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A comprehensive survey of the genes involved in maturation and development of the rainbow trout ovary.

Development and maturation of the ovary requires precisely coordinated expression of specific gene classes to produce viable oocytes. We undertook identification of some of the genes involved in these processes by creating ovary-specific cDNA libraries by suppression subtractive hybridization and by microarray-based analyses. We present 5778 tissue- and sex-specific genes from subtracted ovary and testis libraries, many of which remain unidentified. A microarray containing 3557 salmonid cDNAs was used to compare the transcriptomes of precocious ovary at three different stages during the second year of life with a reference (normal ovary) transcriptome. On average, approximately 240 genes were developmentally regulated during the study period from June to October. Classes of genes maintaining relatively steady-state levels of expression, such as those controlling tissue remodeling, immunoregulation, cell-cycle progression, apoptosis, and growth also were identified. Concurrent expression of various cell division and ubiquitin-mediated proteolysis regulators revealed the utility of microarray analysis to monitor important maturation events. We also report unequivocal evidence for expression of the transcripts that encode the common glycoprotein alpha, LH beta, FSH beta, thyroid-stimulating hormone beta, and retinol-binding protein in both the ovary and testis of trout.

Age Factors↗

Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake.

This study presents an original 18S rRNA PCR survey of the freshwater picoeukaryote community, and was designed to detect unidentified heterotrophic picoflagellates (size range 0.6-5 microm) which are prevalent throughout the year within the heterotrophic flagellate assemblage in Lake Pavin. Four clone libraries were constructed from samples collected in two contrasting zones in the lake. Computerized statistic tools have suggested that sequence retrieval was representative of the in situ picoplankton diversity. The two sampling zones exhibited similar diversity patterns but shared only about 5% of the operational taxonomic units (OTUs). Phylogenetic analysis clustered our sequences into three taxonomic groups: Alveolates (30% of OTUs), Fungi (23%) and Cercozoa (19%). Fungi thus substantially contributed to the detected diversity, as was additionally supported by direct microscopic observations of fungal zoospores and sporangia. A large fraction of the sequences belonged to parasites, including Alveolate sequences affiliated to the genus Perkinsus known as zooparasites, and chytrids that include host-specific parasitic fungi of various freshwater phytoplankton species, primarily diatoms. Phylogenetic analysis revealed five novel clades that probably include typical freshwater environmental sequences. Overall, from the unsuspected fungal diversity unveiled, we think that fungal zooflagellates have been misidentified as phagotrophic nanoflagellates in previous studies. This is in agreement with a recent experimental demonstration that zoospore-producing fungi and parasitic activity may play an important role in aquatic food webs.

Chlorophyll↗

Diverse uncultivated bacterial groups from soils of the arid southwestern United States that are present in many geographic regions.

We have performed a phylogenetic survey of microbial species present in two soils from northern Arizona. Microbial DNA was purified directly from soil samples and subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences (rDNAs). Clone libraries from the two soils were constructed, and 60 clone inserts were partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity. Most of the analyzed sequences (64%) fell into five novel clusters having no known cultured members. Extensive analysis of 10 nearly full-length rDNAs from clones representative of the novel groups indicated that four of the five groups probably cluster into a large "supergroup" which is as distinct from currently recognized bacterial divisions as the latter are from each other. From this we postulate the existence of a major bacterial lineage, previously known only from a single cultured representative, whose diversity and ecology we are only beginning to explore. Analysis of our data and that from other rDNA sequence-based studies of soils from different geographic regions shows considerable overlap of sequence types. Taken together, these groups encompass most of the novel rDNA sequences recovered in each comparable analysis reported to date, despite large differences in soil types and geographic sources. Our results indicate that members of these new groups comprise a phylogenetically diverse, geographically widespread, and perhaps numerically important component of the soil microbiota.

Arizona↗

Language matters: the vocabulary of racism in health care.

Race equality as a matter of governance has gained momentum in most Western countries and is reflected in race/ethnicity data collection in administrative systems and the attention accorded to terminology by census agencies. However, the vocabulary of health care--both in its literature and the language of officialdom--has proved resistant to the use of this lexicon of acceptable terms, as exemplified by the portrayal of peoples as 'Oriental' and 'Negro'. What makes such language racist is the historical legacy it carries--that is, its symbolic importance. Survey evidence shows that the majority of those so described find the terms offensive. Countries have dealt with these linguistic issues in a variety of ways, including the use of the legislature and action by library associations and professional bodies in the USA. In Britain it has fallen upon the judiciary and universities to prohibit such terms. Given the momentum currently being achieved by public authorities in their response to race equality legislation, it is now time for health care to purge its language of these epithets.

Delivery of Health Care↗

Screening of drugs and toxic compounds with liquid chromatography-linear ion trap tandem mass spectrometry.

BACKGROUND: In clinical and forensic toxicology, general unknown screening is used to detect and identify exogenous compounds. In this study, we aimed to develop a comprehensive general unknown screening method based on liquid chromatography coupled with a hybrid triple-quadrupole linear ion trap mass spectrometer. METHODS: After solid-phase extraction, separation was performed using gradient reversed-phase chromatography. The mass spectrometer was operated in the information-dependent acquisition mode, switching between a survey scan acquired in the Enhanced Mass Spectrometry mode with dynamic subtraction of background noise and a dependent scan obtained in the enhanced product ion scan mode. The complete cycle time was 1.36 s. A library of 1000 enhanced product ion-tandem mass spectrometry spectra in positive mode and 250 in negative mode, generated using 3 alternated collision tensions during each scan, was created by injecting pure solutions of drugs and toxic compounds. RESULTS: Comparison with HPLC-diode array detection and gas chromatography-mass spectrometry for the analysis of 36 clinical samples showed that linear ion trap tandem mass spectrometry could identify most of the compounds (94% of the total). Some compounds were detected only by 1 of the other 2 techniques. Specific clinical cases highlighted the advantages and limitations of the method. CONCLUSION: A unique combination of new operating modes provided by hybrid triple-quadrupole linear ion trap mass spectrometers and new software features allowed development of a comprehensive and efficient method for the general unknown screening of drugs and toxic compounds in blood or urine.

Antipsychotic Agents↗

cDNA cloning of androgen-repressed mRNA in rat seminal vesicles: partial characterization of a cDNA clone, pSvr-1.

When the in vitro translation products of mRNAs from castrated animals (48 h) were compared with those from androgen-treated animals (48 h) to survey the molecular mechanism of androgen-responsive gene expressions in the rat seminal vesicles, some peptide bands which were repressed in response to androgen were observed. From these findings, we constructed a partial cDNA library of poly(A+)RNAs which had been isolated from the seminal vesicles of castrated rats (48 h) and modestly enriched with respect to the concentration of androgen-repressed mRNAs by sucrose density gradient centrifugation, and screened by differential colony hybridization. One cDNA clone, pSvr-1, whose mRNA is markedly induced within 24 h after castration of the animal in the seminal vesicles as well as in the ventral prostate, was isolated. pSvr-1 hybridized to a mRNA of 1,700 nucleotides in length. Partial sequence analysis showed that this clone had highly homologous but not identical sequences to those reported for rat sulfated glycoprotein-2. This cDNA clone may provide a useful probe for the study of the negative regulation mechanism of gene expression by androgens.

Androgens↗

Screening for protease substrate by polyvalent phage display.

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.

Antibody Affinity↗

Purification, cDNA cloning, and developmental changes in the steady-state mRNA level of rat testicular tissue inhibitor of metalloproteases-2 (TIMP-2).

Using multiple high-performance liquid chromatography (HPLC) steps and high-performance electrophoresis chromatography (HPEC) in conjunction with an [125I]collagen film assay to identify inhibitors of metalloproteases, we have purified a 22-kDa polypeptide to apparent homogeneity from primary Sertoli cell-enriched culture medium. Partial N-terminal amino acid sequence analysis revealed that this protein is similar to the human tissue inhibitor of metalloproteases-2 (TIMP-2). To determine the similarity of rat testicular TIMP-2 to the human homolog, a full-length cDNA coding for rat testicular TIMP-2 was isolated from a rat Sertoli cell cDNA expression library and sequenced. Analysis of the nucleotide sequence and the deduced amino acid sequence of the rat testicular TIMP-2 cDNA revealed an 84 and 98% homology with the human TIMP-2 nucleotide and amino acid sequences, respectively. A survey of its mRNA transcripts in different tissues by northern blots revealed the presence of two mRNA species of 3.7 and 1.3 kb in the testis and brain but not in the kidney, spleen, epididymis, and liver in adult male rats. Studies using polymerase chain reaction (PCR) and Southern blot to detect the TIMP-2 mRNA using total RNA isolated from germ cells, Sertoli cells, and Leydig cells have shown that only Sertoli and Leydig cells expressed TIMP-2 mRNA. These results indicate that Sertoli cells are the major source of TIMP-2 in the testis behind the blood-testis barrier (seminiferous tubule barrier). During testicular development from 3 to 60 days of age, the testicular steady-state TIMP-2 mRNA level increased steadily with an advancement of age. Such an increase in the steady-state testicular TIMP-2 mRNA level apparently is not the result of an up-regulation by germ cells because germ cells cocultured with Sertoli cells failed to elicit an increase in the Sertoli cell steady-state TIMP-2 mRNA level. The results of this study suggest that TIMP-2 secreted by Sertoli cells may play a role in tissue restructuring and germ cell migration during spermatogenesis.

Animals↗

Sample sequencing of a Salmonella typhimurium LT2 lambda library: comparison to the Escherichia coli K12 genome.

As part of the ongoing sequencing of the complete Salmonella typhimurium LT2 genome, a partly ordered set of 416 lambda clones has been developed, representing over 90% of the genome. The average insert size is 17 kb. Sequences were obtained from both ends of each clone in this set. A total of over 600 kb of sequence has been deposited in the genome survey sequence section of GenBank. This resource of clones is available from the Salmonella Genome Stock Center. A preliminary comparison with the Escherichia coli K12 genome indicates that there are likely to be many hundred insertion deletion events, encompassing more than one gene, that distinguish these genomes. Fully 30% of the S. typhimurium sequences have no close homologs in the GenBank database.

Adult↗

Netherlands Twin Register: a focus on longitudinal research.

In 1986 we began The Netherlands Twin Register (NTR) by recruiting young twins and multiples a few weeks or months after birth. Currently we register around 50% of all newborn multiples in The Netherlands. Their parents receive a questionnaire at registration and afterwards when the children are 2, 3, 5, 7, 10 and 12 years of age. Teachers are asked to rate the behavior of the children at ages 7, 10 and 12 years. Adolescent and young-adult twins were recruited through City Councils in the early 1990s. These twins, their parents and siblings participate in longitudinal survey studies that include items about health, fertility, lifestyle, addiction, personality and psychopathology, religion, socioeconomic status, and educational attainment. The total number of twins and multiples registered with the NTR is currently over 60,000. Subgroups of twins and siblings take part in studies of cognitive development, brain function and neuropsychological indices of attention processes, and molecular genetic studies of classical and behavioral cardiovascular risk factors. DNA samples are currently collected in selected twin families for two large linkage studies, which aim to find QTLs for anxious depression and for nicotine addiction. Sisters who are mothers of DZ twins contribute DNA samples for a linkage study of DZ twinning. Large cohorts of phenotyped family members from the general population are very valuable for genetic epidemiological studies and permit selection of informative families for gene finding studies.

Adolescent↗

The Medical Library Association in retrospect, 1937-1967.

The medical librarian of 1967 lives in a period of changing concepts, dramatic new methods, everwidening scientific horizons. In looking toward the immediate past he may think of the medical librarian of thirty years ago as a complacent follower of accepted procedures, not as a pioneer in a brave new world. Yet the corps of trained medical librarians today and the resources of our collections and their management are dependent upon the efforts of those who were then pioneers in medical librarianship. Training, standards, recruitment, literature control, international relations, all had continuing attention at a time when financial assistance through government funds, support by administrators, concern by scientists was almost nonexistent. In these years the day of the devoted amateur passed; the trained medical librarian came into being and matured.This, the first Janet Doe Lecture, is named for one who illustrates the best in medical librarianship, serving with scholarly distinction. It is a brief survey pointing to some of the Association's significant achievements during the years of Miss Doe's greatest activity when she and her colleagues met their "Challenge of Change."

History, 20th Century↗

The Medical Library Association in retrospect, 1937-1967.

The medical librarian of 1967 lives in a period of changing concepts, dramatic new methods, everwidening scientific horizons. In looking toward the immediate past he may think of the medical librarian of thirty years ago as a complacent follower of accepted procedures, not as a pioneer in a brave new world. Yet the corps of trained medical librarians today and the resources of our collections and their management are dependent upon the efforts of those who were then pioneers in medical librarianship. Training, standards, recruitment, literature control, international relations, all had continuing attention at a time when financial assistance through government funds, support by administrators, concern by scientists was almost nonexistent. In these years the day of the devoted amateur passed; the trained medical librarian came into being and matured. This, the first Janet Doe Lecture, is named for one who illustrates the best in medical librarianship, serving with scholarly distinction. It is a brief survey pointing to some of the Association's significant achievements during the years of Miss Doe's greatest activity when she and her colleagues met their "Challenge of Change".

History, 20th Century↗

A survey of education and research facilities for palliative medicine trainees in the United Kingdom.

In June 1997 a postal questionnaire was sent to all palliative medicine trainees in the United Kingdom to ascertain the resources available to facilitate education and research, and the experiences of trainees who had undertaken research. Questions posed related to protected study time, library facilities, information technology (IT) support, postgraduate educational activities, details of research projects undertaken, level of senior supervision and overall impressions. The response rate was 85%. Thirty-six per cent of respondents were undertaking postgraduate educational courses and 30% of those remaining planned to do so. Protected time for study was allocated to almost all trainees, but access to educational resources was more variable. The availability of several journals and of information technology support was lower than their perceived importance. Satisfaction scores for the availability of books covering areas related to clinical practice were high, but were lower for availability of the internet and of books covering specific training issues. Ninety-one per cent of respondents had been involved in research/audit projects, two-thirds had published at least one article and half had presented an oral or written paper. Details of 162 projects were received, together with problems encountered. The results indicate that many of the foundations for specialist education in palliative medicine are in place and that trainees are highly motivated towards conducting research. The aspects in need of improvement on the educational side include IT facilities and the availability of specific speciality related journals. There is a need for more accessible supervision from senior colleagues and training in research methodology.

Education, Medical, Graduate↗

Computer use in Canadian drug information centres.

In July 1988, a survey was conducted to determine the extent of computer use by Canadian drug information centres. A questionnaire, mailed to 36 major DI centres, yielded an 89% return. Computers were being used by 75% of the responding centres (65.6% personal computers, 9.4% mainframe). Word processing was the most popular computer application, followed by formulary maintenance, online literature searching, compiling workload statistics, pharmacokinetic calculations, storing answered requests, and storing journal article citations. Medline was the most common database accessed; the National Library of Medicine was the most popular vendor. Compact disc read only memory systems were used by 18.75% of centres. Some respondents reported developing internal programs for drug information.

Canada↗

Autoantigenic epitopes on DNA topoisomerase I. Clinical and immunogenetic associations in systemic sclerosis.

OBJECTIVE: To elucidate the clinical and immunogenetic associations with reactivity to autoantigenic epitopes on DNA topoisomerase I (topo I) recognized by sera from patients with systemic sclerosis (SSc). METHODS: Autoantigenic epitopes on topo I were identified by screening an epitope library constructed from topo I complementary DNA restriction fragments using autoimmune anti-topo I-positive sera as a probe. Epitope reactivities of sera from 43 anti-topo I-positive SSc patients were surveyed by immunoblotting, and associations with clinical symptoms and HLA-DR types were examined. RESULTS: Four different epitope regions were identified on the topo I molecule. Immunoreactivity to the region encompassing amino acid residues 658-700, termed ER4, was found to be associated with diffuse cutaneous SSc, progressive pulmonary interstitial fibrosis, and poor prognosis for 15-year survival. SSc patients with ER4 reactivity frequently displayed the DR2/DRw52 phenotype. CONCLUSION: Molecular analysis of precise antigenic epitopes on topo I is helpful in classifying clinical subsets of SSc.

Adult↗

Modeling bacterial species abundance from small community surveys.

Taxon abundance patterns are a central focus in evolution and ecology, providing the basic architecture of natural assemblages and potential clues to their formative processes. To better interpret species abundance patterns in natural microbial communities, we examined the consequences of three fundamental types of abundance patterns--uniform, geometric, and lognormal distributions. Theoretical communities were constructed based on the three pattern types with 2000 to 20,000 species and 10(7) to 10(10) individuals. The "dominant" species (species 1) among models that differed only in pattern type generally varied in abundance by 1 to 3 orders of magnitude. At the extremes among all the models examined, the dominant species comprised as much as 16% and as little as 0.005% of the total community. Analysis of the models and comparison with seven published surveys suggests that entire soil bacterial communities do not routinely exhibit Preston's cannonical subset of lognormal distributions. Use of the models to evaluate survey limitations showed that common diversity indices are generally sensitive to sample size over the range (50 to 200 clones) commonly used for microbial communities, emphasizing the need to compare surveys of similar size. The results collectively demonstrate that uniform, geometric, and lognormal distributions have profoundly different experimental and ecological consequences. Further, defined abundance models provide a simple quantitative tool for evaluating abundance patterns in clone libraries (even small ones) from natural communities.

Bacteria↗

The Xenopus B1 factor is closely related to the mammalian activator USF and is implicated in the developmental regulation of TFIIIA gene expression.

The Xenopus laevis TFIIIA promoter contains a motif that has been implicated in promoter activation in late-stage oocytes and contains the sequence (-269) CACGTG (-264). A cDNA encoding a protein (B1) that binds to this element has been cloned from X. laevis and Xenopus borealis ovarian cDNA libraries. We show that this protein is a member of the helix-loop-helix family of regulatory proteins and contains 80% sequence identity with the human adenovirus major late transcription factor (MLTF or USF). A survey of B1 protein expression during oogenesis and embryogenesis revealed both oocyte-specific and somatic cell-specific B1 protein-DNA complexes. Immunological data, RNA blot analysis, and proteolytic clipping band shift assays indicated that these complexes most likely represent altered forms of a single B1 polypeptide. Implications for TFIIIA gene regulation during development are discussed.

Amino Acid Sequence↗