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PubMed2000

Ets domain transcription factor PE1 suppresses human interstitial collagenase promoter activity by antagonizing protein-DNA interactions at a critical AP1 element.

In MC3T3E1 calvarial osteoblasts, fibroblast growth factor receptor (FGFR) signaling elicits multiple transcriptional responses, including upregulation of the interstitial collagenase/matrix metalloproteinase 1 (MMP1) promoter. FGF responsiveness maps to a bipartite Ets/AP1 element at base pairs -123 to -61 in the human MMP1 promoter. Under basal conditions, the MMP1 promoter is repressed in part via protein-DNA interactions at the Ets cognate, and minimally two mechanisms convey MMP1 promoter upregulation by FGF2: (a) transcriptional activation via Fra1/c-Jun containing DNA-protein interactions at the AP1 cognate and (b) derepression of promoter activity regulated by the Ets cognate. To identify osteoblast Ets repressors that potentially participate in gene expression in the osteoblast, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA isolated from MC3T3E1 cells, using degenerative amplimers to the conserved Ets DNA binding domain to survey the Ets genes expressed by these cells. Six distinct Ets mRNAs were identified: Ets2, Fli1, GABPalpha, SAP1, Elk1, and PE1. Of these, only PE1 has extensive homology to the known Ras-regulated Ets transcriptional repressor, ERF. Therefore, we cloned and characterized PE1 cDNA from a mouse brain library and performed functional analysis of this particular Ets family member. A 2 kb transcript was isolated from brain that encodes a approximately 57 kDa protein; the predicted protein contains the known N-terminal Ets domain of PE1 and a novel C-terminal domain with signficant homology to murine ERF. The murine PE1 open reading frame (ORF) is much larger than the previously reported human PE1 ORF. Consistent with this, affinity-purified rabbit anti-mouse PE1 antibody specifically recognizes an approximately 66 kDa protein present only in the nuclear fraction of MC3T3E1 osteoblasts. Recombinant PE1 binds authentic AGGAWG Ets DNA cognates, and transient transfection studies demonstrate that PE1 represses MMP1 promoter activity. Surprisingly, although deletion of the MMP1 Ets cognate at nucleotides -88 to -83 abrogates FGF2 induction, it does not prevent suppression of the AP1-dependent MMP1 promoter by PE1. PE1 regulation maps to the MMP1 promoter region -75 to -61, suggesting that PE1 suppresses transcription via protein-protein interactions with AP1. Consistent with this, recombinant GST-PE1 specifically inhibits the formation of protein-DNA interactions on the MMP1 AP1 site (-72 to -66) when present in an admixture with MC3T3E1 crude nuclear extract. In toto, these data indicate that PE1 participates in the transcriptional regulation of the MMP1 promoter in osteoblasts. As observed with other transcriptional repressors of MMP1 gene expression, transcriptional suppression by PE1 occurs via inhibition of AP1-dependent promoter activity.

Amino Acid Sequence↗
PubMed1977

Coronary risk factors in flying personnel: a progress report.

Since October 1974, the Flight Surgeon's Office at the USAF Hospital Dover has implemented a program of early detection and treatment of coronary risk factors in aircrew personnel. The program is integrated with USAF periodic physical examinations with interval follow-up of members found to have possible risk factors. A report of initial (baseline) findings was presented at the Aerospace Medical Association meeting in May 1975. During the past year, the base did experience one death due to myocardial infarction in an aircrew member, while two others were grounded due to serial ECG changes, consistent with silent myocardial infarction, confirmed by review at the USAF-SAM ECG Library.

Adult↗
PubMed1980

A survey of career assistance programs at U.S. dental schools.

1. There is a perceived need among the leaders of our dental schools for career assistance programs for dental students. 2. Current programs and services for transmitting career assistance at a majority of dental educational institutions are either nonexistent or sporadic, and handled informally without carefully coordinated activities. 3. The most frequently used career assistance services, where programs do exist, are a library of career informational materials, formalized arrangements with outside professionals in the community, and placement services. 4. Among the variety of individuals coordinating current programs in career assistance in dental schools, the most common is a member of the administration (primarily the director of student affairs).

Career Choice↗
PubMed2001

Plate screening methods for the detection of polysaccharase-producing microorganisms.

Polysaccharide-degrading enzymes (polysaccharases) are widely applied in industry. One of the sources of these enzymes are polysaccharide-degrading microorganisms. To obtain such microorganisms from enrichment cultures, strain collections or gene libraries, efficient plate screening methods are required that discriminate between intact and degraded polysaccharide. This can be achieved by making use of specific physicochemical properties of the polysaccharide, such as complex formation with dyes and gelling capacity, or by the application of dye-labelled polysaccharides. This review presents a survey of plate methods based on these principles. Both theoretical and practical aspects of the methods are discussed.

Bacteria↗
PubMed1999

A computer-assisted personalized approach in an undergraduate plant physiology class

We used Computer-Assisted Personalized Approach (CAPA), a networked teaching and learning tool that generates computer individualized homework problem sets, in our large-enrollment introductory plant physiology course. We saw significant improvement in student examination performance with regular homework assignments, with CAPA being an effective and efficient substitute for hand-graded homework. Using CAPA, each student received a printed set of similar but individualized problems of a conceptual (qualitative) and/or quantitative nature with quality graphics. Because each set of problems is unique, students were encouraged to work together to clarify concepts but were required to do their own work for credit. Students could enter answers multiple times without penalty, and they were able to obtain immediate feedback and hints until the due date. These features increased student time on task, allowing higher course standards and student achievement in a diverse student population. CAPA handles routine tasks such as grading, recording, summarizing, and posting grades. In anonymous surveys, students indicated an overwhelming preference for homework in CAPA format, citing several features such as immediate feedback, multiple tries, and on-line accessibility as reasons for their preference. We wrote and used more than 170 problems on 17 topics in introductory plant physiology, cataloging them in a computer library for general access. Representative problems are compared and discussed.

Journal Article↗
PubMed2004

Developing methods for systematic reviewing in health services delivery and organization: an example from a review of access to health care for people with learning disabilities. Part 2. Evaluation of the literature--a practical guide.

OBJECTIVES: To develop methods to facilitate the 'systematic' review of evidence from a range of methodologies on diffuse or 'soft' topics, as exemplified by 'access to health care'. DATA SOURCES: Twenty-eight bibliographic databases, research registers, organizational websites or library catalogues. Reference lists from identified studies. Contact with experts and service users. Current awareness and contents alerting services in the area of learning disabilities. REVIEW METHODS: Inclusion criteria were English language literature from 1980 onwards, relating to people with learning disabilities of any age and all study designs. The main criteria for assessment was relevance to Guillifords' model of access to health care which was adapted to the circumstances of people with learning disabilities. Selected studies were evaluated for scientific rigour then data was extracted and the results synthesized. Quality assessment was by an initial set of 'generic' quality indicators. This enabled further evidence selection before evaluation of findings according to specific criteria for qualitative, quantitative or mixed-method studies. RESULTS: Eighty-two studies were fully evaluated. Five studies were rated 'highly rigorous', 22 'rigorous', 46 'less rigorous' and nine 'poor' papers were retained as the sole evidence covering aspects of the guiding model. The majority of studies were quantitative but used only descriptive statistics. Most evidence lacked methodological detail, which often lowered final quality ratings. CONCLUSIONS: The application of a consistent structure to quality evaluation can facilitate data appraisal, extraction and synthesis across a range of methodologies in diffuse or 'soft' topics. Synthesis can be facilitated further by using software, such as the microsoft 'access' database, for managing information.

Adult↗
PubMed2002

Random sequencing of Paramecium somatic DNA.

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.

Amino Acid Sequence↗
PubMed2006

In-center hemodialysis patients' use of the internet in the United States: a national survey.

BACKGROUND: Education is critical to help patients with chronic kidney disease self-manage their illness by thoroughly understanding their treatment options, medications, diet and fluids, and symptoms. METHODS: This study assesses in-center hemodialysis patients' use of the Internet in general and for health information seeking, both independently and by proxy-asking a family member or friend. Patients (n = 1,804) were recruited from 37 randomly selected dialysis clinics in 18 End-Stage Renal Disease Networks. Respondents completed a survey in English or Spanish. RESULTS: Across the entire sample, 34.7% had used the Internet themselves compared with 38% of disabled Americans. Internet use was more likely among patients who were younger, non-Hispanic, from the Southeast or Texas, and more highly educated. Median education level of English-reading respondents was 12th grade. Their total Internet health information use was 43.5% (24.7% independently, 18.8% by proxy). Median education level of Spanish-reading respondents was 6th grade; their total Internet health information use was only 25.5% (8.5% independently, 17% by proxy). Reasons for not using the Internet related more to not having access to a computer or knowledge (70.4%) than to lack of interest (21.3%). CONCLUSION: Alerting patients to Internet access at public libraries or providing a computer in dialysis clinic waiting rooms may help overcome this barrier. Proxy use may extend the reach of the Internet to patients who do not have access on their own.

Adolescent↗
PubMed1999

Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon).

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.

Amino Acid Sequence↗
PubMed1996

A survey of the goat genome transcribed in the lactating mammary gland.

To fulfill its primary function, which is to synthesize milk during the course of lactation, the mammary gland requires efficient transcriptional, translational, and secretory machineries involving multiple genes among which promising candidates underlying the genetic variation of milk production have to be found. With the aim of providing a first transcriptional profile of lactating mammary tissue, a non-normalized cDNA library has been constructed from the udder of a lactating goat. After having discarded cDNA clones encoding the major milk proteins the rapid characterization of genes expressed in this tissue, by automated partial cDNA sequencing, was used to analyze a total of 435 cDNA clones. Examination of the Expressed Sequence Tags (ESTs) for similarities with sequence databases identified 234 cDNAs corresponding to 140 unique genes or proteins. Eighty-three clones, not similar to any current database entries, representing 77 novel sequences unrelated to previously described genes, were thus identified. Tissue specificity and relative abundance of 18 of these 77 unidentified clones were examined by dot blot and RT-PCR experiments. Sequence data were subsequently used to assign six genes of unknown localization in the bovine genome, to synteny groups by use of bovine-hamster cell hybrids and PCR.

Animals↗
PubMed2002

Rice genome organization: the centromere and genome interactions.

Over the last decade, many varied resources have become available for genome studies in rice. These resources include over 4000 DNA markers, several bacterial artificial chromosome (BAC) libraries, P-1 derived artificial chromosome (PAC) libraries and yeast artificial chromosome (YAC) libraries (genomic DNA clones, filters and end-sequences), retrotransposon tagged lines, and many chemical and irradiated mutant lines. Based on these, high-density genetic maps, cereal comparative maps, YAC and BAC physical maps, and quantitative trait loci (QTL) maps have been constructed, and 93 % of the genome has also been sequenced. These data have revealed key features of the genetic and physical structure of the rice genome and of the evolution of cereal chromosomes. This Botanical Briefing examines aspects of how the rice genome is organized structurally, functionally and evolutionarily. Emphasis is placed on the rice centromere, which is composed of long arrays of centromere-specific repetitive sequences. Differences and similarities amongst various cereal centromeres are detailed. These indicate essential features of centromere function. Another view of various kinds of interactive relationships within and between genomes, which could play crucial roles in genome organization and evolution, is also introduced. Constructed genetic and physical maps indicate duplication of chromosomal segments and spatial association between specific chromosome regions. A genome-wide survey of interactive genetic loci has identified various reproductive barriers that may drive speciation of the rice genome. The significance of these findings in genome organization and evolution is discussed.

Centromere↗
PubMed1998

Sequence analysis of bacterial DNA in the colon of an Andean mummy.

We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th-11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths. The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved. In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon. This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present.

Cloning, Molecular↗
PubMed1992

A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.

The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2003

A novel type of two-component regulatory system affecting gingipains in Porphyromonas gingivalis.

We surveyed the Porphyromonas gingivalis W83 genome database for homologues of FimS, the first two-component sensor histidine kinase, which could possibly control virulence factors. Including fimS, we found six putative sensor kinase genes in the genome. The gene encoding one of the homologues was cloned from a P. gingivalis plasmid library, sequenced, and analyzed using its mutants. Two gene-disruption mutants were created in strain ATCC 33277 by introducing a drug cassette into the gene. The mutants formed nonpigmented colonies, indicating that they might be defective in proteinase production, a characteristic of this organism. Proteinase activities, measured as arginine- and lysine-specific (Rgp and Kgp gingipains, respectively) activities, of the mutants were almost half those of the parent strain. Unlike the parent and wildtype strains, most of the gingipain activities were detected in the culture supernatant, not in cells, of the mutants. Abnormal production of gingipains was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses. These results strongly suggest that this newly-discovered two-component sensor kinase is involved in maturation and proper localization of gingipains to the outer membrane through an unknown mechanism. The gene encoding the sensor histidine kinase was designated gppX, which represents regulation (X) of gingipains and black pigmentation in P. gingivalis.

Adhesins, Bacterial↗
PubMed1983

Bibliotherapy: an adjunct to care of patients with problems of living.

Bibliotherapy, or the selected use of reading as an adjunct to the treatment of medical and psychological problems, has a long history in the library science literature. However, the use of bibliotherapy by practicing physicians has not been significant. Many patients who see family physicians are candidates for bibliotherapy as adjunctive treatment. In the past five years numerous well-written books that address various patient problems of living have been made available. The authors surveyed a variety of health care specialists in the Denver area to develop a reading list for people with life problems. These problems have been categorized under the following headings: dealing with life crises and transitions, parents and children, parenting, coping with illness and disability, death and dying, lifestyle modification, sexuality, and coping with feelings. The resulting annotated bibliography may be used as a guide for practicing physicians who feel that appropriate reading material may be beneficial to their patients with such problems.

Adjustment Disorders↗
PubMed2006

Assessing computational amino acid beta-turn propensities with a phage-displayed combinatorial library and directed evolution.

Structure propensities of amino acids are important determinants in guiding proteins' local and global structure formation. We constructed a phage display library--a hexa-HIS tag upstream of a CXXC (X stands for any of the 20 natural amino acids) motif appending N-terminal to the minor capsid protein pIII of M13KE filamentous phage--and developed a novel directed-evolution procedure to select for amino acid sequences forming increasingly stable beta-turns in the disulfide-bridged CXXC motif. The sequences that emerged from the directed-evolution cycles were in good agreement with type II beta-turn propensities derived from surveys of known protein structures, in particular, Pro-Gly forming a type II beta-turn. The agreement strongly supported the notion that beta-turn formation plays an active role in initiating local structure folding in proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Access to the internet in an acute care area: experiences of nurses.

The internet is expected to play a key role in delivering evidence-based practice (Department of Health (DoH), 1997, 1998a; Beyea, 2000). In the UK, poor access to the internet and a lack of knowledge and confidence have been identified as barriers to nurses accessing evidence via the internet. The study described here aimed to evaluate how open access to the internet in an acute ward area had an impact upon the evidence-based practice of nurse and allied health professionals. The study was funded by the Department of Health. It incorporated a survey of over 200 staff, semi-structured interviews and monitoring of internet use. The results indicate that the ward-based internet facilities were valued and used by nurses. However, time, support and training are required for nurse to realize the full potential of the internet. Future partnerships between health science libraries and the NHS are recommended to help staff to access and use the internet in order to answer clinically derived questions.

Access to Information↗
PubMed2004

Gene expression analysis in the hippocampal formation of tree shrews chronically treated with cortisol.

Adrenal corticosteroids influence the function of the hippocampus, the brain structure in which the highest expression of glucocorticoid receptors is found. Chronic high levels of cortisol elicited by stress or through exogenous administration can cause irreversible damage and cognitive deficits. In this study, we searched for genes expressed in the hippocampal formation after chronic cortisol treatment in male tree shrews. Animals were treated orally with cortisol for 28 days. At the end of the experiments, we generated two subtractive hippocampal hybridization libraries from which we sequenced 2,246 expressed sequenced tags (ESTs) potentially regulated by cortisol. To validate this approach further, we selected some of the candidate clones to measure mRNA expression levels in hippocampus using real-time PCR. We found that 66% of the sequences tested (10 of 15) were differentially represented between cortisol-treated and control animals. The complete set of clones was subjected to a bioinformatic analysis, which allowed classification of the ESTs into four different main categories: 1) known proteins or genes (approximately 28%), 2) ESTs previously published in the database (approximately 16%), 3) novel ESTs matching only the reference human or mouse genome (approximately 5%), and 4) sequences that do not match any public database (50%). Interestingly, the last category was the most abundant. Hybridization assays revealed that several of these clones are indeed expressed in hippocampal tissue from tree shrew, human, and/or rat. Therefore, we discovered an extensive inventory of new molecular targets in the hippocampus that serves as a reference for hippocampal transcriptional responses under various conditions. Finally, a detailed analysis of the genomic localization in human and mouse genomes revealed a survey of putative novel splicing variants for several genes of the nervous system.

Animals↗