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V Gopalan

Publications and source records attributed to V Gopalan.

At least 19 recordsLinked to original sources
PubMed2004

Enhancement of ferroelectricity in strained BaTiO3 thin films.

Biaxial compressive strain has been used to markedly enhance the ferroelectric properties of BaTiO3 thin films. This strain, imposed by coherent epitaxy, can result in a ferroelectric transition temperature nearly 500 degrees C higher and a remanent polarization at least 250% higher than bulk BaTiO3 single crystals. This work demonstrates a route to a lead-free ferroelectric for nonvolatile memories and electro-optic devices.

Journal Article↗
PubMed2002

Long term follow-up of patients with polycystic ovarian syndrome after laparoscopic ovarian drilling: clinical outcome.

BACKGROUND: Currently, there is an uncertainty about the impact of laparoscopic ovarian drilling (LOD) on the natural history of polycystic ovarian syndrome (PCOS). This longitudinal follow-up study was undertaken to investigate the long-term effects of LOD. METHODS: The study included 116 anovulatory PCOS women who underwent LOD between 1991 and 1999 (study group) and 34 anovulatory PCOS women diagnosed during the same period but who had not undergone LOD (comparison group). The hospital records were reviewed and questionnaires were sent to all the women. In addition, most women attended a follow-up interview. Clinical data recorded at different intervals of follow-up (short-term, <1 year; medium-term, 1-3 years; and long-term, 4-9 years) included: the menstrual pattern, symptoms relating to hyperandrogenaemia and reproductive history. RESULTS: The proportion of women with regular menstrual cycles increased significantly [relative risk (RR) = 1.6, 95% confidence interval (CI) = 1.4-1.9, P < 0.05] from 8% before LOD to 67% post-operatively. The proportion dropped to 37% (RR = 2.6, 95% CI = 1.8-3.8, P < 0.01) at medium-term follow-up and then increased again to 55% (RR = 2.2, 95% CI = 1.7-2.8, P < 0.01) at long-term follow-up. After LOD, 54/110 women (49%) conceived spontaneously during the first year and 42 (38%) during medium- and long-term follow-up. Among women with hirsutism (n = 43) and acne (n = 25), 10 (23%) and 10 (40%) respectively experienced long-term improvement after LOD. CONCLUSION: LOD produces long-term improvement in menstrual regularity and reproductive performance in about one-third of cases. A modest and sustained improvement in acne and hirsutism can be expected in approximately 40 and approximately 25% of patients respectively.

Acne Vulgaris↗
PubMed2001

Use of EPR spectroscopy to study macromolecular structure and function.

Electron paramagnetic resonance (EPR) spectroscopy is now part of the armory available to probe the structural aspects of proteins, nucleic acids and protein-nucleic acid complexes. Since the mobility of a spin label covalently attached to a macromolecule is influenced by its microenvironment, analysis of the EPR spectra of site-specifically incorporated spin labels (probes) provides a powerful tool for investigating structure-function correlates in biological macromolecules. This technique has become readily amenable to address various problems in biology in large measure due to the advent of techniques like site-directed mutagenesis, which enables site-specific substitution of cysteine residues in proteins, and the commercial availability of thiol-specific spin-labeling reagents (Figure 1). In addition to the underlying principle and the experimental strategy, several recent applications are discussed in this review.

Animals↗
PubMed2000

Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe.

The protein subunit of Escherichia coli ribonuclease P (which has a cysteine residue at position 113) and its single cysteine-substituted mutant derivatives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a sulfhydryl-specific iron complex of EDTA-2- aminoethyl 2-pyridyl disulfide (EPD-Fe). This reaction converts C5 protein, or its single cysteine-substituted mutant derivatives, into chemical nucleases which are capable of cleaving the cognate RNA ligand, M1 RNA, the catalytic RNA subunit of E. coli RNase P, in the presence of ascorbate and hydrogen peroxide. Cleavages in M1 RNA are expected to occur at positions proximal to the site of contact between the modified residue (in C5 protein) and the ribose units in M1 RNA. When EPD-Fe was used to modify residue Cys16 in C5 protein, hydroxyl radical-mediated cleavages occurred predominantly in the P3 helix of M1 RNA present in the reconstituted holoenzyme. C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot. However, hydroxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA-Fe, perhaps indicative of Cys113 being distal from the RNA-protein interface in the RNase P holoenzyme. Our directed hydroxyl radical-mediated footprinting experiments indicate that conserved residues in the RNA and protein subunit of the RNase-P holoenzyme are adjacent to each other and provide structural information essential for understanding the assembly of RNase P.

Amino Acid Sequence↗
PubMed1999

Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using electron paramagnetic resonance spectroscopy.

Ribonuclease P (RNase P) is a catalytic ribonucleoprotein (RNP) essential for tRNA biosynthesis. In Escherichia coli, this RNP complex is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein. Using the sulfhydryl-specific reagent (1-oxyl-2,2,5, 5-tetramethyl-Delta3-pyrroline-3-methyl)methanethiosulfonate (MTSL), we have introduced a nitroxide spin label individually at six genetically engineered cysteine residues (i.e., positions 16, 21, 44, 54, 66, and 106) and the native cysteine residue (i.e., position 113) in C5 protein. The spin label covalently attached to any protein is sensitive to structural changes in its microenvironment. Therefore, we expected that if the spin label introduced at a particular position in C5 protein was present at the RNA-protein interface, the electron paramagnetic resonance (EPR) spectrum of the spin label would be altered upon binding of the spin-labeled C5 protein to M1 RNA. The EPR spectra observed with the various MTSL-modified mutant derivatives of C5 protein indicate that the spin label attached to the protein at positions 16, 44, 54, 66, and 113 is immobilized to varying degrees upon addition of M1 RNA but not in the presence of a catalytically inactive, deletion derivative of M1 RNA. In contrast, the spin label attached to position 21 displays an increased mobility upon binding to M1 RNA. The results from this EPR spectroscopy-based approach together with those from earlier studies identify residues in C5 protein which are proximal to M1 RNA in the RNase P holoenzyme complex.

Amino Acid Sequence↗
PubMed1997

Analysis of the functional role of conserved residues in the protein subunit of ribonuclease P from Escherichia coli.

The processing of precursor tRNAs and some other small cellular RNAs by M1 RNA, the catalytic subunit of Escherichia coli ribonuclease P, is accelerated by C5 protein (the protein cofactor) both in vitro and in vivo. In an effort to understand the mechanism by which the protein cofactor promotes and stabilizes certain conformations of M1 RNA that are most efficient for RNase P catalysis, we have used site-directed mutagenesis to generate mutant derivatives of C5 protein and assessed their ability to promote RNase P catalysis in vivo and in vitro. Our results indicate that certain conserved hydrophobic and basic residues in C5 protein are important for its function and that single amino acid residue changes in C5 protein can alter the substrate specificity of the RNase P holoenzyme.

Amino Acid Sequence↗
PubMed1997

Fluorescence properties of a tryptophan residue in an aromatic core of the protein subunit of ribonuclease P from Escherichia coli.

Escherichia coli ribonuclease P (RNase P), a ribonucleoprotein complex which primarily functions in tRNA biosynthesis, is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein. The fluorescence emission spectrum of the single tryptophan residue-containing C5 protein exhibits maxima at 318 nm and 332 nm. Based on a comparison of the emission spectra of wild-type C5 protein and some of its mutant derivatives, we have determined that the 318 nm maximum could be the result of a complex formed in the excited state as a result of hydrophobic interactions between Trp109, Phe18 and Phe73. The analogous tryptophan fluorescence emission spectra of wild-type C5 protein and the barstar mutant W38F/W44F, taken together with the detailed structural information available for barstar, provide a possible explanation for the unusual emission spectrum of C5 protein.

Bacterial Proteins↗
PubMed1997

Schistosomiasis: an unusual cause of right lower quadrant abdominal pain.

Schistosomiasis, although unusual in North America, is a common disease worldwide. Symptoms vary depending on the species involved. Immigrants from endemic regions are the commonly affected patients found in North America. In most cases, schistosomiasis does not present with right lower quadrant pain. Even in endemic regions, this form of presentation is uncommon. In the United States, most cases of right lower quadrant pain often will be treated as appendicitis. Questions remain unanswered as to whether the schistosomes cause appendicitis or are found incidentally in these cases. Stool and urine specimens may be helpful in making a diagnosis. Most cases require operative intervention to rule out appendicitis and to obtain tissue for histopathologic diagnosis. Praziquantel is effective in eradicating infestations.

Abdominal Pain↗
PubMed1992

Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic beta-glucosidase.

We demonstrate that although the guinea pig liver cytosolic beta-glucosidase does not catalyze the hydrolysis of gentiobiose, it does hydrolyze, disaccharide-containing glycosides such as p-nitrophenyl-beta-D-gentiobioside (Glc beta 1----6Glc beta-pNP) and mandelonitrile-beta-D-gentiobioside (amygdalin). Furthermore, we establish that the enzyme attacks disaccharide glycosides exolytically; specifically, we document the exolytic deglucosylation of amygdalin and the generation of the intermediate monosaccharide glycoside mandelonitrile-beta-D-glucoside prior to the formation of the aglycone (mandelonitrile). We also show that the cytosolic beta-glucosidase catalyzes the hydrolysis of various phenolic (e.g. arbutin and salicin) and cyanogenic plant glucosides (e.g. prunasin). Using the everted gut-sack technique, we demonstrate that the plant glucosides, amygdalin, prunasin, and vicine, are transported across the small intestine of the guinea pig efficiently and without being hydrolyzed. Based on these data we speculate that the cytosolic beta-glucosidase may participate in biotransformation of toxic plant glucosides.

Amygdalin↗
PubMed1992

Transglucosylation as a probe of the mechanism of action of mammalian cytosolic beta-glucosidase.

This study establishes that guinea pig liver cytosolic beta-glucosidase generates a common glucosyl-enzyme intermediate from a variety of aryl beta-D-glucoside substrates and that the intermediate can react with various acceptors to form distinct products at rates which are dependent on the structure, nucleophilicity, and concentration of the acceptor. Specifically, we demonstrate that water and linear alkanols will react with the glucosyl-enzyme intermediate to form D-glucose and alkyl-beta-D-glucoside (e.g. octyl-beta-D-glucoside), respectively. The rate of alcoholysis is 24-fold greater than the rate of hydrolysis of the glucosyl-enzyme intermediate and accounts for the increase in steady-state rate of substrate disappearance in the presence of alcohols. In addition, the substrate molecule itself (e.g. p-nitrophenyl-beta-D-galactoside (pNP-Gal)) can serve as an acceptor in the transglycosylation reaction, thereby enabling the enzyme to synthesize disaccharide glycosides (e.g. pNP-beta-Gal(6----1)beta-Gal). The transglycosylation data point to the presence of two hydrophobic subsites in the active site of the cytosolic beta-glucosidase. These data support a model in which the cytosolic beta-glucosidase binds an acceptor and a glycosyl donor simultaneously within its catalytic center and efficiently catalyzes the transfer of a sugar residue from the donor to the acceptor.

1-Butanol↗
PubMed1992

Continuous spectrophotometric assays for beta-glucosidases acting on the plant glucosides L-picein and prunasin.

The neutral pH optimum beta-glucosidases of mammalian liver and almonds are each capable of hydrolyzing a number of plant glucosides, including L-picein (p-hydroxyacetophenone-beta-D-glucoside) and prunasin (D-mandelonitrile-beta-D-glucoside). Taking advantage of the marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile, we have devised spectrophotometric assays that permit the continuous monitoring at pH 7.0 of p-hydroxyacetophenone (piceol) release from L-picein by guinea pig hepatic cytosolic beta-glucosidase and mandelonitrile from prunasin by almond beta-glucosidase. When L-picein hydrolysis was monitored at 320 nm and prunasin at 282 nm, the molar absorption coefficients determined for their products, namely piceol and mandelonitrile, were 3200 and 1360 M-1 cm-1, respectively. The kinetic parameter Km and Vmax values obtained using these spectrophotometric procedures for the guinea pig liver cytosolic beta-glucosidase acting on L-picein were 0.88 mM and 5.29 x 10(5) units/mg protein and for the almond beta-glucosidase acting on prunasin, Km 1.1 mM and Vmax 5.24 x 10(6) units/mg protein. These values agreed well with previously reported values obtained using less convenient, discontinuous assay procedures.

Animals↗
PubMed1991

2,3-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol is a substrate for human glucocerebrosidase.

Glucocerebrosidase, the lysosomal enzyme that is deficient in patients with Gaucher's disease, hydrolyses non-physiological aryl beta-D-glucosides and glucocerebroside, its substrate in vivo. We document that 2,3,-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol (2,3,-di-14:0-beta-Glc-DAG) inhibits human placental glucocerebrosidase activity in vitro (Ki 0.18 mM), and the nature of inhibition is typical of a mixed-type pattern. Furthermore, 2,3-di-14:0-beta-Glc-DAG was shown to be an excellent substrate for the lysosomal beta-glucosidase (Km 0.15 mM; Vmax. 19.8 units/mg) when compared with the natural substrate glucocerebroside (Km 0.080 mM; Vmax. 10.4 units/mg). The observations that (i) glucocerebrosidase-catalysed hydrolysis of 2,3-di-14:0-beta-Glc-DAG is inhibited by conduritol B epoxide and glucosylsphingosine, and (ii) spleen and brain extracts from patients with Gaucher's disease are unable to hydrolyse 2,3-di-14:O-beta-Glc-DAG demonstrate that the same active site on the enzyme is responsible for catalysing the hydrolysis of 4-methylumbelliferyl beta-D-glucopyranoside, glucocerebroside and 2,3-di-14:O-beta-Glc-DAG. With the aid of computer modelling we have established that the oxygen atoms in 2,3-DAG-Glc at the C-1, C-4*, C-5* (the ring oxygen in glucose) and C-2 positions correspond topologically to the oxygens at C-1, C-4* and C-5* and the nitrogen atom attached to C-2 respectively in glucocerebroside (* signifies a carbon atom in glucose); furthermore, all of the distances with respect to overlap of corresponding heteroatoms range from 0.02 A to 0.77 A (0.002-0.077 nm). A root-mean-square deviation of 0.31 A (0.031 nm) was obtained when the energy-minimized structures of 2,3-di-14:O-beta-Glc-DAG and glucocerebroside were compared using the latter four heteroatom co-ordinates.

Binding, Competitive↗
PubMed1991

A case of nonneurologic Gaucher's disease that biochemically resembles the neurologic types.

Systemic findings such as hepatosplenomegaly and typical Gaucher storage cells in a bone marrow aspirate led to the clinical diagnosis of Gaucher's disease in the seven-year old patient described in this report. On the basis of the lack of neurologic involvement the child was classified as having the Type 1, nonneurologic form of Gaucher's disease. After splenectomy glucocerebrosidase was extracted from her spleen for biochemical analysis. As expected, a marked deficiency of glucocerebrosidase activity was evident in the splenic extract, however her enzyme displayed anomalous behavior compared to other identical splenic preparations from documented Type 1 Gaucher's disease patients in that it failed to reconstitute with the acidic lipid phosphatidylserine. Using the polymerase chain reaction (PCR)-based color complementation assay and restriction endonuclease analysis, we compared the mutation genotype of this child with that of five other classical Type 1 patients. This analysis revealed that our patient alone was homoallelic for a T----C transition at position 1448 in the glucocerebrosidase cDNA that results in a 444Leu----Pro substitution in the glucocerebrosidase protein. The latter mutation genotype is normally associated with the neurologic phenotype, namely, the Types 2 and 3 forms of the disease. The relevance of the nature of polarity in clinical and biochemical analyses is discussed with regard to the phenotypic classification and the future clinical course of disease in the child.

Child↗
PubMed1989

Use of 4-heptylumbelliferyl-beta-D-glucoside to identify Gaucher's disease heterozygotes.

Three fluorometric beta-glucosidase assays were compared for their ability to identify Gaucher's disease heterozygotes, using leukocytes as the source of enzyme: the pH 5.5-taurocholate assay of Peters et al.; the conduritol B epoxide dependent variation of that assay; and the newly developed method described herein. While the first two procedures utilize the standard substrate 4-methylumbelliferyl-beta-D-glucopyranoside to estimate beta-glucosidase activity, the new assay uses 4-heptylumbelliferyl-beta-D-glucoside as (C7UGlc) substrate. Use of this substrate enhances the specificity of the method for lysosomal glucocerebrosidase, thereby minimizing the contribution of the nonspecific cytosolic beta-glucosidase to estimates of substrate hydrolysis. Using Student's t test for the three assays examined, the C7UGlc assay procedure was determined to have the lowest p value (p less than 0.001) and highest t value (t = 4.95) for the discrimination between the mean glucocerebrosidase value of control and obligate Gaucher heterozygote samples. The high reliability and simplicity of the C7UGlc assay lends adequate reason to favor this assay for regular clinical diagnosis of Gaucher heterozygotes.

Brain↗
PubMed1989

The dual effects of alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase.

This report demonstrates the effect of primary alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase. Lineweaver-Burk analyses of the kinetic data revealed a biphasic response; at low concentrations the alcohols increased the Vmax 5--7-fold while at higher concentrations they caused a purely competitive type of inhibition. For example, with n-butyl alcohol, increasing the alcohol's concentration in the assay medium from 0 to 0.14 M (0-1% (v/v)) resulted in a progressive increase in Vmax to a value 7-fold above the basal level without affecting the Km. However, between 0.14 and 0.54 M (1 and 4% (v/v)) n-butyl alcohol, the Km for 4-methylumbelliferyl-beta-D-glucopyranoside increased significantly from 0.14 to 0.93 mM. In contrast to n-butyl alcohol or isobutyl alcohol, which are potent activators, structurally related compounds like sec-butyl alcohol, tert-butyl alcohol, butylurea, and butanesulfonic acid did not stimulate the activity of the cytosolic beta-glucosidase. In the concentration range where activation was observed, conventional secondary replots of 1/delta slope versus 1/[alcohol] yielded perfect straight lines, demonstrating that binding of a single molecule of alcohol to the beta-glucosidase was responsible for the initial phase of activation. Furthermore, the glycohydrolase displayed a propensity to bind the longer chain alcohols, as reflected by the KA (binding constant) values of 555, 146, 34.1, and 7.47 mM for ethanol, n-propyl alcohol, n-butyl alcohol, 1-pentanol, respectively. This phenomenon of nonessential activation by alcohols has led us to speculate on the presence of a physiologic activator for the beta-glucosidase in mammalian tissues which contain this enzyme.

Alcohols↗
PubMed1989

Kinetic analysis of the interaction of alkyl glycosides with two human beta-glucosidases.

This paper addresses the similarities and differences in the topology of the catalytic centres of human liver cytosolic beta-glucosidase and placental lysosomal glucocerebrosidase, and utilizes well-documented reversible active-site-directed inhibitors. This comparative kinetic study was performed mainly to decipher the chemical and structural nature of the active site of the cytosolic beta-glucosidase, whose physiological function is unknown. Specifically, analysis of the effects of a family of alkyl beta-glucosides consistently displayed 100-250-fold lower inhibition constants with the cytosolic broad-specificity beta-glucosidase compared with the placental glucocerebrosidase; for example, with octyl beta-D-glucoside the Ki values were 10 microM and 1490 microM for the cytosolic and lysosomal beta-glucosidases respectively. Furthermore the higher affinity of the cytosolic beta-glucosidase than glucocerebrosidase for the amphipathic alkyl beta-D-glucosides was validated by the greater increase in the free energy of binding with increasing alkyl chain length [delta delta G0 (K,)/CH2: lysosomal enzyme, 2.01 kJ/mol (480 cal/mol); cytosolic enzyme, 3.05 kJ/mol (730 cal/mol)]. The implications of the presence of highly non-polar domains in the active site of the cytosolic beta-glucosidase are discussed with regard to its potential physiological substrates.

Binding Sites↗