Biomedical subjects
Publications and source records attributed to C Narasimhan.
We report the case of a young adult with mitral stenosis and pulmonary oedema who did not respond to conventional antifailure therapy, but improved with intravenous propranolol and later underwent successful balloon mitral valvotomy. Intravenous beta blockade, though contraindicated in most cases of pulmonary oedema, may benefit patients with mitral stenosis and tachycardia.
cDNA encoding avian liver hydroxymethylglutaryl-CoA synthase has been cloned into a pET vector, and the resulting expression plasmid has been used to transform Escherichia coli BL21 (DE3). Heterologous expression of hydroxymethylglutaryl-CoA synthase occurs upon growth of this bacterial strain in the presence of isopropyl-1-thio-beta-D-galactopyranoside, with the target enzyme representing over 20% of total cellular protein. Recombinant enzyme is soluble and stable in crude E. coli extracts, facilitating its isolation in homogeneous form. With respect to specific activity, acylation stoichiometry, Km,Ac-CoA, and binding of a spin-labeled substrate analog, the recombinant enzyme is equivalent to avian enzyme, suggesting its utility for mechanistic and structural studies. Our earlier prediction that this avian cDNA encodes the cholesterogenic cytosolic isozyme is supported by a series of experimental observations. Upon SDS-polyacrylamide gel electrophoresis, the recombinant synthase exhibits mobility in agreement with the 57.6-kDa deduced molecular mass, which exceeds the 53-kDa estimate and experimental observation for the ketogenic mitochondrial isozyme. Activity of the recombinant synthase is stimulated by Mg2+, as predicted for the cholesterogenic cytosolic isozyme and in contrast to the inhibition observed for the mitochondrial isozyme. Although antibody prepared against avian mitochondrial synthase effectively detects both avian mitochondrial and recombinant synthases on Western blots, antibody prepared against rodent cytosolic synthase discriminates between the two proteins, sensitively detecting recombinant enzyme while reacting poorly with authentic mitochondrial enzyme. Directed mutagenesis of the recombinant synthase has been performed to produce a C129S variant, in which the sulfhydryl previously implicated in formation of the acetyl-S-enzyme reaction intermediate is replaced by a hydroxyl group. EPR measurements on the binary C129S-spin-labeled acyl-CoA complex demonstrate that the mutant's substrate binding site is unperturbed in comparison with wild-type protein. These data illustrate the utility of spin-labeled substrate analogs as tools to stringently evaluate the structural integrity of engineered proteins. C129S is catalytically inactive (10(5)-fold decrease in kcat) despite retaining the ability to form noncovalent complexes with acetyl-CoA or a spin-labeled acetyl-CoA analog. The demonstrated failure of C129S to form a covalent acyl-O-enzyme species accounts for these observations; data derived from experiments performed with a C129G mutant confirm this conclusion. These results distinguish hydroxymethylglutaryl-CoA synthase from beta-ketoacyl thiolase.(ABSTRACT TRUNCATED AT 400 WORDS)
Pseudohypertension has often been reported in elderly subjects, but is an unusual phenomenon in children. We report the case of a 5-year-old child who presented with features of Williams syndrome (characterized by elfin facies, supravalvar aortic stenosis, and peripheral pulmonary artery stenosis). Repeated blood pressure recordings made with appropriately sized blood pressure cuffs were very high, while simultaneous intraarterial blood pressure was normal, confirming the presence of pseudohypertension. This was shown to be caused by excessively thickened arterial vessels.
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase has been expressed in an active form in Escherichia coli and purified to homogeneity. Enzyme activity in crude extracts is 30-fold higher than reported for a homologous expression system. After Q-Sepharose fast-flow anion-exchange chromatography, the enzyme, which represents the first homogeneous preparation of a prokaryotic form of the protein, exhibits a specific activity of 70 units/mg. The purified enzyme is stable when stored in 20% glycerol at -80 degrees C. The recombinant bacterial enzyme cross reacts with antiserum produced against avian liver lyase, indicating some sequence homology between the two proteins. The enzyme exhibits a Km = 20 microM for (S)-HMG-CoA. Divalent cations (Mg2+ and Mn2+) markedly stimulate the enzyme activity under assay conditions; activity is only modestly increased by exogenous mercaptans. The activator constant, K(a), for Mg2+ (6.9 mM) is 3 orders of magnitude greater than that for Mn2+ (2.0 microM). While EDTA does not affect activity, o-phenanthroline treatment markedly inhibits the enzyme. In contrast, m-phenanthroline is ineffective, suggesting that the ortho isomer's effect is attributable to chelation of a tightly bound metal ion. Atomic absorption and EPR analyses of isolated enzyme indicate the presence of tightly bound copper. In enzyme expressed using standard LB broth, copper is detected at stoichiometries of only 0.07-0.10. When the growth medium is supplemented with 1 mM CuSO4, stoichiometry of copper binding increases to over 0.7 per enzyme subunit. Copper-enriched lyase displays enhanced thermal stability in comparison with enzyme that is low in metal content.(ABSTRACT TRUNCATED AT 250 WORDS)
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase is irreversibly inactivated by the reactive substrate analog 2-butynoyl-CoA. Enzyme inactivation, which follows pseudo-first-order kinetics, is saturable with a KI = 65 microM and a limiting k(inact) of 0.073 min-1 at 23 degrees C, pH 7.2. Protection against inactivation is afforded by the competitive inhibitor 3-hydroxyglutaryl-CoA. Labeling of the bacterial enzyme with [1-14C]-2-butynoyl-CoA demonstrates that inactivation coincides with covalent incorporation of inhibitor, with an observed stoichiometry of modification of 0.65 per site. Avian HMG-CoA lyase is also irreversibly inactivated by 2-butynoyl-CoA with a stoichiometry of modification of 0.9 per site. Incubation of 2-butynoyl-CoA with mercaptans such as dithiothreitol results in the formation of a UV absorbance peak at 310 nm. Enzyme inactivation is also accompanied by the development of a UV absorbance peak at 310 nm indicating that 2-butynoyl-CoA modifies a cysteine residue in HMG-CoA lyase. Tryptic digestion and reverse-phase HPLC of the affinity-labeled protein reveal a single radiolabeled peptide. Isolation and sequence analysis of this peptide and a smaller chymotryptic peptide indicate that the radiolabeled residue is contained within the sequence GGXPY. Mapping of this peptide within the cDNA-deduced sequence of P. mevalonii HMG-CoA lyase [Anderson, D. H., & Rodwell, V. W. (1989) J. Bacteriol. 171, 6468-6472] confirms that a cysteine at position 237 is the site of modification. These data represent the first identification of an active-site residue in HMG-CoA lyase.
A case of polymyositis associated with Bancroftian filariasis in an adult male who presented with generalised painful swelling and weakness of muscles is presented. He had elevated muscle enzymes, a myopathic EMG pattern, focal vasculitis on gastrocnemius muscle biopsy and W. bancrofti in the peripheral blood. There was clinical, biochemical and histopathological evidence of resolution of the disorder and total clearance of microfilaremia with diethyl carbamazine (DEC) therapy.
The clinical features and results of laboratory investigations of the first 19 Indian patients with AIDS seen in our hospital are presented. Weight loss, fever, and diarrhea were the most common symptoms. Tuberculosis (TB) was the most common secondary infectious disease; among 13 patients, seven had only pulmonary TB, five had pulmonary and extrapulmonary TB, and one had only extrapulmonary TB. Oropharyngeal candidiasis was found in 11 patients. Other secondary infections were predominantly by virulent bacteria. Opportunistic infections other than candidiasis were infrequent; one patient had cryptococcosis, two had symptomatic cryptosporidiosis, one had noncoagulase-positive staphylococcus septicemia, and one had cytomegalovirus retinitis. Reduced lymphocyte counts (particularly of the CD4 subset), anemia, hypoalbuminemia, hyperglobulinemia, and elevated liver enzyme levels were frequent laboratory findings. Six patients are under follow-up, two are lost to follow-up, and 11 have died. Lymphocyte counts less than 500/mm3 were only seen in those patients who subsequently died. Response to antituberculosis therapy was good in several patients. Thus, the clinical profile of Indian patients with AIDS is not different from the common picture of patients of low socioeconomic and poor hygienic standards; patients presented with TB, undernutrition, and multiple infections. Therefore, a large population of patients with AIDS in India will not be recognized unless they are tested for evidence of HIV infection.
We report here a novel approach to label specifically one of the two cryptic, free sulfhydryl groups per subunit of human plasma fibronectin with either an 15N,2H-maleimide spin label or a coumarinylphenyl maleimide fluorescent label. This permits the use of electron spin resonance (ESR) or fluorescence techniques to study molecular dynamics of fibronectin with the label attached to a single site per chain on the protein molecule. The method is based on our observation that upon adsorption of fibronectin to a gelatin-coated surface, the SH1 site, located between the DNA-binding and the cell-binding domains, is partially exposed, while the SH2 site, located within the carboxyl-terminal fibrin-binding domain, remains buried and unreactive. The procedures for the preparation of the selectively labeled fibronectins are described in detail. The physicochemical properties of these single-site labeled fibronectins, particularly as affected by high salt, heparin, surface binding, and temperature, were characterized by ESR spin-label and steady-state fluorescence techniques. The steady-state fluorescence measurement indicates that both local environments of SH1 and SH2 sites are relatively hydrophobic, and that the SH2 site is more hydrophobic than the SH1 site. The ESR results show that heparin or high salt induces an increase in the domainal flexibility in both SH1 and SH2 regions, perhaps through the disruption of domain-domain interactions in the fibronectin molecule, and that the former is more effective than the latter in producing such an effect. The observed heparin effect is reversible by addition of calcium ions in the SH2 regions but not in the SH1 regions. In addition, at temperatures above 44 degrees C, both type III homologous regions containing the free sulfhydryl groups are shown to undergo denaturation and aggregation processes. The data presented here suggest that the newly developed method for differential labeling of the free sulfhydryl groups in fibronectin should be useful for mapping the spatial arrangement of structural domains in the protein molecule using spin-label-spin-probe and fluorescence energy transfer techniques.
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An outbreak of epidemic dropsy in Andhra Pradesh was studied during August to September 1987. Thirty cases from 6 families were identified and examined. Ghee and one oil sample were found to be contaminated with the toxic compound sanguinarine. The affected families had purchased ghee from one vendor. The epidemic ended after alerting the villagers of the contaminated ghee, and stopping its further use.
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We report a young male with sickle cell trait who developed severe hypertension and splenic infarction soon after travel to a high altitude. Hypertension persisted for three days after a diagnostic laparotomy. His blood pressure then continued to be normal over the next one and a half years. Red cell sludging in the small vessels of the kidney possibly activated the renin angiotensin system.
We have measured the diffusional water permeability of sickle cell anemia red blood cells under isotonic conditions using pulsed nuclear magnetic resonance (NMR) techniques. We have found that the equilibrium diffusional permeability for sickle cells is about 1.61.10(-3) cm/s, or about 60% of the value measured for normal cells. This abnormality is not related to the heterogeneity generally found in cell populations in sickle red cells with different mean corpuscular hemoglobin concentrations. We speculate that the abnormality of water exchange under isotonic conditions in sickle cells reflects an alteration of membrane proteins responsible for water exchange, possibly caused by oxidation of Band 3 proteins.
Changes in local environment of the free sulfhydryl groups in plasma fibronectin upon adsorption of the protein to polystyrene beads have been examined by electron spin resonance (ESR) spin-label spectroscopy. The two free sulfhydryl groups per subunit of plasma fibronectin were modified chemically with an [15N, 2H]maleimide spin-label. For soluble fibronectin, both free sulfhydryl groups are shown to be in confined environments as evidenced from the labeled protein exhibiting a strongly immobilized ESR spectrum as described previously using [14N, 1H]maleimide spin-labels [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. When the labeled protein was adsorbed to the beads, half of the strongly immobilized component was found to convert into a weakly immobilized component, a result indicating that one of the two labeled sites becomes exposed and exhibit a fast tumbling motion. Experiments conducted using various spin-labeled fibronectin fragments suggest that the newly exposed labeled site is located between the DNA-binding and the cell-binding regions of the molecule. The data obtained indicate that, upon adsorption to polystyrene beads, plasma fibronectin undergoes a conformational change through which the buried free sulfhydryl group near the cell-binding region of the molecule is exposed. This observation may have important implications regarding the expression of cell adhesive properties of the fibronectin molecule.
Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion.
The accessibility in human plasma fibronectin of the two free sulfhydryl groups per chain to sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and a maleimide derivative has been examined. For soluble fibronectin, the free sulfhydryl groups are not accessible to DTNB unless urea or guanidine hydrochloride is added [Smith et al. (1982) J. Biol. Chem. 257, 5831-5838]. Upon binding to polystyrene beads, 0.87 +/- 0.05 sulfhydryl group per chain becomes titratable to DTNB. Experiments using fibronectin fragments demonstrate that this newly exposed sulfhydryl group is located in a Type III homologous unit between the DNA-binding and the cell-binding domains. The results suggest that, upon adsorption to solid substrates, plasma fibronectin undergoes a conformational change, thereby exposing one buried sulfhydryl group. These findings have implications regarding the "surface activation" of adhesion activities of fibronectin.
Cetiedil, an antisickling agent and a vascular smooth muscle relaxant, is an amphiphilic molecule. The critical micelle concentration in 5 mM phosphate buffer with 150 mM NaCl is approximately 8.8 mM. The molecule, as the citrate salt, is highly acidic at millimolar concentrations. The UV absorption extinction coefficient at 233 nm, E233, is 2796 M-1 cm-1. The studies of free cetiedil concentrations in the presence of membrane ghosts show that large amounts of cetiedil associate with membrane samples. Spin label electron paramagnetic resonance experiments showed that the lipids and the proteins of erythrocyte membrane samples were both affected by the addition of cetiedil. However, the cetiedil effects on membrane components are reversible. The protein spin label results demonstrate the binding of cetiedil to the membrane with an apparent equilibrium dissociation constant of approximately 2 mM. The binding is saturable. The apparent half-saturation concentrations for the binding at physiological ionic strength and temperature are in the range of 1-3 mmoles of cetiedil per gram of membrane proteins. Our studies also indicate that binding is not affected by the removal of the spectrin and actin network from the membranes. Interaction of cetiedil with Band 3 molecules in the erythrocyte membrane is suggested. The regions near the lipid head groups in the membrane samples are also affected by cetiedil.
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