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Biomedical subjects

A Madan

Publications and source records attributed to A Madan.

At least 55 records · Page 3Linked to original sources
PubMed1997

Inhibition of coumarin 7-hydroxylase activity in human liver microsomes.

Nine organic solvents and 47 commonly used P450 substrates and inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A6) activity in human liver microsomes. Of the nine organic solvents examined (final concentration 1%, v/v), only methanol did not inhibit the 7-hydroxylation of coumarin (0.5 to 50 microM) by human liver microsomes. Dioxane and tetra-hydrofuran, which are structurally related to coumarin, were the most inhibitory solvents examined. Although the rates of coumarin 7-hydroxylation varied enormously among nine samples of human liver microsomes and cDNA-expressed CYP2A6 (Vmax = 179 to 2470 pmol/ mg protein/min), the Km for coumarin 7-hydroxylation was fairly constant (ranging from 0.50 to 0.70 microM). The following chemicals caused little or no inhibition of CYP2A6 as defined by a Ki > 200 microM: caffeine, chlorzoxazone, cimetidine, dextromethorphan, diazepam, diclofenac, erythromycin, ethinylestradiol, ethynyltestosterone, fluconazole, furafylline, furfural, hexobarbital, itraconazole, mephenytoin, methimazole, metronidazole, naringenin, naringin, nifedipine, norfloxacin, norgestrel, orphenadrine, quinidine, papaverine, phenacetin, pyrimethamine, ranitidine, spironolactone, sulfaphenazole, sulfinpyrazone, testosterone, tolbutamide, troleandomycin, and warfarin. In other words, these chemicals, at a final concentration of 100 microM, failed to inhibit CYP2A6 when the concentration of coumarin was equal to Km (0.50 microM). The following chemicals were classified as strong inhibitors of CYP2A6 (defined by Ki < 200 microM): clotrimazole, diethyldithiocarbamate, ellipticine, ketoconazole, 8-methoxypsoralen, 4-methylpyrazole, metyrapone, miconazole, alpha-naphthoflavone, nicotine, p-nitrophenol, and tranylcypromine. The potency with which each chemical inhibited the 7-hydroxylation of coumarin was independent of which sample of human liver microsomes was studied. One of the most potent inhibitors of coumarin 7-hydroxylase was 8-methoxypsoralen (methoxsalen), which was determined to be a mechanism-based inhibitor (suicide substrate) of CYP2A6 (k(inactivation) 0.5 min-1). With the exception of 8-methoxypsoralen, preincubation of human liver microsomes and NADPH with the aforementioned inhibitors did not increase their ability to inhibit CYP2A6. The most potent competitive inhibitor of CYP2A6 was tranylcypromine (Ki = 0.04 microM). Several of the chemicals that strongly inhibited CYP2A6, such as ketoconazole and tranylcypromine, are often used with the intention of selectively inhibiting human P450 enzymes other than CYP2A6. The results of this study underscore the need for a systematic evaluation of the specificity of commonly used P450 inhibitors.

Adult↗
PubMed1997

Thrombosis of the deep cerebral veins: CT and MRI findings with pathologic correlation.

Deep cerebral vein thrombosis can present with acute, severe neurological symptoms and may be rapidly fatal as in the 20-year-old woman reported here. Although MRI is superior for establishing the diagnosis, CT is usually the first examination performed in the clinical setting. It is therefore important to recognise certain indicators such as extensive bithalamic low density. These and certain other less specific signs are correlated with the MRI and autopsy findings.

Adult↗
PubMed1997

Biophysical investigations on the myb-DNA system.

The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions. The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c-myb and its cognate DNA target sequences. The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has revealed that the domain is largely helical in nature. Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases. Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different. Most of the conserved tryptophans are associated with the specific DNA binding site. Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein. In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.

Amino Acid Sequence↗
PubMed1996

Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity.

The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5 and the activity of NADPH-cytochrome c reductase decreased 20-40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'- hydroxylation), CYP2D6 (dextromethorphan O-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b5 and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH-cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.

Adult↗
PubMed1996

Diagnosis of Castleman's disease by identification of an immunophenotypically aberrant population of mantle zone B lymphocytes in paraffin-embedded lymph node biopsies.

Castleman's disease (CD) is characterized by lymph node enlargement due to hyperplasia of abnormal lymphoid follicles and paracortical lymphocytic hyaline vascular (HV) stroma or plasmacytosis (PC). The lymphoid follicles in CD show involuted germinal centers and prominent mantle zone lymphocytes. Ninety-seven cases clinically suspected to be CD were analyzed according to conventional histologic criteria established by Castleman and Keller for diagnosis. Twenty-two cases were excluded as nonspecific hyperplasia (12); Hodgkin's and non-Hodgkin's lymphoma (9); and multiple myeloma involving lymph node paracortex (1). The 75 remaining cases, consisting of 51 cases of CD and 24 with altered follicles or paracortex suggestive of CD, were further analyzed immunohistologically for changes in follicular dendritic reticulum cells (FDRC) using the monoclonal antibody Ki-M4p, for germinal center proliferation with Ki-S5, for mantle zone immunophenotype with Ki-B3 and Ki-B5, for paracortical plasmacytoid monocytes with Ki-M1p, and for plasma cell clonality by applying antibodies to kappa and lambda immunoglobulin light chains. Lymph nodes showing nonspecific follicular and paracortical hyperplasia were included as controls. Hyaline vascular CD and plasma cell CD showed enlarged, polyploid FDRC with prominent nucleoli, decreased germinal center proliferation, and mantle zone populations of immunophenotypically aberrant, Ki-B3-negative B lymphocytes. Thirty-seven percent of hyaline vascular CD and plasma cell CD contained plasmacytoid monocytes, and 15% showed interstitial areas of lambda predominant plasma cells. Plasmacytoid monocytes were common in hyaline vascular CD but rare in plasma cell CD. Cases suspected to be CD that demonstrated a mantle zone population of Ki-B3-negative B lymphocytes had clinical finding of POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal paraprotein, and skin changes or sclerotic bone lesions) syndrome and were reclassified as hyaline vascular CD, plasma cell CD, and mantle zone CD with an aberrant mantle zone immunophenotype only (lacking follicular center and paracortical histologic or immunohistologic abnormalities). Immunohistochemistry was valuable for identification of dysplastic FDRC, decreased germinal center proliferation, and plasmacytoid monocytes. In addition, immunohistochemistry was essential for detection of plasma cell clonality, an aberrant mantle zone immunophenotype, and mantle-zone-restricted CD that was devoid of diagnostic alterations of germinal center or paracortex.

Antibodies, Monoclonal↗
PubMed1996

Characterization of the NADPH-dependent covalent binding of [14C]halothane to human liver microsomes: a role for cytochrome P4502E1 at low substrate concentrations.

Activation of halothane to trifluoroacetyl halide, followed by covalent binding to proteins (neoantigen formation) has been proposed to be the mechanism by which halothane causes immune hepatitis. The aim of this study was to identify the cytochrome P450 (CYP) enzyme primarily responsible for the NADPH-dependent covalent binding of [14C]halothane to human liver microsomes. Human liver microsomes were incubated in the absence and presence of NADPH with various concentrations of halothane (from 4.6 to 3,300 microM) to examine the effects of substrate concentration on the nonspecific and specific (NADPH-dependent) binding of [14C]halothane to microsomal protein. As a function of substrate concentration, the specific binding of [14C]halothane to human liver microsomes was biphasic, suggesting that the activation of halothane is catalyzed by a high-affinity enzyme(s) at low substrate concentrations (<150 microM) and by a low-affinity enzyme(s) at high substrate concentrations (>150 microM). For the high-affinity enzyme, the apparent KM for the covalent binding of [14C]halothane was approximately 10 microM, and Vmax was approximately 32 pmol equivalents of halothane bound/mg protein/min under conditions where covalent binding was directly proportional to incubation time and protein concentration. Ten individual samples of human liver microsomes were incubated with a low concentration of halothane (35 microM) to determine the sample-to-sample variation in the specific binding of [14C]halothane to microsomal protein. Covalent binding ranged from 10 to 40 pmol equivalents of halothane bound/mg protein/min and was highly correlated (r2 = 0.93) with the sample-to-sample variation in chlorzoxazone 6-hydroxylase activity, which reflects the levels of CYP2E1. These results suggest that CYP2E1 is the high-affinity enzyme in human liver microsomes responsible for activating halothane to a reactive metabolite. This is supported by the observation that 4-methylpyrazole, a CYP2E1 inhibitor, inhibited the NADPH-dependent binding of [14C]halothane to microsomal protein. The sample-to-sample variation in the covalent binding of [14C]halothane at high substrate concentrations did not correlate with any known CYP enzyme activity. This suggests that several enzymes catalyze the oxidation of halothane at higher substrate concentrations. In conclusion, at pharmacologically relevant concentrations, the covalent binding of halothane to human liver microsomes is primarily catalyzed by CYP2E1.

Autoradiography↗
PubMed1995

Bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-Myb DNA-binding protein.

The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1-R2-R3) of 51-53 amino acids towards its N-terminus as its presumptive DNA binding domain. Using PCR amplification and the T7 expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent-exposed residues were involved.

Animals↗
PubMed1995

The DNA-binding domain of Drosophila melanogaster c-Myb undergoes a multistate denaturation.

The DNA-binding domain of Drosophila c-Myb protein has been studied using different spectroscopic probes, namely CD, fluorescence, acrylamide quenching and NMR, to determine the structure of some of its sub-domains and their relative stabilities in aqueous solutions. While CD and fluorescence spectroscopy showed that the protein had completely lost its tertiary and secondary structures in approximately 3 M urea, solvent accessibility of the tryptophan residues was still partial, as determined by acrylamide quenching. This suggested the presence of significant amounts of residual structure which persisted until the urea concentration was raised to approximately 6.0 M. Thermal-denaturation experiments also indicated the presence of an intermediate in the unfolding pathway. The experimental data could be fitted assuming a minimum of three states in both modes of denaturation. The thermodynamic parameters for the apparent three-state transition have been determined. From the protein stability curve, we have determined that Drosophila melanogaster Myb R123 has maximal stability at 16 degrees C and pH 7.0.

Animals↗
PubMed1995

Regulated basal, inducible, and tissue-specific human erythropoietin gene expression in transgenic mice requires multiple cis DNA sequences.

Erythropoietin (Epo) gene expression in kidney and liver is inducible by anemia. To localize the sequences necessary for regulated expression of the Epo gene, we constructed transgenic mice containing five human Epo gene constructs and examined Epo expression under basal conditions and with anemia. Mice containing the Epo gene with 0.3 kb of 5' flanking sequence, 0.7 kb of 3' flanking sequence, and either all introns or only intron I alone were polycythemic, had Epo expression in various tissues (including non-Epo-producing tissues), and induction only in liver. In contrast, mice containing the Epo gene with 8.5 kb of 3' flanking sequence and either 9.5 or 22 kb of 5' flanking sequence had basal expression at low levels in appropriate tissues and were less likely to be markedly polycythemic. Mice with the smaller of these two constructs had induction only in the liver, whereas those with the larger construct had induction in the kidney and liver. These studies indicate that sequences sufficient for induction in the liver are located in close proximity to the Epo gene, including the immediate 5' and 3' flanking sequence and the first intron. They also indicate that sequences required for induction in the kidney are located more than 9.5 kb 5' to the gene. Furthermore, comparison of these and prior transgenic studies suggest that sequences that limit the basal expression of the Epo gene are located downstream of the gene. We conclude that multiple cis DNA sequences are required for regulated Epo gene expression.

Anemia↗
PubMed1995

Solution structure of the conserved segment of the Myb cognate DNA sequence by 2D NMR, spectral simulation, restrained energy minimization, and distance geometry calculations.

Solution structure of a self-complementary DNA duplex d-ACCGTTAACGGT containing the TAACGG recognition segment of Myb protein has been obtained by NMR spectroscopy. Complete resonance assignments of all the protons (except H5', H5" protons) have been obtained following standard procedures based on two-dimensional NMR techniques. Using a total of 72 coupling constants, and 95 NOE intensities, restrained energy minimization has been carried out, with the X-PLOR force field. The distance constraint set has been iteratively refined, for better fits with experimental NOE intensities. Using the final constraint set thus obtained, and explicit H-bond constraints for A.T, G.C base pairs in the duplex, distance geometry calculations have been carried in the torsion angle space with the program TANDY-2S to identify the family of structures consistent with the NMR data. We observe that the constraint set does indeed define a unique structure for the DNA segment. The structural details have been analyzed, and the sequence-dependent variations in torsion angles, base pair geometries, and helicoidal parameters have been documented. We observed that the helix axis displays a nonregular path, and three centered H-bonds have been seen at AA, AC, and CC steps in the major groove of the helix. Substantial variations have been observed for the helix axis and the groove widths at the recognition site. The base pairs exhibit high negative propeller twists. The structure is characterized by O4'-endo geometry for all the sugar rings (expect G10), and the other torsion angles belong to the B-DNA families.

Amino Acid Sequence↗
PubMed1995

Characterization of diethyldithiocarbamate methyl ester sulfine as an intermediate in the bioactivation of disulfiram.

Disulfiram is bioactivated through a series of intermediates, ultimately forming S-methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-Me sulfoxide), the metabolite proposed to be responsible for the in vivo inhibition of rat liver mitochondrial low Km aldehyde dehydrogenase (ALDH2). Diethyldithiocarbamate methyl ester sulfine (DDTC-Me sulfine) also has been recently identified as a possible metabolite of disulfiram (Madan and Faiman, 1994). In the present studies, DDTC-Me sulfine was characterized and was found to inhibit ALDH2 in vivo (ID50 = 57 mumol/kg) but not in vitro. Maximum inhibition of ALDH2 in rats was observed 1 hr after the i.p. administration of DDTC-Me sulfine. Pretreatment of rats with 1-benzylimidazole, a cytochrome P450 inhibitor, blocked the DDTC-Me sulfine-mediated inhibition of ALDH2. This suggested that DDTC-Me sulfine was further bioactivated by a cytochrome P450-dependent mechanism. DDTC-Me sulfine could not be detected in rat plasma after the i.p. administration of disulfiram (75 mg/kg), DDTC-Me (122 mg/kg) or DDTC-Me sulfine (22.6 mg/kg). However, S-methyl N,N-diethylthiolcarbamate (DETC-Me), the desulfurated form of DDTC-Me, was detected as a major metabolite of DDTC-Me sulfine in rat plasma after DDTC-Me sulfine administration. Also, a disulfiram-like-ethanol reaction was observed in rats treated with DDTC-Me sulfine and challenged with ethanol. These data provided additional support for the idea that DDTC-Me sulfine is an intermediate formed after DDTC-Me metabolism and is probably a precursor to DETC-Me in the overall bioactivation of disulfiram to DETC-Me sulfoxide.

Aldehyde Dehydrogenase↗
PubMed1995

Identification of the human and rat P450 enzymes responsible for the sulfoxidation of S-methyl N,N-diethylthiolcarbamate (DETC-ME). The terminal step in the bioactivation of disulfiram.

The present study investigated the role of rat and human cytochrome P450 enzymes in the sulfoxidation of S-methyl N,N-diethylthiolcarbamate (DETC-Me) to S-methyl N,N-diathylthiolcarbamate sulfoxide (DETC-Me sulfoxide), the putative active metabolite of disulfiram. DETC-Me sulfoxidation by microsomes from male and female rats treated with various cytochrome P450-enzyme inducers suggested that multiple enzymes can catalyze this reaction, and these include, CYP1A1/2, CYP2B1/2, and CYP3A1/2. All cDNA-expressed human cytochrome P450 enzymes examined catalyzed the sulfoxidation of DETC-Me. The turnover rates (min-1) of DETC-Me sulfoxidation by the cDNA-expressed cytochrome P450 enzymes ranked as follows: CYP3A4 > CYP2A6 = CYP2C9 > CYP1A2 > CYP2B6 = CYP2E1 > CYP1A1 > CYP2D6. Interestingly, CYP3A4 ranked first or last, depending on whether or not additional NADPH-cytochrome P450 reductase was coexpressed in the lymphoblastoid cells. This complicated estimates of the contribution of CYP3A4 to DETC-Me sulfoxidation by human liver microsomes. The sample-to-sample variation in DETC-Me sulfoxidation by bank of human liver microsomes (N=13) correlated highly with coumarin 7-hydroxylation (r=0.88) and testosterone 6beta-hydroxylation (r=0.90), suggesting that CYP2A6 and CYP3A4/5 contribute to the sulfoxidation of DETC-Me by human liver microsomes. Although, chlorzoxazone 6-hydroxylation (a marker for CYP2E1) correlated poorly with DETC-Me sulfoxidation, the correlation improved from r=0.07 to r=0.44 when DETC-Me sulfoxidation was studied in the presence of the CYP2A6 inhibitor, coumarin. Similarly, when DETC-Me sulfoxidation was studied in the presence of diethyldithiocarbamate (DDTC), the inhibited DETC-Me sulfoxidase activity correlated better (r=0.50) with chlorzoxazone 6-hydroxylase, compared with DETC-Me sulfoxidase activity in the absence of DDTC (r=0.09). Polyclonal antibodies against CYP2E1 caused a modest inhibition (30%) of DETC-Me sulfoxidation by human liver microsomes. Anti-CYP3A1 antibodies completely inhibited DETC-Me sulfoxidation by cDNA-expressed CYP3A4. Under similar conditions, DETC-Me sulfoxidation by human liver microsomes was only partially inhibited by anti-CYP3A1 antibodies. Although studies with the rat and cDNA-expressed cytochrome P450 enzymes suggested that CYP1A2 contributed to DETC-Me sulfoxidation, this reaction was not inhibited by either furafylline ( a mechanism-based inhibitor of CYP1A2) or antibodies against CYP1A1/2. A significant role for CYP2C9 was excluded by the inability of sulfaphenazole to inhibit the sulfoxidation of DETC-Me by human liver microsomes. Collectively, these data suggest that multiple cytochrome P450 enzymes can catalyze the sulfoxidation of DETC-Me. In human liver microsomes the CYP2A6, CYP2E1, and CYP3A4/5 all contribute significantly to the sulfoxidation of DETC-Me. It is interesting to note that DDTC, the reduced metabolite of disulfiram, is known to inhibit these same enzymes. The ability of DDTC to block the formation of DETC-Me sulfoxide may explain why the dose of disulfiram required to produce a disulfiram-ethanol reaction in alcoholics is so variable and often inadequate.

Animals↗
PubMed1995

Conformational dynamism in d-(GAATTCCGTTATT) containing the complementary Myb responsive element d-CCGTTA: NMR and MD investigations.

Conformational features of the DNA segment d-(GAATTCCGTTATT) containing the complementary Myb responsive element CCGTTA has been studied by NMR and molecular dynamics calculations with a view to see the role of 3D structure in specific DNA recognition. From the low field imino proton NMR spectra, the DNA sequence is seen to exist as a duplex with pyrimidine mismatches in the centre. The 2D NMR spectra however show that teh molecule exhibits substantial dynamism even at 1 degree C. Several extra cross peaks, more than the expected number, are seen in particular regions in all the spectra. These observations indicate that the duplex undergoes slow transitions between base-paired and unbase-paired states due to mismatches in the centre. Hence, to characterise those transitions a restrained verlet dynamics has been performed for 50ps using X-PLOR force field. Structural intermediates at regular intervals have been analysed, and we see that the dynamism in the molecule results in substantial fluctuations in the different torsion angles. The mismatch sites are seen to exhibit the highest degree of fluctuations, with the bases stacking in and looping out of the duplex. The sugar geometry is seen to be fairly steady around the S domain for most of the residues.

Base Sequence↗
PubMed1995

Lipid profile norms in Indian children.

OBJECTIVES: To evaluate the lipid parameters in normal Indian children. DESIGN: Cross-sectional. SETTING: Hospital based. METHODS: 410 children (siblings of hospitalized pediatric cases) between 3-12 years were evaluated for total plasma cholesterol (TC), triglycerides (TG) and high density lipoprotein cholesterol (HDL-C). The low density lipoprotein cholesterol (LDL-C) levels were derived from the above parameters using Fredrickson-Friedwald formula. RESULTS: No significant difference was found between the sexes in any of the lipid parameters studied. The mean values were: TC-134.5 mg/ dl, TG-91.1 mg/dl, HDL-C-34.15 mg/dl and LDL-C-80.1 mg/dl. The suggested cut off limits for these parameters were 190 mg/dl, 150 mg/dl, 20 mg/dl and 130 mg/d respectively. CONCLUSIONS: Lipid profile norms and cut off levels to define abnormalities for Indian children were recommended. The HDL-C levels were lower than western data.

Child↗
PubMed1994

Creating new DNA binding specificities in the yeast transcriptional activator GCN4 by combining selected amino acid substitutions.

The specificity of the GCN4/DNA complex is mediated by a complicated network of interactions between the basic regions of both GCN4 monomers and their target halfsites. According to X-ray analyses (1, 2) one particular thymine of the target sequence is recognized by serine -11 and alanine -15 (we define the leucine in the first d-position of the heptad repeats as +1). We replaced serine -11 or alanine -15 with all other amino acids and analysed the DNA binding properties of the resulting stable GCN4 derivatives by electrophoretic mobility shift assays. Among these, mutants with tryptophan in position -11, or glutamic acid and glutamine in position -15, differ significantly from GCN4 in their DNA binding specificities. We then constructed selected double mutants, which differ from GCN4 in positions -11, -15 or -14 (3) of the basic region. The double mutants with tryptophan in position -11 and asparagine or serine in position -14 show drastically altered DNA binding specificities, presumably due to additive effects.

Base Sequence↗
PubMed1994

Sequence-specific DNA displaces 6-p-toluidino-2-naphthalenesulfonate bound to a hydrophobic site on the DNA-binding domain of Drosophila c-myb.

The N-terminal DNA-binding domain of c-myb oncoprotein binds to DNA in a sequence-specific manner. The domain, consisting of three imperfect tandem repeats, has tryptophan residues at very regular intervals and this is believed to be of some significance in the DNA-binding activity of the protein. We have found that the hydrophobic-site-specific probe 6-p-toluidino-2-naphthalenesulfonate (TNS) binds to the bacterially expressed DNA-binding domain of Drosophila c-myb protein (R123). TNS has a single binding site on this protein with an apparent dissociation constant in the range of (5-8) x 10(-7) M. When the TNS-protein complex was treated with an oligomeric DNA duplex having a cognate myb-binding site, the TNS was displaced from the complex. Nonspecific DNA duplex oligomers were ineffective, indicating that TNS displacement was a sequence-specific process. We examined further some features of the TNS-binding site on the protein, taking advantage of the fluorescence properties of the protein and the bound TNS. Our data indicate that the TNS binding occurs in a peripheral site on the protein in a manner that allows the bound TNS to be solvent accessible. Furthermore, there are indications that tyrosine(s) and tryptophans of the protein mediate resonance energy transfer to the bound TNS. From fluorescence-quenching data of the protein and protein-TNS complex, we could assess that both solvent-accessible and internal tryptophans are in the vicinity of the bound TNS. (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

S-methyl N,N-diethylthiolcarbamate sulfone, an in vitro and in vivo inhibitor of rat liver mitochondrial low Km aldehyde dehydrogenase.

S-Methyl N,N-diethylthiolcarbamate sulfone (DETC-Me sulfone) was investigated for its rat liver mitochondrial low Km aldehyde dehydrogenase (ALDH2) inhibitory properties. DETC-Me sulfone inhibited ALDH2 in vitro (IC50 = 3.8 microM) and in vivo (ID50 = 170 mumol/kg; 31 mg/kg). Maximum inhibition (60%) of ALDH2 was observed 8 hr after DETC-Me sulfone administration. In addition, incubation of S-methyl N,N-diethylthiolcarbamate (DETC-Me) or S-methyl N,N-diethylthiolcarbamate sulfoxide (DETC-Me sulfoxide) with rat liver microsomes and an NADPH-generating system failed to produce DETC-Me sulfone. Furthermore, DETC-Me sulfone could not be detected in plasma from rats treated with either DETC-Me sulfoxide or DETC-Me sulfone. In conclusion, DETC-Me sulfone inhibited ALDH2 in vitro and in vivo. However, there was no evidence suggesting that DETC-Me sulfoxide was metabolized to DETC-Me sulfone.

Aldehyde Dehydrogenase↗