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PubMed2003

NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity.

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.

Amides↗
PubMed1998

Analyses of single B cells by polymerase chain reaction reveal rearranged VH with germline sequences in spleens of immunized adult rabbits: implications for B cell repertoire maintenance and renewal.

We used PCR to amplify rearranged VHDJH genes in single cells collected by micromanipulation from splenic germinal centers of immunized adult rabbits. In the course of the study, the objective of which was to analyze diversification of rearranged VHDJH sequences, we were surprised to find cells 7 and 10 days after immunization with rearranged VH1a2 as well as a-negative (y33 and x32) sequences that were identical or close to germline (10 or fewer changes). About 58% (82/140) of the sequences had unique CDR3 regions and were unrelated. In seven different germinal centers, we found one to four different clones with two to seven members. Clonally related cells underwent diversification by hypermutation and gene conversion. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. The attractive idea that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life, is challenged by the current finding. Although a major population of B lymphocytes may be generated early in life, diversified extensively, and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. Although considerable repertoire diversity is generated in young rabbits, mechanisms for continued generation of B cell receptor diversity are retained in adult life, where they may confer survival advantage.

Amino Acid Sequence↗
PubMed1991

[Congenital developmental defects in children and environmental pollution].

In one of the southern regions of the Ukraine, analysis was made over the recent three years of the genetic monitoring data on the incidence and structure of congenital developmental abnormalities in the newborn as related to the status of environmental pollution (the content of dust, carbon black, carbon and nitrogen oxides, nitrogen and sulfur dioxides, hydrogen sulfide, formaldehyde, phenol, fluorine compounds in the atmospheric air; the content of toxic chemicals and pesticides in foods, soil, water and feeds) as well as to the radiation situation. In the region under study, areas with varying degree of environmental pollution were distinguished. That was mainly done in terms of the characteristics of the atmospheric air and (to a less degree) varying annual dose of external radiation on conversion to one inhabitant. Analysis of the genetic monitoring has demonstrated the prevalence of congenital abnormalities, in the genesis of which the mutation component was of paramount importance, to be significantly higher in an ecologically unsafe area. The data obtained can be used in the regional programs aimed at the improvement of the environment, a reduction of the influence of exogenous risk factors implicated in the formation of congenital developmental abnormalities in children.

Abnormalities, Drug-Induced↗
PubMed2004

Gene cloning, expression and functional characterization of a phosphopantetheinyl transferase from Vibrio anguillarum serotype O1.

Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism. Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases). Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V. anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid. In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V. anguillarum serotype O1. A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase. angD was overexpressed in E. coli and the resultant protein, AngD, was purified. Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E. coli and purified. The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture. Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E. coli, modifying PCP and ArCP completely.

Amino Acid Motifs↗
PubMed1989

Chromatographic and spectroscopic properties of hemiacetals of aflatoxin and sterigmatocystin metabolites.

Improved fluorescence detection of aflatoxin B1 by chromatographic analysis is accomplished by conversion to the corresponding hemiacetal, aflatoxin B2a. Because the metabolites aflatoxin M1, aflatoxin P1, aflatoxin Q1, sterigmatocystin, and O-methylsterigmatocystin have the same molecular conversion site, we investigated the chromatographic and spectroscopic properties of hemiacetals of these compounds to assist in confirming aflatoxins and sterigmatocystins in human urine. Nuclear magnetic resonance and infrared absorbance were used to confirm the hemiacetal structure for aflatoxin B1 and sterigmatocystin. The ultraviolet absorbance, fluorescence, and chromatographic properties of the metabolites were investigated. Using these data, we optimized the detection and solvent conditions for high-performance liquid chromatography. We determined that, of the conditions studied, maximum sensitivity and resolution for the native aflatoxins were achieved with a mobile phase of methanol, tetrahydrofuran, and water, a C8 column in series with a C18 column, and fluorescence detection with 365 nm excitation and 430 nm emission wavelengths for aflatoxins B1 and M1 and with 500 nm emission wavelength for aflatoxins P1 and Q1. For the analysis of the hemiacetals, a mobile phase of methanol, acetonitrile, and water provided better chromatography and fluorescence detection. Sterigmatocystin and O-methylsterigmatocystin were readily converted to the hemiacetal forms, which, like the aflatoxins, were more polar and, therefore, earlier eluting by reversed-phase HPLC (methanol, acetonitrile, and water, 236 nm absorbance). These data are important to maximize the sensitivity and confidence for detecting the mycotoxin metabolites in biological specimens.

Acetals↗
PubMed1980

Study of the kinetics of the thyroid hormones and the peripheral conversion rate of thyroxine to triiodothyronine in acute renal insufficiency under experimental conditions.

We studied the kinetic behavior of triiodothyronine (T3) and thyroxine (T4) in two groups of 10 sheep. One was used as the control group (I) and the other consisted of sheep with acute renal failure (A.R.F.) induced by an iv. injection of mercuric dichloride. Five sheep of each group were injected with 125IT3 and the remaining five with 125IT4, later taking periodical extractions throughout the first 96 hours. The radioactive contaminants in the plasma were eliminated by extracting the hormones through immunoextraction. The mathematical calculation of the kinetics was based on the open two-compartment model. The A.R.F. caused a decrease in the disposition constant of the slow phase in the T3 kinetics, while at the same time prolonging the plasma half-life of the phase. The steady state distribution volume increases and the distribution constant K21 decreases in the group suffering from renal insufficiency. As regards the T4, all the kinetic parameters were altered with the exception of the distribution volumes, the most significant changes being the shortening of the plasma half-life in the end phases, the rise of the elimination constant K13 and the increase in the plasma clearance and thyroxine turnover rates. The analysis of the peripheral conversion rate revealed with a great amount of variation, that there is evidence of the conversion of T4 to T3 in the healthy group while this phenomenon remains undetected in the sick animals.

Acute Kidney Injury↗
PubMed2006

Polymorphic variation in the 11beta-hydroxylase gene associates with reduced 11-hydroxylase efficiency.

The -344 C/T and intron 2 conversion variants in the CYP11B2 gene, encoding aldosterone synthase, have been associated with markers of impaired 11beta-hydroxylase activity. We hypothesize that this association is because of variations in the adjacent 11beta-hydroxylase gene (CYP11B1) and arises through linkage disequilibrium between CYP11B1 and CYP11B2. The pattern of variation across the entire CYP11B locus was determined by sequencing 26 normotensive subjects stratified by and homozygous for the -344 and intron conversion variants. Eighty-three variants associated with -344 and intron conversion were identified. Haplotype analysis revealed 4 common haplotypes, accounting for 68% of chromosomes, confirming strong linkage disequilibrium across the region. Two novel CYP11B1 polymorphisms upstream of the coding region (-1889 G/T and -1859 A/G) were identified as contributing to the common haplotypes. Given the potential for such mutations to affect transcriptional regulation of CYP11B1, these were analyzed further. A total of 512 hypertensive subjects from the British Genetics of Hypertension Study population were genotyped for these polymorphisms. A significant association was identified between the -1889 polymorphism and urinary tetrahydrodeoxycortisol/total cortisol metabolite ratio, indicating reduced 11beta-hydroxylase efficiency. A similar pattern was observed for the -1859 polymorphism, but this did not achieve statistical significance. Functional studies in vitro using luciferase reporter gene constructs show that these polymorphisms significantly alter the transcriptional response of CYP11B1 to stimulation by adrenocorticotropic hormone or forskolin. This study strongly suggests that the impaired 11beta-hydroxylase efficiency associated previously with the CYP11B2 -344 and intron conversion variants is because of linkage with these newly identified polymorphisms in CYP11B1.

Adrenal Cortex Hormones↗
PubMed2007

The conversion of chicken manure to biooil by fast pyrolysis II. Analysis of chicken manure, biooils, and char by curie-point pyrolysis-gas chromatography/mass spectrometry (Cp Py-GC/MS).

The initial chicken manure and the three fractions derived from it by fast pyrolysis, that is, the two biooils Fractions I and II as well as the residual char were analyzed by Curie-point pyrolysis-gas chromatography/mass spectrometry (Cp Py-GC/MS). The individual compounds identified were grouped into the following six compound classes: (a) N-heterocyclics; (b) substituted furans; (c) phenol and substituted phenols; (d) benzene and substituted benzenes; (e) carbocyclics; and (f) aliphatics. Of special interest were the relatively high concentrations of N-heterocyclics in biooil Fraction II which was obtained in the highest yield and had the highest calorific value. Prominent N-heterocyclics in biooil Fraction II were methyl-and ethyl-substituted pyrroles, pyridines, pyrimidine, pyrazines, and pteridine. Also noteworthy was the high abundance of aliphatics in biooil Fraction I and the char. The alkanes and alkenes in biooil Fraction I ranged from n-C7 to n-C18 and C7:1 to C18:1, respectively, and those in the char from n-C7 to n-C19 and C7:1 to C19:1, respectively. The N-heterocyclics in the two biooil Fractions came from the chicken manure, from proteinaceous materials during fast pyrolysis or were formed during the fast pyrolysis manure conversion by the Maillard reaction which involved the formation of N-heterocyclics by amino acids interacting with sugars.

Animals↗
PubMed2003

cis-9, trans-11 CLA derived endogenously from trans-11 18:1 reduces cancer risk in rats.

The present study was designed to examine the effects of increasing dietary levels of vaccenic acid (VA) and cis-9, trans-11 conjugated linoleic acid (CLA) on chemically induced mammary carcinogenesis in rats. Both fatty acids were provided as a natural component in butter fat. The conversion of VA to CLA by delta9-desaturase was documented previously in several species, including rats and humans. Specifically, our objective was to determine the relative contribution of dietary VA and CLA to the tissue concentration of CLA and its ability to inhibit the development of mammary carcinomas. A total of 7 diets were formulated with varying levels of CLA and VA. The overall dietary treatment scheme was designed to evaluate the modulation of mammary cancer risk by 1). small increases of CLA in the presence of a low level of VA and 2). more substantial increases of VA against a background of low levels of CLA. As expected, small increases in dietary CLA at the low end of the CLA dose-response range did not reduce tumorigenesis. In contrast, there was a distinct and marked inhibitory response to VA that was dose dependent. The effect of VA was magnified in this experiment because the dose range of VA tested was much broader than that of CLA. Fatty acid analysis showed that the conversion of dietary VA to CLA resulted in a dose-dependent increase in the accumulation of CLA in the mammary fat pad, which was accompanied by a parallel decrease in tumor formation in the mammary gland. The finding confirms that the conversion of VA to CLA is as important for cancer prevention as the dietary supply of CLA. Thus, VA is also anticarcinogenic, and VA and CLA represent functional food components that are present in ruminant fat.

Animals↗
PubMed2006

The common marmoset (Callithrix jacchus) has two very similar semenogelin genes as the result of gene conversion.

The semen coagulum proteins have undergone substantial structural changes during evolution. In primates, these seminal vesicle-secreted proteins are known as semenogelin I (SEMG1) and semenogelin II (SEMG2). Previous studies on the common marmoset (Callithrix jacchus) showed that ejaculated semen from this New World monkey contains semenogelin, but it remained unclear whether it carries both genes or only SEMG1 and no SEMG2, like the closely related cotton-top tamarin (Saguinus oedipus). In this study we show that there are two genes, both expressed in the seminal vesicles. Surprisingly, the genes show an almost perfect sequence identity in a region of 1.25 kb, encompassing nearly half of the genes and containing exon 1, intron 1, and the first 0.9 kb of exon 2. The underlying molecular mechanism is most likely gene conversion, and a phylogenetic analysis suggests that SEMG1 is the most probable donor gene. The marmoset SEMG1 in this report differs from a previously reported cDNA by a lack of nucleotides encoding one repeat of 60 amino acids, suggesting that marmoset SEMG1 displays allelic size variation. This is similar to what was recently demonstrated in humans, but in marmosets the polymorphism was generated by a repeat duplication, whereas in humans it was a deletion. Together, these studies shed new light on the evolution of semenogelins and the mechanisms that have generated the structural diversity of semen coagulum proteins.

Amino Acid Sequence↗
PubMed2006

An improved analytical method for the determination of urea nitrogen isotopomers in biological samples utilizing continuous flow isotope ratio mass spectrometry.

Over the past few years numerous dual inlet isotope ratio mass spectrometry (IRMS) applications have been adapted to continuous flow systems which allow the automation of sample admission and a higher throughput. The isotopomer analysis of urea nitrogen by IRMS requires the offline conversion of urea into nitrogen gas before analysis. The oxidation of urea with LiOBr results in the monomolecular degradation of urea, which preserves the identity of the parent urea molecule, and has to be conducted under vacuum to prevent contamination with atmospheric nitrogen. We have developed an offline system of urea degradation utilizing disposable Exetainers, in which atmospheric nitrogen is displaced by helium. Recovery of urea nitrogen was linear within the range of the standards tested (0 to 420 microg nitrogen) and standard curves for 15N15N-urea standards showed high coefficients of determination (R2 > 0.9998). A small portion of urea degrades in a non-monomolecular fashion and has been shown to depend on the concentration of urea in the sample. Long-term storage of prepared samples showed a decline in 15N15N enrichment, suggesting air contamination. However, samples were stable for 24 h, which allows for the analysis of large sample batches. Interest in urea metabolism, particularly in ruminant species, has increased recently due to the environmental implications of urea and nitrogen excretion by farm animals. This novel analytical method will allow for accurate measurements and the rapid throughput needed in order to support these field studies.

Animals↗
PubMed1979

U.S. Public Health Service Cooperative trial of three rifampin-isoniazid regimens in treatment of pulmonary tuberculosis.

A total of 822 patients with newly diagnosed pulmonary tuberculosis were assigned randomly to one of 3 daily rifampin-isoniazid (RIF-INH) regimens: 450, 600, or 750 mg of RIF in combination with 300 mg of INH. After an initial 20 weeks of therapy with RIF-INH, patients recieved 300 mg of INH and 15 mg of ethambutol (EMB) per kg of body weight for either 12 or 18 months after their sputum cultures became negative. The rate of bacteriologic conversion of sputum among the 3 RIF-INH regimens was compared for 552 patients who completed the 20 weeks of RIF-INH therapy. Apporximately 60 per cent of these patients also completed their assigned INH-EMB therapy and were examined for relapse for at least one year after therapy was stopped. There was no significant difference in the rate of sputum conversion or rate of relapse between the group of patients who received 600 mg of RIF and those who received 750 mg of RIF. However, the 450-mg RIF regimen was significantly less effective than the other 2 regimens, as manifested by a lower rate of sputum conversion and a higher rate of treatment failures. Further analysis showed that RIF dosages of less than 9 mg per kg of body weight per day may be inadequate for treatment of pulmonary tuberculosis. The acceptability of these regimens was high, and the incidence of adverse reactions requiring discontinuation of RIF-INH therapy was quite low (3.3 per cent). A large proportion of patients (44 per cent) developed increased concentrations of transaminase during therapy with RIF-INH. These abnormalities were usually transient and, in most cases, of no clinical significance. In the relapse analysis, 12 months of chemotherapy after sputum conversion was shown to be as effective as 18 months of therapy after conversion of these RIF-containing regimens.

Adolescent↗
PubMed2001

Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography.

BACKGROUND: R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365 through 366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. MATERIALS AND METHODS: DNA samples containing either the known c.365G>A or c.365 through 366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC) assay. RESULTS: DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C). CONCLUSIONS: A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.

Amino Acid Substitution↗
PubMed2003

Recent advances in capillary electrophoresis and capillary electrochromatography of peptides.

An overview of the recent developments in the applications of high-performance capillary electromigration methods, namely zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography, to analysis, preparation, and physicochemical characterization of peptides is presented. New approaches to the theoretical description and experimental verification of the electromigration behavior of peptides and the methodological aspects of capillary electroseparations of peptides, such as rational selection of separation conditions, sample treatment, and suppression of adsorption, are discussed, and new developments in individual separation modes and new designs of detection systems applied to peptide separations are shown. Several types of applications of capillary electromigration methods to peptide analysis are presented: quality control and purity tests, determination in biomatrices, monitoring of physical and chemical changes and enzymatic conversions, amino acid and sequence analysis, and peptide mapping. The examples of micropreparative peptide separations are given and capabilities of capillary electromigration techniques to provide important physicochemical characteristics of peptides are demonstrated.

Animals↗
PubMed1992

Conversion of DNA adducts of antitumour cis-diamminedichloroplatinum(II). Immunochemical analysis.

Polyclonal antibodies that bind selectively to DNA modified by antitumour cisplatin and its analogues were isolated. The reactivity of the antibodies with the epitope was enhanced by thermal denaturation of DNA that had been modified by cisplatin before its denaturation. On the other hand, denaturation of DNA before its modification resulted in considerably less reaction of the antibodies. The conversion of monofunctional cisplatin-DNA adducts to bifunctional lesions increased the capability of the modified DNA to competitively inhibit the antibodies. The double-helical oligonucleotides containing a unique bifunctional adduct formed by cisplatin at the d(GG) site cross-reacted with the antibodies in contrast to the oligonucleotide containing a single monofunctional adduct formed at the d(G) site. In addition, poly(dG-dC) . poly(dG-dC) modified by cisplatin did not react with the antibodies. It was concluded that the antibodies recognized monodentate lesions, intrastrand cross-links between two purine nucleosides separated by one or more nucleosides and interstrand cross-links negligibly. The antibodies apparently recognized a chemical nature of the bifunctional adduct formed between two adjacent purines and not an unusual conformational feature of DNA resulting from the formation of this adduct. The antibodies were used to analyse the adducts formed by cisplatin on DNA of cultured cells exposed to this drug. During the subsequent incubation of the already exposed cells in the drug-free medium, a part of the bifunctional adducts of cisplatin was completely removed from DNA or transformed to the adducts not recognized by the antibodies.

Animals↗
PubMed1998

Placebo-controlled, double-blind, randomized study of aerosolized tobramycin for early treatment of Pseudomonas aeruginosa colonization in cystic fibrosis.

In chronic Pseudomonas aeruginosa pulmonary infection of patients with cystic fibrosis (CF), antibiotic therapy generally fails to eradicate the bacterial pathogen. The mucoid bacterial phenotype, high sputum production by the host, and low airway levels of antibiotics seem to be responsible for the observed decrease in antibiotic efficacy. We hypothesized that early antibiotic treatment by inhalation in CF patients may be able to prevent or at least delay airway infection. In a prospective placebo-controlled, double-blind, randomized multicenter study, 22 CF patients received either 80 mg b.i.d. of aerosolized tobramycin or placebo for a period of 12 months shortly after the onset of P. aeruginosa pulmonary colonization. Two patients in the tobramycin and six patients in the placebo group stopped inhalation before the 12 month treatment period. Using life table analysis, the time to conversion from a P. aeruginosa-positive to a P. aeruginosa-negative respiratory culture was significantly shorter in the tobramycin-treated group than in the placebo group (P < 0.05, log rank test). Lung function parameters and markers of inflammation did not change in either group during treatment. The results of this study suggest that early tobramycin inhalation may prevent and/or delay P. aeruginosa pulmonary infection in CF patients.

Administration, Inhalation↗
PubMed1984

Complete nucleotide sequence of a functional class I HLA gene, HLA-A3: implications for the evolution of HLA genes.

The complete nucleotide sequence of an active class I HLA gene, HLA-A3, has been determined. This sequence, together with that obtained for the HLA-CW3 gene, represents the first complete nucleotide sequence to be determined for functional class I HLA genes. The gene organisation of HLA-A3 closely resembles that of class I H-2 genes in mouse: it shows a signal exon, three exons encoding the three extracellular domains, one exon encoding the transmembrane region and three exons encoding the cytoplasmic domain. The complete nucleotide sequences of the active HLA genes, HLA-A3 and HLA-CW3, now permit a meaningful comparison of the nucleotide sequences of class I HLA genes by alignment with the sequence established for a HLA-B7-specific cDNA clone and the sequences of two HLA class I pseudogenes HLA 12.4 and LN- 11A . The comparisons show that there is a non-random pattern of nucleotide differences in both exonic and intronic regions featuring segmental homologies over short regions, which is indicative of a gene conversion mechanism. In addition, analysis of the frequency of nucleotide substitution at the three base positions within the codons of the functional genes HLA-A3, HLA-B7 and HLA-CW3 shows that the pattern of nucleotide substitution in the exon coding for the 3rd extracellular domain is consistent with strong selection pressure to conserve the sequence. The distribution of nucleotide variation in the other exons specifying the mature protein is nearly random with respect to the frequencies of substitution at the three nucleotide positions of their codons. The evolutionary implications of these findings are discussed.

Amino Acid Sequence↗
PubMed1988

Desmethyldiazepam pharmacokinetics: studies following intravenous and oral desmethyldiazepam, oral clorazepate, and intravenous diazepam.

After single 10-mg intravenous (IV) doses of desmethyldiazepam (DMDZ) to 12 healthy human volunteers, (mean age, 62 years) blood samples were obtained over the next 14 or more days. Mean kinetic variables were volume of distribution (Vd), 90 liters; elimination half-life (t1/2), 93 hours; and clearance, 12.3 mL/min. Vd was significantly correlated with body weight (r = .73, P less than .01) and with percent ideal body weight (r = .91, P less than .001). Eleven of the same subjects also received 5- to 15-mg doses of IV diazepam (DZ). Mean kinetic variables were Vd, 180 liters; t1/2, 83 hours; and clearance, 28 mL/min. Clearances of DZ and DMDZ were significantly correlated (r = .73, P less than .02). Based on area analysis, the extent of conversion of DZ to systemic DMDZ averaged 53%. After oral administration of DMDZ in tablet form (10 mg), or of clorazepate dipotassium in capsule form (15 mg), systemic availability of DMDZ from each of the oral dosage forms was not significantly different from 100%.

Administration, Oral↗