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PubMed2000

Substrate specificity and inhibition studies of human serotonin N-acetyltransferase.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[(3)H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.

Acyl Coenzyme A↗
PubMed1996

Identification of divergent homologs of Chironomus tentans sp185 and its Balbiani ring 3 gene in Australasian species of Chironomus and Kiefferulus.

A 185-kDa silk protein (sp185) from Chironomus tentans, present in both larval and prepupal silks, contains a striking amino acid sequence motif, Cys-X-Cys-X-Cys, which occurs about every 22-26 residues. Homologous proteins have been found in Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220), which apparently differ in size but are very similar in overall composition and sequence. While surveying Australasian species of Chironomus and Kefferulus we obtained evidence for immunologically related silk protein having similar size and amino acid composition, but noticeably less Cys. Interspecies in situ hybridization to polytene chromosomes with C. tentans and C. pallidivittatus cDNA probes indicated that each species had a related gene. One pair of C. tentans cDNA-derived primers enabled polymerase chain reaction amplification of a discrete fragment of this gene from Kiefferulus 'cornishi'. Preliminary sequence information for this fragment confirmed the presence of an encoded Cys-X-Cys-X-Cys motif in what appeared to be a similar protein region containing less Cys. We conclude that homologs of C. tentans sp185 and its gene have been identified which may contain significant deviations in structure. Once suitable libraries are available, probes described here will be useful for selecting cDNA and genomic clones for detailed study.

Amino Acid Sequence↗
PubMed2006

Detection of transgenic and endogenous plant DNA in digesta and tissues of sheep and pigs fed Roundup Ready canola meal.

The persistence of plant-derived recombinant DNA in sheep and pigs fed genetically modified (Roundup Ready) canola was assessed by PCR and Southern hybridization analysis of DNA extracted from digesta, gastrointestinal (GI) tract tissues, and visceral organs. Sheep (n = 11) and pigs (n = 36) were fed to slaughter on diets containing 6.5 or 15% Roundup Ready canola. Native plant DNA (high- and low-copy-number gene fragments) and the cp4 epsps transgene that encodes 5-enolpyruvyl shikimate-3-phosphate synthase were tracked in ruminal, abomasal, and large intestinal digesta and in tissue from the esophagus, rumen, abomasum, small and large intestine, liver, and kidney of sheep and in cecal content and tissue from the duodenum, cecum, liver, spleen, and kidney of pigs. High-copy chloroplast-specific DNA (a 520-bp fragment) was detected in all digesta samples, the majority (89-100%) of intestinal tissues, and at least one of each visceral organ sample (frequencies of 3-27%) from sheep and swine. Low-copy rubisco fragments (186- and 540-bp sequences from the small subunit) were present at slightly lower, variable frequencies in digesta (18-82%) and intestinal tissues (9-27% of ovine and 17-25% of porcine samples) and infrequently in visceral organs (1 of 88 ovine samples; 3 of 216 porcine samples). Each of the five cp4 epsps transgene fragments (179-527 bp) surveyed was present in at least 27% of ovine large intestinal content samples (maximum = 64%) and at least 33% of porcine cecal content samples (maximum = 75%). In sheep, transgene fragments were more common in intestinal digesta than in ruminal or abomasal content. Transgene fragments were detected in 0 (esophagus) to 3 (large intestine) GI tract tissues from the 11 sheep and in 0-10 of the duodenal and cecal tissues collected from 36 pigs. The feed-ingested recombinant DNA was not detected in visceral tissues (liver, kidney) of lambs or in the spleen from pigs. Of note, however, one liver and one kidney sample from the pigs (different animals) were positive for a 278-bp fragment of the transgenic cp4 epsps (denoted F3). Examination of genomic libraries from these tissues yielded no conclusive information regarding integration of the fragment into porcine DNA. This study confirms that feed-ingested DNA fragments (endogenous and transgenic) do survive to the terminal GI tract and that uptake into gut epithelial tissues does occur. A very low frequency of transmittance to visceral tissue was confirmed in pigs, but not in sheep. It is recognized that the low copy number of transgenes in GM feeds is a challenge to their detection in tissues, but there was no evidence to suggest that recombinant DNA would be processed in the gut in any manner different from endogenous feed-ingested genetic material.

Animal Feed↗
PubMed2000

Where do developing World clinicians obtain evidence for practice: a case study on pneumonia.

There are few data on the practice of evidence based medicine in the developing world, nor on the actual sources of evidence that clinicians use in practice. To test the hypothesis that there was variation between and within developing countries in the proposed management of a patient with hospital acquired pneumonia, and that part of the variation can be explained by the sources of evidence used. Questionnaire responses to hypothetical case history. Investigators from 6 centres within the International Clinical Epidemiology Network (INCLEN) in China, Thailand, India, Egypt, and Kenya. Doctors chosen to represent primary and secondary hospital practice in the regions of the study centres. Investigations and initial treatments which would be ordered for a hypothetical 60-year-old woman who develops pneumonia 5 days after hospital admission, whether local data on antibiotic sensitivities are available and where information would be obtained to guide management. Chest x-ray and sputum gram stain/culture were consistently the most commonly ordered investigations, there being much greater variation in the initial treatment choices with either penicillin, a third-generation cephalosporin or aminoglycoside being the most popular choice. Textbooks were the commonest form of information source, and access to a library, textbooks and journals were statistically significantly associated with appropriate choice of investigations, but not treatment. Access to local antibiotic sensitivities was associated with appropriate initial treatment choice. Improving access to information in the literature and to local data may increase the practice of evidence-based medicine in the developing world.

Cross Infection↗
PubMed2004

Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot.

We explore the thermodynamic basis for high affinity binding and specificity in conserved protein complexes using colicin endonuclease-immunity protein complexes as our model system. We investigated the ability of each colicin-specific immunity protein (Im2, Im7, Im8 and Im9) to bind the endonuclease (DNase) domains of colicins E2, E7 and E8 in vitro and compared these to the previously studied colicin E9. We find that high affinity binding (Kd < or = 10(-14) M) is a common feature of cognate colicin DNase-Im protein complexes as are non-cognate protein-protein associations, which are generally 10(6)-10(8)-fold weaker. Comparative alanine scanning of Im2 and Im9 residues involved in binding the E2 DNase revealed similar behaviour to that of the two proteins binding the E9 DNase; helix III forms a conserved binding energy hotspot with specificity residues from helix II only contributing favourably in a cognate interaction, a combination we have termed as "dual recognition". Significant differences are seen, however, in the number and side-chain chemistries of specificity sites that contribute to cognate binding. In Im2, Asp33 from helix II dominates colicin E2 specificity, whereas in Im9 several hydrophobic residues, including position 33 (leucine), help define its colicin specificity. A similar distribution of specificity sites was seen using phage display where, with Im2 as the template, a library of randomised sequences was generated in helix II and the library panned against either the E2 or E9 DNase. Position 33 was the dominant specificity site recovered in all E2 DNase-selected clones, whereas a number of Im9 specificity sites were recovered in E9 DNase-selected clones, including position 33. In order to probe the relationship between biological specificity and in vitro binding affinity we compared the degree of protection afforded to bacteria against colicin E9 toxicity by a set of immunity proteins whose affinities for the E9 DNase differed by up to ten orders of magnitude. This analysis indicated that the Kd required for complete biological protection is <10(-10)M and that the "affinity window" over which the selection of novel immunity protein specificities likely evolves is 10(-6)-10(-10)M. This comprehensive survey of colicin DNase-immunity protein complexes illustrates how high affinity protein-protein interactions can be very discriminating even though binding is dominated by a conserved hotspot, with single or multiple specificity sites modulating the overall binding free energy. We discuss these results in the context of other conserved protein complexes and suggest that they point to a generic specificity mechanism in divergently evolved protein-protein interactions.

Bacterial Proteins↗
PubMed1995

Comparative pharmacology of calcium antagonists.

OBJECTIVE: To compare clinically relevant pharmacokinetic, pharmacodynamic and toxico logical characteristics of calcium-modulating compounds used in ischemic heart disease. DATA SOURCES: A MEDLINE search (1990 pt B to 1991 pt A revised for 1993; 1991 pt B to 1992 revised for 1993; and January to May 1993) combining the search phrases 'calcium channel blockers', 'myocardial ischemia', 'pharmacodynamics' and 'pharmacokinetics', and a search in Compact Cambridge Drug Information Source vol-6 (revised 1992, fourth quarter) using the search phrase 'calcium antagonists' and medical subject headings (MeSH) 'pharmacokinetics' and 'pharmacodynamics' were used to obtain title and abstract information on available current literature. STUDY SELECTION: Review articles, proceedings and studies published in English and available within the University of Saskatchewan library system, as they appeared to relate closely to the objective, were obtained for closer evaluation. In addition, primary references were examined, and journal reprints were selected from the authors' files. DATA EXTRACTION: The focus was on studies and objective reviews that profiled one or more representative compounds in a manner suitable for deriving background and comparative information pertaining to the objective. Data from multiple studies, or from studies that employed multiple methodological approaches, were preferentially extracted and summarized for presentation. DATA SYNTHESIS: The role of calcium in cardiac and vascular smooth muscle physiology was reviewed, highlighting the major mechanisms responsible for maintaining calcium homeostasis in these cells. With a focus on verapamil, diltiazem and 1,4-dihydropyridines currently employed in the treatment of cardiovascular disorders, a general survey of their sites of action, tissue selective pharmacodynamics, pharmacokinetic properties and side effects was undertaken in a comparative context. CONCLUSIONS: Calcium antagonists are employed in the treatment of angina, certain cardiac arrhythmias and hypertension. They are a chemically and pharmacologically heterogeneous group of compounds that act principally to inhibit the influx of calcium across certain voltage-dependent membrane channels. Concepts pertaining to calcium mobilization in the pathophysiology of myocardial ischemia, particularly at the molecular level, have evolved remarkably over the past decade. The repertoire of agents having calcium-regulating properties has expanded in parallel. The task of integrating new knowledge in both of these areas requires further attention in order to determine optimal approaches to treatment.

Animals↗
PubMed1995

Expression of a large number of novel testis-specific genes during spermatogenesis coincides with the functional reorganization of the male germ cell.

Structural and functional changes, essential for the formation of mature male germ cells, are known to take place at specific stages of the mammalian spermatogenic process. To identify novel genes that are involved in this developmental process, we have initiated a large-scale cDNA sequencing project (Hoog+, Nucleic Acids Res. 19: 93-98, 1991; Starborg et al., Mol. Reprod. Dev. 33: 243-251, 1992; Yuan et al., Biol. Reprod., 1995). Five-hundred and forty cDNAs have been isolated from testicular cDNA libraries and partially sequenced, 355 of which were found to represent genes previously not described in the literature. In addition, a number of cDNAs was found to be related to genes previously identified only in lower eukaryotes, suggesting that these murine genes encode functions that are evolutionary conserved. One of these murine cDNAs was related to the Aspergillus nidulans BimE gene, a putative cell cycle checkpoint regulator (Starborg et al., J. Biol. Chem., 1994). Southern blot analysis revealed that the murine BimE-related gene is strongly conserved in mammals. RNA blotting experiments of 361 novel murine cDNAs have identified 52 cDNAs that are expressed only during spermatogenesis, 36 of which are expressed only in spermatids, and 16 cDNAs that are expressed in both spermatocytes and spermatids. A survey of the literature revealed 40 mammalian genes that have previously been shown to be expressed mainly during spermatogenesis, and together with our results, they define three dominating temporal patterns of gene expression during spermatogenesis, each pattern coinciding with known functional or structural changes occurring during this differentiation process.

Aging↗
PubMed1989

MEDLINE-for-Medics: an outline searching course for health-care professionals.

An online searching course for health professionals was initiated at a university medical school. A group of end users were taught to search the MEDLINE database on DATA-STAR and their subsequent online progress was monitored. The preliminary survey by questionnaire, pretraining interviews, the tutorial programme, course evaluation and end-user feedback were described. Logsheets showed that most end-user searches were for research purposes, although some were for patient care. The effects of CD-ROM on online end-user searching were discussed and it was suggested that direct online access to information might be a more appropriate medium for busy clinical and academic staff.

Academic Medical Centers↗
PubMed1997

Mapping the literature of speech-language pathology.

The purpose of this study, part of the Medical Library Association (MLA) Nursing and Allied Health Resources Section's project to map the allied health literature, is to identify the core journals in the field of speech-language pathology and to identify indexing and abstracting services that provide access to these journals. Four representative speech-language pathology journals were selected and subjected to citation analysis to determine which journals were cited and how many times each was cited. Bradford's Law of Scattering was applied to the resulting list of journals to identify the core journals of this discipline. Six indexing and abstracting services were selected and scanned to determine coverage for the speech-language pathology core journals. The core journals received broad coverage in the health sciences and social sciences indexing and abstracting databases surveyed, although there was no one database that provided complete coverage of all core journals. The full Current Contents database provides the most extensive coverage of core journals. For individuals without access to the complete Current Contents database, a combined search of both MEDLINE and PsycINFO provides very comprehensive coverage of core journals.

Abstracting and Indexing↗
PubMed2000

[Tyrosine kinase expression in the inner ear of mice].

Tyrosine-specific phosphorylation events serve to modulate cell to cell interactions during the normal growth and development of many vertebrate tissues. Numerous studies have been performed on the involvement of growth factor receptor tyrosine kinases (RTKs) in inner ear function. Many RTKs, however, have only been recently identified, and their expression patterns have not yet been systematically examined in all tissues. In this study, we surveyed mouse inner ear transcript sequences for the presence of RTKs using a degenerate-PCR based methodology. Isolates encoding RTKs that had and had not been previously identified in the inner ear were obtained. Our data reveals the presence of numerous RTKs in the inner ear that have not yet been studied in the context of inner ear and demonstrates the usefulness of RT-PCR analysis in identifying genes involved in inner ear function.

Animals↗
PubMed1996

ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins.

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.

Amino Acid Sequence↗
PubMedDate not supplied

The hospitalist movement 5 years later.

CONTEXT: We originally described the hospitalist model of inpatient care in 1996; since then, the model has experienced tremendous growth. This growth has important clinical, financial, educational, and policy implications. OBJECTIVES: To review data regarding the effect of hospitalists on resource use, quality of care, satisfaction, and teaching; and to analyze the impact of hospitalists on the health care system and frame key issues facing the movement. DATA SOURCES AND STUDY SELECTION: We searched MEDLINE, BIOSIS, EMBASE, and the Cochrane Library from 1996 to September 2001 for studies comparing hospitalist care with an appropriate control group in terms of resource use, quality, or satisfaction outcomes. DATA EXTRACTION: We extracted information regarding study design, nature of hospitalist and control groups, analytical strategies, and key outcomes. DATA SYNTHESIS: Most studies found that implementation of hospitalist programs was associated with significant reductions in resource use, usually measured as hospital costs (average decrease, 13.4%) or average length of stay (average decrease, 16.6%). The few studies that failed to demonstrate reductions usually used atypical control groups. Although several studies found improved outcomes, such as inpatient mortality and readmission rates, these results were inconsistent. Patient satisfaction was generally preserved, while limited data supported positive effects on teaching. Although concerns about inpatient-outpatient information transfer remain, recent physician surveys indicate general acceptance of the model. CONCLUSIONS: Empirical research supports the premise that hospitalists improve inpatient efficiency without harmful effects on quality or patient satisfaction. Education may be improved. In part catalyzed by these data, the clinical use of hospitalists is growing rapidly, and hospitalists are also assuming prominent roles as teachers, researchers, and quality leaders. The hospitalist field has now achieved many of the attributes of traditional medical specialties and seems destined to continue to grow.

Health Services Research↗
PubMed2004

A survey of the composition and diversity of bacterial populations in bleached kraft pulp-mill wastewater secondary treatment systems.

Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques. 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system. Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters. Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities. There was little overlap in the composition of the culturable community between mills at the genus level. Furthermore, RSGP variation was almost as high within a mill as between mills. Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species. Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown alpha-, beta-, and gamma-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum. These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.

Bacteria↗
PubMed1994

Characterization of the murine beta-hexosaminidase (HEXB) gene.

The murine HEXB gene, encoding the beta-subunit of the lysosomal hydrolase, beta-hexosaminidase, was isolated from a mouse cosmid library as a single cosmid clone. The entire gene spans 22 kb, considerably less than the 40 kb spanned by its human counterpart. It is highly homologous to the human gene. The 14 intron-exon junctions are entirely conserved, although the intron sequences diverge rapidly. Upstream of the coding region, a 1.3 kb segment was sequenced and shown to function as a promoter when fused with a reporter gene and expressed in monkey COS-7 cells. A short sequence (100 bp), near the start of the coding region, exhibits strong homology to the human HEXB promoter. Analysis of the tissue distribution of the HEXB mRNA in 129/Sv male mice revealed up to 28-fold tissue-specific variations in transcript levels. The kidney and the epididymis had the highest mRNA levels consistent with past surveys of enzyme activity.

Amino Acid Sequence↗
PubMed2005

High-throughput target discovery using cell-based genetics.

High-throughput target discovery requires robust disease models and the ability to rapidly survey the genome for function. In the post-genomics era, there has been a strong emphasis placed upon "gene-to-function" approaches that take advantage of the large amount of gene sequence information now available. Here, we advocate a return to "function-to-gene" approaches as a first step in target discovery (and validation), followed by hypothesis-driven research to validate new targets identified by their activity in cell-based disease models.

Animals↗
PubMed2006

The impact of the physical and urban environment on mental well-being.

OBJECTIVES: To examine the strength of association between physical and social factors in the built environment and mental well-being, and to determine which factors are the most important. STUDY DESIGN: A postal survey based on a theoretical model of domains that might link the physical and urban environment with mental well-being was sent to 2696 adults aged 18 years or over, in four areas of Greenwich, London. Mental health was measured using the SF36 subscales for mental health (MH) and vitality (V). Additional household and area level data were appended for each respondent from a range of sources. RESULTS: 1012 questionnaires were returned (38% response rate). At the univariate level significant confounders that were associated with poorer mental well-being were being female, 85+ years, unemployed or retired, on housing benefit, council tenant, two or more children, and having requested re-housing Better mental well-being was associated with being aged 65 years to 84 years (better MH and V). Within domain analysis, adjusting for each of the confounding factors, resulted in the following factors being significantly associated with being in the lowest quartile for MH score: (i) control over the internal environment (damp), (ii) design and maintenance (not liking the look of the estate/road, (iii) noise (neighbour noise), (iv) density and escape (feeling over-crowded in the home, being dissatisfied with green spaces, dissatisfied with social and entertainment facilities) being dissatisfied with community facilities (such as libraries and community centres) was only significant for vitality, (v) fear of crime and harassment (feeling unsafe to go out in the day, feeling unsafe to go out at night, agreeing that needles and syringes left lying around are a problem) (vi) social participation (not enough events to get people together, not enough places to stop and chat). When these 12 factors were entered into a single model with the significant confounders five remained significantly associated with being in the lowest quartile for MH or V: neighbour noise MH OR 2.71 [95% CI 1.48, 4.98]; feeling over-crowded in the home MH OR 2.22 [1.42, 3.48]; being dissatisfied with access to green open spaces MH OR 1.69 [1.05, 2.74]; access to community facilities V OR 1.92, [1.24, 3.00]; feeling unsafe to go out in the day MH OR 1.64 [1.02, 2.64]; V OR 1.58 [1.00, 2.49]. CONCLUSIONS: This study confirms an association between the physical environment and mental well-being across a range of domains. The most important factors that operate independently are neighbour noise, sense of over-crowding in the home and escape facilities such as green spaces and community facilities, and fear of crime. This study highlights the need to intervene on both design and social features of residential areas to promote mental well-being.

Adult↗
PubMed1992

Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin's disease.

We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease.

Amino Acid Sequence↗
PubMed2004

Image informatics at a national research center.

Image informatics at the Communications Engineering Branch of the Lister Hill National Center for Biomedical Communications (LHNCBC), an R&D division of the National Library of Medicine (NLM), includes document and biomedical images. In both domains, research into computer-assisted methods for information extraction, and the implementation of prototype systems incorporating such methods, is central to our mission. Current document image research focuses on extracting bibliographic data from scanned journal articles. Current biomedical imaging work focuses on content-based image retrieval (CBIR) and related problems in segmentation, indexing, and classifying collections of images of the spine and of the uterine cervix.

Diagnostic Imaging↗