addition, the results of these studies suggest that interaction of calcium with the elastica of the arterial wall may represent an important pathogenetic factor in the initiation of giant cell arteritis.
S D Gertz, A Kurgan, D Eisenberg
Autoimmune helper T lymphocytes were selected from the blood of two myasthenic patients of different HLA-DR type, using acetylcholine receptor (AChR) from Torpedo californica. These polyclonal T cell lines were tested for reactivity with three synthetic peptides corresponding to the NH2-terminal region of the human AChR alpha subunit. This segment is a good candidate for T cell epitopes since it has a propensity to form an amphipathic alpha helix. The peptides elicited 10-30% of the response induced by native Torpedo AChR. Different peptides were recognized by the autoreactive T cells of the two patients. These results suggest that the NH2-terminal region of the AChR alpha chain contains T cell-stimulating epitopes, and that the T cell autoimmune response in myasthenia gravis, like the B cell response, is heterogeneous.
R Hohlfeld, K V Toyka, L L Miner, S L Walgrave, B M Conti-Tronconi
Receptor-mediated regulation of prolactin synthesis by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in the pituitary cell strain GH4C1 is dependent on the concentration of extracellular calcium. We have now investigated the actions of 1,25(OH)2D3 on cytosolic free calcium concentrations [( Ca2+]i) in these cells using the fluorescent indicator quin2. Basal resting [Ca2+]i was unchanged in cells treated with 1 nM 1,25(OH)2D3 either acutely (from 0 to 15 min) or for periods of up to 48 h. However, the initial peak of the biphasic change in [Ca2+]i induced by thyrotropin-releasing hormone (TRH) was enhanced more than twofold in cells pretreated for 24 or 48 h with 1,25(OH)2D3. This 1,25(OH)2D3-enhanced calcium response was restricted to the initial phase of TRH action; the secondary plateau phase was unaffected. Neither the affinity nor number of TRH receptors nor the early time course of [3H]MeTRH binding to GH4C1 cells were affected by pretreatment with 1,25(OH)2D3. Because TRH binding was not altered, four sites along the intracellular signal transduction pathway of TRH action were examined. Neither protein kinase C activation nor inositol polyphosphate accumulation were enhanced in response to TRH, in 1,25(OH)2D3 pretreated cells, indicating that phosphatidylinositol hydrolysis was unchanged by pretreatment. A low concentration of ionomycin was used to probe the size of the nonmitochondrial intracellular calcium pool that is sensitive to TRH. Ionomycin was not able to mobilize more calcium from 1,25(OH)2D3 pretreated cells, indicating that TRH-responsive intracellular calcium stores were probably not enhanced by pretreatment. Chelation of extracellular calcium, however, did eliminate enhancement of the TRH response in 1,25(OH)2D3-pretreated cells. We conclude that 1,25(OH)2D3 modulates acute dynamic changes in [Ca2+]i induced by TRH without affecting basal [Ca2+]i. The mechanism of the enhanced response of 1,25(OH)2D3-pretreated cells to TRH appears to depend upon a postreceptor event independent of phosphatidylinositol hydrolysis that involves increased calcium conductance at the level of the plasma membrane. A less likely explanation involves enhancement of intracellular calcium stores in an ionomycin-resistant, EGTA-sensitive, TRH-mobilizable reservoir.
J C Chisholm, S Kim, A H Tashjian Jr
The bare lymphocyte syndrome is a rare combined immunodeficiency disorder associated with the absence of class I and/or class II major histocompatibility (MHC) antigens. Although it has been inferred that the immune deficiency is a consequence of disordered MHC-restricted interactions among otherwise normal cells, the biological capabilities and differentiation of B lymphocytes deficient in class II MHC antigens have not been rigorously analyzed. We have examined the phenotypic and functional attributes of B cells with absent class II MHC antigens. Our data demonstrate that these B cells are intrinsically defective in their responses to membrane-mediated activation stimuli. In addition, virtually all the B cells had phenotypic evidence of arrested differentiation at an immature stage. Finally, these B cells also failed to express the C3d-EBV receptor normally present on all B lymphocytes. These data indicate that class II MHC molecules are vital participants in early events of the B cell activation cascade, and that other non-MHC membrane molecules may also be absent as a consequence of either arrested differentiation or as a result of the basic defect affecting the expression of MHC membrane antigens.
L T Clement, S Plaeger-Marshall, A Haas, A Saxon, A M Martin
The mechanism whereby the human neutrophil membrane heterodimer, CD11b/CD18 (Mac-1, Mo1), mediates neutrophil adherence is not known. We studied the role of CD11b/CD18 surface expression in the promotion of neutrophil adhesiveness. We found that phorbol myristate acetate (PMA), calcium ionophore (A23187), and FMLP caused a three- to sevenfold increase in surface expression of both CD11b (alpha M) and CD18 (beta) as assayed by binding of MAbs 60.1 (anti-CD11b) and 60.3 (anti-CD18). Increased binding of MAbs was temporally associated with the promotion of neutrophil aggregation and adherence to cultured endothelial monolayers. Pretreatment of neutrophils with the anion channel-blocking agent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), inhibited the increased surface expression of CD11b and CD18 after stimulation by PMA, A23187, or FMLP and resulted in nearly complete inhibition of neutrophil aggregation. However, pretreatment with DIDS did not diminish either PMA-, A23187-, or FMLP-stimulated neutrophil adherence to endothelial monolayers. We also observed that stimulation of granule-depleted neutrophil cytoplasts by PMA, A23187, or FMLP induced aggregation and adherence to endothelial monolayers without increasing surface expression of CD11b or CD18. We conclude that the increased surface expression of CD11b/CD18 that occurs after stimulation is neither sufficient nor necessary for enhanced adherence to endothelium. Moreover, though both are CD11b/CD18-dependent, the mechanisms involved in neutrophil aggregation are different from those involved in neutrophil adherence to endothelium.
N B Vedder, J M Harlan
Epidermolysis bullosa acquisita (EBA) is a severe, chronic blistering disease of the skin. EBA patients have circulating and tissue-bound autoantibodies to a large (Mr = 290,000) macromolecule that is localized within the basement membrane zone between the epidermis and dermis of skin, the site of blister formation. The "EBA antigen" is known to be distinct from laminin, heparan sulfate proteoglycan, fibronectin, the bullous pemphigoid antigen, elastin, and collagen types I, II, III, IV, and V. Sera from patients with EBA, two monoclonal antibodies to the EBA antigen, and a monoclonal antibody to the carboxyl terminus of type VII procollagen identically label human amnion and skin by immunofluorescent and immunoelectron microscopy. Western immunoblots of the EBA antigen extracted from skin and of type VII procollagen labeled with the above sera and antibodies are identical. None of the sera or antibodies labels Western blots of pepsinized type VII collagen which is missing the globular amino and carboxyl terminal domains. These data show that the EBA antigen is the carboxyl terminus of type VII procollagen.
D T Woodley, R E Burgeson, G Lunstrum, L Bruckner-Tuderman, M J Reese, R A Briggaman
To assess effects of beta-adrenergic blockade on ventricular tachycardia (VT) of various mechanisms, electrophysiology studies were performed before and after intravenous infusion of propranolol (0.2 mg/kg) in 33 patients with chronic recurrent VT, who had previously been tested with intravenous verapamil (0.15 mg/kg followed by 0.005 mg/kg/min infusion). In the verapamil-irresponsive group, 10 patients (group IA) had VT that could be initiated by programmed ventricular extrastimulation and terminated by overdrive ventricular pacing, and 11 patients (group IB) had VT that could be provoked by isoproterenol infusion (3-8 micrograms/min) but not by programmed electrical stimulation, and that could not be converted to a sustained sinus rhythm by overdrive ventricular pacing. Notably, in the group IA patients, all 10 patients had structural heart disease (coronary arteriosclerosis or idiopathic cardiomyopathy); beta-adrenergic blockade accelerated the VT rate in one patient but exerted no effects on the VT rate in the remaining 9 patients, and VT remained inducible in all 10 patients. By contrast, in the group IB patients, 7 of the 11 patients had no apparent structural heart disease; beta-adrenergic blockade completely suppressed the VT inducibility during isoproterenol infusion in all 11 patients. There were 12 patients with verapamil-responsive VT (group II). 11 of the 12 patients had no apparent structural heart disease. In these patients, the initiation of VT was related to attaining a critical range of cycle lengths during sinus, atrial-paced or ventricular-paced rhythm; beta-adrenergic blockade could only slow the VT rate without suppressing its inducibility. Of note, 14 of the total 33 patients had exercise provocable VT: two in group IA, five in group IB, and seven in group II. Thus, mechanisms of VT vary among patients, and so do their pharmacologic responses. Although reentry, catecholamine-sensitive automaticity, and triggered activity related to delayed afterdepolarizations are merely speculative, results of this study indicate that beta-adrenergic blockade is only specifically effective in a subset group (group IB) of patients with VT suggestive of catecholamine-sensitive automaticity.
R J Sung, E C Keung, N X Nguyen, E C Huycke
The present experiments were designed to characterize the kinetics of [1-14C]arachidonic acid (AA) metabolism as a function of time in hepatocytes obtained from rats infused continuously for 30 h with a nonlethal dose of Escherichia coli endotoxin (ET). Chronic endotoxemia greatly reduces the ability of hepatocytes to utilize [1-14C]AA, which is reflected from the earliest times of incubation in very low labeling of intermediates in the biosynthetic pathways of glycerolipids (phosphatidic acid and diacylglycerol) and slower removal of [1-14C]AA from the free fatty acid pool as compared with saline-infused rats. At later times of incubation, the labeling of phospholipids (especially phosphatidylethanolamine and phosphatidylinositol [PI]), but not of triacylglycerides is decreased. Analysis of fatty acid composition of individual phospholipids from cells of ET-infused rats reveals that the content of AA is significantly reduced only in PI. Hence an impairment in activation/acylation enzymatic mechanisms could affect the turnover of metabolically active phospholipid pools, i.e., PI, involved in signal transmission processes, and result in increased availability of 20:4 for eicosanoid synthesis, contributing to cellular metabolic perturbations in endotoxicosis.
E B Rodriguez de Turco, J A Spitzer
By employing early-passaged rabbit kidney epithelial cells in tissue culture, we demonstrated that angiotensin II (AII) has unique mechanisms of signal transduction. First, unlike its action in other target tissues, micromolar concentrations of AII are required to induce small rises in cytosolic calcium, [Ca2+]i, an action which is not accompanied by the release of inositol phosphates (IP). In contrast, nanomolar bradykinin (BK) mobilizes [Ca2+]i through activation of phospholipase C and release of IP. Neither of these stimulated calcium responses exhibits pertussis toxin (PTx) sensitivity. Secondly, AII and BK at 10(-9) to 10(-7) M stimulate cAMP indirectly through PGE2 production in distal cells. AII- and BK-stimulated PGE2 release is PTx inhibitible, suggestive of the presence of a GTP binding protein mediating the response. By contrast, arginine vasopressin fails to elicit rises in [Ca2+]i but exerts its primary effect on cAMP production in distal cells via direct coupling to a stimulatory GTP binding protein, as evidenced by uncoupling with cholera toxin. Regulation of PGE2 synthesis appears to occur via phospholipase A2, not C, by all three peptides.
C Welsh, G Dubyak, J G Douglas
Changes in arachidonate metabolism were examined in mouse peritoneal macrophages incubated with various types of lipoproteins. Oxidized low density lipoprotein (LDL) was incorporated by macrophages and stimulated macrophage prostaglandin E2 (PGE2) and leukotriene C4 syntheses, respectively, 10.8- and 10.7-fold higher than by the control. Production of 6-keto-PGF1 alpha, a stable metabolite of prostacyclin, was also stimulated. No stimulation was found with native LDL, which was minimally incorporated by the cells. Acetylated LDL and beta-migrating very low density lipoprotein (beta-VLDL), though incorporated more efficiently than oxidized LDL, also had no stimulatory effect. When oxidized LDL was separated into the lipoprotein-lipid peroxide complex and free lipid peroxides, most of the stimulatory activity was found in the former fraction, indicating that stimulation of arachidonate metabolism in the cell is associated with uptake of the lipoprotein-lipid peroxide complex. These results suggest that peroxidative modification of LDL could contribute to the progression of atheroma by stimulating arachidonate metabolism during incorporation into macrophages.
M Yokode, T Kita, Y Kikawa, T Ogorochi, S Narumiya, C Kawai
The effects of sulfonylureas on the production of plasminogen activator (PA) and antiactivator (PAI) were investigated using bovine aortic endothelial cells. All compounds studied stimulated PA release (1.3- to 5.2-fold), with glipizide being the most potent, followed by tolazamide, chlorpropamide, and tolbutamide, in that order, while glyburide was the least effective. Both tissue-type and urokinase-type PA production was enhanced. Studies using metabolic inhibitors indicated that both RNA and protein syntheses are required for the sulfonylurea-mediated stimulation of PA release. In addition to continuous release of the two PAs, there was also a continuous release of a single PAI, which did not show an increase after the sulfonylureas. These results suggest that, in addition to their beneficial effects in the treatment of diabetes mellitus, some sulfonylurea compounds may also have significant thrombolytic effects. These results also suggest that pharmacological enhancement of PA production by vascular endothelial cells may be a promising antithrombotic mechanism.
B S Kuo, G Korner, T D Bjornsson
Lipid metabolism was studied in cultured skin fibroblasts from patients with the inherited disorder, Sjögren-Larsson syndrome (SLS). Intact SLS fibroblasts incubated in the presence of [1-14C]palmitate accumulated more radioactive hexadecanol than did normal cells, whereas incorporation of radioactivity into other cellular lipids was unaltered. The hexadecanol content of SLS fibroblasts was abnormally elevated. Hexadecanol accumulation was not due to increased fatty alcohol synthesis nor its deficient utilization for glycerol ether synthesis. The half-life of intracellular hexadecanol loaded into SLS fibroblasts was increased (70 min) compared with normal (15 min), and intact SLS fibroblasts showed impaired oxidation of [14C]-hexadecanol to fatty acid. Fatty alcohol:NAD+ oxidoreductase, the enzyme catalyzing this reaction, was deficient in SLS fibroblasts. Mean total activity in SLS fibroblasts (n = 5) was 13% of that in normal fibroblasts, and palmitoyl CoA-inhibitable activity was 1% of normal. Fibroblasts from two obligate SLS heterozygotes had enzyme activities intermediate between that in normal fibroblasts and individuals with SLS. These results suggest that the primary defect in SLS is deficiency of fatty alcohol:NAD+ oxidoreductase. SLS represents the first inherited disorder in man associated with an isolated abnormality in fatty alcohol metabolism.
W B Rizzo, A L Dammann, D A Craft
This study related ATP levels with membrane damage, lipid abnormalities, and cell death in energy-depleted LLC-PK1 cells. Oxidative phosphorylation was inhibited by antimycin A, and glycolysis was regulated by graded glucose deprivation to achieve stepwise ATP depletion. Over a range of ATP levels down to approximately equal to 5% of normal, over 5 h, cells were altered only minimally, or injured reversibly. Such cells maintained mitochondrial potential, and retained more K+ than cells without an energy source. Over the same duration, cells without an energy source were lethally injured. Treatment with antimycin induced increments of triglycerides and decreases of phospholipids. With severe ATP depletion (approximately equal to 5-10% of normal after 5 h), decrease of phospholipids was marked. Cells in which ATP was not measurable (or was less than 5% of normal) showed comparable phospholipid declines but, in addition, showed massive and progressive increase of unesterified fatty acids. The results identified a low threshold of ATP, at best 5-10% of normal, which preserved viability in LLC-PK1 cells despite major loss of membrane phospholipids. This threshold also determined the ability of cells to maintain their normally low levels of unesterified fatty acids. Failure of energy-dependent mechanisms that normally metabolize unesterified fatty acids may be a correlate of the extent of energy depletion that determines lethal injury.
M A Venkatachalam, Y J Patel, J I Kreisberg, J M Weinberg
Preincubation of neutrophils with recombinant human tumor necrosis factor-alpha (rH TNF-alpha) enhanced the subsequent release of superoxide anion in response to various concentrations of N-formylmethionylleucylphenylalanine (FMLP). Enhanced superoxide anion production was evident by 5 min and had reached a plateau by 15 min. Not only was the total amount of superoxide anion released greater, but the rate of release was also enhanced threefold by rH TNF-alpha. In contrast, rH TNF-alpha reduced or abolished neutrophil locomotion under agarose in response to a gradient of FMLP. Binding studies of f-Met-Leu-[3H]Phe to purified human neutrophils revealed a heterogeneous binding to unstimulated cells. The high affinity component consisted of approximately 2,000 sites per cell and had an average Kd of 2 +/- 0.7 nM (n = 4). The low affinity component consisted of approximately 40,000 sites per cell and had an average Kd of 180 +/- 50 nM (n = 4). rH TNF-alpha caused conversion to a linear Scatchard plot showing no significant change in total binding sites but a single Kd of 40 +/- 10 nM (n = 4). These data indicate that rH TNF-alpha may influence neutrophil responses to FMLP by regulating the affinity of FMLP receptors.
Y H Atkinson, W A Marasco, A F Lopez, M A Vadas
The diurnal response of ACTH release to intravenously administered arginine vasopressin was tested in normal volunteers given consecutively moderate doses of vasopressin every 15 min (0.1, 0.3, 1.0, and 3.0 IU) at 2200 h and again at 0700 h (PM/AM). This protocol was repeated 4 wk later with the times reversed (AM/PM). A dose-related increase in ACTH secretion was observed in all subjects. When the AM response of the AM/PM protocol was compared with the PM response of the PM/AM protocol, the release of ACTH was greater in the morning (P less than 0.05) as evaluated by the following criteria: peak value of ACTH (129.9 +/- 30.4 pg/ml in the AM vs. 57.1 +/- 20.2 in the PM); area under the curve (689 in the AM vs. 259 in the PM); and, sensitivity of the ACTH dose-response curve (first significant increase in ACTH with 1 IU of vasopressin in the AM but not significant even after 3 IU in the PM). In addition, when the AM vasopressin testing followed a previous evening stimulation (PM/AM protocol), there was a blunted ACTH response compared with the AM/PM protocol. Corticotropin-releasing factor (CRF) is probably the major ACTH secretagogue, but since vasopressin acts synergistically with CRF to produce an augmented release of ACTH, we suggest that the ACTH response to administered vasopressin depends upon the ambient endogenous level of CRF. We interpret our data and published data that CRF produces a lesser release of ACTH in the AM as follows: in the morning endogenous CRF is high and administered CRF produces little further release of ACTH, but administered vasopressin acting synergistically with high endogenous CRF causes a greater release of ACTH; conversely, in the evening endogenous CRF is low and administered CRF causes a greater release of ACTH, but vasopressin (a weak secretagogue by itself) gives a low ACTH response. We conclude that vasopressin stimulation of ACTH secretion can be used as an in vivo bioassay of endogenous CRF, and that there is a diurnal rhythm of CRF in hypophyseal portal blood.
R A Salata, D B Jarrett, J G Verbalis, A G Robinson
T cell lines (TCLs) specific for Schistosoma japonicum egg antigen were established from a patient with chronic schistosomiasis japonica to investigate the regulatory mechanism of S.japonicum egg antigen-driven T cell responses in man. All five TCLs tested were CD2+, CD4+, CD8-, and were strongly proliferative only to S. japonicum egg antigen in the absence of exogenous IL-2. All but one TCL produced IL-2-like lymphokines in vitro, indicating their helper T cell functions. One TCL, SjE-3, failed to produce IL-2-like lymphokines. Moreover, this TCL suppressed the specific proliferation of autologous peripheral blood lymphocytes to S. japonicum egg antigen. This TCL produced a soluble suppressor factor(s). These functional diversities among established TCLs were also confirmed by cloned T cells. Our observations might suggest that the regulatory system through helper and suppressor T-T interactions somehow involved in T cell responses to the egg antigen in human chronic schistosomiasis japonica.
N Ohta, T Itagaki, M Minai, K Hirayama, Y Hosaka
The experimental metastasis of B16-F10 murine melanoma cells is blocked by the anti-cell adhesive pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) derived from the central cell-binding domain of fibronectin. In this report, we show that peptide treatment substantially extends the survival time for mice injected intravenously with B16-F10 cells (8/8 vs. 0/8 mice alive at 150 d), thereby demonstrating the potential efficacy of GRGDS treatment in protection against metastatic colonization. We have also examined the specificity of GRGDS activity by testing a series of related homologues for their effects on experimental metastasis. The overall profile of the relative inhibitory activities of these peptides closely matched their previously established capacity to disrupt adhesion in vitro. Lung retention studies with radiolabeled B16-F10 cells revealed an accelerated rate of cell loss from the lung 0-6 h after coinjection with the active peptide GRGDS. This early effect of GRGDS was consistent with its short circulatory half-life, which was found to be 8 min. Taken together, these results suggest that peptide-mediated inhibition of pulmonary colonization is due to interference with B16-F10 cell adhesion to structures in the target organ. Possible peptide interference in tumor cell-blood cell interactions was examined in order to assess (a) possible biological side-effects of peptide treatment and (b) whether such interactions might be an alternative mechanism for GRGDS-mediated inhibition of pulmonary colonization. GRGDS was found to retain full inhibitory activity when coinjected with B16-F10 cells into mice in which platelet function was impaired by acetylsalicylic acid treatment or into thrombocytopenic mice treated with antiplatelet serum (76-93% inhibition of colony formation). These data suggest that platelet involvement in the effects of the peptide is minimal. Similarly, GRGDS was also found to be a potent inhibitor of experimental metastasis in natural killer (NK) cell-deficient beige mice (86% inhibition), thereby discounting the possibility that GRGDS artifactually enhanced NK cell activity. We conclude as a result of these studies that cell-binding fibronectin peptides are specific inhibitors of experimental metastasis that prolong survival, that they appear to function by blocking the adhesion of B16-F10 cells to structures in the target organ, and that they do not appear to act through side effects on certain metastasis-related blood cell functions. In the future, derivatives of fibronectin peptides may be potentially useful prophylactic agents for interfering with the process of metastasis.
M J Humphries, K M Yamada, K Olden
The effect of biosynthetic recombinant insulin-like growth factor I/somatomedin C (IGF-I/Sm-C) and human growth hormone (hGH) on the in vitro growth and maturation of human marrow myeloid progenitors was investigated. Myeloid colony formation was maximally enhanced by 60 ng/ml IGF-I/Sm-C and by 250 ng/ml hGH, resulting in an increase in colony numbers of 41 +/- 7 and 38 +/- 4%, respectively (P less than 0.001). Both peptides induced a 1.5-2.5-fold increase in the frequency of colonies composed of granulocytes alone, but did not alter the numbers of monocyte/macrophage or mixed granulocyte/macrophage colonies. IGF-I/Sm-C and hGH were also found to enhance myeloid maturation towards mature granulocytes in suspension cultures of human marrow cells. The effect of both peptides on human marrow granulopoiesis was similarly demonstrable in serum-free cultures stimulated with human recombinant granulocyte/macrophage colony-stimulating factor. Enhancement of human marrow granulopoiesis in vitro by hGH required the presence of marrow adherent cells and was abrogated by specific monoclonal antibodies directed against IGF-I/Sm-C receptors. The effect of hGH on marrow myeloid progenitors thus appears to be mediated by paracrine IGF-I/Sm-C.
S Merchav, I Tatarsky, Z Hochberg
Serial studies were designed to characterized changes in osmoregulation throughout gestation. Eight women underwent a 2-h infusion of hypertonic saline before conception, during gestational weeks 5-8, 10-12, and 28-33, and then 10-12 wk postpartum. Basal plasma osmolality (Posmol) was already significantly decreased by 5-8 wk (P less than 0.001) and remained 10 mosmol.kg-1 below nonpregnant values throughout pregnancy. The apparent threshold for AVP release (defined as the abscissal intercept of the regression line relating plasma AVP [PAVP] to Posmol) was also decreased significantly throughout gestation, as was the osmotic threshold for thirst (derived from analogue scales relating desire to drink to Posmol). The decrement in osmotic thirst threshold appeared to precede that for AVP release, and consistent with this 24-h urine volumes were significantly greater at 5-8 wk gestation (P less than 0.05). The slopes of each regression equation defining PAVP vs. Posmol (whose r values ranged from 0.79 to 0.99), very reproducible before and after pregnancy, were similar at 5-8 and 10-12 wk, but were markedly reduced in the third trimester (P less than 0.001). These volunteers had randomly undergone an additional infusion before conception (both tests in the luteal phase of the menstrual cycle) when 10,000 IU of human chorionic gonadotrophin (hCG) had been given intramuscularly over a 5-d period. Serum hCG values between 0.2 and 3.3 U.ml-1 were lower than usually seen in pregnancy, but the osmotic thresholds for AVP release and thirst decreased by 3 and 4 mosmol.kg-1, respectively (P less than 0.05). Finally we studied a patient with a molar pregnancy in whom thresholds for hormone release and thirst were both decreased to values resembling normal gestation and remained so for approximately 6 wk postevacuation, only normalizing when hCG had virtually disappeared from her serum. In contrast, thresholds increased within the first two puerperal weeks in two women with normal pregnancies. These data demonstrate (a) osmotic thresholds for both AVP release and thirst decrease within the very first gestational weeks; (b) increment in PAVP per unit increase in Posmol is reduced late in gestation; and (c) hCG may be involved in the osmoregulatory changes of pregnancy.
J M Davison, E A Shiells, P R Philips, M D Lindheimer
Immunoprecipitations of cultured keratinocyte extracts have shown that pemphigus vulgaris (PV) sera bind a polypeptide of 210,000 mol wt with disulfide-linked chains of 130,000 and 85,000 mol wt. To identify proteins in normal human skin recognized by PV antibodies, we performed immunoprecipitations of normal human epidermal extracts. All 22 PV sera tested immunoprecipitated a complex of polypeptides (PV complex) of 210,000, 130,000, and 85,000 mol wt, after reduction. One- and two-dimensional gel electrophoresis showed that the 130,000- and 85,000-mol-wt polypeptides of the PV antigen from both cultured keratinocytes and epidermis have identical charges and sizes. In addition to precipitating the PV complex, 14 of 22 PV sera also have antibodies to a calcium-sensitive epitope on a different complex of polypeptides (PF complex) which has previously been shown to be precipitated by all pemphigus foliaceus (PF) sera. The PF complex consists of polypeptides of 260,000, 160,000, 110,000, and 85,000 mol wt. Although the majority of PV sera also precipitate the PF complex, no PF sera precipitate the PV complex. Thus, PV and PF can be absolutely distinguished on a molecular level using the patients' autoantibodies. The PV and PF complexes, although distinct, have certain similarities. The 85,000-mol-wt polypeptide of each is identical. The 160,000-mol wt-peptide of the PF complex and the 130,000-mol-wt peptide of the PV complex have the same isoelectric point and both are capable of disulfide linkage to the 85,000-mol-wt polypeptide. The PV and PF complexes are closely related and may prove important in cell adhesion.
R W Eyre, J R Stanley
The etiology of 3-ketothiolase deficiency has been attributed to a defect of mitochondrial acetoacetyl-CoA thiolase because the acetoacetyl-CoA thiolase activity in related materials is not activated by K+, a property characteristic for this enzyme. We studied the enzyme protein and the biosynthesis of mitochondrial acetoacetyl-CoA thiolase, using cultured skin fibroblasts from a 5-yr-old boy with 3-ketothiolase deficiency. The following results were obtained. (a) Activation of acetoacetyl-CoA thiolase activity by K+ was nil; (b) The enzyme activity was not affected by treatment with the antibody against mitochondrial acetoacetyl-CoA thiolase; (c) A signal for mitochondrial acetoacetyl-CoA thiolase protein was not detected in the immunoblot analysis; and (d) Pulse-chase experiments of skin fibroblasts, using [35S]methionine, revealed no incorporation of radioactivity into this enzyme. Therefore, fibroblasts from this patient lacked mitochondrial acetoacetyl-CoA thiolase protein due to a defect in its biosynthesis.
S Yamaguchi, T Orii, N Sakura, S Miyazawa, T Hashimoto
A number of factors have been proposed as potential mediators of the syndrome of humoral hypercalcemia of malignancy (HHM), but to date no firm cause-and-effect relationship has been established. We attempted to establish such a relationship by determining whether the presence or absence of adenylate cyclase-stimulating activity (ACSA) in the media of cultured tumor cells predicted the occurrence of the syndrome of HHM when these cell lines were grown in nude mice in vivo. Conditioned media from 35 human renal carcinoma cell lines were surveyed for ACSA in the PTH-sensitive rat osteosarcoma 17/2.8 cell assay. 12 lines were positive (mean, 13.7-fold stimulation, range, 3.0 to 44.0), and 23 lines were negative (mean, 1.2-fold stimulation, range, 0.9 to 1.5). We were successful in establishing five of the positive and six of the negative lines in three to five nude mice per line. Mice implanted with the positive lines uniformly became hypercalcemic (mean serum calcium, 15.8 mg/dl), whereas mice implanted with the negative lines uniformly remained normocalcemic (mean serum calcium, 9.5 mg/dl), in spite of comparable mean tumor size. Acid-urea tumor extracts from each of four hypercalcemic animals contained potent in vitro ACSA (mean, 15.9-fold stimulation), while 5/5 extracts from normocalcemic animals did not (mean, 1.4-fold stimulation). Our study demonstrates that in this model system in vitro ACSA is a reliable predictive marker for HHM in vivo. Whether the protein responsible for this activity is also the mediator of the bone resorption seen in HHM remains to be demonstrated.
E C Weir, K L Insogna, D G Brownstein, N H Bander, A E Broadus
Application of enkephalins to the luminal surface of the bowel augments intestinal absorption. However, to date, endogenous enkephalins have not been demonstrated within intestinal luminal fluid. To determine whether enkephalins are present in the intestinal lumen, five adult dogs had 25-cm chronic jejunal Thiry-Vella loops constructed. Dogs were studied in the awake, fasted state. Jejunal loops were perfused with isoosmotic, neutral Krebs buffer containing protease inhibitors. After basal sampling, the dogs received a high fat meat meal. Collections were made during the meal and for 60 min postprandially. Luminal met-enkephalin levels were determined by radioimmunoassay and confirmed by HPLC. HPLC separation of luminal samples demonstrated two immunoreactive peaks which co-eluted with pure met-enkephalin and met-enkephalin-sulfoxide. Basal met-enkephalin outputs averaged 52 +/- 13 ng/min. The meal significantly increased mean luminal met-enkephalin output to 137 +/- 71 ng/min. During the initial 20-min postprandial period, output remained elevated (180 +/- 73 ng/min), after which it returned to basal levels. We conclude that met-enkephalin is present in the jejunal lumen, and that luminal release of this opioid is augmented by a meal.
S R Money, A Petroianu, A R Gintzler, B M Jaffe
Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at approximately 9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-pro-ile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence. Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.
S D Revak, T A Merritt, E Degryse, L Stefani, M Courtney, M Hallman, C G Cochrane
Endothelial cells are a known source of hematopoietic growth-enhancing factors, including platelet-derived growth factor (PDGF). In addition, endothelium interacts directly with plasma lipoproteins which have been shown to modulate hematopoiesis. To determine the relationship of these properties, we measured the release of an erythroid growth-enhancing factor from bovine endothelial cells under lipid-loaded and control conditions. Human bone marrow cells cultured under serum-free conditions form more erythroid, granulocyte/macrophage, and mixed hematopoietic colonies when supplemented with endothelial cell-conditioned medium (ECCM) than do controls (P less than 0.05). The activity is expressed over a wide range of erythropoietin, lymphocyte-conditioned medium (LCM), recombinant human interleukin-3, and colony-stimulating factor (CSF) concentrations, and is related to ECCM dose. In contrast, enhancing activity in ECCM prepared with 0-400 micrograms/ml acetylated low density lipoproteins (AcLDL) or native LDL is diminished to 0% in a dose-dependent fashion (relative to ECCM from unexposed cells or from cells incubated with very low density lipoproteins, P less than 0.05). Upon dilution, medium prepared from cells incubated with LDL shows a rightward shift in the dose-response curve for erythroid colony formation, while that prepared from AcLDL loaded cells demonstrates a downward shift, indicating that the inhibitory activities are kinetically distinct. Delipidation of ECCM prior to addition to marrow culture removes the inhibitory action of native LDL (P less than 0.05) but not that of AcLDL (P greater than 0.10). Immunochemical analysis suggests that the erythropoietic activity in ECCM is unrelated to that of PDGF, recombinant human CSF, and erythroid burst-promoting activity (BPA) present in LCM. This conclusion is supported by Northern blot analysis of endothelial cells using a cDNA probe for the v-sis homologue of the PDGF beta chain and by immunoprecipitation of metabolically labeled PDGF. The relative amounts of c-sis transcripts and of secreted PDGF were similar in endothelial cells incubated with or without AcLDL. We conclude that AcLDL impair the synthesis or release of an erythropoietic growth-enhancing factor(s) which is biologically distinct from PDGF and BPA present in LCM.
N Dainiak, H B Warren, S Kreczko, M A Riordan, L Feldman, J Lawler, A M Cohen, P F Davies
The mechanism by which fatty acids enter cardiomyocytes is unclear. Therefore, the influx kinetics of [3H]oleate into isolated rat heart myocytes were examined. Cells were incubated at 37 degrees C with [3H]oleate bound to albumin in various molar ratios and the initial rate of uptake (V0) was determined as a function of the unbound oleate concentration in the medium. V0 was saturable with increasing oleate concentrations incubated (Km 78 nM; Vmax 1.9 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Furthermore, binding of [3H]oleate to isolated plasma membranes of cardiomyocytes was saturable, revealing a KD of 42 nM, and was inhibited by heat denaturation or trypsin pretreatment of the membranes. From these membranes a single 40-kD protein with high affinity for a variety of long chain fatty acids was isolated. With a monospecific antibody to this membrane protein, binding as well as cellular influx of [3H]oleate was selectively inhibited. These data indicate that at least a portion of myocardial fatty acid uptake is mediated by a specific membrane protein.
Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.
R Pannell, J Black, V Gurewich
Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal meningitis, we have studied the presence of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat organs and their binding was assessed by indirect immunofluorescence. In the brain of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and brain ventricles. The binding was completely inhibited by the trisaccharide NeuAc alpha 2-3Gal beta 1-4Glc, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressing S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, whereas the non-fimbriated host strain and a recombinant strain expressing P fimbriae did not adhere to brain tissues. The results suggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatal brain has a pathogenetic role during bacterial invasion from circulation into the cerebrospinal fluid.
J Parkkinen, T K Korhonen, A Pere, J Hacker, S Soinila
Leukotriene C4 (LTC4) synthase, which conjugates LTA4 and LTA4-methyl ester (LTA4-me) with glutathione (GSH) to form LTC4 and LTC4-me, respectively, has been solubilized from the microsomes of guinea pig lung and purified 91-fold in four steps to a specific activity of 692 nmol/10 min per mg protein using LTA4-me as substrate. LTC4 synthase of guinea pig lung was separated from microsomal GSH S-transferase by Sepharose CL-4B chromatography and further purified by DEAE-Sephacel chromatography, agarose-butylamine chromatography, and DEAE-3SW fast-protein liquid chromatography. It was also differentiated from the microsomal GSH S-transferase, which utilized 1-chloro-2,4-dinitrobenzene as a substrate, by its heat lability and relative resistance to inhibition by S-hexyl-GSH. The Km value of guinea pig lung LTC4 synthase for LTA4 was 3 microM and the Vmax was 108 nmol/3 min per microgram; the Km values for LTA3 and LTA5 were similar, and the Vmax values were about one-half those obtained with LTA4. The conversion of LTA4-me to LTC4-me was competitively inhibited by LTA3, LTA4, and LTA5, with respective Ki values of 1.5, 3.3, and 2.8 microM, suggesting that these substrates were recognized by a common active site. IC50 values for the inhibition of the conjugation of 20 microM LTA4-me with 5 mM GSH were 2.1 microM and 0.3 microM for LTC4 and LTC3, respectively. In contrast, LTD4 was substantially less inhibitory (IC50 greater than 40 microM), and LTE4 and LTB4 had no effect on the enzyme, indicating that the mixed type product inhibition observed was specific for sulfidopeptide leukotrienes bearing the GSH moiety.
T Yoshimoto, R J Soberman, B Spur, K F Austen
The pathway for hepatic glycogen synthesis in the postprandial state was studied in meal-fed rats chronically cannulated in the portal vein. Plasma glucose concentration in the portal vein was found to be 4.50 +/- 1.01 mM (mean +/- SE; n = 3) before a meal and 11.54 +/- 0.70 mM (mean +/- SE; n = 4) after a meal in rats meal-fed a diet consisting of 100% commercial rat chow for 7 d. The hepatic-portal difference of plasma glucose concentration showed that liver released glucose in the fasted state and either extracted or released glucose after feeding depending on plasma glucose concentration in the portal vein. The concentration of portal vein glucose at which liver changes from glucose releasing to glucose uptake was 8 mM, the Km of glucokinase [E.C. 188.8.131.52]. The rate of glycogen synthesis in liver during meal-feeding was found to be approximately 1 mumol glucosyl U/g wet wt/min in rats meal-fed a 50% glucose supplemented chow diet. The relative importance of the direct vs. indirect pathway for the replenishment of hepatic glycogen was determined by the incorporation of [3-3H,U-14C]glucose into liver glycogen. Labeled glucose was injected into the portal vein at the end of meal-feeding. The ratio of 3H/14C in the glucosyl units of glycogen was found to be 83-92% of the ratio in liver free glucose six minutes after the injection, indicating that the majority of exogenous glucose incorporated into glycogen did not go through glycolysis. The percent contribution of the direct versus indirect pathway was quantitated from the difference in the relative specific activity (RSA) of [3H] and [14C]-glycogen in rats infused with [3-3H,U-14C]glucose. No significant difference was found between the RSA of [3H]glycogen and [14C]glycogen, indicating further that the pathway for glycogen synthesis in liver from exogenous glucose is from the direct pathway. Our results do not support the thesis that the majority of liver glycogen is synthesized from glucose-6-phosphate derived from gluconeogenesis. Reasons for the discrepancy between current findings and other reports supporting the indirect pathway for glycogen synthesis in the liver are discussed.
M T Huang, R L Veech
This paper addresses the question: in Graves' disease is there a thyroid-growth stimulating IgG (TGI) separate from thyroid-stimulating antibody (TSAb)? Using the functioning rat thyroid line (FRTL5) cells for TGI (incorporation of [3H]-thymidine into DNA) and TSAb (increase in cAMP concentration) assays, we tested IgG from 30 Graves' patients. Positive TGI assay occurred only if cAMP increased in the cells and responses correlated, i.e., r = 0.95, P less than 0.001. With one very potent TSAb-IgG we showed that Fab was active as TGI and TSAb, IgG with pI of 8.5-9.0 was the most potent fraction in both systems and an inhibitory IgG prevented the action of both TSAb-IgG and TSH in both the TSAb and TGI assays. In the last example, the action was on the cell membrane and not on the TSH or IgG. These data are entirely compatible with the view that in Graves' disease, at least as tested in FRTL5 cells, the same IgG is active in stimulating both growth and adenylate cyclase.
M Zakarija, S Jin, J M McKenzie
This study investigates the effect of variations in mineralocorticoid as well as cell sodium delivery and uptake on Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle (mTALH). Pharmacologic doses of the mineralocorticoid deoxycorticosterone acetate (DOCA) resulted in a 28% increase of Na-K-ATPase activity. Furosemide-induced inhibition of sodium uptake by the mTALH cell also resulted in Na-K-ATPase activity reduction (45%). Sodium deprivation did not cause a clear change in enzyme activity, either at 3 d or 2 wk, likely reflecting the result of the opposing influences of decreased sodium delivery and increased endogenous aldosterone. Finally, the behavior of Na-K-ATPase activity at 3 d of sodium deprivation in the mTALH contrasted with a 60% increase in activity observed in the cortical collecting tubule, a nephron segment known to be responsive to mineralocorticoid, and this heterogeneity of response may suggest an important role for the mTALH in maintaining salt homeostasis.
E B Grossman, S C Hebert
A patient with a mild hemolytic anemia and osmotically fragile, spherocytic erythrocytes was studied. Analysis of the erythrocyte membrane proteins by SDS-PAGE revealed a deficiency of protein 4.2 (less than 0.10% of normal). The protein 4.2-deficient erythrocytes contained normal amounts of all other membrane proteins, although the amount of band 3 was slightly reduced and the amount of band 6 (G3PD) was slightly elevated. The spectrin content of these cells was normal, as measured by both SDS-PAGE and radioimmunoassay. Erythrocytes from the patient's biologic parents were hematologically normal and contained normal amounts of protein 4.2. Immunological analysis using affinity purified antibodies revealed that the patient's protein 4.2 was composed of equal amounts of a 74-kD and 72-kD protein doublet, whereas the normal protein was composed primarily of a 72-kD monomer. Proteolytic digestion studies using trypsin, alpha-chymotrypsin and papain demonstrated that the patient's protein 4.2 was similar but not identical to the normal protein. Binding studies showed that the protein 4.2-deficient membranes bound purified protein 4.2 to the same extent as normal membranes, suggesting that the membrane binding site(s) for the protein were normal. Depleting the protein 4.2-deficient membranes of spectrin and actin resulted in a loss of nearly two-thirds of the membrane ankyrin, whereas similar depletion of normal membranes resulted in no loss of ankyrin. Repletion of the protein 4.2-deficient membranes with purified protein 4.2 before spectrin-actin extraction partially prevented the loss of ankyrin. These results suggest that protein 4.2 may function to stabilize ankyrin on the erythrocyte membrane.
A C Rybicki, R Heath, J L Wolf, B Lubin, R S Schwartz
The thymus is believed to play a central role in the pathogenesis of Myasthenia gravis (MG). According to a previous hypothesis, MG is initiated within the thymus by immunogenic presentation of locally produced nicotinic acetylcholine receptor (AChR) to potentially autoimmune T cells. Data of 10 consecutive MG patients demonstrate two critical features of MG thymuses that support the concept of intrathymic activation of autoreactive, AChR-specific lymphocytes. Morphologically, the thymuses showed lympho-follicular hyperplasia in nine cases and benign thymoma in one case. The paramount feature revealed by immunohistological double marker analyses was the intimate association of myoid cells (antigen producing) with interdigitating reticulum cells (potentially antigen presenting cells), both of which were surrounded by T3+ lymphocytes in thymus medulla. All 10 thymuses contained T lymphocytes reactive with AChR. This was in contrast to the peripheral immune compartment (blood) where in only 3 of 10 patients, significant T cell responses to AChR were observed. AChR-specific T cell lines could be established from 8 of 10 thymuses, all members of the helper/inducer subset as indicated by the expression of markers T3 and T4.
A Melms, B C Schalke, T Kirchner, H K Müller-Hermelink, E Albert, H Wekerle
The low density lipoprotein (LDL) receptors in fibroblasts from 132 subjects with the clinical syndrome of homozygous familial hypercholesterolemia were analyzed by immunoprecipitation with an anti-LDL receptor monoclonal antibody. 16 of the 132 cell strains (12%) synthesized no immunodetectable LDL receptor protein, indicating the presence of two mutant genes that failed to produce cross-reacting material (crm- mutations). DNA and mRNA from 15 of the 16 crm- patients, representing 30 crm- genes, were available for further study. Haplotype analysis based on 10 restriction fragment length polymorphisms (RFLPs) suggested that the 30 crm- genes represent 13 mutant alleles. Four of the alleles produced no mRNA. Three of these four mRNA- alleles had large deletions ranging from 6 to 20 kb that eliminated the promoter region of the gene. The fourth mRNA- allele did not contain any deletion or alteration in the promoter sequence; the reason for the mRNA- phenotype was not apparent. Nine alleles were positive for mRNAs, of which three encoded mRNAs of abnormal size. One of the abnormal mRNAs was produced by a gene harboring a deletion, and another was produced by a gene with a complex rearrangement. The third abnormal-sized mRNA (3.1 kb larger than normal) was produced by an allele that had no detectable alterations as judged by Southern blotting. The other six mRNA+ alleles appeared normal by Southern blotting and produced normal-sized mRNA but no receptor protein. The current studies demonstrate that mRNA analysis coupled with haplotype determination by Southern blot analysis can be used to classify crm- mutations at a genetic locus where multiple alleles exist.
H H Hobbs, E Leitersdorf, J L Goldstein, M S Brown, D W Russell
The ability of purified anaphylatoxins to induce human lung mast cell mediator release was investigated. In eight anti-IgE responsive (histamine release = 22 +/- 5%, mean +/- SEM) mast cell preparations of 1-96% purity, C5a and C5a des Arg (0.55 pg/ml to 55 micrograms/ml), failed to elicit or potentiate histamine release; lung fragments were similarly unresponsive. The related peptide C3a was also inactive. All anaphylatoxins failed to induce mast cell leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) release. LTC4 release was also negligible from basophils where C5a was a potent histamine release stimulus. Supernatants from C5a-challenged mast cells remained fully active on basophils, excluding carboxypeptidase inactivation of C5a as an explanation for the lung mast cell results. In contrast to lung, skin mast cells were C5a-responsive (histamine release = 8 +/- 1%, at 55 micrograms/ml, n = 2). We conclude that C5a, though devoid of activity on the human lung mast cell, is a human basophil and skin mast cell secretagogue. These findings demonstrate significant organ-specific heterogeneity in mast cell responsiveness.
E S Schulman, T J Post, P M Henson, P C Giclas
A tumor-derived factor believed to cause hypercalcemia by acting on the parathyroid hormone (PTH) receptor was recently purified, cloned, and found to have NH2-terminal sequence homology with PTH. The 1-34 region of this protein was synthesized, evaluated for its postreceptor effects on the ROS 17/2.8 cell line, and its properties were compared to 1-34 PTH. Both 1-34 human humoral hypercalcemia factor (HCF) and 1-34 PTH stimulated adenylate cyclase with an effective concentration (EC)50 of approximately 1 nM. The extent of stimulation by both peptides was equally enhanced by dexamethasone. They both had a pronounced inhibitory effect on growth in the presence of dexamethasone, with an EC50 of approximately 0.1 nM, reduced alkaline phosphatase (AP) activity by approximately 70% in the absence of dexamethasone and by approximately 80% in the presence of dexamethasone with an EC50 of 0.03 nM, and when present at a concentration of 10 nM, reduced AP mRNA levels (estimated by Northern analysis) by approximately 80% in the presence or absence of dexamethasone. Thus, in addition to similar dose-response curves for adenylate cyclase stimulation, both HCF and PTH produced identical postreceptor effects in ROS 17/2.8 cells. These effects of HCF are probably mediated by the interaction of the tumor-derived factor with the PTH receptor.
S B Rodan, M Noda, G Wesolowski, M Rosenblatt, G A Rodan
BB rats serve as a model for human insulin-dependent diabetes mellitus (IDDM), since without insulin treatment, most 60-140-d-old animals die within 1 to 2 wk of developing polyuria, polydypsia, hyperglycemia, and hypoinsulinemia. Lymphoid cells accumulate in the islets of Langerhans and beta cells undergo destruction. We report that inoculation of such BB rats with lymphocytic choriomeningitis virus (Armstrong strain, clone 13) reduces over a prolonged period the incidence of IDDM, normalizes the concentration of blood sugar and pancreatic insulin, prevents the mononuclear cell infiltration in the islets of Langerhans, and for a short time after inoculation alters T lymphocyte subsets. Thus, a virus might be programmed to carry out useful functions.
T Dyrberg, P L Schwimmbeck, M B Oldstone
A synthetic peptide corresponding to the first 34 amino acids of the parathyroid hormone-related protein (PTH-rP) produced by a human tumor associated with hypercalcemia was examined for skeletal and renal effects on calcium metabolism in vivo and in vitro. These effects were compared with those of human parathyroid hormone (1-34), hPTH (1-34). Equal doses of PTH-rP(1-34) and hPTH(1-34) produced equivalent stimulation of adenylate cyclase in vitro in bone cells and kidney cells and tubules. Subcutaneous injection of PTH-rP(1-34) in mice caused a significant dose-related increase in blood ionized calcium similar to that seen with hPTH(1-34) at equivalent doses. Repeated injections of equal doses of both peptides caused sustained hypercalcemia which was significantly greater in PTH-rP(1-34)-treated mice, although each induced comparable increases in histomorphometric indices of osteoclastic bone resorption. PTH-rP(1-34) and hPTH(1-34) also caused similar increases in bone resorption when incubated with fetal rat long bones in organ culture. Infusion of either peptide into thyroparathyroidectomized rats suppressed urinary calcium excretion and increased urinary excretion of cyclic AMP. PTH-rP appears to have similar effects to those of PTH on the skeleton, the kidney, and overall calcium homeostasis.
A J Yates, G E Gutierrez, P Smolens, P S Travis, M S Katz, T B Aufdemorte, B F Boyce, T K Hymer, J W Poser, G R Mundy
Leukocytes have been shown to play an important role in the development of isolated organ injury after experimental ischemia and reperfusion. To examine the role of leukocytes in generalized ischemia-reperfusion injury we used the MAb 60.3 (directed to the human leukocyte adherence glycoprotein, CD18) to block leukocyte adherence functions in a rabbit model of hemorrhagic shock and resuscitation. In control animals subjected to 1 h of shock (mean blood pressure 45 torr and mean cardiac output 30% of baseline) followed by resuscitation, only 29% survived 5 d. All had gross and histologic evidence of injury to lungs, liver, and gastrointestinal mucosa. In contrast, 100% of the MAb 60.3-treated animals survived 5 d (P less than 0.01) and organ injury was absent or markedly attenuated. The control animals also had a persistent acidosis, lost more weight, and had evidence of continued gastrointestinal bleeding in contrast to MAb 60.3-treated animals. We conclude that increased leukocyte adhesiveness plays an important role in the development of multiple organ injury and death after generalized ischemia-reperfusion and that this injury may be significantly reduced by blocking leukocyte adherence functions with the MAb 60.3.
N B Vedder, R K Winn, C L Rice, E Y Chi, K E Arfors, J M Harlan
Complete adenine phosphoribosyltransferase (APRT) deficiency causes 2,8-dihydroxyadenine urolithiasis. In previous reports, analysis of the kinetic properties of APRT from APRT-deficient Japanese subjects revealed strikingly similar abnormalities suggesting a distinct "Japanese-type" mutation. In this paper, we report studies of 11 APRT-deficient lymphoblast cell lines. Nucleotide sequence analysis of APRT genomic DNA from WR2, a Japanese-type homozygote, identified a T to C substitution in exon 5, giving rise to the substitution of threonine for methionine at position 136. RNase mapping analysis confirmed this mutation in WR2 and revealed that six other Japanese-type homozygotes carry the same mutation on at least one allele. The remaining Japanese subject, who does not express the Japanese-type phenotype, did not demonstrate this mutation. Southern blot analysis showed that all seven Japanese-type subjects were confined to one TaqI restriction fragment length polymorphism (RFLP) haplotype. These studies provide direct evidence for the nature of the mutation in the Japanese-type APRT deficiency.
Y Hidaka, S A Tarlé, S Fujimori, N Kamatani, W N Kelley, T D Palella
The CD34 antigen is present on 1-4% of human marrow cells including virtually all hematopoietic progenitors detected by in vitro assays. Since the anti-CD34 monoclonal antibody 12-8 reacts with a similar marrow population in baboons, it was possible to test whether this antigen is expressed by stem cells responsible for hematopoietic reconstitution in vivo. CD34+ cells were enriched from marrows of five baboons using avidin-biotin immunoadsorption. After lethal irradiation, the five animals were given 15-27 X 10(6) autologous marrow cells (3.2-4.4 X 10(6) cells/kg) containing 65-91% CD34+ cells. All animals achieved granulocyte counts greater than 1,000/mm3 and platelet counts greater than 20 X 10(3)/mm3 by 13-24 d posttransplant and subsequently developed normal peripheral blood counts. Two additional animals received 184 and 285 X 10(6) marrow cells/kg depleted of CD34+ cells. One animal died at day 29 without engraftment, while the other had pancytopenia for greater than 100 d posttransplant. The data suggest that stem cells responsible for hematopoietic reconstitution are CD34+.
R J Berenson, R G Andrews, W I Bensinger, D Kalamasz, G Knitter, C D Buckner, I D Bernstein