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Biomedical subjects

J Carey

Publications and source records attributed to J Carey.

At least 19 recordsLinked to original sources
PubMed2006

Detecting the signature of selection on immune genes in highly structured populations of wild sheep (Ovis dalli).

The confounding effects of population structure complicate efforts to identify regions of the genome under the influence of selection in natural populations. Here we test for evidence of selection in three genes involved in vertebrate immune function - the major histocompatibility complex (MHC), interferon gamma (IFNG) and natural resistance associated macrophage polymorphism (NRAMP) - in highly structured populations of wild thinhorn sheep (Ovis dalli). We examined patterns of variation at microsatellite loci linked to these gene regions and at the DNA sequence level. Simple Watterson's tests indicated balancing selection at all three gene regions. However, evidence for selection was confounded by population structure, as the Watterson's test statistics from linked markers were not outside of the range of values from unlinked and presumably neutral microsatellites. The translated coding sequences of thinhorn IFNG and NRAMP are fixed and identical to those of domestic sheep (Ovis aries). In contrast, the thinhorn MHC DRB locus shows significant evidence of overdominance through both an excess of nonsynonymous substitution and trans-species polymorphism. The failure to detect balancing selection at microsatellite loci linked to the MHC is likely the result of recombination between the markers and expressed gene regions.

Animals↗
PubMed2006

Evidence for cryptic glacial refugia from North American mountain sheep mitochondrial DNA.

The separation of populations by ice sheets into large refugia can account for much of the genetic diversity found in present day populations. The evolutionary implications of small glacial refugia have not been as thoroughly explored. To examine refugial origins of North American mountain sheep Ovis spp., we analyzed a 604 bp portion of the mitochondrial DNA (mtDNA) control region from 223 O. dalli and O. canadensis. Major refugia were identified in eastern Beringia and southern North America, and we found evidence for two smaller refugia situated between the Laurentide and Cordilleran glaciers. Our results are the first to demonstrate support for survival of any organism in the latter two refugia. These refugia also appear to have conserved a genetic signal that confirms past hybridization of O. dalli and O. canadensis.

Altitude↗
PubMed2006

Density conversion factor determined using a cone-beam computed tomography unit NewTom QR-DVT 9000.

OBJECTIVE: The purpose of this study was to determine a conversion coefficient for Hounsfield Units (HU) to material density (g cm(-3)) obtained from cone-beam computed tomography (CBCT-NewTom QR-DVT 9000) data. METHODS: Six cylindrical models of materials with different densities were made and scanned using the NewTom QR-DVT 9000 Volume Scanner. The raw data were converted into DICOM format and analysed using Merge eFilm and AMIRA to determine the HU of different areas of the models. RESULTS: There was no significant difference (P = 0.846) between the HU given by each piece of software. A linear regression was performed using the density, rho (g cm(-3)), as the dependent variable in terms of the HU (H). The regression equation obtained was rho = 0.002H-0.381 with an R2 value of 0.986. The standard error of the estimation is 27.104 HU in the case of the Hounsfield Units and 0.064 g cm(-3) in the case of density. CONCLUSION: CBCT provides an effective option for determination of material density expressed as Hounsfield Units.

Humans↗
PubMed2004

Abnormal motor behavior and vestibular dysfunction in the stargazer mouse mutant.

In stargazer mutant mice, a mutation in the gene encoding stargazin results in absence epilepsy, cerebellar ataxia, and a characteristic abnormal motor syndrome. The main goal of the current studies was to characterize the nature and source of the abnormal motor behavior. Because the stargazer motor syndrome resembles that of other rodents with vestibular dysfunction, the motor abnormalities were compared with those of normal mice treated with toxins known to damage the vestibular system. Quantitative open field assessments revealed that the stargazer mice display a motor syndrome very similar to that exhibited by mice with toxin-induced vestibulopathy. However, stargazer mice also displayed several additional behaviors, such as ataxic gait and sustained extensor movements of the neck. In addition, stargazer mice performed worse than mice with toxin-induced vestibulopathy in most standard tests of motor function. Motor function was also impaired on each of four behavioral tests sensitive to vestibular function. Because of the close associations between the vestibular and auditory systems, tests of auditory function were also employed. The stargazer mutants exhibited relatively normal auditory brainstem evoked responses but no apparent acoustic startle reflex. Histological examination of vestibular sensory epithelium at the light and electron microscopic levels confirmed the existence of abnormalities in the stargazer mutants. These results imply a previously unrecognized role for stargazin in the normal functions of the vestibular system and indicate that some, but not all, of the abnormal motor syndrome of stargazer mice can be attributed to vestibular dysfunction.

Acoustic Stimulation↗
PubMed2004

Population genetic structure of North American thinhorn sheep (Ovis dalli).

The thinhorn sheep (Ovis dalli ssp.) provides a rare example of a North American large mammal that occupies most of its native range and maintains close to ancestral population size. There are currently two recognized subspecies, Dall's sheep (O. d. dalli) and Stone's sheep (O. d. stonei), the validity of which remains uncertain. We investigated the spatial genetic structure of thinhorn sheep populations representing both subspecies by genotyping individuals (n = 919) from across the species range at 12 variable microsatellite loci. We found high levels of genetic diversity within (HE = 0.722) and significant genetic structure among the 24 sampled areas (FST = 0.160). Genetic distance measures and Bayesian clustering analyses revealed the presence of at least eight subpopulations that are delineated by mountain range topology. A strong overall pattern of isolation-by-distance is evident across the sampling range (r = 0.75, P < 0.001) suggesting limited dispersal and extensive philopatry. Partial Mantel tests of this relationship showed mountain range distinctions represent significant barriers to gene flow (P = 0.0001), supporting the Bayesian analyses. Genetic structure was more strongly pronounced in southern Yukon and Alaska than elsewhere. We also show evidence for genetic differences between the two currently recognized thinhorn subspecies.

Alaska↗
PubMed2002

5-HT(2B) receptors play a key role in mediating the excitatory effects of 5-HT in human colon in vitro.

1. 5-Hydroxytryptamine (5-HT) is known to produce a number of different effects in the gastrointestinal tract of various species, and has been proposed to play a key role in a number of intestinal disorders in man, including irritable bowel syndrome (IBS), although the receptors involved have yet to be established. The aim of the present study was to investigate the distribution and function of 5-HT(2B) receptors in human colon, and to establish their possible role in the aetiology of IBS. 2. The distribution of 5-HT(2B) receptor mRNA and protein were investigated by quantitative RT - PCR, Western analysis and immunocytochemistry. High levels of both mRNA and protein for 5-HT(2B) receptors were found throughout the human gastrointestinal tract, and in particular in colon, where 5-HT(2B) receptors were found predominantly in the longitudinal and circular smooth muscle layers within the muscularis externa, and in the myenteric nerve plexus lying between these two layers. 3. Electrical field stimulation of longitudinal muscle preparations of human colon mounted in organ baths resulted in neuronally-mediated contractile responses, that were significantly potentiated by application of 5-HT (up to 10(-7) M), with a pEC(50) of 8.2 +/- 0.1 (n=49 donors). The response to 5-HT was inhibited by a number of selective 5-HT(2B) receptor antagonists. 4. This study has shown for the first time that, in contrast to animal studies, the excitatory effects of 5-HT in human colon are mediated by 5-HT(2B) receptors. It is proposed that these receptors contribute to the putative 5-HT-induced colonic smooth muscle hypersensitivity associated with IBS.

Colon↗
PubMed2001

Quantitative analysis of DNA binding by the Escherichia coli arginine repressor.

Allosteric activation of the hexameric arginine repressor (ArgR) for specific operator DNA binding appears to involve alteration in its quaternary structure. Current models for activation include subunit assembly and/or domain rearrangements in response to binding of the coeffector l-arginine. To investigate the molecular basis for ArgR operator interactions, we have carried out a series of quantitative analyses of ArgR subunit assembly and of the affinity, stoichiometry, cooperativity, and l-arginine- and DNA sequence-dependence of ArgR-DNA binding. The results indicate that subunit assembly plays no role in activation, although communication among subunits of the ArgR hexamer is required for specific DNA binding. The data suggest that DNA is also an allosteric effector of ArgR.

Allosteric Regulation↗
PubMed2001

A functioning chimera of the cyclic nucleotide-binding domain from the bovine retinal rod ion channel and the DNA-binding domain from catabolite gene-activating protein.

The eukaryotic cyclic nucleotide-gated (CNG) ion channels are a family of large membrane proteins activated by cytoplasmic cGMP or cAMP. Their cyclic nucleotide-binding domain is structurally homologous with that of the catabolite gene-activator protein (CAP), a soluble Escherichia coli transcription factor. Differences in ligand activation among sensory channels suggest differences in the underlying molecular mechanisms of signal readout. To study the structural, functional, and conformational consequences of nucleotide binding, we fused the cyclic nucleotide-binding domain from the bovine retinal rod CNG channel alpha subunit (Bralpha) to the DNA-binding domain from CAP. The chimera forms a soluble dimer that binds both cGMP and cAMP with association constants of 3.7 x 10(4) M(-1) for [(3)H]cGMP and 3.1 x 10(4) M(-1) for [(3)H]cAMP. The binding of cAMP, but not cGMP, exposes a chymotrypsin cleavage site in the chimera at a position similar to the site in the CAP exposed by cAMP binding. At high cAMP concentrations, a biphasic pattern of cleavage is seen, suggesting that the low-affinity cAMP binding sites are also occupied. Cyclic AMP promotes specific binding to a DNA fragment encoding the lac operator region; the K(d) for the protein-DNA binding is approximately 200 nM, which is 2-fold higher than the K(d) for CAP under identical conditions. A 7 A crystal structure shows that the overall secondary and tertiary structure of Bralpha/CAP is the same as that of CAP with two cAMP molecules bound per dimer. The biochemical characterization of the chimera suggests it will be a useful system for testing hypotheses about channel activation, providing further insight into channel function.

Amino Acid Sequence↗
PubMed2001

Photosynthetic and phylogenetic primers for detection of anoxygenic phototrophs in natural environments.

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.

Anaerobiosis↗
PubMed2000

In situ compressive properties of the glenoid labrum.

The purpose of this study was to measure the structural stiffness (load/displacement response) and elastic modulus (stress-strain response) of the glenoid labrum at different locations throughout the tissue. It was intended that the results of this work would serve to define the properties of the labrum. This in turn may permit a better understanding of labral function and of the mechanics of injury or degeneration and may ultimately contribute to improving the design of future labral reconstruction procedures. In addition, these data may allow incorporation of labral properties into a glenoid arthroplasty component. The testing procedure consisted of rapid compression of the labrum using a flat indentor. Stiffness and modulus results demonstrated differences between the superior and inferior portions of the labrum. The elastic moduli findings for the labrum were 0.18 +/- 0.17, 0.11 +/- 0.16, and 0.23 +/- 0.20 MPa for the inferior anterior, inferior, and inferior posterior sections, respectively. The superior anterior, superior, and superior posterior sections were respectively 0.19 +/- 0.09, 0. 32 +/- 0.22, and 0.41 +/- 0.32 MPa. These results are similar to those of knee menisci. The modulus findings for the substrate cartilage were 1.92 +/- 0.78, 1.99 +/- 0.70, and 2.00 +/- 1.33 MPa for the inferior anterior, inferior, and inferior posterior sections, respectively. The superior anterior, superior, and superior posterior sections were respectively 1.60 +/- 0.79, 1.29 +/- 0.75, and 1.42 +/- 0.54 MPa, which are comparable to previous cartilage findings.

Arthroplasty, Replacement↗
PubMed2000

Corneal insult affects the production and distribution of FGF-2 within the lacrimal gland.

The purpose of this study was to determine the distribution of FGF-2 within rabbit lacrimal glands and to determine whether corneal insult affects that distribution. The scarified corneas of experimental animals were inoculated either with adenovirus type 5 or buffer. Control animals were either untreated, or animals whose corneas were scarified. Twenty-one days later all animals were killed and the lacrimal glands were studied by immunocytochemistry and Western blotting to detect FGF-2. In untreated control animals, FGF-2 was immunolocalized predominantly within a population of elongated cells in the basal epithelium of ducts, and to a lesser degree in the basal epithelium of the acini. The elongated immunopositive cells appear to be myoepithelial cells known to be present at these sites. Interstitial cells around ducts and acini, and the basement membranes of the ducts and acini, were also immunopositive for FGF-2. Twenty-one days after adenovirus inoculation and scarification of the cornea, immunopositivity for FGF-2 was dramatically decreased in basement membranes, but increased within myoepithelial cells of the duct epithelium. These myoepithelial cells were frequently enlarged, bulging toward the duct lumen. In animals whose corneas were inoculated with buffer and scarified, or animals whose corneas were simply scarified, the changes in the lacrimal gland were similar, but somewhat less pronounced, to those of adenovirus-inoculated animals. Western blots confirmed the presence of FGF-2 immunoreactivity in all groups. The major band in untreated controls was at 24 kD, whereas all animals with corneal scarification had major bands at 38 kD. Densitometry of Western blots demonstrated that the amount of 24 kD FGF-2 present within the lacrimal gland after corneal scarification was at least 50% less than in untreated controls, whereas 38 kD FGF-2 was at least ten-fold greater. Our findings indicate that corneal scarification results in an altered distribution of FGF-2 within the lacrimal gland, which involves a decrease in low molecular weight FGF-2 and a dramatic increase in a higher molecular weight isoform of FGF-2. FGF-2 may be released from myoepithelial cells apically (exocrine) into the tear fluid and basally (autocrine/paracrine) into the connective tissue, as well as from extracellular complexes within basal laminae.

Adenoviridae Infections↗
PubMed2000

Distribution of growth factors and immune cells are altered in the lacrimal gland during pregnancy and lactation.

We have undertaken a series of studies to elucidate the roles of growth factors (FGF-2, EGF, TGF-beta1) and prolactin (PRL) in lacrimal gland function during pregnancy and lactation, and to better understand the status of the immune system within the lacrimal gland during those physiological states. In this initial study, lacrimal glands of pregnant (d15, d29), lactating (9d, 22d), and adult female control rabbits, were evaluated by immunohistochemistry, Western blotting and image analysis. In control rabbits EGF, TGF-beta1, and PRL, were immunolocalized primarily in the apical cytoplasm of intralobular ductal epithelial cells, and acini demonstrated a basement membrane-associated immunopositivity for TGF-beta1. FGF-2 immunolocalized in myoepithelial cells in the basal ductal epithelium and complexed to the basement membrane enclosing ducts and acini. Cells immunopositive for immune cell markers (RTLA and CD18) were apparent primarily around interlobular ducts. In d29 pregnant rabbits immunopositivity for EGF and TGF-beta1 was increased within intralobular ducts, both apically and basally, and within some interlobular ductal epithelial cells. Immunopositivity for PRL was strongest in d29 pregnant rabbits within the apical and basal cytoplasm of intralobular ductal epithelial cells. Immunopositivity for FGF-2 in myoepithelial cells was strong in d15 and d29 pregnant rabbits, although basement membrane-associated immunopositivity around acini was often decreased. Immunostaining for EGF and TGF-beta1 in lactating rabbits was similar to that in d29 pregnant rabbits, although basement membrane-associated immunopositivity around acini was more comparable to controls. By 22d lactation immunopositivity for FGF-2 closely resembled that in controls. Image analysis of pregnant and lactating rabbits demonstrated that cells immunopositive for RTLA and CD18 were less abundant around ducts and more abundant between acini, although in 22d lactating rabbits the size of periductal foci was increased to nearly that of controls. Western blots correlated well with the immunohistochemistry. Our findings demonstrate that pregnancy and lactation are accompanied by a shift in the distributions of growth factors and PRL, suggestive of increased release both apically into the lacrimal fluid and basally into the interstitium. Additional shifts in the distributions of cells of the immune system from periductal foci to interacinar sites suggest that there is a recruitment of immune cells away from ducts and toward the connective tissue interstitium surrounding the acini, possibly as part of a heightened state of immune readiness during pregnancy and lactation.

Animals↗
PubMed2000

Anticipation of oxidative damage decelerates aging in virgin female medflies: hypothesis tested by statistical modeling.

Empirical analysis of survival data obtained from large samples of Mediterranean fruit flies shows that the trajectory of the mortality rate for virgin females departs from that for females maintained in mixed sex cages. It increases, decelerates, reaches its maximum, declines and then increases again within the reproductive interval. Non-virgin females, however, display an early-age plateau instead of this dip. We assume that these deviations are produced by the interplay between changes in oxygen consumption associated with reproductive behavior and the antioxidant defense that acts against anticipated oxidative damage caused by reproduction. Since there are no data on antioxidant mechanisms in medflies available that explain the observed patterns of mortality, we develop a model of physiological aging based on oxidative stress theory, which describes age-related changes in oxygen consumption and in antioxidative capacity during the reproductive period. Using this model, we simulate virtual populations of 25,000 virgin and non-virgin flies, calculate the respective mortality rates and show that they practically coincide with those of experimental populations. We show that the hypothesis about the biological support of reproduction used in our model does not contradict experimental data. The model explains how the early-age dip and plateau might arise in the mortality rates of female medflies and why the male mortality pattern does not exhibit such deviations.

Aging↗
PubMed2000

Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques.

PURPOSE: Activation of muscarinic receptors has been implicated in an increased risk of cataract after anticholinesterase treatment for glaucoma. The purpose of the present study was to determine the acetylcholine muscarinic receptor subtype(s) present in native human lens epithelial cells (NHLECs) and a human lens cell line, HLE-B3, and to compare the distribution in other ocular cells. METHODS: Human lens cells were perfused with artificial aqueous humor (35 degrees C) after fura-2 incorporation, and calcium levels were measured using a fluorometric single-cell digital imaging system. Acetylcholine was the primary muscarinic agonist, and the receptor subtypes were elucidated by determining the relative effectiveness of pirenzepine and AF-DX 384 in blocking the agonist-induced response. The levels of expression of mRNA for the receptor subtypes M1 through M5 were determined by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) using a sequence detection system (ABI Prism 7700; Perkin-Elmer, Foster City, CA). This was performed using total RNA extracted from native lens, retina, iris, and sclera and also cultured lens cells. RESULTS: Acetylcholine induced a similar concentration-dependent increase in peak-amplitude cytosolic calcium in the range 100 nM to 100 microM in both native and HLE-B3 cells. However, the kinetics of the response waveforms to 30-second pulses of acetylcholine were different in the two cell types. At higher concentrations (> 1 microM), a second phase appeared in the HLE-B3 cells that was absent in the NHLEC response. The 50% inhibitory concentration (IC50) values for blockade of a 1 microM acetylcholine response by pirenzepine and AF-DX 384 were 30 nM and 230 nM, respectively, for NHLECs, and 300 nM and 92 nM, respectively, for HLE-B3 cells. The QRT-PCR data showed that more than 90% of the total muscarinic receptor mRNA from NHLEC was of M1 origin. In the HLE-B3 cells, however, more than 95% of the mRNA was of M3 origin. mRNA for M3 was also in greatest abundance in other eye tissues, although there was a significant contribution from M1 in iris and sclera. CONCLUSIONS: Both NHLECs and HLE-B3 cells express muscarinic receptors that produce significant changes in cytosolic calcium in response to acetylcholine. Both pharmacologic and QRT-PCR evidence shows that whereas the M1 subtype predominates in NHLECs, M3 is the major contributor in HLE-B3 cells. In all other eye tissues, M3 appears to be the major contributor. These data should be taken into account when choosing particular models to investigate cataract mechanisms and also when designing muscarinic agonists to treat glaucoma.

Acetylcholine↗