Spectrin, either in the form of unfractionated low ionic strength extracts of erythrocyte membranes or purified by chromatography on Sepharose (CL)4B, was subjected to tryptic digestion at 0 degrees C. Four patients, each with a different variant of hereditary elliptocytosis, were studied. In one patient, whose erythrocytes showed significant fragmentation on heating on 45 degrees C, such preparations generated a remarkably different pattern of polypeptide fragments on tryptic digestion at low ionic strength. In this patient 32P was released at a slower rate on tryptic digestion of labeled band 2, and an unusual 32P-labeled peptide fragment was also generated, in contrast to control preparations in which such a peptide could not be easily distinguished. There was increased susceptibility of this patient's spectrin to tryptic digestion at physiological ionic strength, but the qualitative pattern of polypeptide fragments was normal. Phosphorylation of spectrin by membrane protein kinase was markedly impaired in this patient, whereas phosphorylation of casein ws unimpaired. However, the phosphorylation of spectrin in her intact erythrocytes was normal. Our findings suggest an abnormality of spectrin structure which we postulate is causally related to the predisposition to hemolysis in this patient, but do not distinguish whether this is a primary abnormality or a post-translational modification of the spectrin molecule. The other three patients showed normal tryptic digestion of spectrin.
T Coetzer, S S Zail
Streptococcus pyogenes, bearing M-protein on its surface, resists opsonization by normal human serum and subsequent phagocytosis by human polymorphonuclear leukocytes. Previous studies have shown that M-protein positive organisms are poorly opsonized by the alternate pathway of complement. In an attempt to define further the role of the surface components of S. pyogenes in this process, we examined the ability of clindamycin, an antibiotic that inhibits protein biosynthesis, to alter bacterial opsonization.
Curtis G. Gemmell, Phillip K. Peterson, David Schmeling, Youngki Kim, John Mathews, Lewis Wannamaker, Paul G. Quie
Patients with recessive X-linked ichthyosis, one of the inherited types of excessive stratum corneum cohesion, have deficient steroid sulfatase in fibroblasts grown from their dermis. Because of the expense and long period required to grow such cells, we have assayed this enzyme in peripheral blood leukocytes and found it to be undetectable in those from patients with this type of ichthyosis, but normal in those from patients with other hereditary or acquired types of ichthyosis. In addition, steroid sulfatase activity is less in leukocytes from women who are carriers of this disease than normal women, and this assay can be used to detect such carriers. Despite previous studies demonstrating that the gene for this enzyme escapes the inactivation of other x-chromosome genes, normal women have leukocyte steroid sulfatase activity only 1.3 times that of normal men, suggesting that some gene dosage compensation occurs. Normal human epidermis, the tissue most affected clinically, also expresses steroid sulfatase activity. The epidermal enzyme is similar in its subcellular localization, its molecular size, and kinetically to that of placenta, leukocytes, and fibroblasts.
E H Epstein Jr, M E Leventhal
Inhibitors of erythropoiesis have been found in the blood of uremic patients but their nature has not been identified. These patients have excess blood levels of parathyroid hormone (PTH) and it is possible that PTH inhibits erythropoiesis. The present study was undertaken to examine the effect of intact PTH molecules and some of its fragments on human peripheral blood and mouse bone marrow burst-forming units-erythroid (BFU-E), on mouse bone marrow erythroid colony-forming unit (CFU-E), and granulocyte macrophage progenitors (CFU-GM), and evaluate the interaction between PTH and erythropoietin (Ep) on human BFU-E. Intact PTH (1-84 bPTH) in concentrations (7.5-30 U/ml;) comparable to those found in blood of uremic patients produced marked and significant (P less than 0.01) inhibition of BFU-E and mouse marrow GFU-GM, but not of mouse marrow CFU-E. Inactivation of 1-84 bPTH abolished its action on erythropoiesis. Increasing the concentration of Ep in the media from 0.67 to 1.9 U/ml overcame the inhibitory effect of 1-84 bPTH on BFU-E. The N-terminal fragment of PTH (1-34 bPTH) and 53-84 hPTH had no effect on BFU-E. The results demonstrate that (a) either the intact PTH molecule or a C-terminal fragment(s) bigger than 53-84 moiety exerts the inhibitory effect on erythropoiesis, and (b) adequate amounts of Ep can overcome this action of PTH. The data provide one possible pathway for the participation of excess PTH in the genesis of the anemia of uremia.
D Meytes, E Bogin, A Ma, P P Dukes, S G Massry
In man, use of the general anesthetic nitrous oxide, N2O, is associated with hematologic and neurologic abnormalities that mimic those seen in cobalamin (Cbl, vitamin B12) deficiency. We have measured a number of aspects of Cbl metabolism in rts exposed to various concentrations of N2O for various periods of time. As little as 2% N2O given for 15 h resulted in 30% inhibition of methionine synthetase (MS) in rat liver. With 50% N2O, inhibition of 70% occurred with 1 h and did not change during the next 48 h. Under these conditions, no inhibition of methylmalonyl-CoA mutase (MMCoAM) was observed. The recovery of MS activity was slow and was only 80% of control values 72 h after N2O was stopped. Studies employing rats previously injected with [57Co]Cbl showed that N2O displaced [57Co]Cbl from MS in a manner that temporally and quantitatively paralleled the loss of MS activity. Recovery of MS activity paralleled the reappearance of [57Co]Cbl on MS. N2O also caused the hepatic content of CH3-[57Co]Cbl to decrease by 20-60%. When [57Co]-Cbl was extracted from liver and analyzed by paper chromatography, [57Co]Cbl analogues were present (10-40% of total [57Co]Cbl) in rats exposed to N2O, but not in control animals. When rats were exposed to 50% N2O for 33 d, the total of endogenous Cbl and Cbl analogues in liver decreased to 35% of control values and endogenous Cbl decreased to 10% of control values. At this time, MS activity was 15% of control values and MMCoAM was only 26% of control values. We conclude that N2O causes multiple defects in Cbl metabolism that include the following: (a) rapid inhibition of MS activity with a slow recovery when N2O is stopped; (b) displacement of Cbl from MS; (c) decreased CH3-Cbl; (d) conversion of Cbl to Cbl analogues; (e) the gradual development of Cbl deficiency and (f) an eventual decrease in MMCoAM activity with a further decrease in MS activity.
H Kondo, M L Osborne, J F Kolhouse, M J Binder, E R Podell, C S Utley, R S Abrams, R H Allen
We have studied erythrocytes from homozygous CC patients in vitro and in perfused rat mesoappendix vasculature to answer some long-standing questions. By examination of wet whole blood preparations, and by comparing the cell distribution on isopycnic continuous density gradients of whole blood samples from a splenectomized CC patient with those from three intact CC patients, we have demonstrated the presence of a distinct crystal-containing band of cells that is present in the former, but totally absent from the latter. We conclude that Hb CC cells containing crystals circulate in Hb CC individuals, but in intact patients they are effectively removed by the spleen. By use of 31P nuclear magnetic resonance and viscosity measurements on cells, we have demonstrated that intracellular aggregation of hemoglobin C occurs on deoxygenation even when no crystal formation is detectable by morphological methods. These two observations are in apparent contradiction with the absence of clinical microcirculatory impairment found in both intact and splenectomized CC patients. The contradiction was resolved by rheological studies on isolated rat mesoappendix preparations and erythrocyte diameter measurements that lead to the conclusion that the hemorheological properties of CC cells in the microcirculation are nearly normal because their increased viscosity is offset by their smaller diameter and size.
M E Fabry, D K Kaul, C Raventos, S Baez, R Rieder, R L Nagel
Bovine aortic endothelial and smooth muscle cells in culture were incubated with arachidonic acid or prostaglandin H2. The amount of prostacyclin nd thromboxane A2 synthesized ws then determined by specific radioimmunoassay for 6-keto-prostaglandin F1 alpha and thromboxane B2. Although smooth muscle cells produced only 6-keto-prostaglandin F1 alpha and thromboxane B2 in a ratio of 5:1 to 10:1. The same ratio of these metabolites of arachidonic acid ws also found when prostaglandin production from endogenous arachidonic acid was stimulated in endothelial cells by the ionophore A23187. Cyclooxygenase inhibitors inhibited the production of both metabolites equally, whereas thromboxane synthetase inhibitors selectively inhibited the production of thromboxane B2. Cells in culture were also incubated with [1-14C]arachidonic acid and the extracted products were identified by two-dimensional thin-layer chromatography. 6-Keto-prostaglandin F1 alpha was the only metabolite produced by smooth muscle cells, but endothelial cells synthesized 6-keto prostaglandin F1 alpha, thromboxane B2, prostaglandin E2, and prostaglandin F2 alpha.
C Ingerman-Wojenski, M J Silver, J B Smith, E Macarak
Regulation of serum anti-DNA antibody in systemic lupus erythematosus (SLE) by an antiidiotypic antibody was evaluated. Various sera from SLE patients in active and inactive states of their disease, as well as sera from normal individuals, were first completely depleted of anti-DNA and of DNA by affinity chromatography. The suppressive capacity of equimolar concentrations of the various depleted sera (blocking sera) on target lupus sera were determined. The target sera were from lupus patients with known DNA-binding capacity. Blocking sera from inactive SLE suppressed the binding of autologous anti-DNA antibody to [3H]DNA (n = 19,P < 0.01). Blocking sera from active SLE (n = 19), as well as human serum albumin, did not suppress. Sera from normal donors who had no contact with lupus patients or with lupus sera did not suppress (n = 14, P > 0.5), whereas those from normal donors who had contact with lupus patients or sera did suppress the binding (n = 5,P < 0.02). The anti-anti-DNA antibody suppressive activity in the inactive lupus serum was shown to be localized within the F(ab′)2 portion of immunoglobulin (Ig)G and could not be removed upon adsorption by normal human gammaglobulin. Furthermore, immune complexes could be detected by a Clq binding assay when the inactive lupus blocking sera were incubated with the anti-DNA antibody containing target sera. The specificity of the suppressive serum factor was shown by its inability to block the binding of tetanus toxoid to antitetanus antibody and its ability to block the binding of DNA to F(ab′)2 fragments of active lupus IgG.
Nabih I. Abdou, Helen Wall, Herbert B. Lindsley, John F. Halsey, Tsuneo Suzuki
The effect of the charge of circulating immune complexes on glomerular localization was studied in a model of passive serum sickness. Preformed immune complexes of heterogeneous or restricted charge, prepared in vitro from isoelectrically focused or chemically modified proteins, were injected intravenously into mice. The distribution of immune complexes in the kidney was compared by immunofluorescence and electron microscopy. Cationic but not anionic or electrophoretically heterogeneous immune complexes gave rise to diffuse localization in the glomerular basement membrane. The binding in subepithelial and subendothelial sites correlated with the known distribution of structural anionic sites. The observations suggest that electrostatic interactions between fixed anionic sites and immune complexes may be an important factor in glomerular trapping. Alternative mechanisms based on initial localization of excess free cationic antigen cannot be completely excluded and are also considered.
G R Gallo, T Caulin-Glaser, M E Lamm
Measurements of right coronary artery blood flow, aortic and right ventricular (RV) pressures and heart rate were radiotelemetered during strenuous, spontaneous exercise in normal dogs and dogs with severe RV hypertrophy induced by chronic (5-6 mo) pulmonary artery stenosis. With fixed pulmonic stenosis, dogs with RV hypertrophy exhibited a decrease (P less than 0.01) in arterial pressure during exercise. Under these conditions, exercise increased right coronary artery blood flow and decreased right coronary vascular resistance less (P less than 0.05) in dogs with RV hypertrophy compared with normal. This attenuated response of right coronary artery blood flow of dogs with RV hypertrophy was not observed when arterial pressures remained at preexercise values during exercise. However, regardless of changes in arterial pressures during exercise, all dogs with RV hypertrophy demonstrated a striking postexercise coronary hyperemia (P less than 0.01), suggesting a perfusion deficit of the hypertrophied right ventricle during exercise. These results imply a fundamental defect in the ability of the coronary circulation of the severely hypertrophied right ventricle to provide sufficient nutrient supply in the face of elevated metabolic demands of exercise.
P A Murray, S F Vatner
Tumor-promoting phorbol diesters were shown to suppress natural killing in vitro by human peripheral blood mononuclear cells. The inhibitory effect of different phorbol diesters and their analogues correlated with their potency as tumor promoters, the most effective agent being 12-O-tetradecanoylphorbol-13-acetate (TPA). Both peripheral blood cells and targets specifically bound TPA, and natural killing could be inhibited by pretreatment of either cell population with TPA, though this was less effective than direct addition of TPA to the assay. Cells that had been pretreated with TPA released TPA and metabolites of tPA during subsequent incubation in fresh medium. This release of tPA was evidently responsible for the inhibition of natural killing by pretreated target cells; in experiments where labeled and unlabeled target cells were mixed, pretreatment of unlabeled targets with TPA inhibited killing of labeled targets. Suppression of natural killing by TPA was greatly reduced when adherent cells were removed from the peripheral blood cells, suggesting that monocytes mediate suppression. Inhibition of natural killing by TPA provides a model for examining the regulation of natural killing. Suppression of natural killing by phorbol diesters may contribute to their activity as tumor promoters.
W E Seaman, T D Gindhart, M A Blackman, B Dalal, N Talal, Z Werb
The roles of glomerular functional and morphologic changes were examined in the acute renal failure associated with generalized Shwartzman reaction in postpartum Munich Wistar rats. The susceptibility of postpartum rats to acute deterioration in renal function after a 2-h endotoxin infusion was found to be greater than in virgin litter mates: glomerular filtration rate fell by 93% in the former vs. 24% in the latter group (P less than 0.001). In postpartum rats there were marked changes in platelet count and fibrinogen level (P less than 0.025) compatible with consumption coagulopathy. Renal blood flow and glomerular filtration rate fell from 5.5 +/- 0.9 and 0.74 +/- 0.12 to 2.0 +/- 0.7 and 0.12 +/- 0.01 ml/min, respectively (both P less than 0.001). Blood pressure did not change. Results of glomerular dynamics studies showed decreases in single nephron filtration rate from 28 +/- 7 to 6 +/- 4 nl/min and in glomerular plasma flow rate from 77 +/- 26 to 23 +/- 12 nl/min (both P less than 0.001). Afferent net ultrafiltration pressure fell from 20 +/- 3 to 5 +/- 4 mm Hg due to a fall in glomerular capillary hydraulic pressure from 47 +/- 1 to 29 +/- 5 mm Hg (P less than 0.001). There were four- and twofold increases in afferent and efferent arteriolar resistances, respectively. Less than 20% of glomeruli had evidence of fibrin deposition after 2 h of endotoxin infusion, a time when glomerular filtration rate was reduced by greater than 90%. [1-Sar, 5-Ile, 8-Gly] angiotensin II infusion before endotoxin significantly protected glomerular filtration rate, 62 vs. 7% of control in rats with no preinfusion (P less than 0.01) despite consumption coagulopathy and glomerular fibrin deposition similar to rats without pretreatment. These data suggest that the early deterioration in renal function in the generalized Shwartzman reaction in the postpartum rat is due to major changes in glomerular dynamics induced by neurohumoral agents and that glomerular fibrin deposition plays a lesser pathogenetic role at this time in this disorder. The study does not address the pathogenesis of renal failure in pregnancy nor peripartum renal failure in another species.
J D Conger, S A Falk, S J Guggenheim
In these experiments we investigated whether NAD could serve as an intracellular modulator of the brush border membrane (BBM) transport of inorganic phosphate (Pi). NAD, both oxidized (NAD+) and reduced (NADH) form, inhibited the Na+-dependent uptake of 32Pi in the concentration range of 10-300 microM NAD when added in vitro to BBM vesicles isolated from rat kidney cortex, but did not inhibit BBM uptake of D-[3H]glucose or BBM uptake of 22Na+. Neither nicotinamide (NiAm) nor adenosine alone influenced BBM uptake of 32Pi. NAD had a similar relative effect (percent inhibition) in BBM from rats stabilized on low Pi diet (0.07% Pi), high Pi diet (1.2% Pi), or normal Pi diet (0.7% Pi). Subsequently, we examined the renal effects of changing the tissue NAD level in vivo. Rats stabilized on low Pi diet were injected intraperitoneally with NiAm (0.25-1.0 g/kg body wt); urinary excretions of Pi (UPiV), of fluid, and of other solutes were measured before and after NiAm injection, then renal cortical tissue nucleotide content was determined, and a BBM fraction was isolated for transport measurements. In BBM from NiAm-treated rats, the Na+-dependent uptake of 32Pi was decreased, but BBM uptake of D-[3H]glucose and BBM uptake of 22Na+ were not changed. NiAm injection elicited an increase in NAD+ (maximum change, 290%), a lesser increase in NADH (maximum change, +45%), but no change in the content of ATP or cyclic AMP in the renal cortex. Na+-dependent BBM uptake of 32Pi ws inversely correlated with NAD+ content in renal cortex (r = -0.77 +/- 0.1; P less than 0.001) and with UPiV (r = -0.67 +/- 0.13; P less than 0.01). NAD+ in renal cortex was positively correlated with UPiV (r = 0.88 +/- 0.05; P less than 0.001). Injection of NiAm elicited a marked increase in UPiV, but no change in excretions of creatinine or K+, or in urine flow; excretion of Na+ and Ca declined. NiAm injection caused similar renal responses, in normal and in thyroparathyroidectomized rats, as well as in rats on normal Pi diet and low Pi diet. We conclude that NAD can serve as an intracellular modulator (inhibitor) of Na+-dependent transport of Pi across the renal luminal BBM and across the proximal tubular wall by its direct interaction with BBM. We propose that at least some hormonal and/or metabolic stimuli elicit phosphaturia by increasing NAD+ in cytoplasm of proximal tubular cells.
S A Kempson, G Colon-Otero, S Y Ou, S T Turner, T P Dousa
Growth hormone (GH) release was studied in adults of normal stature, ages 21-86 yr. The subjects were 85-115% of ideal body weight, between the 5th and 95th percentiles in height, and free of active or progressive disease. 9 to 12 individuals in each decade from thirds to ninth were evaluated. The following criteria of GH status were measured: serum GH concentration, analyzed by radioimmunoassay at half-hour intervals for 4 h after onset of sleep, and at 1-h intervals from 8 a.m. to 4 p.m. in 52 subjects; daily retention of N, P, and K in response to 0.168 U human (h)GH/kg body wt3/4/day in 18 subjects; and plasma somatomedin C (SmC) level before and during exogenous hGH treatment in 18 subjects. All 10 individuals, 20-29 yr old, released substantial amounts of endogenous GH during both day and night (average peak serum GH obtained during day and night was 7.3 and 20.3 ng/ml, respectively); average plasma SmC was 1.43 U/ml (95% tolerance limits, 0.64-2.22 U/ml). There was no significant effect of exogenous hGH on elemental balances or on plasma SmC. In contrast, 6 of 12 individuals 60-79 yr old showed the following evidences of impaired GH release; peak waking and sleeping serum GH less than 4 ng/ml; plasma SmC less than 0.38 U/ml; a significant retention in N, P, and K; and a significant rise in plasma SmC, in response to exogenous hGH. Plasma SmC, serum GH during sleep, serum GH during the day, retentions of N, P, and K in response to exogenous hGH, and rise in plasma SmC in response to hGH were all intercorrelated (P less than 0.05). Plasma SmC less than 0.38 U/ml corresponded to peak nocturnal serum GH less than 4 ng/ml. The prevalence of plasma SmC less than 0.38 U/ml increased progressively from age 20 to 90: third decade, 0%; fourth, 11%; fifth, 20%; sixth, 22%; seventh, 42%; eight, 55%; and ninth, 55%. Within each decade, plasma SmC was inversely related to adiposity.
D Rudman, M H Kutner, C M Rogers, M F Lubin, G A Fleming, R P Bain
Radionuclide and contrast ventriculography were evaluated for their ability to estimate myocardial ischemia. In 14 closed-chest, sedated dogs, a small and larger region of ischemia were produced by inflating balloon occluders on the left anterior descending coronary artery. The systemic arterial pressure, atrial-paced heart rate, global ejection fraction by radionuclide and contrast ventriculography, regional wall-motion abnormalities (as the percentage of abnormally contracting segments), and regional myocardial blood flow (using the microsphere technique) were measured during an initial control period, two separate ischemic periods, and a final control period. The regional ischemic weights based on myocardial blood flow ranged from 0 to 38.5 g and were grouped as zero, small (range 0 to less than 10 g, mean 3.40 g), and large regions of ischemia (greater than 10 g, mean 24.8 g). Regional wall-motion abnormalities were sensitive qualitative indicators of ischemia. Receiver operating characteristic analysis showed that both ventriculographic methods were highly sensitive, specific, and accurate for detecting regional ischemia. Contrast ventriculography was slightly superior for detecting small regions less than 4 g, but the methods were equal for regions greater than 4 g. The arterial pressure and heart rate were unchanged during ischemia. For small regions of ischemia, the global ejection fraction did not fall using either the contrast or radionuclide technique, but it fell significantly when large regions were produced. There was a quantitative relationship between the percentage of abnormally contracting segments and the grams of myocardial ischemia (for radionuclide ventriculography, r = 0.65, P = 0.003, and for contrast ventriculography, r = 0.75, P less than 0.001), but for many small regions of ischemia, wall-motion changes were greater than anticipated, suggesting hypofunction of the continguous normal tissue. This study demonstrated that both radionuclide and contrast ventriculography were quite sensitive and specific for detecting measured amounts of regional ischemia. The functional changes resulting from ischemia are quantitatively related to the extent of regional ischemia, small areas resulting in regional wall motion abnormalities, and large areas producing both reduced global ejection fraction and wall motion changes.
M W Kronenberg, M L Born, C W Smith, L Brorson, J C Collins, S B Higgins, W K Vaughn, F D Rollo, G C Friesinger, S S Pearson, J L Norris, O H Wolfe
The transient granulocytopenia of hemodialysis results indirectly from plasma complement activation by dialyzer cellophane membranes. The C5adesarg so produced can induce reversible granulocyte aggregation in vitro and in vivo, and we hypothesized that the pulmonary leukostasis responsible for the granulocytopenia results from embolization of aggregates formed under the influence of C5adesarg produced in the dialyzer. These studies were designed to measure C5adesarg generation during dialysis by granulocyte aggregometry and to determine the reason for the transience of the leukostasis. C5adesarg generation was equally evident throughout dialysis, persisting well after granulocytopenia had reversed, and dialyzer-induced complement activation was insufficient to produce significant depletion of plasma complement titers. That granulocyte deactivation might be responsible for the transience was suggested by the absence of the usual granulocytopenia in a patient with uniquely high levels of C5adesarg in his predialysis plasma. Granulocytes drawn from seven stable uremic patients after granulocytopenia had reversed exhibited a dose-related, selective and irreversible refractoriness to stimulation with C5adesarg, but their responses to n-formyl-Met-Leu-Phe remained normal. Identical deactivation was produced in normal cells by short- or long-term exposure of C5adesarg in vitro. These studies suggest that C5adesarg is indeed generated by the dialyzer throughout hemodialysis and that the transience of the leukostasis and granulocytopenia is due to selective down-regulation of cellular responses to C5adesarg—a phenomenon that hitherto has been described only in vitro and that may be important in limiting the deleterious effects of adherent granulocytes on the endothelium in patients with intravascular complement activation.
Keith M. Skubitz, Philip R. Craddock
Increased numbers of circulating granulocyte-monocyte precursor cells (CFUc) have been observed in the peripheral blood of man after antineoplastic chemotherapy. We have developed a canine model to study the biologic significance of this phenomenon for hematopoietic reconstitution following hematopoietically lethal exposure to total body irradiation (TBI). After cyclophosphamide administration, a 16-fold expansion of circulating CFUc numbers was observed during the period of rapid leukocyte recovery that occurred after the chemotherapy-induced leukocyte nadir. We had previously noted this association between leukocyte recovery and CFUc expansion in our human studies. After 900 rad TBI hematopoietic reconstitution was attempted with autologous, cryopreserved collections of peripheral blood mononuclear cells obtained either at times of post-cyclophosphamide CFUc expansion (group A, 14 dogs) or without CFUc expansion (group B, 12 dogs). Asd compared to group B collections, group A collections contained 11-fold more CFUc and were 12.5-fold more potent in fostering hematopoietic recovery after TBI. These results suggest that the expansion of CFUc numbers we observed was accompanied by a similar expansion of more primitive hematopoietic stem cell numbers. We conclude that chemotherapy-induced expansion of circulating CFUc numbers appears to be of substantial import in effecting hematopoietic reconstitution--an observation that may be of significance for further studies of autologous hematopoietic reconstitution in man.
R A Abrams, K McCormack, C Bowles, A B Deisseroth
During the course of Plasmodium berghei malaria in the rat, splenic clearance of damaged uninfected erythrocytes (heated or Heinz body-containing) underwent changes strikingly similar to those of infected erythrocytes. Splenic trapping of abnormal erythrocytes was impaired during the period of rising parasitemia but became supernormal just before the onset of resolution of the acute infection. These changes could be related to the development of splenomegaly and alterations in splenic cordal microcirculation during infection. The relative distribution of flow through the cords was decreased during rising parasitemia and was restored before the onset of resolution. Together, our observations support the hypothesis that altered rheologic properties of infected erythrocytes are a major determinant of their removal by the spleen. These data suggest that the alterations in splenic microcirculation that occur during malaria may have important implications for host defense.
D J Wyler, T C Quinn, L T Chen
The purpose of the present experiments was to evaluate the role of circulating antibodies in the rejection of human renal allografts and to study the apparent target(s) for antibody binding. Eluates obtained from surgical biopsy and nephrectomy specimens of rejecting, cadaveric human renal allografts were tested for antibodies directed to structural antigens of normal kidney and for cytotoxic antibody activity against mononuclear cell populations. By indirect immunofluorescence 23 of 35 eluates contained immunoglobulin that bound to normal kidney. Staining was in smooth muscle only in 10 patients, in smooth muscle and other structures such as tubular basement membranes, proximal cells, or brush border in 9 patients, and in structures other than smooth muscle in 4 patients. All 16 eluates tested contained antibodies cytotoxic for cells derived from a panel of normal volunteers. Six were cytotoxic to T cells and 10 to B cell and monocyte-enriched preparations. Absorption of eluates with pooled buffy coat cells, platelet concentrates and packed, cultured B cells removed antibodies reactive with vascular wall smooth muscle and endothelium, but not antibodies to tubular basement membranes, proximal or distal tubular cells, brush border, or other structures of kidney sections. Two of five eluates containing antikidney antibodies were found to bind to rat kidneys in vivo. These results suggest that circulating antibodies participate in cadaveric renal allograft destruction and demonstrate that they can be recovered directly from the allograft. Moreover, the data indicate that there are different antibody populations involved: some clearly directed to allo-specific differences and others that are apparently kidney-specific.
John J. McPhaul Jr.
The intracellular distribution of glutathione into kinetically distinct pools and the determinants of glutathione turnover were examined in vivo. Glutathione turnover was measured in individual, restrained rats with a biliary fistula by administration of acetaminophen to trap the previously labeled hepatic glutathione as an excretable acetaminophen adduct. Fasting for 48 h resulted in a decrease of hepatic glutathione from 4.7±0.9 to 3.6±0.8 μmol/g liver and a marked increase in the fractional rate of glutathione turnover from 0.19±0.04 to 0.43±0.07/h. Within 6 h following refeeding, the rate of glutathione turnover and the hepatic glutathione concentration returned to normal. The simultaneously determined specific activities of free intrahepatic glutathione and the acetaminophen-glutathione adduct in bile were identical, indicating that the hepatic glutathione pool is kinetically homogeneous. The synthesis of glutathione could, therefore, be estimated from the rate constant and the intrahepatic glutathione concentration. During fasting hepatic synthesis of glutathione increased from 0.86±0.17 to 1.50±0.23 μmol/g per h. In fed animals the administration of dibutyryl cyclic adenosine monophosphate and theophylline stimulated the rate of hepatic glutathione turnover similar to fasting. In contrast, glucose given intraduodenally to fasted animals decreased the rate of glutathione turnover. These data are consistent with the view that the increased glutathione turnover that occurs during fasting results from two mechanisms. Because of a decrease in the intrahepatic free glutathione/mixed disulfide ratio, which is apparently mediated by cyclic adenosine monophosphate, the free glutathione pool contracts and turns over more rapidly in order to maintain glutathione synthesis. In addition, glutathione consumption via the gamma-glutamyl cycle apparently is increased, which may be related to the increased uptake of amino acids for gluconeogenesis during fasting.
Bernhard H. Lauterburg, Jerry R. Mitchell
Obese subjects have elevated adipose tissue lipoprotein lipase activity per fat cell when compared with lean control subjects. This enzyme, which is rate limiting for the uptake and storage of lipoprotein triglyceride in adipose tissue, has been shown to be further elevated in a group of previously obese subjects who had been weight stable at a reduced weight for 4-28 mo. In the present prospective study of eight obese subjects, adipose tissue lipoprotein lipase activity was demonstrated to increase after weight stabilization at a reduced weight (0.33 mU/10(6) cells). In three subjects who lost weight and subsequently regained their lost weight, the enzyme activity increased after weight loss and then returned toward the original basal level with weight gain. One subject who maintained his weight loss for 10 mo. continued to have an elevated level of enzyme activity. Because adipose tissue lipoprotein lipase activity does not "normalize" after weight loss, we hypothesize that this enzyme may play a counterregulatory role in resisting deviation from a "set point" for fat mass or fat cell size and thereby predispose to reattainment of the original obese state.
R S Schwartz, J D Brunzell
Previous reports have described conflicting results concerning the glycoprotein (GP) and protein composition of Bernard-Soulier platelets. In view of this controversy we have analyzed the platelets of four Bernard-Soulier patients using improved single and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis procedures. An absence of staining for carbohydrate of membrane GP Ib was characteristic for the platelets of each patient. Major periodate-Schiff staining bands corresponding to membrane GP IIb, IIIa, and IIIb were clearly detected and their presence was confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis. The protein content of the Bernard-Soulier platelets was increased two- to fourfold. However, analysis of their protein composition using 7-12% acrylamide gradient gels showed normal polypeptide profiles. Lactoperoxidase-catalyzed 125I-labeling of the Bernard-Soulier platelet surface proteins was followed by SDS-polyacrylamide gel electrophoresis and autoradiography. No labeling in the Ib position was detected whereas the other major membrane GP, including Ia and IIa, were normally located. In contrast, GP Ib was clearly detected by periodate-Schiff staining and autoradiography when normal human platelets that had been exhaustively treated with neuraminidase before the lactoperoxidase-catalyzed iodination were analysed. No abnormalities were detected in the GP patterns of membranes isolated from the patients' erythrocytes. Only a severe molecular abnormality or possible deletion of GP Ib could account for this major platelet lesion in the Bernard-Soulier syndrome.
A T Nurden, D Dupuis, T J Kunicki, J P Caen
In the two genetic forms of abetalipoproteinemia described previously, recessive abetalipoproteinemia and homozygous hypobetalipoproteinemia, all lipoproteins that normally contain apolipoprotein B are absent from plasma. We describe here a new disorder in which normal low density and very low density lipoproteins are absent, but in which triglycerides are absorbed from the intestine and chylomicrons are present in plasma. The underlying molecular defect appears to be selective deletion of the hepatogenous B-100 apolipoprotein. The B-48 apolipoprotein found in chylomicrons is spared. These findings suggest that the two species of apolipoprotein B are under separate genetic control and that low density lipoproteins are not normally derived from chylomicrons.
M J Malloy, J P Kane, D A Hardman, R L Hamilton, K B Dalal
It is well accepted that the C cells of the thyroid contain somatostatin, but the role in local endocrine function has not yet been firmly established in this organ, and it has not been proved that thyroidal somatostatin is released into the circulation. We have measured the contents of somatostatin-like immunoreactivity in the effluent of canine thyroid glands perfused without recirculation with a synthetic buffer medium. During basal conditions a definite release was consistently found in the order of 10 pg/ml corresponding to 12 pg/min. The somatostatin-like immunoreactivity was studied in dilution experiments and by gel-filtration chromatography, and found to have properties identical to those of synthetic cyclic somatostatin, which was also recovered quantitatively when added to sampling tubes. Various compounds were infused in concentrations that are highly active in pancreas perfusion experiments. 14-min infusion of arginine, 5 and 11.5 mmol/liter; isoproterenol, 10 and 23.7 nmol/liter and 68.7 mumol/liter; acetylcholine, 5 mumol/liter, carbamylcholine, 10 and 100 mumol/liter; glucagon, 1 and 30 nmol/liter; and porcine calcitonin, 1 and 100 ng/ml did not affect the basal release of somatostatin-like immunoreactivity significantly. Neither did an increase from the control level of 4 mmol/liter glucose of 10 or 20 mmol/liter, nor an increase in the control level of 4.4 mmol/liter K+ to 7.5 or 14.4 mmol/liter. Each of these compounds were tested in three or four dogs. The effect of an increase in Ca++ from the control level of 1.5 mmol/liter to 2.25, 3.0, and 4.5 mmol/liter was tested in random order in five thyroid lobes. All three doses elicited an immediate increase in effluent somatostatin-like immunoreactivity. In most experiments the response was biphasic with an early spike, followed by a stable level that was maintained during prolonged Ca++ infusion. The secretory response was not diminished through a series of repeated short pulses of calcium infusion. The response to 3.0 mmol/liter Ca++ (control period 8.4 +/- 1.5, test period 337 +/- 110 pg/ml, mean +/- SE) and 4.5 mmol/liter Ca++ (control period 9.5 +/- 1.4, test period 386 +/- 125) were significantly higher than 2.25 mmol/liter Ca++ (control period 7.2 +/- 1.0 test period 140 +/- 39), while there was no significant difference between responses to the two high doses. Infusion of salmon calcitonin, 10 ng/ml and 1 microgram/ml; or porcine calcitonin, 1 microgram/ml during calcium stimulation (2.25 mmol/liter of Ca++) did not induce alterations in the release of somatostatin-like immunoreactivity. The results demonstrate that thyroidal somatostatin is mobilizable, and it appears to be selectively sensitive to calcium stimulation, indicating a possible role in calcitonin release control.
P Laurberg, H Orskov
The suppression of collagen production by increasing the cyclic (c) AMP content of cultured cells was examined vis-à-vis the β-adrenergic system. Cultured human fetal lung fibroblasts incubated for 6 h with the β-agonists isoproterenol or epinephrine produced ∼30% less collagen per cell than in the absence of the hormones. To demonstrate that the β-agonists were operating by their interaction with the β-receptor to stimulate adenylate cyclase to increase the intracellular content of cAMP, d- and l-isoproterenol were incubated separately with the cultured cells. Only l-isoproterenol increased intracellular cAMP and decreased collagen production. While 20 nM l-isoproterenol was effective, the d-isomer was ineffective even at 2μM. An increase in cAMP from 40 to 73 pmol/mg protein was effective in suppressing collagen production; increasing the cAMP content to much higher levels had little additional effect on collagen production. 3-Isobutyl-1-methylxanthine, an analog of theophylline that inhibits phosphodiesterase, potentiated the effect of isoproterenol in suppressing collagen production. Further support for the concept that isoproterenol suppressed collagen production by acting through the β-receptor was provided by the finding that only the l-isomer of propranolol, a β-blocker, was effective in blocking both the increase in intracellular cAMP and the suppression of collagen production caused by isoproterenol. These results demonstrate that collagen production in human fibroblasts can be regulated by the β-adrenergic system and indicate that when the cAMP content is increased beyond a threshold value, collagen production is suppressed. Since collagen production is sensitive to the small changes of cAMP content of cells brought about by β-stimulation in cultured cells, the results point to a possibly important mechanism for the regulation of collagen production in the body.
Richard A. Berg, Joel Moss, Bruce J. Baum, Ronald G. Crystal
Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37°C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-14C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of 14CO2 production on the presence of thiamin-PP, coenzyme A (CoA), Mg++, and NAD+. Also, it has been shown that acetyl-CoA and 14CO2 are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg++ and Ca++, or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity.
Kwan-Fu Rex Sheu, Chii-Whei C. Hu, Merton F. Utter
Supernatant fluids from the cultures of bone marrow cells from 10 of 12 patients with multiple myeloma (MM) caused bone resorption in organ cultures of fetal rat calvaria. In four patients, the marrow cells were cultured with and without indomethacin (1 μM). The supernatant fluids from indomethacintreated marrow cultures caused significantly less bone resorption than supernatant fluids of cell cultures without indomethacin. This inhibition of release of bone resorbing factor(s) by myeloma cultures is similar to the previously observed indomethacin-induced inhibition of osteoclast-activating factor (OAF) production by activated human leukocytes. None of the MM supernatants had any effect on cyclic (c)AMP accumulation in resorbing bone in vitro.
Robert G. Josse, Timothy M. Murray, Gregory R. Mundy, Donna Jez, Johan N. M. Heersche
The possibility that mucosal antibody is produced as a host response to Haemophilus influenzae type b (Hib) infection was examined in this study. 17 of 18 prospectively evaluated children ranging in age from 2 mo to 7 yr developed a detectable level of anticapsular antibody in their nasopharyngeal secretions after systemic Hib infection. The mean concentration of nasal anti-capsular antibody of the 18 children was 554 ng/mg IgA (SD = 35-8,863) during the acute phase of illness and declined to 224 ng/mg IgA (SD = 19-2,688) in convalescence. Some children had mucosal antibody detectable at least 10 mo after infection. The mucosal antibody levels were not affected by the length of illness before diagnosis, type of disease, age of the patient, sex, or presence of detectable capsular antigen or viable bacteria in the nasopharynx. The mucosal antibody was predominantly of the IgA class and occurred independent of the serum antibody. Six of the children aged less than 1 yr who did not produce and/or sustain a serum antibody level correlated with protection demonstrated a persistent mucosal antibody response. These findings suggest that the mucosal immune system may have the ability to respond at an earlier age than the serum immune system and lead us to postulate that protective secretory antibodies to prevent systemic Hib disease may be inducible in young infants in spite of the poor serum antibody response occurring at this age.
M E Pichichero, C B Hall, R A Insel
Mitogen-stimulated human T cell activation is absolutely dependent on the participation of a nonresponding accessory cell. In populations of human peripheral blood mononuclear cells, monocytes function as the requisite accessory cells. The possibility that cultured endothelial cells (EC) might also function as accessory cells was studied by examining the potential of endothelial cells to restore mitogen responsiveness to monocyte-depleted human T cells. Highly purified T cells were prepared by isolating cells rosetting with sheep erythrocytes and removing monocyte contamination by glass adherence and nylon wool column passage. When cultured at low cell density, T cells failed to respond to stimulation with various mitogenic lectins, whereas co-culture with monocytes restored responsiveness. Similarly, EC obtained from umbilical vein, pulmonary artery, and ovarian vein restored the capacity of T cells to respond to mitogens. Mitogen-stimulated T cell activation required viable endothelial cells. Moreover, effective endothelial T cell cooperation appeared to involve the establishment of cell-to-cell contact between EC and responding T cells. Accessory cell function was not a nonspecific property of all tissue culture cells as evidenced by the finding that human foreskin fibroblasts, lung fibroblasts, and HeLa cells were unable to restore responsiveness to monocyte-depleted T cells. These observations indicate that endothelial cells can support the induction of mitogen-induced T cell activation and suggest that cells lining blood vessels may play an active role in the initiation of immune responses in vivo.
E R Ashida, A R Johnson, P E Lipsky
Prompted by recognition of the similar structures of riboflavin (vitamin B2), phenothiazine drugs, and tricyclic antidepressants, our studies sought to determine effects of drugs of these two types upon the conversion of riboflavin into its active coenzyme derivative, flavin adenine dinucleotide (FAD) in rat tissues. Chlorpromazine, a phenothiazine derivative, and imipramine and amitriptyline, both tricyclic antidepressants, each inhibited the incorporation of [14C]riboflavin into [14C]FAD in liver, cerebrum, cerebellum, and heart. A variety of psychoactive drugs structurally unrelated to riboflavin were ineffective. Chlorpromazine, imipramine, and amitriptyline in vitro inhibited hepatic flavokinase, the first of two enzymes in the conversion of riboflavin to FAD.
John Pinto, Yee Ping Huang, Richard S. Rivlin
Because successful allotransplantation of islets of Langerhans isolated by collagenase digestion has been difficult in many animal species, we asked whether isolated islet preparations might have tissue specific determinants conferring amplified immunogenicity in vitro. Lymphocyte proliferative responses ([3H]thymidine uptake) were studied in beagle dogs in mixed culture combinations of lymphocyte vs. lymphocyte (MLC) and lymphocyte vs. islet (MLIC). In five MLC responder and five nonresponder pairs, peripheral blood lymphocytes of dogs A and B were used as responding cells, and dog B x-irradiated lymphocytes (Bx), x-irradiated (or nonirradiated) islets (BI), or hepatic cells (BH) were used as stimulating cells in primary and secondary reactions. For the secondary reactions, A + Bx, A + BI, or B + BI were incubated for 9 d (A′B, A′BI, B′BI, respectively) before addition of new stimulating cells.
Alexander Rabinovitch, Laphalle Fuller, Daniel Mintz, Walter Severyn, Jack Noel, Cathy Flaa, George Kyriakides, Joshua Miller
Perfused rat liver removes 97% of the taurocholate from the afferent circulation when the perfusate albumin concentration is 0.5 g/dl. Increasing the albumin concentration 10-fold reduces the concentration of free taurocholate by a factor of five but produces only a 50% reduction in the apparent uptake coefficient. A similar discrepancy is evident from a model-independent analysis of the extraction fractions. From these observations we argue that uptake is not driven solely, or even predominantly, by the plasma concentration of free taurocholate but also depends on interaction between albumin and the cell surface. Nonequilibrium binding, saturation kinetics, and an inhomogeneous population of liver cells are considered as alternative explanations and excluded. The possibility that albumin exerts its effect by enhancing the diffusion of taurocholate across an unstirred layer in the Disse space appears improbable but cannot be eliminated.
E L Forker, B A Luxon
Seminal plasma diluted 1:5-1:1,000 gave marked inhibition of serum antibody complement-mediated bactericidal and opsonic effects against Neisseria gonorrhoeae and other gram-negative organisms. Serum that was bactericidal at a dilution of 1:5,120 was not bactericidal at a dilution of 1:10 when seminal plasma was added. Bactericidal action of immune human or rabbit sera, or purified immunoglobulin (Ig)G or IgM plus complement for six strains of N. gonorrhoeae, serogroups A, B, C, and Y of Neisseria meningitidis, Escherichia coli and other gram-negative rods was inhibited by seminal plasma. Using C8- or C7-deficient sera as antibody and complement sources, opsonization, phagocytosis, and killing of N. gonorrhoeae and E. coli 014-K7 were inhibited by seminal plasma. Opsonization, phagocytosis, and killing of Staphylococcus aureus 502A was not inhibited. For the gram-negative organisms, the early phase of the opsonization process, probably complement activation, appeared to be inhibited rather than the ingestion or polymorphonuclear leukocyte killing steps; addition of seminal plasma yielded a significant reduction in the percentage of polymorphonuclear cells with associated bacteria. Seminal plasma did not prevent attachment of IgG, IgM, or IgA antibodies to gonococci. It reduced serum hemolytic whole complement activity by 25%. The seminal plasma inhibitor was of low molecular weight and was stable at 56 degrees C for 30 min, but inhibitory activity was lost after heating to 100 degrees C for 10 min. It is likely that the inhibitory factor(s) is a low-molecular weight protease or protease inhibitor. Seminal plasma probably has an important role in inhibition of complement and antibody functions in the genital tract. It may enhance pathogenesis of agents of sexually transmitted diseases.
G F Brooks, C J Lammel, B H Petersen, D P Stites
We studied biochemical genetics of low density lipoprotein (LDL) receptor mutations in fibroblasts from six homozygous and five heterozygous patients with familial hypercholesterolemia (FH). Three of six homozygotes are receptor-negative type and the other three homozygotes are receptor-defective type. In the cells from three receptor-negative homozygotes, the receptor binding, internalization, and degradation of 125I-LDL were 0.5±0.3 ng/mg protein (mean±SEM), 14±8 and 8±6 ng/mg protein per 6 h (four normal cells; 44±3, 386±32, and 1,335±214 ng/mg protein per 6 h), respectively. In the cells from three receptor-defective homozygotes, the receptor binding, internalization, and degradation of 125I-LDL were 6±2, 29±8, and 90±32 ng/mg protein per 6 h, respectively. In these six homozygotes, two pairs of siblings are included. Two siblings in the same family were classified as receptor-negative and two siblings in another family were classified as receptor-defective. The receptor-negative phenotypes and the receptor-defective phenotypes bred true in individual families. The cells from five heterozygotes showed ∼46% of the normal activities of receptor.
Toshihiro Haba, Hiroshi Mabuchi, Akira Yoshimura, Akira Watanabe, Takanobu Wakasugi, Ryozo Tatami, Kosei Ueda, Ryosei Ueda, Tomio Kametani, Junji Koizumi, Susumu Miyamoto, Ryoyu Takeda
During phagocytosis, neutrophils generate reactive oxygen metabolites and release lysosomal enzymes into the extracellular medium. We have investigated the possibility that these enzyme are inactivated by the oxygen compounds. Phagocytosing neutrophils from 12 patients with chronic granulomatous disease, which do not generate these oxygen metabolites, released two to three times more activity of lysozyme and beta-glucuronidase than did normal neutrophils. This difference proved to be due to a decrease of approximately 20% of the total activity of these enzymes in normal neutrophils, but not in neutrophils of patients with chronic granulomatous disease. This inactivation of enzymes took place during phagocytosis of opsonized zymosan particles as well as during stimulation of normal cells with phorbol myristate acetate. The inactivation was not due to formation of inhibitors. The lysosomal enzymes were not activated when the neutrophils were stimulated under anaerobic conditions. Addition of catalase, superoxide dismutase, or albumin gave no protection against the oxidative damage; reduced glutathione gave partial protection. The oxidative inactivation was more pronounced in the presence of azide. Measurement of the activity and the amount of protein of acid alpha-glucosidase in the cells showed that the specific activity of this enzyme decreased by approximately 50% during 30 min of phagocytosis. This indicates that the inactivation of the lysosomal enzymes takes place in the phagolysosomes, before the enzymes have leaked into the extracellular medium.
A A Voetman, R S Weening, M N Hamers, L J Meerhof, A A Bot, D Roos
The metabolic disposition of the plasma binding protein (DBP) for vitamin D and its metabolites was studied in adult rabbits. Apo-DBP was purified from rabbit plasma and enzymatically labeled with radioiodine. The radioiodine-labeled protein retained its ability to bind vitamin D sterols and its physicochemical properties. When 125I-labeled DBP and 131I-labeled rabbit albumin were simultaneously injected intravenously, the 125I was cleared from plasma at a faster rate (t 1/2 = 1.7 d) than 131I (t 1/2 = 5 d) and 125I was present in excess of 131I in kidney, liver, skeletal muscle, heart, lung, intestine, testis, and bone 1 h after injection. In contrast to DBP, 25(OH)D3 was cleared more slowly (t 1/2 = 10.7 d). Compared to albumin, DBP radioactivity appeared earlier and in greater quantity in the urine of catheterized rabbits. Gel filtration analyses of plasma revealed most of the 125I to elute in the position of DBP, with only small amounts in the less than 1,000-dalton region. In contrast, almost all of the urine 125I eluted in this small molecular weight fraction. The molar ratio of DBP to 25(OH)D3 in normal rabbit plasma was 138/1. The extravascular pool of DBP was calculated to be 1.5-2.4 times larger than the intravascular DBP pool, and the molar replacement rate of DBP was 1,350-fold higher than that of 25(OH)D3. The plasma disappearance curves of holo-DBP, prepared either by saturating with 25(OH)D3 or by covalently linking 3 beta-bromoacetoxy-25(OH)D3, were very similar to that of apo-DBP. Neuraminidase treatment of DBP did not alter its plasma survival. These studies indicate that DBP or DBP-25(OH)D3 complex is removed from plasma by a variety of tissues, that the DBP moiety is degraded during this process, and that a significant recirculation of 25(OH)D3 probably occurs. The molar excess of DBP to 25(OH)D3 in plasma, and the relatively rapid turnover of DBP indicate that a high capacity, high affinity, and dynamic transport mechanism for vitamin D sterols exists in rabbit plasma.
J G Haddad, D R Fraser, D E Lawson
The relationship between platelet release and fibrinopeptide A cleavage from fibrinogen to form fibrin I in vitro was examined in blood allowed to clot undisturbed or with gentle agitation. In undisturbed or agitated blood platelet release and fibrin I formation occurred simultaneously. When hirudin was added to undisturbed blood it prevented platelet release as well as fibrin I formation. In contrast, hirudin added to agitated blood had little effect on platelet release despite complete inhibition of fibrin I formation. Collagen added to either undisturbed or agitated blood increased platelet release and then fibrin I formation, and ADP added to undisturbed blood caused an initial burst of platelet release followed by slight acceleration of fibrinopeptide A cleavage. Prostaglandin E1 and theophylline prevented platelet release in both undisturbed and agitated blood, but did not affect fibrin I formation. The results with inhibitors in agitated blood suggest that fibrin I formation and platelet release can occur independently in the presence of the increased interactions induced by agitation. Addition of thrombin or tissue thromboplastin to undisturbed blood accelerated fibrin I formation with little effect on platelet release. Finally, initial thrombin formation in undisturbed blood appeared to be associated with the platelet surface. These relationships suggest that thrombin formation via the intrinsic system leads to thrombin generation on the platelet surface and simultaneous platelet release and fibrin I formation, while thrombin generated via tissue thromboplastin leads to thrombin formation in the plasma and fibrin I formation preceding platelet release. Activation by interaction of blood with collagen causes initial acceleration of platelet release and later acceleration of fibrin I formation.
K L Kaplan, M Drillings, G Lesznik
The fetal rat mobilizes liver glycogen during parturition for use as a glucose source until the onset of gluconeogenesis at 2 h after birth. A rat strain (NZR/Mh) unable to mobilize liver glycogen because of a phosphorylase b kinase deficiency has been used to assess the importance of liver glycogen in glucose homeostasis of the newborn. In normal rats the mean blood glucose concentration of the fetus measured at various times up to 24 h after natural birth ranged between 3.7 and 5.4 mM. In contrast, fetuses of the affected rats were hypoglycemic before birth (2.02 +/- 0.15 mM), and by 1 h after birth the blood glucose had decreased to 0.74 +/- 0.14 mM. Concentrations increased by 4 h to 1.48 +/- 0.17 mM and by 24 h reached values not significantly different from the normal newborn rats. Changes in plasma insulin over the perinatal period were similar in both groups although concentrations were always significantly lower in the affected rts. The findings demonstrate the crucial role of the fetal liver glycogen store in the maintenance of normoglycemia in the newborn. The normal rat does not develop hypoglycemia when born naturally and left with the mother after birth (in contrast to other studies in which the newborn were taken by cesarian delivery 1 d prematurely and kept in an artificial environment without food). The rats with the glycogen storage disorder experienced severe hypoglycemia without any apparent effects, which raises questions concerning alternative fuels available to and utilized by the newborn.
K R Gain, R Malthus, C Watts
Newly synthesized acid hydrolases, destined for transport to lysosomes, acquire a phosphomannosyl targeting signal by the transfer of N-acetylglucosamine 1-phosphate from uridine 5'-diphosphate (UDP)-N-acetylglucosamine to a mannose residue of the acid hydrolase followed by removal of the outer, phosphodiester-linked N-acetylglucosamine to expose 6-phosphomannose. This study demonstrates that fibroblasts from patients with the lysosomal enzyme storage diseases, I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III), are severely deficient in UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminylphosphotransferase, the first enzyme of the sequence. The N-acetylglucosaminylphosphotransferase activity (assayed using endogenous acceptors) in cultures from six normal subjects ranged from 0.67 to 1.46 pmol N-acetylglucosamine-1-phosphate transferred/mg protein per h, whereas five pseudo-Hurler polydystrophy and five I-cell disease cultures transferred less than 0.02 pmol/mg protein per h. The activity in five other pseudo-Hurler cultures ranged from 0.02 to 0.27 pmol transferred/mg protein per h. The activity of alpha-N-acetylglucosaminyl phosphodiesterase, the enzyme responsible for phosphomonoester exposure, is normal or elevated in cultured fibroblasts from both I-cell disease and pseudo-Hurler polydystrophy patients. The deficiency of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminylphosphotransferase explains the biochemical abnormalities previously observed in I-cell disease and pseudo-Hurler polydystrophy.
M L Reitman, A Varki, S Kornfeld
The potential role of epidermal growth factor (EGF) in the regulation of rat liver regeneration was examined by assessing the binding of 125I-EGF to hepatic membranes isolated at various times after partial hepatectomy. The results demonstrated a fall in 125I-EGF binding detectable as early as 8 h after partial hepatectomy. The nadir in EGF binding, less than 40% of that observed in sham-operated control rats, was seen 36 and 48 h after partial hepatectomy. Scatchard analysis showed that the decrease in binding capacity was due to a fall in receptor number. The specificity of the observed loss of EGF receptors was substantiated in parallel studies of 125I-insulin and 125I-wheat germ lectin binding; the binding of these ligands did not decrease appreciably during liver regeneration. The data are consistent with the hypothesis that EGF or a similar substance is one component of the complex humoral signal that regulates liver regeneration.
H S Earp, E J O'Keefe
Products of the lipoxygenation of arachidonic acid that express neutrophil chemotactic activity were examined in vitro for effects on the uptake of 45Ca by rabbit peritoneal neutrophils. At optimally chemotactic concentrations, 5- and 11-hydroperoxyeicosatetraenoic acid, 11- and 12L-hydroxyeicosatetraenoic acid, and leukotriene B4 enhanced 45Ca uptake within 1 min by a mean of 212-694% of the values for control neutrophils incubated in buffer alone, as compared with 75% for 5(S)-hydroxyeicosatetraenoic acid and no effect for 15-hydroperoxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid. The approximate rank of potency of the factors stimulating 45Ca uptake was similar to that observed for chemotaxis. Leukotriene B4, in addition to stimulating the rate of 45Ca uptake into rabbit neutrophils, also displaced intracellular calcium. The net result of the leukotriene B4-induced changes in calcium homeostasis ws to elevate transiently the intracellular level of exchangeable calcium. That neutrophil lipoxygenase metabolites of endogenous arachidonic acid rapidly enhance the influx of 45Ca supports a possible role for such metabolites in general, and leukotriene B4 in particular, in the regulation of the intracellular levels of free calcium in the neutrophils and possibly in other hormonally sensitive cells in which this cation is a second messenger.
P H Naccache, R I Sha'afi, P Borgeat, E J Goetzl
Two distinct types of colony-stimulating factor (CSF) have recently been described in human tissues and cultured cell lines. Antisera to purified type I and II CSF were prepared in rabbits. Anti-CSF I antibody inhibits CSF I, but has no effect on CSF II. It cross-inhibits CSF I from several other human sources, but does not inhibit CSF from mouse lung or mouse L cells. Anti-CSF II antibody inhibits the activity of CSF II, but has no effect on CSF I. A radioimmunoassay for CSF I has been established. Competitive binding assay further demonstrated the immunological differences between CSF I and II. When CSF II is used to stimulate human marrow cells fractionated by sedimentation velocity, two populations of CFU-C are separated, one sedimenting at 8 mm/h and forming colonies by day 7, and a second sedimenting at 6.8 mm/h and forming colonies by day 13. In contrast, CSF I does nt stimulate colony growth by day 7 but does do so by day 13 in cells sedimenting between 7.2-8.5 mm/h. These results indicate that CSF I and II are distinct in their biochemical, immunological, and functional properties.
M C Wu, A M Miller, A A Yunis