Papillary and surface micropuncture was used to assess the effects of a chronic metabolic acidosis on the renal tubular handling of ammonium by surface nephrons, juxtamedullary nephrons, and the terminal segment of collecting duct. Rats chronically fed ammonium chloride had an expected decline in arterial pH and bicarbonate concentration associated with a doubling in the amount of ammonium excreted and a decline in urine pH. The glomerular filtration rate and absolute delivery of water and sodium to micropuncture sites of surface and deep nephrons was not measurably altered. Ammonium delivery to the end of the proximal tubule increased from 853±102% to 1,197±142% (SE) of the filtered load of ammonium after the induction of metabolic acidosis. This increase was due to a rise in tubular fluid ammonium content from 2.31±0.23 to 4.06±0.28 mM/liter. After the induction of acidosis, absolute and fractional delivery of ammonium ion to the end of the distal tubule was less than to the end of the accessible portion of the proximal tubule. These findings indicate that ammonium is lost in the intervening segment.
John Buerkert, Daniel Martin, David Trigg
To determine whether production or catabolism of low density lipoprotein (LDL) is the major factor controlling LDL concentrations in subjects with plasma cholesterol levels from low-normal to mildly elevated, measurements of apoprotein of LDL (apoLDL) turnover were performed in 16 patients with various plasma cholesterol concentrations. Cholesterol balance studies were done simultaneously in 13 of these patients. Plasma concentrations of apoLDL and LDL-cholesterol were positively correlated with synthetic rates of apoLDL (r = 0.74, P less than 0.001; r = 0.50, P less than 0.05, respectively). No correlation was noted between the fractional catabolic rate for apoLDL and apoLDL levels (or LDL-cholesterol). For further analysis, the patients were divided into three groups with stepwise increases in apoLDL concentrations. When apoLDL levels rose significantly, from 83 +/- 5 SEM to 122 +/- 2 to 149 +/- 5 mg/dl, synthetic rates for apoLDL also increased significantly from 11.6 +/- 12. to 17.0 +/- 0.9 to 23.8 +/- 1.8 mg/d/kg ideal weight. In contrast, the fractional catabolic rate of apoLDL was not different among the three groups (0.32 +/- 0.03 vs. 0.29 +/- 0.02 vs. 0.33 +/- 0.03/d). No relation was noted between synthesis of total body cholesterol (or bile acids) and concentrations, production rates, or removal of apoLDL. Thus, concentrations of apoLDL and LDL-cholesterol in these subjects with plasma cholesterol levels from low-normal to mildly elevated were regulated mainly by synthetic rates of apoLDL and not by LDL catabolism.
Y A Kesaniemi, S M Grundy
Formation of heme, bilirubin, and bilirubin conjugates has been examined in mucosal cells isolated from the rat upper small intestine. Intact, viable cells were prepared by enzymatic dissociation using a combined vascular and luminal perfusion and incubated with an isotopically labeled precursor, delta-amino-[2,3-3H]levulinic acid. Labeled heme and bile pigment were formed with kinetics similar to those exhibited by hepatocytes. Moreover, the newly formed bilirubin was converted rapidly to both mono- and diglucuronide conjugates. In addition, cell-free extracts of small intestinal mucosa from rats or humans exhibited a bilirubin-UDP-glucuronyl transferase activity that was qualitatively similar to that present in liver. The data suggest that the small intestinal mucosa normally contributes to bilirubin metabolism.
F Hartmann, D M Bissell
Human milk fat globules require colipase to be hydrolyzed by pancreatic lipase in the presence of bile salts. This is contrary to a recent report in this Journal (J. Clin. Invest. 67: 1748-1752.) according to which inhibition of lipase by bile salt could be overcome by the addition of colipase or phospholipase A2. This latter finding is shown to be due to contamination of commercially available pancreatic phospholipase A2 by colipase.
B Borgström, C Erlanson-Albertsson
Sera from 35 men were collected before and at timed intervals subsequent to vasectomy and examined for the presence of (a) antibody reactive with human spermatozoa, (b) sperm-related antigen, and (c) circulating immune complexes (CIC). Fewer than 10% of the men examined were ever positive for antisperm antibodies. However, sperm-related antigens were elevated in the sera of 18, 18, and 26% of the mean at 2 wk, 2 mo, and 4 mo postvasectomy, respectively. CIC were detected in the sera of some vasectomized men by three different assays. The CIC in patients' sera were precipitated with polyethylene glycol, dissociated, and the individual CIC components identified by an enzyme-linked immunosorbent assay. Most, but not all, of the CIC contained antigen reactive with antisperm immunoglobulin (Ig)G and some also contained complement components C3 and/or Clq. IgA was identified in some of the CIC positive for IgG and sperm antigen and two men had IgM-containing CIC. Analysis of the CIC by sucrose gradient centrifugation revealed them to be heterogeneous in size.
S S Witkin, G Zelikovsky, A M Bongiovanni, N Geller, R A Good, N K Day
Viable rat islet cells were used to detect islet cell surface antibodies (ICSA) in the sera of diabetic and control patients. ICSA were present in almost all recent-onset insulin-dependent diabetics younger than 30 yr (15/16); their incidence in other diabetics (6/22) was also higher than in normal controls (1/18) or in patients with autoimmune thyroiditis (1/12). The varying specificity of the ICSA for the different islet cell types led to the recognition of class I sera, whose ICSA bind exclusively to B cells, class II sera, binding only to A and pancreatic polypeptide (PP) cells and class III sera, reacting with the three islet cell types but not with D cells. Most recent-onset insulin-dependent diabetics younger than 30 contained class I-ICSA, which is consistent with an autoimmune basis of their disease and with an involvement of surface antibodies in the B cell destruction. The presence of class II ICSA in three older diabetics and in one normal control raises the question whether autoimmune reactions against A and PP cells exist and are associated with a distinct entity in islet disease.
M. Van De Winkel, G. Smets, W. Gepts, D. Pipeleers
To determine if environmental factors could effect the switchover from fetal hemoglobin (HbF) to adult hemoglobin (HbA) synthesis, studies were carried out on blood samples from eight infants born at less than 1,000 g, when they had reached their postconceptional age corresponding to term. All of these infants required prolonged intensive care, multiple blood transfusions, and two required exchange transfusions. Several were ventilated mechanically for 60 d and two infants had bronchopulmonary dysplasia at the time of the study. The blood samples were incubated in an amino acid mixture containing [14C]leucine followed by column chromatography on DEAE Sephadex for separation of radioactive HbA and HbF. In spite of the extreme prematurity and poor growth of these sick infants, the proportional synthesis of HbF and HbA, as determined by the incorporation of [14C]leucine during the erythrocyte incubations, was characteristic of the period of human development from which the erythrocytes were obtained.
H Bard, J Prosmanne
These studies examine the effects of acute changes in the availability of inorganic phosphate on the function of isolated proximal renal tubules from rabbit kidney. We removed phosphate from the extracellular fluids and measured fluid absorption rates in isolated perfused tubules and oxygen consumption rates in suspensions of cortical tubules. In proximal convoluted tubules, the selective removal of phosphate from the luminal fluid reduced fluid absorption rates from 1.11±0.12 to −0.01±0.08 nl/mm · min. This effect on fluid absorption was dependent on the presence of glucose transport and metabolism. The addition of phlorizin to the phosphate-free luminal fluid preserved fluid absorption rates (1.12±0.12 nl/mm · min) as did the substitution of nonmetabolized α-methyl d-glucopyranoside for glucose (1.05±0.21 nl/mm · min) or the addition of 2-deoxyglucose, an inhibitor of glycolysis, to the bathing medium (1.01±0.15 nl/mm · min). There was no effect on fluid absorption if phosphate was removed from the bath only. Additionally, removal of phosphate from the luminal fluid of proximal straight rather than convoluted tubules had no effect on fluid absorption rates. Oxygen consumption rates in suspensions of cortical tubules were reduced from 18.9±0.6 to 10.6±0.6 nmol O2/mg tubular protein · min by the removal of phosphate from the medium. This inhibition was prevented by the substitution of α-methyl d-glucopyranoside for glucose in the phosphate-free medium. The data indicate that under certain conditions, proximal convoluted tubules require the presence of phosphate in the luminal fluid to preserve tubular function. In the absence of intraluminal phosphate, glucose metabolism causes a reduction in both oxidative metabolism and fluid absorption. This response is analogous to the Crabtree effect and suggests limitations on the intracellular availability of inorganic phosphate.
Peter C. Brazy, Steven R. Gullans, Lazaro J. Mandel, Vincent W. Dennis
The effect of insulin on hepatic triglyceride synthesis and secretion is controversial. Previously, we have described a cell culture system of adult rat hepatocytes that synthesize and secrete very low density lipoprotein (VLDL) triglycerides with small and irreproducible effects of insulin on triglyceride metabolism. To study the primary effects of insulin on hepatic triglyceride metabolism a method was developed utilizing fibronectin-coated culture dishes that allowed adhesion, spreading, and maintenance of hepatocytes for 2-3 d in the absence of serum and insulin. This culture system allowed mass measurements of both cellular and secreted VLDL triglycerides for long time periods after the addition of physiological concentrations of insulin to hormone-free culture medium. In the absence of insulin and after an initial 4 h in culture, the medium was replenished and triglyceride mass was measured at the end of 18-h incubations. VLDL triglyceride accumulated in the culture medium at a linear rate over this time-course with increasing accumulation as the medium glucose concentration was raised from 2.5 to 25 mM glucose (1.77±0.24 to 3.09±0.76 μg triglyceride/mg cell protein per h). There was no apparent significant lipolysis or hepatocellular reuptake of secreted VLDL triglycerides. In the absence of insulin cellular triglyceride levels were unchanged between 3 and 24 h in culture while insulin (50-500 μU/ml) significantly increased cellular triglyceride content at all glucose concentrations tested (0-25 mM). The addition of insulin to the culture medium progressively reduced the rate of VLDL triglyceride secretion accompanied by an increase in cellular triglyceride at insulin concentrations > 50 μU/ml. Most or all of the observed increase in cell triglyceride content could in all experiments be accounted for by the insulin-induced inhibition of VLDL secretion. Incorporation of [2-3H]glycerol into cellular and VLDL triglycerides as a function of insulin concentration was also measured. Glycerol incorporation data at 20-22 h after plating of the cells closely paralleled the insulin-induced changes in cellular and VLDL triglyceride as determined by mass analysis. The observed effects of insulin occurred at concentrations close to the physiological range and suggest that the direct hepatic effect is to suppress VLDL secretion although the net effect in vivo will clearly reflect many additional accompanying changes.
Paul N. Durrington, Roger S. Newton, David B. Weinstein, Daniel Steinberg
The detection of an elevation in neurotensinlike immunoreactivity in peripheral plasma for several hours after a meal has been confirmed and shown to be primarily due to the presence of aminoterminal fragments of neurotensin (NT) rather than to NT itself. We have developed a procedure to separate and characterize these N-terminal cross-reacting substances, and to estimate the contributions of these constitutents to plasma neurotensinlike immunoreactivity. Gel chromatography of pooled plasma extracts on Sephadex G-25 followed by reverse-phase high pressure liquid chromatography indicated that peptides coeluting with NT and its N-terminal partial sequences NT(1-8) and NT(1-11) were present in plasma. Comparison of plasmas collected before and 1 h after a defined meal, in five experiments, demonstrated no change in C-terminal immunoreactivity and an 8- to 10-fold rise in N-terminal immunoreactivity. Chromatographic analysis of pooled pre- and postmeal plasma in four experiments showed that essentially all of this elevation in neurotensinlike immunoreactivity measured with an N-terminal directed antiserum was due to increases in NT(1-8) and NT(1-11), while NT itself, measured using a C-terminal directed antiserum, did not increase appreciably in peripheral plasma 1 h after the meal. Generation of tritiated substances with the same elution times as NT(1-8) and NT(1-11) did occur after incubation of [3H]NT with whole blood in vitro, providing supporting evidence that these fragments are metabolites of NT. The marked elevation in the circulating levels of these fragments reflects that an increased secretion of NT occurred in response to the test meal. The secreted NT may have acted as a hormone before it was metabolized, or it may only have had a local (paracrine) effect.
Robert A. Hammer, Robert E. Carraway, Susan E. Leeman
Leishmania are obligate intracellular parasites of mononuclear phagocytes in the mammalian host. To more clearly define features of the early events in host-parasite interaction, human monocytes were so-cultured with Leishmania tropica amastigotes in vitro. Infection of monocytes was time dependent and inhibited at 4 degrees C and in the presence of cytochalasin B. Pretreatment of amastigotes with cytochalasins prevented their attachment to normal monocytes. Untreated amastigotes attached normally but could not enter cytochalasin-pretreated monocytes. This suggests that amastigotes actively participate in attachment but require host cell participation for interiorization.
D J Wyler
[14C]Stearic acid-labeled cerebroside sulfate (CS) was presented to cultured skin fibroblasts in the media. After endocytosis into control cells 86% was readily metabolized to galactosylceramide, ceramide, and stearic acid, which was reutilized in the synthesis of the major lipids found in cultured fibroblasts. Uptake and metabolism of the [14C]CS into cells from typical and atypical patients and carriers of metachromatic leukodystrophy (MLD), Krabbe disease, and Farber disease were observed. Cells from patients with late infantile MLD could not metabolize the CS at all, while cells from an adult MLD patient and from a variant MLD patient could metabolize ∼40 and 15%, respectively, of the CS taken up. These results are in contrast to the in vitro results that demonstrated a severe deficiency of arylsulfatase A in the late infantile and adult patient and a partial deficiency (21-27% of controls) in the variant MLD patient. Patients with Krabbe disease could metabolize nearly 40% of the galactosylceramide produced in the lysosomes from the CS. This is in contrast to the near zero activity for galactosylceramidase measured in vitro. Carriers of Krabbe disease with galactosylceramidase activity near half normal in vitro and those with under 10% of normal activity were found to metabolize galactosylceramide in cells significantly slower than controls. This provides a method for differentiating affected patients from carriers with low enzyme activity in vitro. Cells from patients with Farber disease could catabolize only ∼15% of the ceramide produced from galactosylceramide. This technique provides a method for the identification of typical and atypical patients and carriers of three genetic diseases using one substrate.
Tooru Kudoh, David A. Wenger
T cells proliferate in response to autologous non-T cells in the autologous mixed lymphocyte reaction (AMLR). AMLR was impaired in the peripheral blood of patients with advanced lung cancer (4,159 +/- 3,878 delta cpm vs. 11,221 +/- 4,156 delta cpm for normal donors) but normal or even higher in their malignant pleural effusions (13,257 +/- 7,075 delta cpm vs. 10, 870 +/- 5,013 delta cpm for nonmalignant control effusions). Blood T cells also failed to respond to autologous effusion non-T cells, while effusion T cells strongly responded to autologous erythrocytes blood non-T cells. The presence of blood T cells did not inhibit effusion AMLR of the same patients. A subset of T cells that form rosettes with autologous erythrocytes if found to proliferate in AMLR. The number of autorosette-forming cells was lower in blood T cells of cancer patients than in blood T cells of normal donors and in effusion T cells of the patients. After enrichment of autorosette-forming cells, there was no difference in AMLR of normal blood and cancer blood and effusions. These results indicate that the loss of AMLR in the blood of cancer patients is due to a reduction of number of autoreactive T cells and not to a defect of autologous stimulator non-T cells.
A Uchida, M Micksche
This study was designed to provide direct information on the in vivo metabolism in man of free (unesterified) cholesterol in the major lipoprotein classes. Five human subjects were administered one or two (simultaneous) of the following; [2-14C] mevalonic acid, high density lipoprotein (HDL)-free [14C] cholesterol, low density lipoprotein (LDL)-free [14C] cholesterol, and very low density lipoprotein (VLDL)-free [3H]cholesterol. Blood was then obtained at frequent intervals for at least 9 h, and the α(HDL) and β(LDL + VLDL) lipoproteins were quickly separated by heparin-manganese precipitation to prevent ex vivo exchange of free cholesterol. After the administration of [14C]mevalonic acid the specific activity (disintegrations per minute/micromole) of free cholesterol in the α- and β-lipoproteins increased for 3 h. During this period the α-free cholesterol specific activity was higher than the β specific activity. After administration of VLDL and LDL labeled with free cholesterol, the α-free cholesterol specific activity reached a peak value within 20 min, at which time it was considerably lower than the β-free cholesterol specific activity. When HDL labeled with free cholesterol was administered, a precursor product relationship was observed between the α-free cholesterol (precursor) and β-free cholesterol (product) specific activities.
Charles C. Schwartz, Z. Reno Vlahcevic, Mones Berman, John G. Meadows, Richard M. Nisman, Leon Swell
The present study was designed to investigate the mechanisms by which insulin regulates the disposal of an intravenous glucose load in man. A combined tracer-hepatic vein catheter technique was used to quantitate directly the components of net splanchnic glucose balance (NSGB), i.e., splanchnic glucose uptake and hepatic glucose output, and peripheral (extrasplanchnic) glucose uptake. Four different protocols were performed: (a) intravenous infusion of glucose alone (6.5 mg kg−1 min−1) for 90 min (control group); (b) glucose plus somatostatin (0.6 mg/h) and glucagon (0.8 ng kg−1 min−1; (c) glucose plus somatostatin, glucagon, and insulin (0.15 mU kg−1 min−1); and (d) glucose plus somatostatin, glucagon, and insulin (0.4 m U kg−1 min−1). In groups 2-4, arterial blood glucose was raised to comparable levels to those of controls (≃170 mg/dl) by a variable glucose infusion. In the control group, plasma insulin levels reached 40 μU/ml at 90 min. NSGB switched from a net output of 1.71±0.13 to a net uptake of 1.5-1.6 mg kg−1 min−1 due to a 90-95% suppression of hepatic glucose output (P < 0.01) and a 105-130% elevation of splanchnic glucose uptake (from 0.78±0.13 to 1.6-1.8 mg kg−1 min−1; P < 0.01). Peripheral glucose uptake rose by 150-160% (P < 0.01). In group 2, plasma insulin fell to <5 μU/ml. Net splanchnic glucose output initially rose twofold but later returned to basal values. This response was entirely accounted for by similar changes in hepatic glucose output since splanchnic glucose uptake remained totally unchanged in spite of hyperglycemia. In contrast, peripheral glucose uptake rose consistently by 100% (P < 0.01) despite insulin deficiency. In an additional group of experiments, glucose metabolism by the forearm muscle tissue was quantitated during identical conditions to those of group 2 (hyperglycemia plus insulin deficiency). Both the arterial-deep venous blood glucose difference and forearm glucose uptake increased markedly by 300-400% (P < 0.05 - <0.01). In group 3, plasma insulin was maintained at near-basal, peripheral levels (12-14 μU/ml). Hepatic glucose output decreased slightly by 35-40% (P < 0.05) while splanchnic glucose uptake remained unchanged. Consequently, the net glucose overproduction seen in group 2 was totally prevented although NSGB still remained as a net output. In group 4, peripheral insulin levels were similar to those of the control group (35-40 μU/ml). The suppression of hepatic glucose output was more pronounced (60-65%) and splanchnic glucose uptake rose consistently by 65% (P < 0.01). Consequently, NSGB did not remain as a net output but eventually switched to a small uptake (0.3 mg kg−1 min−1). Peripheral glucose uptake rose to the same extent as in controls.
Luigi Saccà, Marco Cicala, Bruno Trimarco, Biagio Ungaro, Carlo Vigorito
An endothelial cell-associated cofactor that greatly enhances the rate of protein C activation by thrombin has recently been described. The observation that the cofactor binds thrombin with unusually high affinity (Kd = 0.5 nM) suggested that low level thrombin infusion into dogs might lead to the selective activation of protein C.
Philip C. Comp, Rene M. Jacocks, Gary L. Ferrell, C. T. Esmon
Effect of metabolic acidosis on two distinct 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) systems was studied in the kidneys of vitamin D-deficient rats; one is localized in the proximal convoluted tubule (PCT), is activated in vitamin D deficiency, and is regulated primarily by parathyroid hormone (PTH) via cyclic AMP; the other is localized in the proximal straight tubule (PST), is latent in vitamin D deficiency, and is selectively stimulated by calcitonin via a cyclic AMP-independent mechanism. The 1α-hydroxylase activities were measured in the PCT and PST microdissected from the kidney of vitamin D-deficient rats with or without metabolic acidosis of varying duration. The 1α-hydroxylase activity decreased in the PCT from 0.74±0.07 fmol/mm per h to 0.24±0.02 at day 3 of metabolic acidosis without a further decline at day 7. Neither metabolic acidosis of 16 h duration nor reduction of the incubation medium pH from 7.4 to 7.0 affected the enzyme activity in the PCT. To examine the underlying mechanism for the suppression of 1α-hydroxylase activity, PTH, cyclic AMP, or calcitonin was given to rats with metabolic acidosis of 3 d duration. Although PTH failed to augment the suppressed 1α-hydroxylase activity in the PCT, cyclic AMP restored it to the level of control rats. The 1α-hydroxylase activity in the PST remained undetectable in control rats and in acidotic rats with or without PTH or cyclic AMP treatments. However, calcitonin stimulated the 1α-hydroxylase activity in the PST equally from undetectable to 0.75±0.09 fmol/mm per h in control and to 0.78±0.10 in acidotic rats. The data suggests that metabolic acidosis suppresses 1α-hydroxylase only in the PCT by inhibiting PTH-dependent adenylate cyclase, and that cellular events beyond cyclic AMP in the PCT and the events responsive to calcitonin in the PST are unaffected. The results show the definite advantage of using defined single nephron segments to study the hormonal and ionic control of the 1α-hydroxylase system in the kidney.
Hiroyuki Kawashima, Jeffrey A. Kraut, Kiyoshi Kurokawa
We characterized the RNA-containing antigens precipitated by sera from 260 patients with positive antinuclear antibodies. 49 individuals, most of whom had systemic lupus erythematosus or Sjögren's syndrome, possessed antibodies that precipitated the previously identified RNP, Sm, Ro, and La antigens either singly or in combinations. These antigens, which are located on discrete sets of small nuclear or cytoplasmic RNA-protein particles, exhibited a number of antigenic interrelationships. One patient's serum recognized a new particle containing a small RNA which we have called Th; it also precipitated the Ro complexes. Other patients with systemic lupus erythematosus, hepatitis B virus infection, juvenile rheumatoid arthritis, myositis, and rheumatoid arthritis had antibodies that precipitated specific subsets of ribosomal RNA and transfer RNA. One patient's serum contained a monoclonal immunoglobulin G that precipitated ribosomes. Most of these antibodies identified antigenic determinants constituted at least in part of protein. The specificity of the proteins bound to particular cellular RNA, probably explains the exquisite precision with which antibodies from rheumatic disease patients discriminate among RNA subsets. Such sera should be useful probes for investigating specific roles that different RNA and RNA-protein complexes play in cellular metabolism.
J A Hardin, D R Rahn, C Shen, M R Lerner, S L Wolin, M D Rosa, J A Steitz
The nature and mechanism of the pancreatic exocrine dysfunction in diabetes mellitus were evaluated in vitro using isolated pancreatic acini prepared from streptozotocin-induced diabetic rats. The content of amylase and ribonuclease in diabetic acini was ∼0.5 and 50% of the normal content, respectively. Further, reduced amounts of both enzymes were secreted by diabetic acini in response to both cholecystokinin (CCK) and carbamylcholine. However, when enzyme secretion was normalized relative to initial acinar contents, both normal and diabetic acini released enzymes at a comparable maximal rate. The time course of the release of these enzymes, and newly synthesized protein were similar in both acini. In normal acini, the effect of CCK was maximal at a concentration of 100 pM; higher concentrations led to submaximal enzyme release. The dose-response curve in diabetic acini was similarly shaped, but shifted three-fold towards higher concentration. The mobilization of cellular Ca2+ in response to CCK was also shifted. In contrast to these results with CCK, the dose-response curve to carbamylcholine was unaltered by diabetes. The observed effects were confirmed to be due to insulin deficiency and not due to direct toxic effect of streptozotocin on acinar cells or malnutrition. Streptozotocin had no acute effect on acini when measured 24 h after administration, and alloxan, another beta cell toxin, induced similar changes in acinar enzyme content and secretory response. Moreover, the administration of exogenous insulin to diabetic rats returned the content of pancreatic amylase and the secretory response to CCK towards normal. Starvation for 48 h, although inducing a significant weight loss, did not mimic the effects of diabetes. The present studies demonstrate two major abnormalities in pancreatic exocrine secretion in the diabetic rat: (a) the content of certain digestive enzymes is markedly altered, leading to an altered amount of zymogen secretion, (b) the sensitivity to CCK is selectively reduced, most likely related to a defect in receptor activated transmembrane signaling.
Makoto Otsuki, John A. Williams
Neisseria gonorrhoeae isolated from patients with disseminated infection (DGI) often resist complement (C′)-dependent killing by normal human serum (NHS) and less commonly by convalescent DGI serum. 7 of 10 NHS specimens completely inhibited killing of serum-resistant (serr) gonococci by convalescent or immune DGI serum. Immunoglobulin G (IgG) purified from NHS was shown to be the blocking agent. In addition, IgM (plus C′) purified from NHS was shown to be fivefold more effective (wt/wt) in killing serum-sensitive (sers) gonococci than equivalent amounts of IgM tested in the presence of IgG (whole serum). Although inhibition of NHS killing of sers gonococci required a 640% excess of IgG, only a 40% excess was required to block immune serum killing of serr gonococci. F(ab′)2 prepared from IgG also blocked killing of serr gonococci by immune serum indicating antigenic specificity of blocking IgG.
Peter A. Rice, Dennis L. Kasper
Triglyceride-rich lipoproteins may be responsible for the lipid accumulation in macrophages that can occur in hypertriglyceridemia. Chylomicrons and very low density lipoproteins (VLDL, total and with flotation constant [Sf] 100-400) from fasting hypertriglyceridemic subjects induced a massive accumulation of oil red O-positive inclusions in unstimulated peritoneal macrophages. Cell viability was not affected. The predominant lipid that accumulated in cells exposed to hypertriglyceridemic VLDL was triglyceride. Hypertriglyceridemic VLDL stimulated the incorporation of [14C]oleate into cellular triglyceride up to ninefold in 16 h, but not into cholesteryl esters. Mass increase in cellular triglyceride was 38-fold. The stimulation of cellular triglyceride formation was dependent on time, temperature, and concentration of hypertriglyceridemic VLDL. By contrast, VLDL, low density, and high density lipoproteins from fasting normolipemic subjects had no significant effect on oleate incorporation into neutral lipids or on visible lipid accumulation.
Sandra H. Gianturco, William A. Bradley, Antonio M. Gotto Jr., Joel D. Morrisett, Duane L. Peavy
Lactic acid represents a major exogenous nutrient for the developing fetal lamb in utero. Our study was undertaken (a) to quantitate the net consumption of lactate by the fetus, (b) to quantitate the net lactate production and metabolism by the placenta, and (c) to compare the net fetal lactate consumption with fetal lactate use, measured simultaneously with radioactive tracers. 14 pregnant sheep were prepared with catheters in the maternal femoral artery and uterine vein and in the fetal aorta and umbilical vein. By simultaneous application of the Fick principle to the uterine and umbilical circulations, placental glucose consumption and placental lactate production were rapid, averaging 39.8 +/- 5.1 and 11.8 +/- 0.7 mg.min-1. Net lactate umbilical uptake averaged 1.95 +/- 0.16 mg-1.kg.min-1. During infusion of L-[14C(U)]lactate, fetal lactate turnover was much more rapid, averaging 6.5 +/- 0.8 mg.kg-1.min-1, and lactate utilization within the anatomic fetus was 5.9 +/- 0.7 mg.kg-1.min-1. During infusion of tracer glucose, endogenous fetal lactate production from glucose and nonglucose substrates averaged 3.0 and 1.5 mg.kg-1.min-1, respectively. The present studies have quantitated under well oxygenated, steady-state conditions, the rapid placental metabolism and production of lactate, the net fetal consumption of lactate, and the rapid endogenous fetal lactate production from glucose and nonglucose substrates.
J W Sparks, W W Hay Jr, D Bonds, G Meschia, F C Battaglia
Children with the Chediak-Higashi (CH) syndrome are known to have abnormalities of natural killer (NK) cell function. We used the HNK-1 monoclonal antibody that reacts specifically with human NK and K cells to distinguish whether this abnormality was due either to a numerical deficiency of NK cells or a defect in their ability to function. In eight CH patients, a significant proportion of their blood mononuclear cells (10--19%) expressed the HNK-1 differentiation antigen. The level of NK cells in the five children with CH syndrome was higher than for age-matched normal controls (15.8% vs. 5.8%, P less than 0.001). When HNK-1+ cells were isolated with a fluorescence-activated cell sorter, the NK cells from CH patients were a homogeneous population of lymphocytes with a single large granule rather than the multiple small granules seen in Nk cells from normal individuals. The purified HNK-1+ cells from the CH patients had minimal NK or K cell function. The CH syndrome thus includes a functionally defective population of NK cells that retain the capability of expressing the HNK-1 differentiation antigen.
T Abo, J C Roder, W Abo, M D Cooper, C M Balch
In livers of fetuses of 220--340 g body wt, total cytosolic tyrosine aminotransferase activity was 1.0 nmol of product/mg of protein per min, and the corresponding values for autopsy livers of newborns of 740--1,475 g and 2,600--3,650 g were 1.5 and 5.7, respectively, as compared with the adult value of 12.7. On the other hand, para-hydroxyphenylpyruvate dioxygenase activity is at adult level already in fetuses less than 340 g body wt. The Km value for tyrosine of tyrosine aminotransferase (1 mM) was considerably higher than the corresponding value for para-hydroxyphenylpyruvate of para-hydroxyphenylpyruvate dioxygenase (50 micro M). These results suggest that tyrosine aminotransferase is the rate limiting enzyme in the catabolism of tyrosine in premature infants.
J J Ohisalo, T Laskowska-Klita, S M Andersson
The null cell compartments of human bone marrow and mouse spleen were arbitrarily divided into three subpopulations based upon the ability of cells to acquire T or B cell membrane markers when incubated with poly A:U or ubiquitin. There was an accumulation of T cell precursors with congenital absence of the thymus. In contrast, T cell precursors were reduced and there was an accumulation of uninduced null cells with old age. These observations suggest that there is an intrinsic defect of null cell differentiation with a drift towards more differentiated precursors in T cell differentiation with aging. This could result in a diminution in the range of responses by their progeny, mature T lymphocytes.
J J Twomey, R J Luchi, N M Kouttab
Peripheral blood lymphocytes and splenocytes of patients with autoimmune disease were used to prepare human-human hybridomas that produce autoantibodies. Because exogenous immunization was not used, the hybridoma antibodies were derived from B cells that spontaneously produced autoantibodies. 108 hybrids grew from 4,254 wells (2.5%). Optimal conditions for obtaining hybridomas with the GM 4672 myeloma line included initial growth in 2-ml wells, the use of 44% polyethylene glycol, a mononuclear cell/GM 4672 cell ratio 5:1, and prior stimulation of the B lymphocytes with pokeweed mitogen. Hybridoma supernatants had activity against ssDNA, platelets, and erythrocytes. The results demonstrate the feasibility of producing human-human hybridomas from lymphocytes of patients with various autoimmune diseases.
Y Shoenfeld, S C Hsu-Lin, J E Gabriels, L E Silberstein, B C Furie, B Furie, B D Stollar, R S Schwartz
[3H]Nitrendipine, a potent calcium channel antagonist [3-ethyl-5-methyl-1-1,4-dihydro-2,6 - dimethyl - 4 - (3 - nitrophenyl) - 3,5 - pyridine carboxylate], was used to label high affinity binding sites on membranes prepared from bovine aortic smooth muscle. The binding of [3H]nitrendipine is rapid (t1/2 < 5 min) and reversible at 37°C. The binding sites have a high affinity for [3H]nitrendipine with an equilibrium dissociation constant of 2.1 nM. The density of sites is 40-60 fmol/mg of membrane protein. Analogues of nitrendipine compete for the binding sites with affinities consistent with their known biological effects as calcium antagonists. Nisoldipine, [isobutyl methyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine carboxylate], a calcium antagonist more potent than nifedipine [2,6-dimethyl-3,5-dicarbomethoxy-4-(2-nitrophenyl)-1,4-dihydropyridine] in relaxing vascular smooth muscle, has an affinity three-fold higher than that of nifedipine in competing for the binding sites. A biologically inactive derivative of nifedipine does not compete for [3H]nitrendipine binding. Verapamil (α-isopropyl-α[(N-methyl - N-homoveratryl) -α-aminopropyl]-3,4-dimethyoxyphenyl acetonitrile), a structurally different calcium antagonist, only partially (25%) inhibits binding at high concentrations (1 μM). Prazosin, an alpha adrenergic antagonist does not compete for [3H]nitrendipine binding sites. The binding of [3H]nitrendipine is not affected by 1.5 mM calcium. Canine cardiac membranes also have high affinity [3H]nitrendipine binding sites, (KD = 6 nM) but bovine erythrocytes do not. The relative affinities of nisoldipine and nifedipine for the cardiac membrane binding sites reflect the relative activities of these compounds as calcium channel antagonists. These results suggest that the [3H]nitrendipine binding sites are the sites through which dihydropyridines act as calcium channel antagonists.
Lewis T. Williams, Patrice Tremble