Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l-phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values.
Timothy M. Cox, Martin W. O'Donnell, Michael Camilleri, Arthur H. Burghes
The sickle erythrocyte (RBC) is a pathologic RBC that contains multiple membrane abnormalities. Some of these abnormalities have been implicated in the pathophysiology of vasoocclusive crises characteristic of sickle cell disease; others have yet to be defined in terms of their clinical significance. Recent information has shown that sickle RBC adhere abnormally to cultured endothelial cells yet little is known about the ways in which sickle cells interact with model membranes of defined size and lipid composition. We investigated this phenomenon by interacting sickle RBC with artificial lipid vesicles (liposomes) containing acidic phospholipids. Our results demonstrate that sickle disease (hemoglobin SS) RBC bind more of these liposomes than do normal or sickle trait (hemoglobin AS) RBC and that these differences are accentuated by hypoxia-induced sickling. Binding of liposome phospholipid to sickled RBC was not attributable to phospholipid exchange between liposomes and RBC and was consistent with a mechanism involving both membrane fusion and a stable reversible adhesion of liposomes to the RBC membrane.
Robert S. Schwartz, Nejat Düzgüneş, Danny Tsun-Yee Chiu, Bertram Lubin
The role of prostaglandins (PG), renin-angiotensin system (RAS) and calcium (Ca) in the control of renal hemodynamics and glomerular filtration rate (GFR) in chronic hypercalcemia (serum Ca 12.8 mg%) was studied. Renal blood flow (RBF, 6.39 ml/min per gram kidney weight [gkw]) and GFR (0.52 ml/min per gkw) were significantly decreased in hypercalcemic rats when compared with normocalcemic rats (7.15, P < 0.001 and 0.74, P < 0.05, respectively). These changes in RBF and GFR occurred independent of any significant alterations in systemic hemodynamics, blood and plasma volume. Inhibition of the renal PG with indomethacin resulted in marked decrements in both RBF (6.39-4.12 ml/min per gkw, P < 0.01) and GFR (0.52-0.19 ml/min per gkw, P < 0.01) in hypercalcemic rats, whereas there was no significant alterations in normocalcemic rats. Inhibition of the RAS with captopril resulted in marked increments in both RBF (6.39-7.35 ml/min per gkw, P < 0.05) and GFR (0.52-0.74 ml/min per gkw, P < 0.05) in hypercalcemic rats. In fact, there was no significant difference from the RBF and GFR of similarly treated normocalcemic rats. Similar results were also obtained with the competitive angiotensin II (AII) antagonist (sarcosyl1-isoleucyl5-glycyl8) AII. Since both the renal PG and the RAS are involved in the control of RBF and GFR in hypercalcemia, the role of each is best revealed in the absence of the other. Hence, comparison of the RBF and GFR in the PG-inhibited hypercalcemic rats in the presence of AII (4.12 and 0.19 ml/min per gkw, respectively) and absence of AII (5.99 and 0.53 ml/min per gkw, P < 0.01 for both) reveals the vasoconstrictive role for AII in hypercalcemia. On the other hand, comparison of the RBF and GFR in the AII-inhibited hypercalcemic rats in the presence of PG (7.35 and 0.74 ml/min per gkw, respectively) and absence of PG (5.99 and 0.53 ml/min per gkw, P < 0.01 and P < 0.05, respectively) reveals the vasodilatory role for PG in hypercalcemia. Finally, comparison of the RBF and GFR in both PG- and AII-inhibited hypercalcemic rats (5.99 and 0.53 ml/min per gkw, respectively) with similarly treated normocalcemic rats (7.30 and 0.94 ml/min per gkw, P < 0.001 and P < 0.005, respectively) reveals the vasoconstrictive role for Ca in chronic hypercalcemia. Our study therefore demonstrates that in chronic hypercalcemia the RBF and GFR are controlled by an active interplay of the vasoconstrictive effect of AII, the vasodilatory effect of renal PG, and the direct vasoconstrictive effect of Ca, independent of either AII or PG. The sum total of these forces produces a modest but significant decrease in RBF and GFR.
Moshe Levi, Marilyn A. Ellis, Tomas Berl
Papillary and surface micropuncture were used to study the handling of ammonium and the formation of net acid by surface nephrons, deep nephrons, and the terminal segment of collecting duct (CD) after renal mass was reduced by two-thirds. Net acid excretion by the remnant kidney (RK) was significantly reduced, averaging 794±81 neq/min (SE) compared with 1,220±105 neq/min after sham operation (P < 0.001), due to a decrease in ammonium excretion (494±54 vs. 871±79 nmol/min in controls, P < 0.001). Urinary pH and titratable acid excretion were not different in the two groups of animals. After RK formation, ammonium delivery to the end of the proximal tubule increased nearly threefold and averaged 66.2±5.6 compared with 18.4±2.9 pmol/min in controls, (P < 0.001). This greater delivery of ammonium was primarily due to renal tubule entry rather than to changes in the filtered load and was only partially related to the differences in flow rate. Ammonium processing by deep nephrons was profoundly affected by a reduction in renal mass. Although absolute delivery of ammonium was greater to the bend of Henle's loop (BHL), the difference could be accounted for on the basis of an increase in nephron size. Thus, fractional delivery (FDNH+4) to this site was not different for the two groups of animals, averaging 1,567±180% in controls and 1,400±181% in the group with the RK. Hydrogen secretion in the proximal segments of deep and surface nephrons did not increase in proportion to the decrease in renal mass and as a consequence bicarbonate delivery to the end of the proximal tubule of surface nephrons and to the BHL of deep nephrons was increased.
John Buerkert, Daniel Martin, David Trigg, Eric Simon
The present studies examined whether vasopressin increases prostaglandin biosynthesis in isolated rabbit cortical collecting tubules (CCT) and whether endogenous prostaglandin biosynthesis plays a role in modulating the response of this nephron segment to vasopressin. Three groups of studies were performed. In the first group, CCT and proximal straight tubules (PST) were incubated with [3H]arachidonic acid, and metabolites were separated and identified using silica gel thin-layer chromatography. CCT were capable of producing all of the major prostaglandins (PG) (PGE2 > thromboxane B2[TxB2] > PGF2α > PGI2). PST produced significantly lesser quantities of these lipids. In the second group, radiolabeled arachidonic acid was incorporated into the phospholipid pool of both CCT and PST, vasopressin was added to the incubation medium, and metabolities were separated and identified as above. Vasopressin stimulated the release of all of the major prostaglandins in CCT but had no effect on PST. PGE release into the incubation medium, as assessed by a radioreceptor assay, increased 108%, and a vasopressin analogue, 1-desamino-8-d-arginine vasopressin, had a quantitatively similar effect. In the third group, a submaximal dose of vasopressin was administered to isolated, perfused CCT studied in the presence and absence of indomethacin to assess whether endogenous prostaglandins play a role in modulating the antidiuretic response to vasopressin. Studies were performed in rabbits on a normal diet and in desoxycorticosterone acetate (DOCA)- or KCl-loaded animals. In the state of mineralocorticoid excess, basal prostaglandin synthesis was 63% lower, and vasopressin-stimulated prostaglandin synthesis 76% lower, than the synthesis observed in rabbits on a normal diet. Cyclooxygenase inhibition exposed a significant hydroosmotic response to a submaximal dose of vasopressin in CCT from DOCA- or KCl-loaded animals. With arachidonic acid in the bath, the same dose of vasopressin failed to elicit a hydroosmotic response in CCT from rabbits on a normal diet even in the presence of a cyclooxygenase inhibitor. However, removal of exogenous arachidonic acid, with a consequently lower rate of prostaglandin synthesis, allowed the cyclooxygenase inhibitor to enhance the hydroosmotic response to vasopressin in these tubules.
Michael A. Kirschenbaum, Andrew G. Lowe, Walter Trizna, Leon G. Fine
In hereditary pyropoikilocytosis (HPP) the erythrocyte membrane skeleton exhibits mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To detect structural changes in the functional domains of HPP spectrin we have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from two families that contain three HPP patients. Limited tryptic digestion of all three HPP patients revealed a similar and reproducible decrease in the staining intensity of an 80,000-, and 22,000-, and an 88,000-dalton polypeptide with a concomitant increase in a 74,000- and a 90,000-dalton polypeptide as compared with controls. Only changes in the 80,000-, and 74,000-, and 22,000-dalton polypeptides could be correlated to defective spectrin self-association and the amount of spectrin dimers in 0°C extracts of the HPP patients and their affected kindred. Similar results were obtained when the tryptic digests were analyzed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the affected 74,000- and 80,000-dalton polypeptides focusing into multiple spots ranging in isoelectric point from 5.3-5.4. When HPP spectrin dimers and tetramers were separated and subjected to trypsin digestion, changes in the 80,000-, 74,000-, and 22,000-dalton polypeptides were found predominantly in the spectrin dimer pool. Similar results were obtained for spectrin from two of the probands' mother, whom we have identified as an HPP carrier. We conclude that these HPP patients contain a population of normal, (principally tetrameric) and mutant (principally dimeric) spectrin. The latter is characterized by a defective spectrin dimer self-association due to conformational changes that affect the 80,000-dalton domain.
Jack Lawler, Shih-Chun Liu, Jiri Palek
These studies were carried out to investigate the mechanism of neutralization of purified Epstein-Barr virus (EBV) by fresh human serum from normal individuals lacking antibody to the EBV viral capsid (VCA) and nuclear antigens (EBNA). Such individuals thus lack serological evidence of immunity to EBV. Although an enzyme-linked immunosorbent assay (ELISA) with highly purified immobilized EBV detected low levels of IgG antibody reactive with EBV in these normal nonimmune sera, this antibody failed to neutralize EBV in the absence of complement. Studies with depleted sera and mixtures of purified complement proteins at physiologic concentrations showed that the IgG antibody and C1, C4, C2, and C3 of the classical pathway were able to fully neutralize EBV. Mixtures of the purified components of the alternative pathway at physiologic concentrations failed to neutralize purified EBV in the presence or absence of the antibody and the alternative pathway did not potentiate classical pathway-mediated neutralization. No evidence for a requirement for C8 was obtained, precluding lysis as the mechanism of neutralization. Since C3 deposition on the viral surface accompanied classical pathway activation, viral neutralization is most likely secondary to the accumulation of complement protein on the viral surface. A coating of protein on the virus could interfere with attachment to, or penetration of potentially susceptible cells.
Glen R. Nemerow, Fred C. Jensen, Neil R. Cooper
Recent studies have shown that ∼75% of the nuclear 3,5,3′-triiodothyronine (T3) present in adult rat cerebral cortex (Cx) derives from 5′-deiodination of thyroxine (T4) within this tissue. The activity of iodothyronine 5′-deiodinase (I 5′D), the enzyme catalyzing T4 to T3 conversion, increases rapidly after thyroidectomy, suggesting that this could be a compensatory response to hypothyroxinemia. To evaluate this possibility during the period of central nervous system maturation, we studied several thyroid hormone-responsive enzymes (aspartic transaminase [AT], succinic dehydrogenase [S.D.], and Na/K ATPase) in the Cx of 2-, 3-, and 4-wk-old rats. The rats were made congenitally hypothyroid by placing 1, 2, 5, and 20 mg methimazole (MMI) in 100 ml of the mothers' drinking water from day 16 of gestation throughout the nursing period and to the litters after weaning. In addition, serum thyroid hormones, I 5′D, and, in some experiments, in vivo T4 to T3 conversion in Cx were measured in the same pups. Serum T4 concentrations varied from <1 to 40 ng/ml and were generally inversely related to maternal MMI dose. Serum T3 was less affected by MMI than was T4. At 2 wk, decreases in AT, S.D., and ATPase were present in the 20-mg-MMI group but not in the 5-mg-MMI pups despite low serum T4 (<10 ng/ml) in the latter. At 3 and 4 wk, both 5- and 20-mg-MMI groups had significant reductions in these cortical enzymes despite a normal serum T3 in the 5-mg-MMI rats. Cortical I 5′D activity was 10-fold the control value in 5- and 20-mg-MMI animals at 2 wk but increased only three- to fivefold at 3 and 4 wk. I 5′D correlated inversely with serum T4 (r ≥ 0.96) at all ages, but the less marked elevation of this enzyme in 3- and 4-wk-old pups was not accompanied by an increase in serum T4. Serum T3 increased or remained the same between 2 and 3 wk. These results suggested that the 10-fold increase in I 5′D at 2 wk protected the 5-mg-MMI group from tissue hypothyroidism, but that the three- to fivefold increase at 3 and 4 wk could not. Injection of ∼250 ng T4/100 g body weight to 2-wk-old, 20-mg-MMI pups (one-sixth the normal T4 production rate) led to both a 1.8-ng/g cortical tissue increment in cortical T3 and a significant increase in AT at 24 h, compared with a 0.38-ng/g cortical tissue T3 increment and no change in AT in euthyroid controls. The larger increment in T3 of the 20-mg-MMI pups was due in great part to increased fractional T4 to T3 conversion. Although the latter resulted in greater serum T3 concentrations, three-fourths of the newly formed T3 in the cortex was generated in situ, and it was blocked by iopanoic acid as was the increase in AT. We conclude that 70-80% of the T3 in the Cx of the neonatal rat is produced locally. Serum T4 appears to serve both as a precursor for T3 and as a critical signal for increases in cortical I 5′D. The increased I 5′D can result in normal or near-normal cerebrocortical T3 concentrations despite marked reductions in serum T4. This mechanism seems to be particularly effective around 2 wk of age when many thyroid-hormone-dependent maturational changes occur in the rat Cx.
J. Enrique Silva, P. Reed Larsen
As a prerequisite to studies of whether the plasma membrane of the rat thymocyte contains specific, saturable binding sites for the thyroid hormone 3,5,3′-triiodothyronine (T3), a method was developed for the isolation of a plasma membrane fraction from these cells. As judged from both electron microscopic and marker enzyme studies, the fraction was composed principally of plasma membrane vesicles, was free of nuclear contaminants, and was only slightly contaminated with other subcellular components. At 37°C and pH 7.4, binding of [125I]T3 by the fresh membrane preparation was rapid, reaching a maximum at 5 min and then declining with time, so that by 60 min binding was virtually nil. Decreased binding with time was due to a loss of functional binding sites, but did not reflect desensitization, since the decrease in binding activity with time was independent of the presence or absence of T3. Scatchard analysis of saturation studies revealed the presence of two binding sites, one with an apparent dissociation constant (Kd) of 0.95 nM and a maximum capacity of 5.3 × 1010 sites/100 μg protein, and the other with an apparent Kd of 25 nM and a binding capacity of 1.4 × 1012 sites/100 μg protein. Measurement of the ability of several thyronine analogues to inhibit the binding of [125I]T3 revealed the following rank order of potency: l-T3 > l-T4 > d-T3 = d-T4 > l-3,5-T2 > rT3 > d,l-thyronine. Binding of T3 was inhibited by the omission of calcium from the medium or by the addition of the beta adrenergic antagonist alprenolol. As judged from studies of the lower affinity binding site, these manipulations decreased the affinity, but not the number, of binding sites for T3. The relative potencies of thyronine analogues to inhibit the binding of [125I]T3 were generally parallel to their previously reported potencies in stimulating the uptake of the sugar analogue 2-deoxy-glucose (2-DG) in intact rat thymocytes in vitro. Further, the inhibition of T3-binding produced by l-alprenolol or by excluding calcium from the medium resembled the previously reported inhibition that these manipulations produce with respect to T3-induced enhancement of 2-DG uptake. These findings suggest that the binding sites for T3 present in the plasma membrane of rat thymocytes act as functional receptors linked to the stimulation of 2-DG uptake that T3 induces in these cells.
Joseph Segal, Sidney H. Ingbar
Tangier disease is a rare familial disorder characterized by enlarged orange tonsils, transient peripheral neuropathy, hepatosplenomegaly, and lymphadenopathy, as well as striking reductions in plasma high density lipoproteins (HDL) and their major protein constituents, apolipoproteins (apo)A-I and A-II. In order to test the hypothesis that Tangier patients have abnormal apoA-I or apoA-II, the in vitro lipoprotein binding and in vivo metabolic characteristics of these proteins isolated from normal and Tangier plasma, were studied in normal subjects and patients with Tangier disease.
Ernst J. Schaefer, Linda L. Kay, Loren A. Zech, H. Bryan Brewer Jr.
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