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PubMed1995

Molecular cloning and characterization of the nontypeable Haemophilus influenzae 2019 rfaE gene required for lipopolysaccharide biosynthesis.

The lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae (NTHi) is an important factor in pathogenesis and virulence. In an attempt to elucidate the genes involved in LOS biosynthesis, we have cloned the rfaE gene from NTHi 2019 by complementing a Salmonella typhimurium rfaE mutant strain with an NTHi 2019 plasmid library. The rfaE mutant synthesizes lipopolysaccharide (LPS) lacking heptose, and the rfaE gene is postulated to be involved in ADP-heptose synthesis. Retransformation with the plasmid containing 4 kb of NTHi DNA isolated from a reconstituted mutant into rfaE mutants gave wild-type LPS phenotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed the conversion of the rfaE mutant LPS to a wild-type LPS phenotype. Sequence analysis of a 2.4-kb BglII fragment revealed two open reading frames. One open reading frame encodes the RfaE protein with a molecular weight of 37.6 kDa, which was confirmed by in vitro transcription and translation, and the other encodes a polypeptide highly homologous to the Escherichia coli HtrB protein. These two genes are transcribed from the same promoter region into opposite directions. Primer extension analysis of the rfaE gene revealed a single transcription start site at 37 bp upstream of the predicted translation start site. The upstream promoter region contained a sequence (TA AAAT) homologous to the -10 region of the bacterial sigma 70-dependent promoters at an appropriate distance (7 bp), but not sequence resembling the consensus sequence of the -35 region was found. These studies demonstrate the ability to use complementation of defined LPS defects in members of the family Enterobacteriaceae to identify LOS synthesis genes in NTHi.

Amino Acid Sequence↗
PubMed2002

Genomic analysis of the murine odorant receptor MOR28 cluster: a possible role of gene conversion in maintaining the olfactory map.

Genomic analysis was performed for the murine odorant receptor (OR) genes. The MOR28 cluster on chromosome 14 was extensively studied. It contains six OR genes, MOR28, 10, 83, 29A, 29B and 30. The human homolog of this cluster is located on the human chromosome 14, and contains five OR genes, HOR28/10, 83, 29A, 29B and 30. Sequence comparison of these OR gene paralogs and orthologs suggests that the coding homologies are accounted for not only by recent gene duplication, but also by gene conversion among the coding sequences within the cluster. A possible role of gene conversion in the olfactory system is discussed in the context of the olfactory map.

Animals↗
PubMed2003

Bioinformatics support for high-throughput proteomics.

In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteome data. The rapid advancement of this technique in combination with other methods used in proteomics results in an increasing number of high-throughput projects. This leads to an increasing amount of data that needs to be archived and analyzed. To cope with the need for automated data conversion, storage, and analysis in the field of proteomics, the open source system ProDB was developed. The system handles data conversion from different mass spectrometer software, automates data analysis, and allows the annotation of MS spectra (e.g. assign gene names, store data on protein modifications). The system is based on an extensible relational database to store the mass spectra together with the experimental setup. It also provides a graphical user interface (GUI) for managing the experimental steps which led to the MS data. Furthermore, it allows the integration of genome and proteome data. Data from an ongoing experiment was used to compare manual and automated analysis. First tests showed that the automation resulted in a significant saving of time. Furthermore, the quality and interpretability of the results was improved in all cases.

Algorithms↗
PubMed2000

Standardization of capillary zone electropherograms obtained by using field enhanced sample stacking.

Migration times in a capillary zone electropherogram obtained by using the field enhanced sample stacking technique are strongly affected by the injected sample volume. That is, the migration times significantly decrease with the increase of the sample volume. To avoid inaccurate qualitative analysis due to the above phenomena, the time axis of the electropherograms was converted into an effective mobility axis using our conversion method taking account of the temperature increase in the separation tube and relaxation of the potential gradient of the separation field. After the conversion, accurate qualitative analysis was possible in spite of drastic change of the migration time, suggesting our conversion method could be successfully used for the standardization of electropherograms obtained even by using the stacking effect. The cause of the decrease of the migration time in the stacking process was briefly discussed.

Electrophoresis, Capillary↗
PubMedDate not supplied

Methadone conversion to EDDP during GC-MS analysis of urine samples.

During validation of a gas chromatography-mass spectrometry (GC-MS) method for the methadone metabolite 2-ethylidine-1,5dimethyl-3,3-diphenylpyrrolidine (EDDP), it was noted that detectable levels of EDDP were found during analysis of extracts from drug-free urine samples spiked with methadone. Different amounts of EDDP were detected by GC-MS during confirmation analysis; however, levels consistently exceeded 50 ng/mL at methadone concentrations > 10,000 ng/mL. Quantitation of EDDP was determined by the addition of EDDP-d3 to methadone-spiked urine samples. Subsequent analysis of methadone-spiked urine extracts by high-performance liquid chromatography (HPLC) indicated no EDDP as a result of contaminated standard or conversion during solid-phase extraction. Reducing the GC injector-port temperature from 260 degrees C to 180 degrees C reduced the observed EDDP concentration in one sample from 201 ng/mL to 53 ng/mL at the initial methadone concentration of 10,000 ng/mL. These results indicate GC injector-port temperature induces thermal conversion of methadone to EDDP as an artifact. When confirmation of methadone and EDDP is critical to determining individual compliance with maintenance programs, alternative chromatographic methods (e.g., capillary electrophoresis, HPLC, or liquid chromatography-mass spectrometry) should be considered.

Artifacts↗
PubMed2001

A robust multipoint linkage statistic (tlod) for mapping complex trait loci.

Classical parametric two-point linkage analysis is a powerful analysis tool, however there are clear disadvantages too, including the sensitivity to allele frequency mis-specification. Conversely, multipoint linkage analysis is not sensitive to allele frequency mis-specification, but it is sensitive to genetic model mis-specification. Göring and Terwilliger [Am J Hum Genet 66:1095-106, 2000] proposed a new robust multipoint statistic that increased the robustness of multipoint analyses. In this paper we have referred to this new statistic as the tlod. We applied this new statistic to the Genetic Analysis Workshop (GAW) 12 data using affected status (AFF) as the phenotype of interest. The heterogeneity tlod and two-point hlod scores correlated highly across the genome (p < 0.0001), as expected, but the het-tlod had a lower number false positives. In addition, the tlod analysis handled missing data better, as would be expected for a multipoint method. When one-third of the genotype data was removed (dead people) the tlod analysis was less affected than the two-point analysis. When tlod scores were compared with multipoint lod scores in true gene locations, the robustness of the tlod to model mis-specification was clearly evident. When the "best" replicate from the general population was analyzed, a borderline genome-wide significant two-point hlod result (3.6) was found 4 cM from MG6 and MG7 on chromosome 6. The heterogeneity tlod score was lower than the two-point hlod score (1.8), but greater than the heterogeneity multipoint lod score (0.4). However, when replicate 1 of the isolated population was analyzed none of the true gene locations were identified with either statistic.

Chromosome Mapping↗
PubMedDate not supplied

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H.

3beta-hydroxysteroid dehydrogenase 5-ene isomerase (3betaHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3betaHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K(m)=0.17+/-0. 03 and 1.02 microM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V(max) while K(m) was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K(i) was calculated from replot of Dixon's slope for each substrate concentration. K(i) from the intercept and the slope of this replot were similar (0.276+/-0.01 and 0.263+/-0.02 microM) and higher than the K(m) for DHEA. The K(m) and K(i) values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V(max) while K(m) values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.

Animals↗
PubMed2004

Conversion regular patterns of acetic acid, propionic acid and butyric acid in UASB reactor.

On the basis of continuous tests and batch tests, conversion regular patterns of acetate, propionate and butyrate in activated sludge at different heights of the UASB reactor were conducted. Results indicated that the conversion capacity of the microbe is decided by the substrate characteristic when sole VFA is used as the only substrate. But when mixed substrates are used, the conversion regulations would have changed accordingly. Relationships of different substrates vary according to their locations. In the whole reactor, propionate's conversion is restrained by acetate and butyrate of high concentration. On the top and at the bottom of the reactor, conversion of acetate, but butyrate, is restrained by propionate. And in the midst, acetate's conversion is accelerated by propionate while that of butyrate is restrained. It is proved, based on the analysis of specific conversion rate, that the space distribution of the microbe is the main factor that affects substrates' conversion. The ethanol-type fermentation of the acidogenic-phase is the optimal acid-type fermentation for the two-phase anaerobic process.

Acetic Acid↗
PubMed1989

[Interaction between the veterinarian, the client and the patient].

From the literature it has been established that pets can fulfill specific functions with regard to their owners. The purpose of this investigation is to highlight the interaction which takes place between pet owners and their pets as observed during consultations with veterinarians. The emphasis is however on the meaning of the emotional content of the interaction rather than on the actual functioning of the interaction. The interactions were monitored by means of natural observation, information gained through the completion of questionnaires, information about the client obtained from the veterinarian and content analysis of taped conversations which took place during consultations. The results indicate that the interaction between a pet owner and a pet is one of need fulfillment. Content analysis indicated that the owners attempt to fulfill certain needs through their pets, or conversely attempt to project their own emotions onto their pets. Loneliness and aggression are typical examples of this. A veterinarian who is able to identify his client's emotional needs, can alleviate his problems by giving unconditional support, referring the client with serious emotional problems to a psychologist or consulting a psychologist on behalf of the client.

Animals↗
PubMed1995

Psychotherapeutic discourse analysis.

Psychotherapeutic discourse analysis is a significant, largely unexplored, tool for psychotherapist and an ideal data sight for linguists. Increased multidisciplinary research in this area would be particularly fruitful. Conversations, including therapeutic conversations, are far from transparent conduits of information from one person to another. Complicating surface communication is "metacommunication," which takes the form of unconscious conversational styles--culturally influenced rules, norms, and expectations of how a conversation should proceed. If the therapist and the patient are working with different conversational styles, then blocks in communication and understanding are inevitable. Through discourse analysis conversational styles can become more available to awareness for use in the service of therapeutic goals. This paper is an example of a discourse analysis of an individual therapy session. I attempt to bridge the gap between broad psychodynamic treatment strategies and minute micro-conversational strategies that can be used to enhance the therapeutic process.

Adult↗
PubMed2003

Conversion of 2-fluoromuconate to cis-dienelactone by purified enzymes of Rhodococcus opacus 1cp.

The present study describes the (19)F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.

Adipates↗
PubMed2003

The CD44 receptor is a molecular predictor of survival in ovarian cancer.

PURPOSE: CD44 is a cell surface receptor implicated in cancer progression and metastases. Malignant tumors may show a loss of CD44 splice control mechanisms. We investigated the role of CD44 splice variant expression in ovarian tumors and metastases, and its association with survival. EXPERIMENTAL DESIGN: We tested CD44 expression in 142 cases of epithelial carcinoma of the ovary and 265 metastatic sites by immunohistochemistry. RESULTS: Survival analysis showed that the expression of CD44s, CD44-v4, -v5, -v6, -v9, and -v10 are significant predictors for survival in univariate analysis. After stage, the expression of CD44-v10 in metastases was the strongest predictor of decreased survival in multivariate analysis (p = 0.0009). Conversely, CD44-v10 expression in the primary tumor was an independent predictor of improved survival in multivariate analysis (p = 0.0002). The expression of CD44s in the tumor/stroma interface of the primary tumor was associated with improved survival (p < 0.0001). CONCLUSIONS: CD44 variant expression is a molecular prognostic maker for epithelial ovarian carcinomas. CD44-v10 expression is an independent prognostic indicator and the site of expression determines a positive or negative influence in survival. Our results also indicate that CD44 may be involved in important tumor/stroma interactions.

Adenocarcinoma, Mucinous↗
PubMed1996

Absolute structural determination of stevastelin B.

Stevastelin B, obtained from a culture of a Penicillium sp. NK374186, is a novel depsipeptide containing three amino acids and 3,5-dihydroxy-2,4-dimethylstearic acid. The stereochemistry of the three amino acids was determined by HPLC analysis, and the relative configuration of the 3,5-dihydroxy-2,4-dimethylstearic acid was elucidated by chemical conversion and NMR analysis. The absolute stereochemistry of stevastelin B was determined by synthetic methods.

Anti-Bacterial Agents↗
PubMed2005

Molecular evolution of cadherin-related neuronal receptor/protocadherin(alpha) (CNR/Pcdh(alpha)) gene cluster in Mus musculus subspecies.

The mouse cadherin-related neuronal receptor/protocadherin (CNR/Pcdh) gene clusters are located on chromosome 18. We sequenced single-nucleotide polymorphisms (SNPs) of the CNR/Pcdh(alpha)-coding region among 12 wild-derived and four laboratory strains; these included the four major subspecies groups of Mus musculus: domesticus, musculus, castaneus, and bactrianus. We detected 883 coding SNPs (cSNPs) in the CNR/Pcdh(alpha) variable exons and three in the constant exons. Among all the cSNPs, 586 synonymous (silent) and 297 nonsynonymous (amino acid exchanged) substitutions were found; therefore, the K(a)/K(s) ratio (nonsynonymous substitutions per synonymous substitution) was 0.51. The synonymous cSNPs were relatively concentrated in the first and fifth extracellular cadherin domain-encoding regions (ECs) of CNR/Pcdh(alpha). These regions have high nucleotide homology among the CNR/Pcdh(alpha) paralogs, suggesting that gene conversion events in synonymous and homologous regions of the CNR/Pcdh(alpha) cluster are related to the generation of cSNPs. A phylogenetic analysis revealed gene conversion events in the EC1 and EC5 regions. Assuming that the common sequences between rat and mouse are ancestral, the GC content of the third codon position has increased in the EC1 and EC5 regions, although biased substitutions from GC to AT were detected in all the codon positions. In addition, nonsynonymous substitutions were extremely high (11 of 13, K(a)/K(s) ratio 5.5) in the laboratory mouse strains. The artificial environment of laboratory mice may allow positive selection for nonsynonymous amino acid variations in CNR/Pcdh(alpha) during inbreeding. In this study, we analyzed the direction of cSNP generation, and concluded that subspecies-specific nucleotide substitutions and region-restricted gene conversion events may have contributed to the generation of genetic variations in the CNR/Pcdh genes within and between species.

Animals↗
PubMed2003

Ammonia cleaves polypeptides at asparagine proline bonds.

Polypeptides that contain the sequence Asn-Pro undergo complete cleavage at this amide bond with ammonia. One cleavage product possesses Pro as the new amino terminus and the other Asn or isoAsn as the new C-terminus, the formation of the latter probably arising by way of a cyclic succinimide intermediate. Other Asn-X bonds where X = Tyr, Gln, Ile, Glu, Ala, Gly, Asn or Phe did not exhibit any peptide bond cleavage, whereas when X = Leu, Thr and Ser partial cleavage was observed. Asn residues not involved in chain-cleavage underwent deamidation to Asp as shown by MALDI-ToF mass spectrometry (MS) analysis. The partial conversion of in-chain Asp residues to isoAsp under the reaction conditions was inferred from RP-HPLC and MS analysis of reaction mixtures.

Amino Acid Sequence↗
PubMed1997

Conditions affecting rural hospital specialization, conversion, and closure: a case-based analysis of threat and change.

The purpose of this analysis is to increase understanding of how and why rural hospitals change, with an eye toward the relevance of these questions to overall access and quality in the rural community. This study reports the threats that precipitated three major classes of organizational change (specialization, conversion, and closure) in 16 rural hospital cases. The authors identify the types and levels of threat faced by the case hospitals and examine how different threat situations may lead toward different classes of change. Conversions and closures typically seem to result from moderate- or high-threat situations. Specializations seem to result from low- or moderate-threat situations.

Decision Making, Organizational↗
PubMed2003

Conversion in off-pump coronary artery bypass grafting: an analysis of predictors and outcomes.

BACKGROUND: The incidence, predictive factors, and outcomes related to conversion from off-pump coronary artery bypass (OPCAB) to on-pump coronary artery bypass grafting (ONCAB) have not been well defined. We sought to determine the incidence of conversion, predictive factors, and any associated adverse consequences. METHODS: From January 2000 through June 2002, 1,644 patients underwent nonemergent OPCAB with 61 patients requiring conversion from OPCAB to ONCAB. These groups were retrospectively compared by univariate and multivariate regression analysis. The converted group was then computer matched 1:3, to a cohort of ONCAB patients to determine differences in outcomes. RESULTS: The overall conversion rate was 3.71%. Converted patients compared with a computer-matched ONCAB patients had a higher incidence of operative mortality (18.0% versus 2.7%, p < 0.001). Urgently converted patients had a higher incidence of postoperative cardiac arrest (25% versus 1.1%, p < 0.001), multisystem organ failure (10.7% versus 0.6%, p < 0.001), vascular complications (7.1% versus 1.1%, p = 0.03), and perioperative myocardial infarction (10.7% versus 1.1%, p = 0.02). Predictive factors for conversion were surgeon early in OPCAB experience (odds ratio [OR] 4.4), previous CABG (OR 2.8), and congestive heart failure (OR 2.0). The need for urgent-emergent conversion was highly predictive for operative mortality (OR 7.3) compared with elective conversion. CONCLUSIONS: Patients undergoing urgent-emergent but not elective conversion from OPCAB to ONCAB had a significantly higher risk of mortality and morbidity compared with patients whose procedure was initially ONCAB. Variables predictive of conversion included previous CABG, congestive heart failure, and surgeons early in OPCAB experience.

Cardiopulmonary Bypass↗