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Biomedical subjects

J Crabtree

Publications and source records attributed to J Crabtree.

At least 19 recordsLinked to original sources

Plasma leptin concentration in fetal sheep during late gestation: ontogeny and effect of glucocorticoids.

The ontogeny and developmental control of plasma leptin concentration in the fetus are poorly understood. The present study investigated plasma leptin concentration in chronically catheterized sheep fetuses near term, and in neonatal and adult sheep. The effect of glucocorticoids on plasma leptin in utero was examined by fetal adrenalectomy and exogenous cortisol or dexamethasone infusion. In intact, untreated fetuses studied between 130 and 140 d (term, 145 +/- 2 d), plasma leptin concentration increased in association with the prepartum cortisol surge. Positive relationships were observed between plasma leptin in utero and both gestational age and plasma cortisol. Plasma leptin was also inversely correlated with fetal p(a)O(2). The ontogenic rise in plasma leptin was abolished by fetal adrenalectomy. In intact fetuses at 123-127 d, plasma leptin was increased by infusions of cortisol (3-5 mg kg(-1)d(-1), +127 +/- 21%) for 5 d and dexamethasone (45-60 microg kg(-1)d(-1), +268 +/- 61%) for 2 d. However, the cortisol-induced rise in plasma leptin was transient; by the fifth day of infusion, plasma leptin was restored to within the baseline range. These findings show that, in the sheep fetus, an intact adrenal gland is required for the normal ontogenic rise in plasma leptin near term. Furthermore, fetal treatment with exogenous and endogenous glucocorticoids increases circulating leptin concentration in utero.

Adrenalectomy↗

Leptin secretion to both the maternal and fetal circulation in the ex vivo perfused human term placenta.

The contribution of placental leptin, if any, to both the fetal and maternal circulation and its role in pregnancy remains to be determined. In an experiment to investigate this, 27 placentae from term pregnancies were perfused ex vivo (gestational age=39.5 s.d. 1.2; range=38-42 weeks: fetal weight=3285 s.d. 482; range=2480-4420; birthweight centile range=4th to the 98th) at both the maternal and fetal interface. Placental leptin was exported into both the maternal and fetal circulations. The log leptin production by the maternal side of the placenta was significantly greater (P=0.001) than that for the fetal side (5.193 s.d.1.049 versus 4.387 s.d. 0.768 ng/placenta/min). There was no significant relationship between maternal and fetal log leptin production and maternal body mass index, birthweight, birthweight centile, ponderal index or gestational age or with cord blood pO(2), pCO(2) and pH. There was however, a significant increase in the maternal log leptin production with increasing fetal to placental weight ratio (P=0.017; r(2)=20.7 per cent) but no corresponding relationship for fetal leptin production. It is proposed that such a mechanism would allow the placenta to modulate fat supply to the fetus in response to the fetal demand relative to placental supply.

Body Mass Index↗

A relational schema for both array-based and SAGE gene expression experiments.

MOTIVATION AND RESULTS: A relational schema is described for capturing highly parallel gene expression experiments using different technologies. This schema grew out of efforts to build a database for collaborators working on different biological systems and using different types of platforms in their gene expression experiments as well as different types of image quantification software. The tables are conceptually organized into three categories of information: Platform, Experiment (which includes image scanning and quantification), and Data. The strengths of the schema are: (i) integrating information on array elements using a gene index; (ii) describing samples using ontologies; (iii) reducing an experiment to a single RNA source for precise descriptions yet not losing the relationships between experiments done at the same time or for the same project; and (iv) maintaining both raw and processed (e.g. cleansed and normalized) data and recording how the data is processed. The result is a novel schema, which can hold both array and non-array data, is extensible for detailed experimental descriptions that are precise and consistent, and allows for meaningful comparisons of genes between experiments.

Databases, Factual↗

High-resolution BAC-based map of the central portion of mouse chromosome 5.

The current strategy for sequencing the mouse genome involves the combination of a whole-genome shotgun approach with clone-based sequencing. High-resolution physical maps will provide a foundation for assembling contiguous segments of sequence. We have established a bacterial artificial chromosome (BAC)-based map of a 5-Mb region on mouse Chromosome 5, encompassing three gene families: receptor tyrosine kinases (PdgfraKit-Kdr), nonreceptor protein-tyrosine type kinases (Tec-Txk), and type-A receptors for the neurotransmitter GABA (Gabra2, Gabrb1, Gabrg1, and Gabra4). The construction of a BAC contig was initiated by hybridization screening the C57BL/6J (RPCI-23) BAC library, using known genes and sequence tagged sites (STSs). Additional overlapping clones were identified by searching the database of available restriction fingerprints for the RPCI-23 and RPCI-24 libraries. This effort resulted in the selection of >600 BAC clones, 251 kb of BAC-end sequences, and the placement of 40 known and/or predicted genes within this 5-Mb region. We use this high-resolution map to illustrate the integration of the BAC fingerprint map with a radiation-hybrid map via assembled expressed sequence tags (ESTs). From annotation of three representative BAC clones we demonstrate that up to 98% of the draft sequence for each contig could be ordered and oriented using known genes, BAC ends, consensus sequences for transcript assemblies, and comparisons with orthologous human sequence. For functional studies, annotation of sequence fragments as they are assembled into 50-200-kb stretches will be remarkably valuable.

Animals↗

bioWidgets: data interaction components for genomics.

MOTIVATION: The presentation of genomics data in a perspicuous visual format is critical for its rapid interpretation and validation. Relatively few public database developers have the resources to implement sophisticated front-end user interfaces themselves. Accordingly, these developers would benefit from a reusable toolkit of user interface and data visualization components. RESULTS: We have designed the bioWidget toolkit as a set of JavaBean components. It includes a wide array of user interface components and defines an architecture for assembling applications. The toolkit is founded on established software engineering design patterns and principles, including componentry, Model-View-Controller, factored models and schema neutrality. As a proof of concept, we have used the bioWidget toolkit to create three extendible applications: AnnotView, BlastView and AlignView.

Amino Acid Sequence↗

Multiple endocrine neoplasia type 1: clinical and genetic features of the hereditary endocrine neoplasias.

MEN1 is a syndrome of parathyroid adenomas, gastrinomas, prolactinomas, and other endocrine tumors. Collagenomas and facial angiofibromas are newly recognized but common skin expressions. Many tumors in MEN1 are benign; however, many entero-pancreatic neuroendocrine tumors and foregut carcinoid tumors are malignant. MEN1 is thus the expression of a cancer gene but without available prevention or cure for malignancy. Hereditary (as compared to sporadic) endocrine tumors show early onset age and multiplicity, because each cell of the body has "one hit" by inheritance. Multiple neoplasia syndromes with endocrine tumor(s) all include nonendocrine components; their known defective genes seem mainly to disturb cell accumulation. Hereditary neoplasia/hyperplasia of one endocrine tissue reflects a defect that is tissue selective and directed at cell secretion. Though the hereditary endocrine neoplasias are rare, most of their identified genes also contribute to common sporadic endocrine neoplasms. Hereditary tumors may be caused by activation of an oncogene (e.g., RET) or, more often, by inactivation of a tumor suppressor gene (e.g., P53, MEN1). Recently, MEN1 was identified by positional cloning. This strategy included narrowing the gene candidate interval, identifying many or all genes in that interval, and testing the newly identified candidate genes for mutation in MEN1 cases. MEN1 was identified because it showed mutation in 14 of 15 MEN1 cases. NIH testing showed germline MEN1 mutations in 47 of 50 MEN1 index cases and in seven of eight cases with sporadic MEN1. Despite proven capacity to find germline MEN1 mutation, NIH testing found no MEN1 mutation among five families with isolated hyperparathyroidism, suggesting that this often arises from mutation of other gene(s). Analogous studies in Japan found that familial isolated pituitary tumors also did not show MEN1 germline mutation. MEN1 mutation testing can now be considered for cases of MEN1 and its phenocopies and for asymptomatic members of families with known MEN1 mutation. Germline MEN1 testing does not have the urgency of RET testing in MEN2a and 2b, as MEN1 testing does not commonly lead to an important intervention. Somatic MEN1 mutation was found in sporadic tumors: parathyroid adenoma (21%), gastrinoma (33%), insulinoma (17%), and bronchial carcinoid (36%). For each of these, MEN1 was the known gene most frequently mutated. MEN1 has a widely expressed mRNA that encodes a protein (menin) of 610 amino acids. The protein sequence is not informative about domains or functions. The protein was mainly nuclear. Menin binds to JunD, an AP-1 transcription factor, inhibiting JunD's activation of transcription. Most of the germline and somatic MEN1 mutations predict truncation of menin, a likely destructive change. Inactivating MEN1 mutations in germline and in sporadic neoplasms support prior predictions that MEN1 is a tumor suppressor gene. Germline MEN1 mutation underlies all or most cases of MEN1 (familial or sporadic). Somatic MEN1 mutation is the most common gene mutation in many sporadic endocrine tumor types.

Amino Acid Sequence↗

GAIA: framework annotation of genomic sequence.

As increasing amounts of genomic sequence from many organisms become available, and as DNA sequences become a primary reagent in biologic investigations, the role of annotation as a prospective guide for laboratory experiments will expand rapidly. Here we describe a process of high-throughput, reliable annotation, called framework annotation, which is designed to provide a foundation for initial biologic characterization of previously unexamined sequence. To examine this concept in practice, we have constructed Genome Annotation and Information Analysis (GAIA), a prototype software architecture that implements several elements important for framework annotation. The center of GAIA consists of an annotation database and the associated data management subsystem that forms the software bus along which other components communicate. The schema for this database defines three principal concepts: (1) Entries, consisting of sequence and associated historical data; (2) Features, comprising information of biologic interest; and (3) Experiments, describing the evidence that supports Features. The database permits tracking of annotation results over time, as well as assessment of the reliability of particular results. New framework annotation is produced by CARTA, a set of autonomous sensors that perform automatic analyses and assert results into the annotation database. These results are available via a Web-based query interface that uses graphical Java applets as well as text-based HTML pages to display data at different levels of resolution and permit interactive exploration of annotation. We present results for initial application of framework annotation to a set of test sequences, demonstrating its effectiveness in providing a starting point for biologic investigation, and discuss ways in which the current prototype can be improved. The prototype is available for public use and comment at http://www.cbil.upenn.edu/gaia.

Amino Acid Sequence↗

Managed care in rural Minnesota. Family physicians' attitudes and perceptions.

Prepaid managed care medicine has become dominant in urban Minnesota and is making its way into the rural setting. This study assesses the attitudes of rural family practice physicians in Minnesota toward managed care. A survey, consisting primarily of five-point Likert scale statements, was mailed to 798 rural Minnesota family practice physicians, with a response rate of 35% (281 respondents). We tabulated overall responses and made comparisons based on practice characteristics and years in practice. Twenty physicians participated in a follow-up telephone interview. We also conducted telephone interviews with 10 representatives from managed care organizations. Both positive and negative attitudes toward managed care emerged. Two-thirds of respondents did not feel that their time with patients was diminished under managed care. However, 67% of respondents felt that managed care organizations had failed to incorporate rural patients' specific needs into their policies. Only 7% of respondents felt that managed care organizations adequately explained their benefits packages to enrollees. Rural family practitioners' apparent disillusionment with current managed care models merits the attention of those concerned with medical care in rural areas.

Attitude of Health Personnel↗

The GAIA software framework for genome annotation.

We describe a software framework, GAIA, that supports semi-automated annotation of uncharacterized sequence data. The annotation framework incorporates annotation by data source integration, data analysis, and manual data entry. Components of the system include a configurable, open data analysis pipeline, a relational information storage manager, and Java-based graphical user interfaces. We discuss design decisions and tradeoffs in building such a system, and policies and strategies for producing consistent, uniform, high quality annotation.

Base Sequence↗

Sequestration of p56(lck) by gp120, a model for TCR desensitization.

Ligation of the CD4 receptor by HIV envelope glycoprotein gp120 inhibits T cell activation and signaling through the TCR complex. Recent reports suggest CD4 ligation by gp120 + anti-gp120 Abs uncouples protein tyrosine kinases (PTKs) from the TCR signal-transduction cascade. This finding and other observations led us to hypothesize that the effects of gp120 are mediated through p56(lck), a PTK noncovalently associated with CD4. To test this hypothesis, we first examined the kinetics of gp120/anti-gp120-induced TCR signaling defects in the Jurkat T cell line. Pretreating cells with gp120/anti-gp120 for 1 to 4 h before stimulation prevented TCR-directed PTK activation. Coincident with TCR desensitization, pretreatment with gp120/anti-gp120 also decreased the amount of p56(lck) that could be immunoprecipitated from the Nonidet P-40 detergent-soluble fraction of cellular lysates, while simultaneously increasing the recovery of p56(lck) from the Nonidet P-40 detergent-insoluble fraction (or cytoskeleton). To assess the potential role of the actin in this process, experiments were conducted in the presence of cytochalasin D. Cytochalasin D restored TCR signaling in cells previously desensitized with gp120/anti-gp120 and prevented translocation of p56lck from the Nonidet P-40 detergent-soluble fraction of cell lysates. Furthermore, p56(lck) was found to coimmunoprecipitate with anti-actin. These data suggest that gp120/anti-gp120 may inhibit TCR signaling by sequestering p56(lck) to the cytoskeleton.

Actins↗

Lovastatin inhibits T-cell antigen receptor signaling independent of its effects on ras.

Lovastatin, a cholesterol-lowering drug, has antiproliferative properties that may be related to its inhibition of protein isoprenylation. We examined the effects of lovastatin on signal transduction via the T-cell antigen receptor (TCR). Lovastatin inhibited both proximal and distal TCR-mediated signaling events in a time- and concentration-dependent manner in the human Jurkat T-cell line. Upregulation of CD69 surface expression after TCR stimulation was blocked by lovastatin, although no inhibition of phorbol ester-induced CD69 expression was noted. Proximal TCR-mediated signaling events, including intracellular calcium mobilization, inositol phosphate production, and tyrosine phosphorylation of phospholipase Cgamma1, were similarly inhibited by lovastatin, although global protein tyrosine kinase activity remained intact. In a Jurkat variant transfected with the human type-1 muscarinic receptor, lovastatin also inhibited TCR-mediated calcium mobilization and inositol phosphate production but failed to affect muscarinic receptor-induced responses. Lovastatin, at similar doses, also disrupted post-translational processing of ras and inhibited ras-dependent signals, including phosphorylation and activation of mitogen-associated protein kinase after TCR stimulation. These findings suggest that the antiproliferative properties of lovastatin may be independent of ras and could result from uncoupling protein tyrosine kinases from distinct signal transduction pathways.

Antigens, CD↗

Sequence and analysis of the human ABL gene, the BCR gene, and regions involved in the Philadelphia chromosomal translocation.

The complete human BCR gene (152-141 nt) on chromosome 22 and greater than 80% of the human ABL gene (179-512 nt) on chromosome 9 have been sequenced from mapped cosmid and plasmid clones via a shotgun strategy. Because these two chromosomes are translocated with breakpoints within the BCR and ABL genes in Philadelphia chromosome-positive leukemias, knowledge of these sequences also might provide insight into the validity of various theories of chromosomal rearrangements. Comparison of these genes with their cDNA sequences reveal the positions of 23 BCR exons and putative alternative BCR first and second exons, as well as the common ABL exons 2-11, respectively. Additionally, these regions include the alternative ABL first exons 1b and 1a, a new gene 5' to the first ABL exon, and an open reading frame with homology to an EST within the BCR fourth intron. Further analysis reveals an Alu homology of 38.83 and 39.35% for the BCR and ABL genes, respectively, with other repeat elements present to a lesser extent. Four new Philadelphia chromosome translocation breakpoints from chronic myelogenous leukemia patients also were sequenced, and the positions of these and several other previously sequenced breakpoints now have been mapped precisely, although no consistent breakpoint features immediately were apparent. Comparative analysis of genomic sequences encompassing the murine homologues to the human ABL exons 1b and 1a, as well as regions encompassing the ABL exons 2 and 3, reveals that although there is a high degree of homology in their corresponding exons and promoter regions, these two vertebrate species show a striking lack of homology outside these regions.

Animals↗

The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin.

The gene for Clostridium septicum alpha-toxin was cloned and expressed in Escherichia coli from C. septicum BX96. The toxin was determined to be 443 amino acids in length, with a 31-residue signal peptide that was removed from the toxin during secretion. No extended hydrophobic regions were observed in the mature toxin sequence. Expression of alpha-toxin in E. coli BL21 resulted in the production of ATpro, which was identical to native toxin from C. septicum with respect to activity and activation. The proteolytic activation site for alpha-toxin was determined to be on the carboxy-terminal side of arginine 398, which lies within the sequence KKRRGKR-398SVD. Previous work showing similarities in activation and mechanism between alpha-toxin and Aeromonas hydrophila aerolysin was extended to the primary structures of both toxins. The DNA-derived primary sequence of alpha-toxin exhibited 27% identity and 72% similarity over a 387-residue region with the primary structure of the A. hydrophila aerolysin toxin, a level of similarity heretofore unobserved between toxins produced by a gram-positive organism and a gram-negative organism.

Aeromonas hydrophila↗

The association between CD45 and lck does not require CD4 or CD8 and is independent of T cell receptor stimulation.

CD45, the major transmembrane tyrosine phosphatase of lymphoid cells, is required for optimal signaling via a number of receptors. A model for how CD45 regulates signaling is that it controls phosphorylation of the COOH-terminal tyrosine of src family kinases. We have shown that CD45 physically associates with lck, one src kinase. Others have shown that CD45 also interacts with the CD4 and CD8 surface antigens expressed on many T cells. In this report we examine further the relationship between CD45 and lck in a CD4+ T cell line and in peripheral T cells. We show now that CD45 associates with lck independently of both CD4 and CD8. We show also the time course of an association between CD45 and a form of lck that migrates at an apparent higher molecular mass. Finally, we demonstrate that the interaction between CD45, lck, and a previously reported 32-34 kD protein is stable after stimulation of T cells.

Antigens, CD↗

Tumour necrosis factor does not increase during routine cuprophane haemodialysis in healthy well-nourished patients.

Serum tumour necrosis factor (TNF) was measured by both bioassay and immunoassay (ELISA) during routine cuprophane acetate haemodialysis in 17 asymptomatic patients. In 14 (82%) there was no change in the serum values during haemodialysis. TNF was found to increase, using both assays, in three patients, the responders. These patients differed from the others in terms of body mass index, mean index 16.8 kg/m2 (range 14.8-18.2), compared to the non-responders, mean 24.1 kg/m2 (range 19.6-33.1), P less than 0.05, and had an increased serum calcium, mean 2.9 mmol/l (range 2.6-3.2) compared to the non-responders, mean 2.4 mmol/l (range 1.7-2.8), P less than 0.05. Two of the TNF responders subsequently died of cachexia and respiratory infection. The third underwent a successful parathyroidectomy, and when retested after an increase in body-weight with a normal serum calcium concentration no longer showed an increase in TNF during haemodialysis.

Adult↗

Death during recreational exercise in the State of Rhode Island.

From January 1, 1975 to May 1, 1982, 81 individuals died during or immediately after recreational exercise in the State of Rhode Island. Deaths occurred during a variety of activities, but the majority of deaths occurred during golf (23%), jogging (20%), and swimming (11%). Atherosclerotic coronary heart disease (ASHD) was the presumed cause of 88% of the deaths, primarily in subjects over age 29 with known cardiac abnormalities. Only 7% of ASHD victims had no relevant medical history or ASHD risk factors and were considered healthy by their families and physicians. In contrast, deaths in young subjects were rarely associated with ASHD or prior knowledge of cardiovascular disease. Only six deaths in individuals aged 29 or younger occurred during the study period. These deaths were associated with congenital cardiovascular disease (N = 2), valvular heart disease (N = 1), hemorrhagic gastritis (N = 1), idiopathic myocardial hypertrophy (N = 1), and hypertrophic cardiomyopathy with ASHD (N = 1). A diagnosis was made before death only in the individual with valvular disease. We conclude that death during recreational exercise is predominantly due to ASHD and occurs in men with recognized ASHD risk factors, relevant medical histories, or known disease. Death during exercise in asymptomatic subjects is rare and relatively more frequent in younger age groups.

Adolescent↗