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Biomedical subjects

M R Meyer

Publications and source records attributed to M R Meyer.

At least 19 recordsLinked to original sources

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Algorithms↗

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Cells, Cultured↗

Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast.

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Delta strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4Delta mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.

Amino Acids↗

A cGMP-signaling pathway in a subset of olfactory sensory neurons.

It is well established that signal transduction in sensory neurons of the rat olfactory epithelium involves a cAMP-signaling pathway. However, a small number of olfactory neurons specifically express cGMP-signaling components, namely a guanylyl cyclase (GC-D) and a cGMP-stimulated phosphodiesterase (PDE2). Here, we show that this subset of olfactory neurons expressing GC-D and PDE2 does also express the subunit of a cGMP-selective cyclic nucleotide-gated (CNG) channel that has been previously identified in cone photoreceptors. Further, components of the prototypical cAMP-signaling pathway could not be detected in this subpopulation of cells. These results imply that these neurons use an alternative signaling pathway, with cGMP as the intracellular messenger, and that, in these cells, the receptor current is initiated by the opening of cGMP-gated channels.

Amino Acid Sequence↗

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Cell Wall↗

Reduced prevalence of AD in users of NSAIDs and H2 receptor antagonists: the Cache County study.

OBJECTIVE: To test the hypothesis that nonsteroidal anti-inflammatory drugs (NSAIDs) and histamine H2 receptor antagonists (H2RAs) are associated with a decreased risk of AD in late life. BACKGROUND: Sustained use of non-aspirin NSAIDs has been repeatedly associated with a reduced occurrence of AD. Similar effects with aspirin have been weaker. One prior study showed a strong association between use of H2RAs and reduced AD prevalence. METHODS: In a population study of AD in Cache County, UT, we used a sequenced plan of sampling and case ascertainment to identify 201 cases of AD and 4425 participants with no indication of cognitive impairment. Independently, an interview and medicine chest inventory assessed use of several medicines including aspirin, non-aspirin NSAIDs, H2RAs, and three classes of "control" drugs not thought to be associated with AD. Follow-up questioning probed possible indications for use of these drugs. RESULTS: Compared with cognitively intact individuals, the AD cases had significantly less reported current use of NSAIDs, aspirin, and H2RAs. Stronger associations appeared when subjects reported use of both NSAIDs and aspirin (no H2RAs), two different NSAIDs (no H2RAs), or two different H2RAs (with neither aspirin nor NSAIDs). There was little or no such association with use of the control medicines. Adjustment for usage indication did not influence these findings, and there was no appreciable variation with number of APOE epsilon4 alleles. CONCLUSIONS: As predicted, use of NSAIDs and aspirin were specifically associated with reduced occurrence of AD. Notably, a previous observation of an inverse association of AD and use of H2RAs was also affirmed. Definitive evidence for a preventive action of these agents will require randomized prevention trials.

Aged↗

Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Cell Cycle Proteins↗

Widespread aneuploidy revealed by DNA microarray expression profiling.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

Aneuploidy↗

Relationship of pulmonary arterial pressure to pulmonary haemorrhage in exercising horses.

Exercise-induced pulmonary haemorrhage (EIPH) is characterised by blood in the airways after strenuous exercise and results from stress failure of the pulmonary capillaries. The purpose of this experiment was to establish a threshold value of transmural pulmonary arterial pressure at which haemorrhage occurs in the exercising horse. Five geldings, age 4-14 years, were run in random order once every 2 weeks at 1 of 4 speeds (9, 11, 13, 15 m/s); one day with no run was used as a control. Heart rate, pulmonary arterial pressure and oesophageal pressure were recorded for the duration of the run. Transmural pulmonary arterial pressure was estimated by electronic subtraction of the oesophageal pressure from the intravascular pulmonary arterial pressure. Within 1 h of the run, bronchoalveolar lavage was performed and the red and white blood cells in the fluid were quantified. Red cell counts in the lavage fluid from horses running at 9, 11 and 13 m/s were not significantly different from the control value, but after runs at 15 m/s, red cell counts were significantly (P<0.05) higher. White cell counts were not different from control values at any speed. Analysis of red cell count vs. transmural pulmonary arterial pressure indicated that haemorrhage occurs at approximately 95 mmHg. Red cell lysis in the lavage fluid was also apparent at transmural pulmonary arterial pressures above 90 mmHg. We conclude that, in the exercising horse, a pulmonary arterial pressure threshold exists above which haemorrhage occurs, and that pressure is often exceeded during high speed sprint exercise.

Animals↗

APOE-epsilon4 count predicts age when prevalence of AD increases, then declines: the Cache County Study.

OBJECTIVE: To examine the prevalence of Alzheimer's disease (AD) and other dementias in relation to age, education, sex, and genotype at APOE. Recent studies suggest age heterogeneity in the risk of AD associated with the APOE genotype and a possible interaction between APOE-epsilon4 and female sex as risk factors. We studied these topics in the 5,677 elderly residents of Cache County, Utah, a population known for long life expectancy and high participation rates. METHODS: We screened for dementia with a brief cognitive test and structured telephone Dementia Questionnaire, then examined all individuals with apparent cognitive symptoms and a sample of others. We estimated age-specific prevalence of AD and other dementias and used multiple logistic regression models to describe relation of AD prevalence to age, sex, education, and APOE genotype. RESULTS: We found 335 demented individuals, 230 (69%) with definite, probable, or possible AD (positive predictive value versus autopsy confirmation 85%). The adjusted prevalence estimate for AD was 6.5% and for all dementias 9.6%. After age 90, the adjusted prevalence estimate for AD was 28% and for all dementias 38%. Regression models showed strong variation in AD prevalence with age, sex, education, and number of epsilon4 alleles (effect of epsilon2 not significant). Models were improved by a term for age-squared (negative coefficient) and by separate terms for interaction of age with presence of one or two epsilon4 alleles. An association of AD with female sex was ascribable entirely to individuals with epsilon4. CONCLUSIONS: In participants with no epsilon4 alleles, the age-specific prevalence of AD reached a maximum and then declined after age 95. In epsilon4 heterozygotes a similar maximum was noted earlier at age 87, in homozygotes at age 73. Female sex was a risk factor for AD only in those with epsilon4. The epsilon4 allele accounted for 70% of the population attributable risk for AD.

Age Distribution↗

Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development.

Rho GTPases play an important role in diverse biological processes such as actin cytoskeleton organization, gene transcription, cell cycle progression and adhesion. They are required during early Drosophila development for proper execution of morphogenetic movements of individual cells and groups of cells important for the formation of the embryonic body plan. We isolated loss-of-function mutations in the Drosophila Rho1 (Rho1) gene during a genetic screen for maternal-effect mutations, allowing us to investigate the specific roles Rho1 plays in the context of the developing organism. Here we report that Rho1 is required for many early events: loss of Rho1 function results in both maternal and embryonic phenotypes. Embryos homozygous for the Rho1 mutation exhibit a characteristic zygotic phenotype, which includes severe defects in head involution and imperfect dorsal closure. Two phenotypes are associated with reduction of maternal Rho1 activity: the actin cytoskeleton is disrupted in egg chambers, especially in the ring canals and embryos display patterning defects as a result of improper maintenance of segmentation gene expression. Despite showing imperfect dorsal closure, Rho1 does not activate downstream genes or interact genetically with members of the JNK signaling pathway, used by its relatives dRac and dCdc42 for proper dorsal closure. Consistent with its roles in regulating actin cytoskeletal organization, we find that Rho1 interacts genetically and physically with the Drosophila formin homologue, cappuccino. We also show that Rho1 interacts both genetically and physically with concertina, a G(alpha) protein involved in cell shape changes during gastrulation.

Actins↗

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Calcineurin↗

Corticotropin-releasing factor (CRF) agonists stimulate testosterone production in mouse leydig cells through CRF receptor-1.

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nM for hCRF, antagonized by the CRF antagonist alpha-helical CRF9-41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to [11-D-Thr,12-D-Phe]- and [13-D-His,14-D-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2alpha or -R2beta. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.

Adrenocorticotropic Hormone↗

Neurogenesis in adult insect mushroom bodies.

The occurrence of neurogenesis in mushroom bodies of adult insects belonging to several orthopteroid and coleopteran families is described. Using injections of 5-bromo, T2'-deoxyuridine, we showed that neuroblasts, which are progenitors of Kenyon cells during preimaginal instars, continue to divide in adult Acheta domesticus. Their progeny constitute a central column in mushroom body cortices of 3-week-old females. Other Gryllidae, Gryllus bimaculatus and Gryllomorpha dalmatina, show the same pattern of neuroblast activity and migration of their progeny. Immunocytochemical staining of glial cells failed to reveal any immunoreactivity, either in proliferating regions or in the resulting cells. In another orthopteran, Locusta migratoria, discrete clusters of cells, located dorsolateral to the Kenyon cells, incorporated 5-bromo, 2'-deoxyuridine, but we could not detect any neuronal progeny migrating to the mushroom body cortices. These cells were strongly labeled with an antiglial antibody, indicating that the replicating cells are glioblasts rather than neuroblasts. In Periplaneta americana (Dictyoptera), cells replicating their DNA were similarly shown to immunoreact with glial antibodies. In contrast, three coleopterans (Tenebrio molitor, Zophobas species, Harmonia axyridis) have two large neuroblasts located in the middle of the mushroom body cortices. These produce cells which migrate within the group of Kenyon cells, their nuclei having the same shape and size as those of surrounding Kenyon cells. In adult insects, neurogenesis in mushroom bodies occurs in Gryllidae and several coleopteran families, but could not be demonstrated in Dictyoptera and Acrididae. Its occurrence and distribution raise the issue of unexpected plasticity in the adult insect brain.

Animals↗

Long-term durability of the interface in FRP composites after exposure to simulated physiologic saline environments.

Fiber/matrix interfacial bond strength significantly influences the mechanical behavior of fiber-reinforced polymer (FRP) composites. Interfacial bond strength durability is therefore particularly important in the development of FRP composites for implant applications where diffused moisture may potentially weaken the material over time. In this study, the long-term durability of interfacial bonding in carbon fiber/380 grade polyetheretherketone (C/PEEK) and carbon fiber/polysulfone (C/PSF) composites was investigated after exposure to hygrothermal environments. A single fiber pull-out test was used to quantitatively determine the ultimate bond strength (UBS) of the samples following exposure. Samples were tested at three temperatures (37, 65, and 95 degrees C) for six time periods (0-5000 h) and in two environments (dry and physiologic saline-immersed). A mathematical model based on nth order chemical reaction kinetics was applied to describe the long-term durability of the interface. The results of this study indicate that interfacial bond strengths in C/PSF and C/PEEK (380 grade) composites are significantly decreased by exposure to physiologic saline and are functions of both time and temperature. For each material, the kinetics of degradation analysis predicts further bond strength losses following initial saturation, which then stabilizes at temperature-dependent equilibrium bond strength levels.

Benzophenones↗

Selective expression of glionexin, a glial glycoprotein, in insect mechanoreceptors.

The distribution of a glial cell-associated glycoprotein, glionexin (GX), on sensory receptors of the adult cricket Acheta domesticus is described, using the monoclonal antibody 5B12 as an immunohistochemical probe. GX was previously shown to be widely distributed in the embryo and to persist in the postembryonic to adult central nervous system. Here we demonstrate that it is restricted in the adult periphery to three subclasses of mechano-receptor sensilla: large socketed hair mechanoreceptors, their associated campaniform sensilla, and chordotonal organs. GX was not detected in photoreceptors, chemoreceptors, or other mechanoreceptors. The pattern of distribution differs significantly within the three subclasses of mechanoreceptors. In the hair and campaniform receptors GX is restricted to the extracellular space among glial cells clustered around the axon hillock region, but in chordotonal organs it surrounds the scolopidium at the tip of dendrites. The highly restricted distribution of GX in the periphery suggests possible functions that include mechanical stability of the sensory apparatus and ionic homeostasis in the respective neuronal spike-generating regions. The developmental modulation of GX expression is taken to imply multiple functions for the molecule during the life of the insect.

Animals↗

Mandating vitamin K prophylaxis for newborns in New York State.

New York State's infant deaths and hospitalizations attributed to hemorrhagic disease of the newborn and other neonatal hemorrhagic conditions were reviewed. In 65% of 34 deaths reviewed, vitamin K was not documented as given or was given only after the onset of hemorrhage. Vitamin K was not included in standing orders in any of 22 hospitals contacted. As a result of this review, vitamin K prophylaxis was made a mandatory newborn care procedure in the State Public Health Code.

Cerebral Hemorrhage↗