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PubMed1999

Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols.

The aim of this project was to devise and test improved protocols of the 32P-postlabelling assay for the detection of carcinogen-DNA adducts. The intention was to reverse the drift of different investigators using increasingly divergent experimental conditions. This would lead to a more standardized assay that can be used in future applications by different investigators for the monitoring of human exposure to genotoxic agents, permitting more meaningful comparisons between different studies or between different participants in the same study. As part of this process, there was perceived to be a need for carcinogen-modified DNA standards of known levels of adducts for use as positive controls, as standards for normalization of results with unknown samples and to assist interlaboratory comparisons. The preparation of characterized DNA standards modified by benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), 4-aminobiphenyl (ABP), an aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine, and N-methyl-N-nitrosourea (MNU), a methylating agent yielding DNA containing O6-methylguanine, was carried out. A critical appraisal of all aspects of the 32P-postlabelling procedure and investigations to examine the influence of a number of key variations on the assay were conducted. There followed testing of a consensus protocol in a first interlaboratory trial involving 25 participants in Europe and the USA, conducted on the prepared synthetic DNA standards, the assessment of interlaboratory variability and the reasons for it. Revision of the protocols was followed by further testing in a second interlaboratory trial in which liver DNA from mice treated with BaP or ABP were assayed together with the synthetic DNA standards. Adduct levels were found to be significantly lower by 32P-postlabelling than by 3H incorporation. A recommended set of procedures has been developed for the detection and quantitation of DNA adducts formed by PAHs, aromatic amines and methylating agents. These trials have led to a much clearer idea as to what are the critical features and procedures of the 32P-postlabelling assay and there is a set of standard DNA samples for use in quality control and against which biological samples can be normalized. Use of these standards and procedures has reduced interlaboratory variability in quantitation of DNA adducts.

DNA Adducts↗

PubMed2003

Differences between lovastatin and simvastatin hydrolysis in healthy male and female volunteers:gut hydrolysis of lovastatin is twice that of simvastatin.

The aim of this pharmacokinetic evaluation was to show the effect of the extra methyl group in simvastatin on esterase hydrolysis between lovastatin and simvastatin in male and female volunteers. This study was based on the plasma concentration-time curves and the pharmacokinetics of lovastatin and simvastatin with its respective active metabolite statin-beta-hydroxy acid obtained from two different bioequivalence studies, each with 18 females and 18 males. Results were: The group of female volunteers showed a higher yield of the active metabolite beta-hydroxy acid than the group of males (p < 0.002) for both lovastatin and simvastatin. This difference was not related to the body weight of both groups. In the male/female groups, subject-dependent yield of active metabolite beta-hydroxy acid was demonstrated, which was independent of the formulation. The variation in plasma/liver hydrolysis resulted in a fan-shaped distribution of data points when the AUCt lovastatin was plotted vs. that of the beta-hydroxy acid metabolite. In the fan of data points, subgroups could be distinguished, each showing a different regression line and with a different Y-intercept (AUCtbeta-hydroxy acid). Lovastatin hydrolysis was higher than simvastatin hydrolysis. It was possible to discriminate between hydrolysis of both lovastatin and simvastatin by plasma/liver or tissue esterase activity. The three subgroups of subjects (males/females) showing different but high yield of statin beta-hydroxy acid can be explained by variable hydrolysis of plasma and hepatic microsomal and cytosolic carboxyesterase activity. This study showed clearly that despite the subject-dependent hydrolysis of lovastatin/simvastatin to the active metabolite, males tend to hydrolyse less than females. The extra methyl group in simvastatin results in less hydrolysis due to steric hindrance.

Adult↗

PubMed2003

O6-methylguanine-DNA methyltransferase activity and promoter methylation status in pediatric rhabdomyosarcoma.

OBJECTIVES: To determine the activity of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) and MGMT promoter methylation status of pediatric rhabdomyosarcoma (RMS) and examine MGMT in RMS tumors from different prognostic groups. METHODS: Fifteen samples each of the alveolar (ARMS) and embryonal (ERMS) subtypes were obtained for analysis of MGMT activity and promoter methylation status. MGMT activity was assayed by measuring the removal of O6-[3H] methylguanine from [3H]-methylated substrate by a tumor extract containing the enzyme. Promoter methylation status was examined using methylation-specific polymerase chain reaction (PCR). RESULTS: MGMT activity was successfully assayed from 25 samples, 10 ERMS and 15 ARMS. All ERMS and 11 of the 15 ARMS samples displayed high activity levels. There was significant intertumor variability among both subtypes but no significant difference in mean activity between the two histologic groups. There were trends toward increased activity in ERMS tumors and tumors from anatomically unfavorable locations. Only one tumor was hypermethylated at the MGMT promoter region. CONCLUSIONS: This analysis suggests that a low percentage of RMS samples are hypermethylated at the MGMT promoter and that most have significant MGMT activity, implying that clinical trials with MGMT-modulating agents may have a role in the treatment of these tumors. This analysis does not support MGMT activity as an explanation of the differential response to chemotherapy demonstrated by ARMS and ERMS, but does suggest that MGMT may be involved in RMS treatment failure regardless of subtype and in the poorer response shown by tumors from unfavorable locations.

Biopsy↗

PubMed2000

Sequencing of anti-thyroxine monoclonal antibody fab fragment by ion trap mass spectrometry.

A comprehensive mass spectrometric strategy is described for the sequencing of anti-thyroxine monoclonal antibody Fab region (48 000 Da). After reduction and S-carboxymethylation of the Fab, the modified light chain and Fd fragment were separated and subjected to multiple proteolytic digestions. The resulting digests were characterized by on-line microbore liquid chromatography/electrospray ionization ion trap mass spectrometry. Database search against published immunoglobulins (IgGs) allowed identification of all the peptides in constant domains. The homologous framework residues in the IgGs were utilized as 'sequence maps' for the sequence determination of variable domains. S-Carboxymethylation with an isotopic-enriched moiety greatly facilitated the recognition and data elucidation of cysteinyl peptides through the unique isotopic distribution patterns specific to the modified peptides. Methylation of peptide mixtures provided additional information for the interpretation of MS/MS spectra, allowing easy differentiation of Asp/Asn and Gln/Glu pairs. This study clearly demonstrates the power of mass spectrometry for the sequencing of antibodies without knowing the corresponding DNA sequences.

Amino Acid Sequence↗

PubMed2005

Physical controls on total and methylmercury concentrations in streams and lakes of the northeastern USA.

The physical factors controlling total mercury (HgT) and methylmercury (MeHg) concentrations in lakes and streams of northeastern USA were assessed in a regional data set containing 693 HgT and 385 corresponding MeHg concentrations in surface waters. Multiple regression models using watershed characteristics and climatic variables explained 38% or less of the variance in HgT and MeHg. Land cover percentages and soil permeability generally provided modest predictive power. Percent wetlands alone explained 19% of the variance in MeHg in streams at low-flow, and it was the only significant (p < 0.02) predictor for MeHg in lakes, albeit explaining only 7% of the variance. When stream discharge was added as a variable it became the dominant predictor for HgT in streams, improving the model r2 from 0.19 to 0.38. Stream discharge improved the MeHg model more modestly, from r2 of 0.25 to 0.33. Methylation efficiency (MeHg/HgT) was modeled well (r2 of 0.78) when a seasonal term was incorporated (sine wave with annual period). Physical models explained 18% of the variance in fish Hg concentrations in 134 lakes and 55% in 20 reservoirs. Our results highlight the important role of seasonality and short-term hydrologic changes to the delivery of Hg to water bodies.

Animals↗

PubMed1978

Therapeutic abortion in late first trimester. Prostaglandin pretreatment compared with primary surgery.

One hundred randomly selected nulliparous women, 10-11 weeks pregnant, were submitted to either primary surgery (50 cases) or pretreatment overnight with vaginal suppositories of 15(S)15 methyl prostaglandin F2 alpha methyl ester (50 cases) followed by vacuum aspiration. No peroperative complications were recorded in either of the two groups. Thirtyfive of the 50 pretreated women aborted during the pretreatment period and a variable cervical dilatation was observed in the remaining 15 women of this group. The incidence of early postoperative pelvic infection tended to be less in the pretreated group as compared with the other group.

Abortifacient Agents, Nonsteroidal↗

PubMed2004

Experimental design-based development of a rapid capillary electrophoresis method for determining impurities in the tetrapeptide H-Tyr-(D)Arg-Phe-Phe-NH2.

A capillary electrophoresis (CE) method has been developed and validated for separating the tetrapeptide H-Tyr-(D)Arg-Phe-Phe-NH2 and nine related substances. The method was developed using experimental design in a four-step procedure, in which eight variables were investigated in a total of 47 experiments. The preferred background electrolyte (BGE) consisted of 0.1M malonic acid at pH 2.5 with 7 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin (2,6-DM-beta-CD). The separation of H-Tyr-(D)Arg-Phe-Phe-NH2 and the related substances was accomplished within 15 min, with a resolution greater than 1.5 between all peaks. The method was then investigated with respect to its selectivity, linearity, precision, detection limit (LOD) and quantitation limit (LOQ). In addition, a system suitability test was performed and response factors were determined, essentially following International Conference of Harmonization guidelines for the validation of analytical methods. LOD and LOQ for the related substance H-Arg-Phe-NH2 were found to be 0.3 and 0.8 microg/ml, respectively, at a target H-Tyr-(D)Arg-Phe-Phe-NH concentration of 1mg/ml. The method performed well with respect to all of the validation parameters.

Electrophoresis, Capillary↗

PubMed1997

Hemodynamic effects of the intravenous administration of cyclovirobuxine D [correction of cyclorirobuxine D] in anesthetized pigs.

The present study was undertaken in anesthetized pigs to determine the primary effects of cyclovirobuxine D [corrected] given intravenously on hemodynamic variables. In eight pigs, the administration of 1.5 mg/kg of cyclovirobuxine D [corrected] caused a small increase in aortic blood pressure. When this response was prevented, a decrease in heart rate was obtained in each of the eight pigs. When this response was also prevented, an increase in the maximum rate of change of left ventricular systolic pressure (left ventricular dP/dtmax) was observed. In four pigs, the decrease in heart rate and the increase in left ventricular dP/dtmax were progressively augmented by graded increases in the dose of cyclovirobuxine D [corrected]. In six pigs, the responses of hemodynamic variables to cyclovirobuxine D [corrected] were not affected by blockade of cholinergic and adrenergic receptors. In a further six pigs, blockade of nitric oxide synthase with N omega-nitro-L-arginine methyl ester did not affect the decrease in heart rate caused by the drug, but abolished the increases in left ventricular dP/dtmax and aortic blood pressure. The present study showed that intravenous administration of cyclovirobuxine D [corrected] primarily caused a decrease in heart rate and an increase in left ventricular inotropic state, which secondarily determined an increase in aortic blood pressure, and suggested that the response of heart rate involved a direct effect of the drug on the heart, while the response of left ventricular contractility was related to mechanisms dependent on the release of nitric oxide.

Anesthesia↗

PubMed2002

Optimisation of biodiesel production by sunflower oil transesterification.

In this work the transformation process of sunflower oil in order to obtain biodiesel by means of transesterification was studied. Taguchi's methodology was chosen for the optimisation of the most important variables (temperature conditions, reactants proportion and methods of purification), with the purpose of obtaining a high quality biodiesel that fulfils the European pre-legislation with the maximum process yield. Finally, sunflower methyl esters were characterised to test their properties as fuels in diesel engines, such as viscosity, flash point, cold filter plugging point and acid value. Results showed that biodiesel obtained under the optimum conditions is an excellent substitute for fossil fuels.

Biotechnology↗

PubMed1979

Nuclear magnetic resonance studies on the tertiary folding of transfer ribonucleic acid: assignment of the 7-methylguanosine resonance.

Analysis of the low-field nuclear magnetic resonance (NMR) spectra of several class 1 D4V5 transfer ribonucleic acid (tRNA) species containing 7-methylguanosine in their variable loops reveals a set of six to seven tertiary base pair resonances, one of which is always located at ca. --13.4 ppm. Other tRNA species which do not contain 7-methyl-guanosine do not contain the tertiary resonance at --13.4 ppm. Chemical removal of 7-methylguanosine from several tRNAs containing the same dihydrouridine (DHU) helix sequence as yeast tRNAPhe results in the loss of the --13.4-ppm tertiary resonance. In the initiator methionine tRNA, which contains a different DHU helix sequence, the 7-methylguanosine hydrogen bond has been assigned at --14.55 ppm by chemical removal of this residue. In these experiments the aromatic C8H proton of 7-methylguanosine was also assigned (--9.1 ppm). The unexpectedly low-field position of the 7-methylguanosine resonance is explained by the deshielding effect of the delocalized positive charge in this nucleoside.

Base Composition↗

PubMed2006

A model iridium hydroformylation system with the large bite angle ligand xantphos: reactivity with parahydrogen and implications for hydroformylation catalysis.

Iridium complexes containing the large bite angle bisphosphine ligand xantphos have been synthesized and their reactivity studied. Several of these complexes are the first reported Ir(xantphos) systems to be characterized by X-ray diffraction. Variable-temperature NMR spectroscopic studies of IrI(CO)2(xantphos) (1-I) and Ir(COEt)(CO)2(xantphos) (8) show two separate dynamic processes in which the phosphorus donors and the backbone methyl groups of the xantphos ligand are exchanged. The addition of parahydrogen (p-H2) to 1-I leads to the formation of two dihydride isomers including one in which both hydride ligands are trans to the phosphorus donors, suggestive of an Ir(I) xantphos intermediate with the ligand chelated in a trans-spanning fashion (2b). The bromide and chloride Ir(I) analogues (1-Br and 1-Cl) also form this isomer upon reaction with parahydrogen, with 1-Cl yielding only this dihydride species. The trihydride complex IrH3(CO)(xantphos) (7) has been prepared, and its exchange with free hydrogen at elevated temperature is confirmed by reaction with p-H2. The hydride complexes IrH(CO)2(xantphos) (6) and IrH3(CO)(xantphos) (7), as well as the propionyl complex 8, are modest catalysts for the hydroformylation of 1-hexene and styrene under mild conditions. The addition of p-H2 to 8 permits direct observation of the propionyl dihydride species IrH2(COEt)(CO)(xantphos) (9) under both thermal and photolytic conditions, as well as unusual but weak polarization of the aldehydic proton of the propanal product that forms upon reductive elimination from 9.

Alkylation↗

PubMed1999

Role of nitric oxide in pathogenesis underlying ischemic cerebral damage.

1. Based upon the intriguing report that nitric oxide synthase (NOS) inhibitor dose-dependently reverses N-methyl-D-aspartate (NMDA)-induced neurotoxicity observed in primary cortical cell cultures, many laboratories have investigated whether NOS inhibition is beneficial as a treatment for cerebral ischemia. 2. Although the results are variable, it is likely thought that nitric oxide plays a key role in pathomechanism underlying ischemic brain damage. 3. We review the experimental studies on effects of NOS inhibition on cerebral ischemia and measuring nitric oxide produced in the brain subjected to cerebral ischemia. 4. Finally, the possibility of NOS inhibitors as a therapeutical tool is discussed.

Animals↗

PubMed1980

Reduced sensitivity of cholinesterase as a factor of resistance in leptophos selected strain in the Egyptian cotton leafworm.

Larvae of a leptophos resistant strain of the Egyptian cotton leafworm Spodoptera littoralis (Boisd) showed varying levels of cross resistance to several organophosphorus insecticides and to the carbamate matacil. Enzymatic determinations revealed that levels of cholinesterase activity were almost identical in larvae of susceptible and resistant strains. Resistant larvae, which remained susceptible to the carbamate methomyl possessed cholinesterase of nearly normal sensitivity to this compound. On the other hand, there was a considerable degree of insensitivity of cholinesterase to inhibition by leptophos-oxon, methyl paraoxon, cyolane, cytrolane, monocrotophos, and matacil in larvae of the resistant strain. It was concluded that reduced sensitivity of cholinesterase enzyme could be considered as an important factor which complements other variables contributing to the mechanism of resistance in larvae of the Egyptian cotton leafworm.

Animals↗

PubMed2006

Hypomethylation of MB-COMT promoter is a major risk factor for schizophrenia and bipolar disorder.

The variability in phenotypic presentations and the lack of consistency of genetic associations in mental illnesses remain a major challenge in molecular psychiatry. Recently, it has become increasingly clear that altered promoter DNA methylation could play a critical role in mediating differential regulation of genes and in facilitating short-term adaptation in response to the environment. Here, we report the investigation of the differential activity of membrane-bound catechol-O-methyltransferase (MB-COMT) due to altered promoter methylation and the nature of the contribution of COMT Val158Met polymorphism as risk factors for schizophrenia and bipolar disorder by analyzing 115 post-mortem brain samples from the frontal lobe. These studies are the first to reveal that the MB-COMT promoter DNA is frequently hypomethylated in schizophrenia and bipolar disorder patients, compared with the controls (methylation rate: 26 and 29 versus 60%; P=0.004 and 0.008, respectively), particularly in the left frontal lobes (methylation rate: 29 and 30 versus 81%; P=0.003 and 0.002, respectively). Quantitative gene-expression analyses showed a corresponding increase in transcript levels of MB-COMT in schizophrenia and bipolar disorder patients compared with the controls (P=0.02) with an accompanying inverse correlation between MB-COMT and DRD1 expression. Furthermore, there was a tendency for the enrichment of the Val allele of the COMT Val158Met polymorphism with MB-COMT hypomethylation in the patients. These findings suggest that MB-COMT over-expression due to promoter hypomethylation and/or hyperactive allele of COMT may increase dopamine degradation in the frontal lobe providing a molecular basis for the shared symptoms of schizophrenia and bipolar disorder.

Alcoholism↗

PubMed2004

Complex patterns of transcription at the insertion site of a retrotransposon in the mouse.

Here we report that transcriptional effects of the insertion of a retrotransposon can occur simultaneously both upstream and downstream of the insertion site. We have identified an intra-cisternal A particle (IAP) retrotransposon in intron 6 of a gene that we have named Cabp (CDK5 activator binding protein). The presence of the IAP is associated with an aberrant transcript initiating from a cryptic promoter in the IAP, reading out into the adjacent Cabp gene sequence. The expression of this transcript is highly variable among isogenic mice within the C57BL/6J strain and so Cabp(IAP) can be classified as a metastable epiallele. As expected, the presence or absence of the transcript correlates with differential DNA methylation of the 5' LTR of the IAP. More surprisingly, in mice where the retrotransposon is unmethylated and presumably transcriptionally active, we find a number of short Cabp transcripts which initiate at the normal 5' end of the gene but terminate prematurely, just 5' of the retrotransposon. This is the first report of a retrotransposon having both upstream and downstream effects on transcription at the site of insertion and it suggests that alternative polyadenylation may sometimes be caused by a downstream convergent transcription unit.

Alleles↗

PubMed2004

Stunning the neural nexus: mechanisms of general anesthesia.

General anesthesia requires managing a complex array of anesthetic agents that act on an intricate web of neural connections or neural nexus. Both inhaled and intravenous anesthetics must intervene at some level of the neural nexus that provides for amnesia, immobility, hypnosis, and suppression of noxious reflexes. These interactions occur at the spinal and supraspinal level and involve spinal pathways and centers of arousal and memory formation centrally. Current research does not support the notion of a unitary mechanism of action for general anesthetics, but rather that anesthetics act by altering neuronal ion channels and neural communication. In general, anesthetics act by either enhancing inhibitory transmission or blocking excitatory conduction in neural impulses. The potent inhaled agents and most intravenous agents enhance the inhibitory lambda-aminobutyric acid subtype A (GABAA) and glycine channels and depress the excitatory neuronal nicotinic acetylcholine (nnACh) receptors. Nitrous oxide and ketamine act primarily by depressing the excitatory N-methyl-D-aspartate (NMDA) receptors and enhancing the opioid mu receptors. The extent, distribution, and subunit composition of these receptors determine the effects of various anesthetic agents on an individual patient. This variability, both within the patient and among the mechanisms of action of anesthetics, provides a reasonable degree of flexibility to the clinical practice of anesthesia.

Anesthesia, General↗

PubMed1991

Methyl alcohol poisoning: an autopsy study of 28 cases.

Twenty eight fatal cases out of the 97 cases of methyl alcohol (MA) poisoning admitted to K. E. M. Hospital in August 1988 during a single outbreak were studied. The latent period between consumption of the drink and development of symptoms was variable (range 7 1/2 hours to 60 hours). CNS symptoms were predominant (75% of cases) and shock either at admission or as a late event was seen in 89% of the cases. Blurring of vision was seen in 42.8% and blindness in 10.7% of cases. Minimum fatal period was 7 1/2 hours and maximum was 12 days. Methanol levels in blood and viscera were variable (average 155.8 mg%, max. 420.4 mg%). In 4 cases, significant amount of MA was detected in stomach contents 5-12 days after consumption of MA, raising the question of resecretion of MA in stomach. Shrinkage and degeneration of neurons in the parietal cortex was seen in 85.7% of cases. Putamental degeneration and necrosis was seen in 7.14%. Haemorrhage (3.5%) and sponginess (7.14%) was observed in the optic chiasma. Severe renal tubular degeneration and patchy necrosis was seen in all cases. Fatty changes (67.8%) and microvesicular fat (42.5%) were seen in the hepatocytes.

Adult↗

PubMed1988

[Chromatographic separation of activated proteases from human plasma].

After separation of aceton and dextran sulfate activated human plasma by column chromatography on DEAE-cellulose three esterolytically and amidolytically active fractions, respectively, were obtained, which were assigned to the following species: plasma kallikrein (PK), PK.alpha-macroglobulin.HMW-Kininogen. Their percentage in the whole activity is variable. The proportion of free PK is low (0.11). For characterization of the products we studied inhibition by different polyvalent inhibitors. The Michaelis constant (Km) with p-toluene-sulfonyl-L-arginine methyl ester (TAME) were determined. For simulation of in vivo conditions dextran sulfate activated plasma was inactivated at 37 degrees C. The residual activity and the spontaneous activity in plasma from patients with shock are produced by different active protease inhibitor complexes.

Chromatography, DEAE-Cellulose↗