Biomedical subjects
Publications and source records attributed to I Singh.
We studied the oxidation of [1-14C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts. The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.
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The different topology of palmitoyl-CoA ligase (on the cytoplasmic surface) and of lignoceroyl-CoA ligase (on the luminal surface) in peroxisomal membranes suggests that these fatty acids may be transported in different form through the peroxisomal membrane (Lazo, O., Contreras, M., and Singh, I. (1990) Biochemistry 29, 3981-3986), and this differential transport may account for deficient oxidation of lignoceric acid in X-adrenoleukodystrophy (X-ALD) (Singh, I., Moser, A. B., Goldfisher, S., and Moser, H. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4203-4207). To define the transport mechanism for these fatty acids through the peroxisomal membrane and its possible implication to lignoceric acid metabolism in X-ALD, we examined cofactors and energy requirements for the transport of palmitic and lignoceric acids in isolated peroxisomes from rat liver and peroxisomes isolated from X-ALD and control fibroblasts. The similar rates of transport of palmitoyl-CoA (87.6 +/- 6.3 nmol/h/mg protein) and palmitic acid in the fatty acid activating conditions (83.4 +/- 5.1 nmol/h/mg protein) and lack of transport of palmitic acid (4% of palmitoyl-CoA transport) when ATP and/or CoASH were removed or substituted by alpha,beta-methyleneadenosine-5'-triphosphate (AMPCPOP) and/or desulfoCoA-agarose from assay medium clearly demonstrate that transport of palmitic acid requires prior synthesis of palmitoyl-CoA by palmitoyl-CoA ligase on the cytoplasmic surface of peroxisomes. The 10-fold higher rate of transport of lignoceric acid (5.3 +/- 0.6 nmol/h/mg protein) as compared with lignoceroyl-CoA (0.41 +/- 0.11 nmol/h/mg protein) and lack of inhibition of transport of lignoceric acid when ATP and/or CoASH were removed or substituted with AMPCPOP or desulfoCoA-agarose suggest that lignoceric acid is transported through the peroxisomal membrane as such. Moreover, the lack of effect of removal of ATP or substitution with AMPOPCP (a nonhydrolyzable substrate) demonstrates that the translocation of palmitoyl-CoA and lignoceric acid across peroxisomal membrane does not require energy. The transport, activation, and oxidation of palmitic acid are normal in peroxisomes from X-ALD. The deficient lignoceroyl-CoA ligase (13% of control) and oxidation of lignoceric acid (10% of control) as compared with normal transport of lignoceric acid into peroxisomes from X-ALD clearly demonstrates that pathogenomonic accumulation of very long chain fatty acids (greater than C22) in X-ALD is due to the deficiency of peroxisomal lignoceroyl-CoA ligase activity.
Exogenously supplied catalase, a peroxisomal enzyme, has been found to be of therapeutic value in ischemic injury. Therefore, we examined the effect of ischemic-reperfusion injury on the structure and function of kidney peroxisomes. Ischemic injury changed the density of peroxisomes from 1.21 g/cm3 (peak I) to a lighter density of 1.14 g/cm3 (peak II). The number of peroxisomes moving from the normal density population (peak I) to a lower density population (peak II) increased with an increase in ischemic injury. Latency experiments indicated both populations of peroxisomes to be of intact peroxisomes. Immunoblot analysis with antibodies against peroxisomal matrix and membrane proteins demonstrated that after 90 min of ischemia a significant number of matrix proteins were lost in the peak II population, suggesting that functions of these peroxisomes may be severally affected. Reperfusion following ischemic injury resulted in loss of peroxisomal matrix proteins in both peaks I and II, suggesting that peroxisomal functions may be drastically compromised. This change in peroxisomal functions is reflected by a significant decrease in peroxisomal catalase activity (35%) and beta-oxidation of lignoceric acid (43%) observed following 90 min of ischemia. The decrease in catalase activity was more pronounced in reperfused kidneys even after a shorter term of ischemic injury. Reperfusion restored the normal peroxisomal beta-oxidation in kidneys exposed up to 60 min of ischemia. However, 90 min of ischemia was irreversible as there was a further decrease in beta-oxidation upon reperfusion. The decrease in catalase activity during ischemia alone was due to the formation of an inactive complex, whereas during reperfusion, following 90 min of ischemia, inactivation and proteolysis or decreased synthesis of catalase contributed equally toward the injury. The observed changes in the structure and function of peroxisomes as a result of ischemic-reperfusion injury and the ubiquitous distribution of peroxisomes underlines the importance of this organelle in the pathophysiology of vascular injury in general.
Peroxisomal activities were examined in liver and brain of lactating neonatal rats of mothers maintained on ciprofibrate, a peroxisomal proliferator. The activities of DHAP-acyltransferase, alkyl-DHAP synthetase and the beta-oxidation of palmitic and lignoceric acids increased in brain by 3.9, 2.2, 4.3 and 3.2 fold and in liver by 3.2, 2.6, 6.2 and 2.5 fold, respectively, of lactating pups from mothers on ciprofibrate diet as compared to controls. Ciprofibrate treatment increased the peroxisomal enzyme activities in both brain and liver of lactating neonatal rats demonstrating that ciprofibrate or its metabolite(s) transmitted through the mother's milk can effectively induce peroxisomal proliferation.
In this study, by using highly purified rat liver peroxisomes, we provide evidence from analytical cell fractionation, Western blot, and immunocytochemical analysis that Cu-Zn superoxide dismutase is present in animal peroxisomes. Treatment with ciprofibrate, a peroxisome proliferator, increased the peroxisomal superoxide dismutase activity by 3-fold with no effect on mitochondrial activity but a marked decrease in cytosolic superoxide dismutase activity, further supporting that besides cytosolic and mitochondrial localization, Cu-Zn superoxide dismutase is present in peroxisomes also. Demonstration of superoxide dismutase in peroxisomes suggests a new role for this organelle in pathophysiological conditions, such as ischemia-reperfusion injury.
Thirty-seven patients with intractable bladder hemorrhage were treated with intravesical epsilon aminocaproic acid (EACA). Radiation cystitis and cyclophosphamide-induced cystitis were the two most common causes of intractable bladder hemorrhage. Thirty-four patients responded to treatment. No side effects were noted. Intravesical EACA appears to be a safe and effective method to control intractable bladder hemorrhage.
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Complications of a 3-piece inflatable penile prosthesis reservoir are rare, with rupture and fluid leakage being the most common. We recently encountered a patient in whom the reservoir migrated twice into the bowel. A review of the literature suggests that a previous major pelvic or abdominal operation might contribute to erosion of the reservoir into the adjacent viscera.
A solid-phase, enzyme-linked immunoassay was used to quantitate the soluble fraction of HLA-class I. The sera of 318 individuals were studied, as well as the urine of six individuals with normal renal function. The stability of blood concentrations of the soluble HLA was also evaluated. The data justify the following six conclusions. (1) All normal people have circulating HLA (mean = 357 ng/ml). (2) The population can be divided into one group of low secretors (mean = 162.4 +/- 65.2 ng/ml) and another group of high secretors (mean = 540.7 +/- 185.9 ng/ml) (P less than 0.01). (3) Blood levels in each individual are reasonably consistent over short (days) and long (years) periods of time. (4) The mean concentration of soluble HLA-class I in all renal failure patients was 590 ng/ml, significantly higher than normal (P = less than 0.05); it was highest in patients on peritoneal dialysis (mean = 683 ng/ml) in spite of substantial chronic loss in peritoneal dialysate. (5) Renal allograft recipients with stable allograft function also had mean values greater than normal at 554 ng/ml (P less than 0.05). (6) Soluble HLA-class I was not detected in the urine of individuals with normal renal function.
Fifty-two mentally-handicapped in-patients with associated behavioural disorders were entered into this double-blind trial comparing zuclopenthixol dihydrochloride tablets (10 mg) with placebo. The study consisted of a 6-week open phase, in which all patients were treated with zuclopenthixol tablets, followed by a 12-week double-blind phase where approximately half of the patients were transferred to placebo tablets. Demographic data was obtained from the patients at entry to the study. Clinical assessments were performed at weeks 6, 7, 8, 10, 14 and 18 using the Clinical Global Assessment (CGA), a Behavioural Disorder Assessment prepared specially for this study, and a side-effect check list. Analyses of the rating scales showed significant differences in favour of zuclopenthixol at end-point on the item 'improvement in behaviour disorder' from the CGA and at week 14 and end-point on the Assessment of Behavioural Disorder. All other results demonstrated a favourable trend towards the zuclopenthixol treatment. Side-effects were reported slightly more frequently in the zuclopenthixol group, but generally, they were not a problem.
The rapid mitochondrial uptake of calcium followed by slow release in certain pathophysiological states associated with an increase in intracellular calcium, to normalize the cytoplasmic levels of free calcium, provides an important protective mechanism against calcium cellular toxicity. Salicylic acid, an in vivo metabolite of aspirin, inhibits the uptake and enhances the release of calcium by mitochondria, thereby increasing the levels of cytoplasmic free calcium. The Ca2+ induced mitochondrial swelling is enhanced in the presence of salicylic acid and in which turn leads to loss of biosynthesis of ATP. These results suggest that salicylic acid may promote cellular damage in pathophysiological states associated with increase in intracellular free calcium.
The mechanism by which Semliki Forest virus nucleocapsids are uncoated was analyzed in living cells and in vitro. In BHK-21 cells, uncoating occurred with virtually complete efficiency within 1 to 2 min after the nucleocapsids entered the cytoplasm. It was inhibited by monensin, which blocks nucleocapsid penetration from endosomes. As previously shown for Sindbis virus (G. Wengler and G. Wengler, Virology 134:435-442, 1984), the capsid proteins from incoming nucleocapsids became associated with ribosomes. The ribosome-bound capsid proteins were distributed throughout the cytoplasm, while the viral RNA remained associated with vacuolar membranes. Using purified nucleocapsids and ribosomes in vitro, we established that ribosomes alone were sufficient for uncoating. Their role was to release the capsid proteins from nucleocapsids and irreversibly sequester them, in a process independent of energy and translation. The process was stoichiometric rather than catalytic, with a maximum of three to six capsid proteins bound to each ribosome. More than 80% of the capsid proteins could thus be removed from the viral RNA, resulting in the formation of nucleocapsid remnants whose sedimentation coefficients progressively decreased from 140S to 80S as uncoating proceeded.
We have localized the gene (FACL1) encoding human long chain fatty acid-coenzyme A ligase, E.C.6.2.1.3, also known as palmitoyl-CoA ligase. Using in-situ hybridization, we have mapped this gene to chromosome region 3q13 in the human karyotype.
The Zellweger syndrome is characterized by a defect which results in the abnormal biogenesis of peroxisomes. As a consequence, metabolic activities associated with peroxisomes such as the oxidation of very long chain fatty acids, the synthesis of plasmalogens, and the catabolism of phytanic and pipecolic acids are impaired. Since this disorder is genetically heterogeneous and several complementation groups are known, we were able to study the normalization of peroxisomal activity during the process of complementation. The restoration of catalase and dihydroxyacetone phosphate acyltransferase activities peaked within 3-4 days postfusion while the oxidation of lignoceric acid was much delayed (7-8 days). Electron microscopy indicated that by 6 days following hybridization, peroxisome structure and density in heterokaryons was comparable to normal control cells. The heterogenous biochemical response during peroxisome normalization could be due to several factors including a possible requirement for restoration of peroxisomal structural integrity for maximum activation of certain metabolic pathways.
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A 32-year-old chronically relapsing depressed male patient with a mild mental handicap had tried different forms of pharmacotherapy which were either not tolerated or failed to prevent recurrences of episodes of psychotic depression. The use of maintenance ECT as the mainstay of the therapeutic regime led to a marked consistent clinical improvement.