Dissociation of the tetrameric form of beta-D-galactosidase by inactivating antibodies.
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Antibodies↗
Biomedical subjects
R A Roth
Publications and source records attributed to R A Roth.
Inactivation of normal beta-D-galactosidase by antibodies to defective forms of the enzyme.
A counterpart of the antibody-mediated activation of genetically defective enzymes is reported here. Antibodies elicited by certain mutant forms of beta-D-galactosidase (EC 3.2.1.23) of Escherichia coli were found to inactivate the normal form of the enzyme. (Antibodies elicited by normal beta-D-galactosidase do not affect the enzyme's catalytic activity.) We present evidence that the inactivating antibodies are directed against one or a few determinants of the enzyme. The level of inactivation caused by the antibodies was independent of temperature below 25 degrees and increased with temperature above 25 degrees. The inactivation was proportional to the concentration of antiserum until a maximum level of 50% inactivation was reached. Antibodies capable of inactivating up to 87% of the activity were obtained after the antiserum was partially absorbed in an affinity column. This antibody preparation showed a 10-fold enrichment of inactivating antibodies over other antibodies direct against the enzyme. The antibody-mediated inactivation caused a reduction in the Vmax of beta-D-galactosidase without affecting the apparent Km of the enzyme. In contrast to antibodies to normal beta-D-galactosidase, inactivating antibodies changed the response of the enzyme to cations. To explain these results, we present a model in which there is a temperature-dependent equilibrium between two active forms of beta-D-galactosidase. Inactivation results from a conformational change induced by the binding of inactivating antibodies to only one of these two forms.
Antibodies↗
Letter: Congenital absence of vasa.
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Congenital Abnormalities↗
Modification of ureteral catheter for removal of bladder foreign bodies.
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Foreign Bodies↗
Analysis of the rate of ischemic injury in rat kidney utilizing survival statistics.
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Cell Survival↗
Selective embolization of bilateral arterial cavernous fistulas for posttraumatic penile arterial priapism.
A 22-year-old man suffered a hiking accident with perineal trauma and developed a nonpainful priapism secondary to bilateral arterial-cavernosal fistulas. To minimize the risk of impotence in this young patient, successive selective embolizations with autologous blood clot were performed to close the fistulas. This led to an uncomplicated full recovery. No fistula was detectable on Doppler ultrasonography at 1-year follow-up. Review of the literature confirms the safety of embolization with autologous clot.
Adult↗
Nerve growth factor binding sites on hepatic parenchymal cells.
Nerve growth factor (NGF) binding sites on rat hepatocytes (HCs) in culture for 24 to 48 h were characterized using 125I-NGF. Specific binding of 125I-NGF to HCs was saturable. Scatchard analysis indicated a single population of binding sites with a Kd of 5.5 nM and a Bmax of 540 fmol/mg protein. In isolated hepatocyte membranes, specific binding of 125I-NGF was also apparent with Kd and Bmax values of 10.8 nM and 3740 fmol/mg protein, respectively. Specific binding of 125I-NGF to HCs was displaced by excess, unlabeled NGF but not by up to 1000-fold excess of either insulin or epidermal growth factor. Internalization/sequestration of 125I-NGF into HCs was measured as radioactivity present in solubilized cells after exposure to high salt and acid. These studies indicated 83 +/- 11% of 125I-NGF was accumulated by internalization/sequestration at a concentration of 1 nM 125I-NGF. Internalization was reduced to 43 +/- 4% when incubations were carried out at 4 degrees C. These results indicate the presence of a specific, low-affinity binding site for NGF on hepatocytes in culture.
Animals↗
Lack of protection by ascorbic acid from lethality resulting from exposure to 1 atm O2.
The role of ascorbic acid in protecting animals against the toxic effects of exposure to 1 atm. of O2 was examined. The mean time until death resulting from exposure to one atm. O2 was not different in rats treated intraperitoneally 3 times per day with 1.5 g/kg ascorbic acid compared to saline injected controls. Exposure of rats to one atm. O2 for 48 hr did not alter ascorbic acid or non-protein thiol concentration in lung or liver compared to room air controls. Lung ascorbic acid concentration was markedly depressed in guinea pigs fed an ascorbic acid-deficient diet for 6 days prior to O2 exposure. However, the mean time until death of ascorbic acid-deficient guinea pigs did not significantly differ from those fed an ascorbic acid-supplemented diet. Exposure to one atm. O2 of guinea pigs fed an ascorbic acid-deficient diet or an ascorbic acid-supplemented diet failed to decrease the concentration of ascorbic acid or of non-protein thiol in lung. Results from this study do not support an involvement of ascorbic acid in protection from pulmonary oxygen toxicity.
Animals↗
Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin.
The formation of disulphide bonds is essential to the structure and function of proteins. These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum. Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological. To account for these observations, Anfinsen et al. proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement. Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed. The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation. Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions. Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange. The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein. PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.
Amino Acid Sequence↗
Insulin-like growth factor II receptor as a multifunctional binding protein.
The primary structure of human insulin-like growth factor II receptor, predicted from the complementary DNA sequence, reveals a transmembrane receptor molecule with a large extracellular domain made up of fifteen repeat sequences and a small region homologous to the collagen-binding domain of fibronectin. The structural and biochemical features of the IGF-II receptor appear identical to those of the cation-independent mannose-6-phosphate receptor.
Amino Acid Sequence↗
Chronic pulmonary hypertension--the monocrotaline model and involvement of the hemostatic system.
Monocrotaline (MCT) is a toxic pyrrolizidine alkaloid of plant origin. Administration of small doses of MCT or its active metabolite, monocrotaline pyrrole (MCTP), to rats causes delayed and progressive lung injury characterized by pulmonary vascular remodeling, pulmonary hypertension, and compensatory right heart hypertrophy. The lesions induced by MCT(P) administration in rats are similar to those observed in certain chronic pulmonary vascular diseases of people. This review begins with a synopsis of the hemostatic system, emphasizing the role of endothelium since endothelial cell dysfunction likely underlies the pathogenesis of MCT(P)-induced pneumotoxicity. MCT toxicology is discussed, focusing on morphologic, pulmonary mechanical, hemodynamic, and biochemical and molecular alterations that occur after toxicant exposure. Fibrin and platelet thrombosis of the pulmonary microvasculature occurs after administration of MCT(P) to rats, and several investigators have hypothesized that thrombi contribute to the lung injury and pulmonary hypertension. The evidence for involvement of the various components of the hemostatic system in MCT(P)-induced vascular injury and remodeling is reviewed. Current evidence is consistent with involvement of platelets and an altered fibrinolytic system, yet much remains to be learned about specific events and signals in the vascular pathogenesis.
Animals↗
Hypertrophy and prolonged DNA synthesis in smooth muscle cells characterize pulmonary arterial wall thickening after monocrotaline pyrrole administration to rats.
Monocrotaline pyrrole (MCTP) is a highly reactive pneumotoxic metabolite of the pyrrolizidine alkaloid plant toxin monocrotaline. When administered to rats, it causes a delayed and progressive lung injury, vascular remodeling, and pulmonary hypertension. Structural remodeling consists of endothelial cell swelling followed by increased thickness of the vascular media in small pulmonary arteries and muscularization of normally nonmuscular arteries. Experiments were performed to characterize DNA synthesis and cell proliferation in vascular smooth muscle cells (VSMCs) after MCTP and to determine their relationship to changes in the thickness of the arterial medial layer of pulmonary resistance vessels. Male Sprague-Dawley rats were treated with MCTP (3.5 mg/kg, intravenously) or its vehicle (dimethylformamide). To label cells actively synthesizing DNA, rats were given the thymidine analog, bromodeoxyuridine (BrdU), 3 times by intraperitoneal injection during the 24 hr preceding euthanasia. Using immunohistochemistry, BrdU incorporation was quantified as a ratio of labeled nuclei to total nuclei. Within 5 days after MCTP administration, the thickness of the medial smooth muscle layer in arteries 60-250 microm in diameter was increased, prior to evidence of right heart hypertrophy. BrdU incorporation by VSMCs in pulmonary arteries was not different in vehicle- and MCTP-treated rats for the first 48 hr after treatment. However, MCTP caused a significant increase in DNA synthesis in VSMC on days 3-8 in arteries up to 250 microm in diameter. Although increased DNA synthesis precedes cell proliferation, the relative number of medial VSMCs did not increase over 8 days, suggesting that hypertrophy alone was responsible for the increased thickness of the arterial media. These results demonstrate that MCTP causes thickening of the media of pulmonary vessels through VSMC hypertrophy and that the prolonged DNA synthesis that accompanies VSMC hypertrophy is not followed by proliferation.
Animals↗
Role of the lung in total body clearance of circulating drugs.
Attention in recent years has focused upon the ability of lung to accumulate and/or to metabolise circulating hormones, drugs and other xenobiotic agents. Although often studied in broken cell preparations, such functions must be examined in intact lung before extrapolation to the situation in vivo is possible. The role of lung in total body clearance of xenobiotic agents is often considered to be small, as compared with the liver it usually has much lower concentrations of degradative enzymes and a smaller mass. However, consideration of such factors as organ blood flow or stimulation of drug metabolising enzymes suggests that the contribution of the lung to total body clearance of some drugs is greater than previously recognised. This may explain some of the alterations in drug clearance seen clinically. For example, cigarette smoking may increase the clearance of certain xenobiotic compounds by stimulating pulmonary drug metabolising enzymes. Pulmonary drug disposition may also be altered by conditions affecting cardiac output, such as exercise, hypoxia, and circulatory shock, or those affecting acid-base balance, such as hyperventilation. In addition, pneumotoxicants may affect pulmonary clearance of circulating drugs and xenobiotics.
Animals↗
Neutrophil- and glutathione-mediated hepatotoxicity of alpha-naphthylisothiocyanate.
In summary, both glutathione and blood neutrophils contribute to ANIT hepatotoxicity. Glutathione contributes by virtue of its ability to form a reversible S-conjugate with ANIT that is critical in shuttling ANIT into bile. Where it is released in large and probably toxic concentrations. The possibility remains that this conjugate may be bioactivated by secondary mechanisms, but no evidence for a toxic glutathionyl conjugate of ANIT currently exists. Neutrophils and platelets both appear to play important roles in ANIT hepatotoxicity. The role of platelets is currently unknown, but studies in vitro raise the possibility that neutrophils may be activated during ANIT exposure to release cytotoxic proteases that cause injury to target cells. Although ANIT activates neutrophils in vitro, the mechanisms by which neutrophils are recruited into the periportal region and activated in vivo remain unknown.
1-Naphthylisothiocyanate↗
Role of blood flow in carbon monoxide- and hypoxic hypoxia-induced alterations in hexobarbital metabolism in rats.
The flow dependency of hepatic hexobarbital metabolism was examined in the isolated perfused rat liver. At low flow rates (0.5-1.0 ml/min/g of liver) hexobarbital clearance was found to depend on perfusion fluid flow, whereas at higher flow rates drug clearnace approached flow independence. Calculation of the in vivo hepatic blood flow rate suggested that hexobarbital metabolism in vivo should be highly dependent upon flow. Blood flow in the conscious rat was measured by use of radiolabeled microspheres during acute exposure to levels of hypoxic hypoxia (lowered pO2) or carbon monoxide which resulted in equal alterations in arterial oxyhemoglobin content (approximately 65% oxyhemoglobin). Hypoxic hypoxia (8% O2) caused a massive redistribution of flow away from the splanchnic area, resulting in a 45% decrease in hepatic blood flow. Carbon monoxide (500 ppm) was without significant effect on hepatic blood flow. These data would appear to explain the relatively greater inhibitory potency of hypoxic hypoxia on drug metabolism in vivo, since drug delivery to the liver is depressed by hypoxic hypoxia but unaffected by carbon monoxide exposure.
Animals↗