CYP1A1 and CYP2D6 polymorphism and risk of lung cancer in a North Indian population.

This case-control study was conducted to examine the association between the CYP1A1 and CYP2D6 genotypes and lung cancer risk among North Indians. The estimated relative risk for lung cancer associated with the CYP1A1 Val/Val allele was 2.68, and was four-fold when cases with small cell lung cancer (SCLC) were considered alone. With regard to the metabolism of debrisoquine, no poor metabolizers were found amongst the subjects. The odds ratio of risk with the heterozygous extensive metabolizer (HEM) genotype was 1.5. However, in the presence of at least a single copy of the variant CYP1A1 MspI allele and the CYP2D6 HEM genotype, the risk was two-fold for squamous cell carcinoma (SQCC). When the CYP1A1 Val/Val and CYP2D6 HEM genotypes were taken together, the risk for SCLC was four-fold. Stratified analysis indicated an interaction between bidi smoking and variant CYP1A1 genotypes on the risk for SQCC and SCLC. Heavy smokers (Brinkman index>400) with Val/Val genotypes were at a very high risk of developing lung cancer (odds ratio 29.30, 95% confidence interval 2.42-355, p=0.008). Heavy smokers with CYP1A1 MspI (CYP1A1*1/2A or CYP1A1*2A/*2A) genotype had a seven-fold risk for SCLC compared with non-smokers. This study is the first to be carried out on a North Indian population, and, although small, suggests that CYP1A1 and CYP2D6 polymorphisms might have a role in determining the risk for lung cancer and should be investigated further.     

Rat kidney porphobilinogen deaminase kinetics. Detection of enzyme-substrate complexes

BACKGROUND AND AIMS: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties, under normal conditions. METHODS: Rat kidney PBG-D was purified to homogeneity and initial reaction velocities were calculated by measuring uroporphyrinogen I formation at pH 8.2 for different incubation times (0-20 min) and over a wide range of substrate concentrations (0.8-66 microM). RESULTS: Purified rat kidney PBG-D is a monomeric enzyme showing only a single protein band after SDS-PAGE, Western blot and isoelectric focusing (pI 4.9). Its molecular mass is 40 +/- 2.3 kDa, determined by SDS-PAGE and 39.8 +/- 2 kDa by gel filtration chromatography. Rat kidney PBG-D has an unusual kinetic behaviour, exhibiting a deviation from the Michaelis-Menten hyperbola. PBG-D kinetic data required a fitting to an equation of higher degree, leading to the following apparent kinetic constants: K(1) = 2.08 +/- 0.01 microM and K(2) = 0.102 +/- 0.003 microM. CONCLUSION: The values of these constants fulfil the restriction 4K(2) < or = K(1)(2), necessary for the occurrence of isoenzymes, interpreted in this work as enzyme-substrate intermediates. The initial reaction velocity expression here defined, correlates with an enzyme carrying only one active site but allowing, through conformational changes, the detection of at least two enzyme-substrate intermediates formed during PBG-D reaction.     

Expression of 93D heat shock puff of Drosophila melanogaster in deficiency genotypes and its influence on activity of the 87C puff

Using the overlapping deficiencies Df(3R)GC14 and Df(3R)e ^Gp 4 of the 93D region of Drosophila melanogaster, the benzamide (BM)-inducible site in polytene chromsomes was localised to the 93D6-7 region, which had earlier been identified as heat inducible. The normal developmental and BM-induced 93D6-7 puff was found to be dosage compensated since in larvae heterozygotus for a deficiency, with one dose of 93D6-7, the rate of ³H-uridine incorporation in this puff was the same as in the wild type with two doses. Curiously, however, heat shock (37° C) caused regression of the 93D6-7 puff on the normal chromosome in heterozygotes. In agreement with earlier results from our laboratory, the non-inducibility of the single-dose 93D locus by heat shock in the heterozygotes, caused the 87C puff to be nearly half as active as the 87A puff at 37° C. However, in e ^Gp 4/GC14 larvae, lacking the 93D6-7 locus on both homologues, the 87C puff was less active than 87A in some heat-shocked larvae but in other larvae 87A and 87C were equally active. Possible reasons for this inter-larval variability are discussed.     

Inhibition of porphobilinogenase by porphyrins in Saccharomyces cerevisiae

The biosynthesis of uroporphyrinogen III, the precursor of hemes, chlorophylls, corrins and related structures, is catalyzed by the porphobilinogenase system (PBGase), a complex of two enzymes, PBG-Deaminase (PBG-D) and Isomerase. Although the separate enzymes have been studied in some detail less work has been performed on the properties of the complex.In this study the kinetic behaviour of the enzyme PBGase in a normal yeast strain, D273-10B, and its derivative B231 has been investigated. Uroporphyrinogen formation was linear with time up to 2 hr at 37°C. The enzyme complex shows classical Michaelis-Menten kinetics. From the double reciprocal plots kinetic parameters were estimated for PBGase and PBG-D.Porphyrins were found to be competitive inhibitors with respect to porphobilinogen (PBG) and these compounds appeared to act as inhibitors by forming dead-end complexes with the free enzyme. 5-Aminolevulinic acid (ALA) also inhibited PBGase and this inhibition was overcome by addition of levulinic acid (2μM). These results indicate that ALA, is not an inhibitor but acts through its conversion into porphyrins which are the true inhibitors.     

Integrin alphaIIbbeta3:ligand interactions are linked to binding-site remodeling

This study tested the hypothesis that high-affinity binding of macromolecular ligands to the alphaIIbbeta3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between alphaIIbbeta3 and two model ligands-echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's gamma-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site-were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the alphaIIb/beta3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming--i.e., ectodomain conformational changes and oligomerization induced by incubation at 35 degrees C with the ligand-mimetic peptide cHarGD--promotes complex formation with fibrinogen's gamma-module. We also observed that the gamma-module's flexible carboxy terminus was not required for alphaIIbbeta3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.     

CYP27B1 polymorphisms variants are associated with type 1 diabetes mellitus in Germans

CYP27B1 (25-hydroxyvitamin D(3)-1alpha-hydroxylase) catalyzes the metabolization of 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) the most active natural Vitamin D metabolite. 1,25(OH)(2)D(3) plays a role in the regulation of autoimmunity and cell proliferation and prevents the development of autoimmune diabetes mellitus in animal models besides other autoimmune disorders. One hundred and eighty-seven families with one offspring affected with type1diabetes mellitus were genotyped for the polymorphisms in the promoter region (-1260 C/A) and intron 6 (2338 T/C) of the CYP27B1 gene on chromosome 12 q13.1-13.3 and extended transmission disequilibrium tests (ETDT) were performed. The haplotype CT (-1260 A/2338 T) was significantly more often transmitted to affected offspring (96 transmitted (T) versus 63 not transmitted (NT), P = 0.0089). While the AT (-1260 C/2838 T) was significantly less often transmitted (37 T versus 60 NT, P = 0.0195). This study suggests that CYP27B1 haplotypes may confer susceptibility to type 1 diabetes mellitus in Germans.     

Synthesis, biological evaluation and molecular modelling studies of methyleneimidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17). Part I: Heterocyclic modifications of the core structure

Novel chemical entities were prepared via Suzuki and S(N) reaction as AC-ring substrate mimetics of CYP17. The synthesised compounds 1-31 were tested for activity using human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against hepatic CYP enzymes (3A4, 2D6, 1A2, 2C9, 2C19, 2B6). Two potent inhibitors (27, IC50 = 373 nM/28, IC50 = 953 nM) were further examined in rats regarding their effects on plasma testosterone levels and their pharmacokinetic properties. Compound 28 was similarly active as abiraterone and showed better pharmacokinetic properties (higher bioavailability, t(1/2) 9.5 h vs 1.6 h). Docking studies revealed two new binding modes different from the one of the substrates and steroidal inhibitors.     

Mechanism of substrate inhibition in Escherichia coli phosphofructokinase

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites, which bind the substrates fructose-6-phosphate (Fru-6-P) and MgATP. In the presence of low concentrations of Fru-6-P, MgATP displays substrate inhibition. Previous proposals to explain this substrate inhibition have included both kinetic and allosteric mechanisms. We have isolated hybrid tetramers containing one wild type subunit and three mutated subunits (1:3). The mutated subunits contain mutations that decrease affinity for Fru-6-P (R243E) or MgATP (F76A/R77D/R82A) allowing us to systematically simplify the possible allosteric interactions between the two substrates. In the absence of a rate equation to explain the allosteric effects in a tetramer, the data have been compared to simulated data for an allosteric dimer. Since the apparent substrate inhibition caused by MgATP binding is not seen in hybrid tetramers with only a single native MgATP binding site, the proposed kinetic mechanism is not able to explain this phenomenon. The data presented are consistent with an allosteric antagonism between MgATP in one active site and Fru-6-P in a second active site.     

12(R)-hydroxyeicosatrienoic acid: a vasodilator cytochrome P-450-dependent arachidonate metabolite from the bovine corneal epithelium

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, two biologically active metabolites of arachidonic acid were formed. The structure of one of the metabolites, compound C, was previously reported to be 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid and was found to be a potent inhibitor of the Na+/K+-ATPase in the cornea. The second metabolite, compound D, was found to be a potent vasodilator as well as having the property of stimulating protein influx into the aqueous humor of the eye. Following purification of compound D by thin layer chromatography and high pressure liquid chromatography, it was found to lack a UV chromophore in contrast to the previously reported cytochrome P-450-dependent metabolite. Mass spectrometric analysis using positive and negative ionization modes was carried out on derivatized compound D that had been synthesized from a mixture of labeled [( 5,6,8,9,11,12,14,15-2H8]) and unlabeled arachidonic acid incubated with corneal microsomes. The novel arachidonate metabolite had abundant fragment ions consistent with compound D being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon 12 of the eicosanoid backbone; only seven deuterium atoms from [2H8]arachidonate were retained in the structure. Oxidative ozonolysis yielded a product indicating that the double bonds in metabolite D resided between carbons at positions 8 and 9 and positions 14 and 15 of the 20-carbon chain. Compound D was therefore characterized as 12-hydroxy-5,8,14-eicosatrienoic acid. Model compounds were synthesized from dimethyl malate with the hydroxy at the 12 position with both the R and S absolute configuration and with all double bonds of the cis configuration. Only the 12(R) isomer was found to be a potent vasodilator and to increase aqueous humor protein concentration, suggesting that the biologically active compound D was 12(R)-hydroxy-5,8,14-(Z,Z,Z)-eicosatrienoic acid. As this compound possesses proinflammatory properties, it may play a role in the wound-healing processes of corneal injury.     

Expression of human CYP27B1 in Escherichia coli and characterization in phospholipid vesicles

CYP27B1 is a mitochondrial cytochrome P450 that catalyses the hydroxylation of 25-hydroxyvitamin D3 at the C1α-position to give the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3. We successfully expressed human CYP27B1 in Escherichia?coli and partially purified this labile enzyme and carried out a detailed characterization of its kinetic properties in a reconstituted membrane environment. The phospholipid concentration did not affect the enzyme activity in the vesicle-reconstituted system, although it was influenced by the phospholipid composition, with the addition of cardiolipin lowering the K(m) for 25-hydroxyvitamin D3. These data are consistent with the enzyme accessing substrate from the hydrophobic domain of the vesicle membrane. Cardiolipin also caused the appearance of inhibition of activity at high substrate concentrations. This substrate inhibition fitted a model for one catalytic and two inhibitory sites on the enzyme for the binding of substrate. The K(m) for human adrenodoxin was observed to decrease with decreasing substrate concentration, with the catalytic efficiency (k(cat) /K(m) ) being largely independent of adrenodoxin concentration. Human CYP27B1 was also active on 25-hydroxyvitamin D(2) and on intermediates of the CYP24A1-mediated inactivation pathway, 24R,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, with all these substrates showing comparable k(cat) values of 50-71?min(-1) , similar to 25-hydroxyvitamin D3. The latter two substrates gave higher K(m) values than that for 25-hydroxy-vitamin D3. The present study shows that human CYP27B1 can be partially purified in an active form with the enzyme displaying high activity towards a range of substrates in a phospholipid vesicle-reconstituted system that mimics the inner-mitochondrial membrane.     

The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity.

Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.     

The unique pentagonal structure of an archaeal Rubisco is essential for its high thermostability

We have previously determined the crystal structure of a novel pentagonal ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. Here we have carried out biochemical studies to identify the necessities and/or advantages of this intriguing pentagonal structure. The structure indicated the presence of three neighboring residues (Glu-63, Arg-66, and Asp-69), participating in ionic interactions within unique dimer-dimer interfaces. We constructed three single mutant proteins (E63S, R66S, and D69S) and one triple mutant protein (E63S/R66S/D69S) by replacing the charged residues with serine. The wild type (WT) and all mutant proteins were purified and subjected to gel permeation chromatography at various temperatures. WT and D69S proteins were decameric at all temperatures examined between 30 and 90 degrees C. The majority of E63S and R66S were decamers at 30 degrees C but were found to gradually disassemble with the elevation in temperature. E63S/R66S/D69S was found in a dimeric form even at 30 degrees C. An interesting correlation was found between the subunit assembly and thermostability of the proteins. Circular dichroism and differential scanning calorimetry analyses indicated that the denaturation temperatures of dimeric enzymes (E63S, R66S, and E63S/R66S/D69S) were approximately 95 degrees C, whereas those of the enzymes retaining a decameric structure (WT and D69S) were approximately 110 degrees C. Disassembly into tetramer or dimer units did not alter the slopes of the Arrhenius plots, indicating that the decameric structure had no effect on catalytic performance per se. The results indicate that the decameric assembly of Tk-Rubisco contributes to enhance the thermostability of the enzyme. Taking into account the growth temperature of strain KOD1 (65-100 degrees C), the decameric structure of Tk-Rubisco can be considered essential for the stable presence of the enzyme in the host cells. This study provides an interesting example in which the thermostability of a protein can be enhanced by formation of a unique quaternary structure not found in mesophilic enzymes.     

Metabolism of ochratoxin A by rats.

Albino rats were given ochratoxin A (6.6 mg/kg body weight) intraperitoneally or per os. Independent of route administration, 6% of a given dose was excreted as the toxin, 1 to 1.5% as (4R)-4-hydroxyochratoxin A, and 25 to 27% as ochratoxin alpha in the urine. The metabolite (4S)-4-hydroxyochratoxin A, which is formed by rat liver microsomes in the presence of NADPH, was not detected. Only traces of ochratoxins A and alpha were found in feces. Identical experiments were carried out with brown rats, since the Km value for the formation of the 4S epimer was considerably lower when brown rat microsomes were used. About the same ratios of metabolites and metabolite recoveries as those found for albino rats were found for brown rats. Brown rats were also given the two hydroxylated metabolites and ochratoxin alpha (0.66 mg/kg body weight) intraperitoneally. The three compounds were excreted in the urine; within 48 h, 90% recovery of ochratoxin alpha and 54 and 35%, respectively, of the 4R and 4S isomers were observed.     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline nucleic acid constituents and their halogenated derivatives

Free radicals in gamma-irradiated polycrystalline nucleic acid constituents and their 5-halogenated derivatives have been studied by e.s.r. and spin-trapping. After gamma-irradiation at room temperature, the polycrystalline samples were dissolved in aqueous solutions of t-nitrosobutane (tNB) in the absence or presence of oxygen. For many of the nucleic acid constituents, two types of radicals, -C(5)RH-C(6)H- and -C(5)R-C(6)H2-, formed by H-addition to the double bond [-C(5)R=C(6)H-] of the base, were observed, where R is -CH3 or -H. In addition, radicals formed on the sugar moiety were found for some nucleosides. When oxygen was present in the tNB solution, the relative stability of trapped radicals was changed, and thus the presence of more than one radical species could be established. For halogenated bases, the radical produced by H-abstraction from N(1) was observed, and an additional radical species formed by H-addition to the C(6) position was found for 5-fluorouracil. For halogenated nucleosides, the same spectrum was observed in all compounds except the 5-fluoroderivatives, and was assigned to the radicals produced on the sugar moiety. For 5-fluorodeoxyuridine and 5-fluorouridine, the radical formed by H-addition to the C(6) position of the base was observed. In general, the present results are in good agreement with those of previous single crystal studies, but in the case of halogenated compounds other than the 5-fluoroderivatives, it was not possible to spin-trap the alpha-halo radicals which were the most prominent radicals formed from gamma-irradiation of single crystals at room temperature.     

Cystic fibrosis gene mutations and linked RFLPs in the Slovenian population.

The authors used polymerase chain reaction to analyse 56 Slovenian cystic fibrosis (CF) chromosomes for the presence of delta F508 and eight other most frequent mutations located in exons 7,11 and 20 (R347P, R334W, G551D, R553X, S549RA, S549RT, S549I and S1255X) of the CF gene. We also determined the frequency of haplotypes associated with CF for six linked RFLP markers (MetD/TaqI, MetH/TaqI, XV-2c/TaqI, KM-19/PstI, MP6d9/MspI and J3.11/MspI) in 27 Slovenian CF families. delta F508 mutation was present in 55.4 percent of the CF chromosomes. No case of the other mutations were detected in the sample of tested CF chromosomes. A very high degree of association (0.88) has been found between DNA marker MetH and CF (as measured by the Yule's association coefficient) in our population. Using the RFLP markers XV-2c and KM-19, we found that 85% of delta F508 mutated chromosomes have a single 1 2 (B) haplotype, and that this haplotype is present on only 15.4 percent of CF chromosomes without this deletion.