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Biomedical subjects

A Castle

Publications and source records attributed to A Castle.

12 recordsLinked to original sources
PubMed2002

Quantification of microcystin-producing cyanobacteria and E. coli in water by 5'-nuclease PCR.

AIMS: 5'-Nuclease (real-time, quantitative) PCR methodologies were developed and applied as diagnostic tools for the detection of microcystin-producing cyanobacteria and Escherichia coli in water. METHODS AND RESULTS: PCR was used to detect regions of the lacZ gene in E. coli, and the microcystin synthetase gene in microcystin-producing cyanobacteria. In environmental water samples, natural inhibitors to PCR were effectively removed with a prefiltration step and an EDTA wash. A lower detection limit of 10 cells ml(-1) was obtained with endpoint PCR detection. 5'-Nuclease PCR was used for microbial quantification of 1 ml inoculated water samples. We were able to detect down to three copies of our target genes per sample within about 2 h (post-DNA isolation) for both E. coli and microcystin-producing cyanobacteria. CONCLUSIONS: 5'-Nuclease PCR offers a rapid and sensitive method of bacterial quantification in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 5'-Nuclease PCR can be adopted as an effective diagnostic tool for monitoring microbiological water quality, through coliform quantification, and detection of other waterborne microbial pathogens.

Cyanobacteria↗
PubMed2001

Attenuation of insulin resistance by chronic beta2-adrenergic agonist treatment possible muscle specific contributions.

A possible mechanism by which chronic clenbuterol treatment causes multiple physiological changes in skeletal muscle that leads to reduced insulin resistance in the obese Zucker rat (falfa) was investigated. Animals were gavaged with clenbuterol (CB) (0.8 mg x kg(-1) day(-1)), terbutaline (TB) (1.0 mg x kg(-1)day(-1)), or control (CT) vehicle for six weeks. Oral glucose tolerance and insulin responses were markedly improved in CB rats and impaired in TB rats. CB treatment caused a 24-34% gain in muscle mass in all muscle fiber types, and increases in 3-O-methyglucose transport (2-fold) and GLUT4 concentration (57%) in fast twitch glycolytic (FG) muscle. Oxidative capacity was reduced in both FG (47%) and fast twitch oxidative (FO) muscle (30%), but not in slow twitch oxidative (SO) muscle. Null model analysis for receptor occlusion demonstrated that most functional beta-adrenoceptors were lost in FO (82%) and FG (89%) fibers, but not in SO fibers. We propose that hypertrophy is the result of continuous direct activation of beta-adrenoceptors while loss in oxidative capacity may be the result of receptor down regulation. Improvements in insulin resistance may have been due, in part, to both increases in lean body mass and specific adaptations in FG muscle.

3-O-Methylglucose↗
PubMed2001

Increased insulin sensitivity in Gsalpha knockout mice.

The stimulatory guanine nucleotide-binding protein (G(s)) is required for hormone-stimulated cAMP generation. Gnas, the gene encoding the G(s) alpha-subunit, is imprinted, and targeted disruption of this gene in mice leads to distinct phenotypes in heterozygotes depending on whether the maternal (m-/+) or paternal (+/p-) allele is mutated. Notably, m-/+ mice become obese, whereas +/p- mice are thinner than normal. In this study we show that despite these opposite changes in energy metabolism, both m-/+ and +/p- mice have greater sensitivity to insulin, with low to normal fasting glucose levels, low fasting insulin levels, improved glucose tolerance, and exaggerated hypoglycemic response to administered insulin. The combination of increased insulin sensitivity with obesity in m-/+ mice is unusual, because obesity is typically associated with insulin resistance. In skeletal muscles isolated from both m-/+ and +/p- mice, the basal rate of 2-deoxyglucose uptake was normal, whereas the rate of 2-deoxyglucose uptake in response to maximal insulin stimulation was significantly increased. The similar changes in muscle sensitivity to insulin in m-/+ and +/p- mice may reflect the fact that muscle G(s)alpha expression is reduced by approximately 50% in both groups of mice. GLUT4 expression is unaffected in muscles from +/p- mice. Increased responsiveness to insulin is therefore the result of altered insulin signaling and/or GLUT4 translocation. This is the first direct demonstration in a genetically altered in vivo model that G(s)-coupled pathways negatively regulate insulin signaling.

Animals↗
PubMed2000

Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein.

Phosphorylation of many secreted salivary proteins is necessary for their biological functions. Identification of the kinase, which is responsible for in vivo phosphorylation, is complicated, because several of the protein phosphorylation sites conform both to the recognition sequence of casein kinase 2 (CK2) and Golgi kinase (G-CK), which both are found in the secretory pathway. This study was undertaken to determine the kinase recognition sequence in a secreted proline-rich salivary protein, PRP1, and thereby identify the responsible kinase. This was done by transfecting a human submandibular cell line, HSG, and a kidney cell line, HEK293, with expression vectors encoding wild-type or mutated PRP1. It was shown that phosphorylation occurred only at the same sites, Ser8 and 22, as in PRP1 purified from saliva. Phosphorylation at either site did not depend on the other site being phosphorylated. The sequence surrounding Ser8 has characteristics of both CK2 and G-CK recognition sequences, but destruction of the CK2 recognition site had no effect on phosphorylation, whereas no phosphorylation occurred if the G-CK recognition sequence was altered. The sequence surrounding Ser22 did not conform to any known kinase recognition sites. If Ser22 was mutated to Thr, no phosphorylation was seen, and a cluster of negatively charged residues at positions 27-29 was identified as part of the enzyme recognition site. Ser22 may be phosphorylated by a G-CK that recognizes an atypical substrate sequence or by a novel kinase. No difference in phosphorylation was seen between undifferentiated and differentiated HSG cells.

Amino Acid Sequence↗
PubMed1998

Morphological and molecular identification of Trichoderma isolates on North American mushroom farms.

Green mold disease (causal agent, Trichoderma) has resulted in severe crop losses on mushroom farms worldwide in recent years. We analyzed 160 isolates of Trichoderma from mushroom farms for morphological, cultural, and molecular characteristics and classified these isolates into phenotypic groups. The most common group comprised approximately 40% of the isolates and was identified as a strain of Trichoderma harzianum. This group was consistently recovered from farms with severe green mold disease but not from farms with little or no problem. In addition, the strain identified as the major cause of green mold disease in Ireland and the United Kingdom grouped with these North American isolates in having very similar randomly amplified polymorphic DNA patterns.

Basidiomycota↗
PubMed1998

Intracellular transport and secretion of salivary proteins.

Intracellular transport and secretion of salivary proteins are major activities of salivary acinar cells. While the major intracellular pathway followed by salivary proteins following their synthesis has been described previously, there is only limited understanding of how this process is regulated at the molecular level. Studies of salivary proteins, especially proline-rich proteins, expressed in an endocrine cell line have begun to provide insight regarding intermolecular interactions during transport and the role played by structural signals during intracellular sorting. Analysis of the secretion of newly synthesized salivary proteins in parotid tissue has shown that there are multiple pathways of discharge from acinar cells. While granule exocytosis is the major pathway, at least two other pathways that export salivary proteins have been found to originate from maturing secretion granules. These pathways may contribute to other acinar cell functions, including secretion of proteins in the absence of acute stimulation and support of the secretory process for fluid and electrolytes.

Animals↗
PubMed1993

"High-tech" home care for children with chronic health conditions: a pilot study.

The purposes of this study were to pilot test the instrument and methods for a major study of caregivers of children who are technology dependent and to test constructs proposed for the major study in a discrete sample. The major study will be implemented in 13 cities in the United States. The pilot was conducted in three small- to moderate-size metropolitan areas in the Southeast and Midwest. Seventy-three primary caregivers provided data during a structured telephone interviews. The children represented four groups of children dependent on technology as defined by the Office of Technology Assessment (OTA) (1987). The findings supported the methodology and the viability of the instrument. Analysis suggests that caring for a child who is technology dependent affects family functioning and increases family stress levels in some families. The financial burden is also significant. It is suggested that nurses can be a critical source of support and information to these families.

Adaptation, Psychological↗
PubMed1992

Transmission of mitochondrial DNA in Ustilago violacea.

Mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were used as genetic markers for following mitochondrial transmission in the basidiomycete Ustilago violacea. Yeast-like cells of opposite mating types (a1 and a2) were mated on 2% water agar and were treated with alpha-tocopherol to induce formation of dikaryotic hyphae. Upon depletion of the alpha-tocopherol, the hyphae budded off haploid cells with parental nuclear genotypes. These cells were examined for mitochondrial RFLP phenotype. In progeny expressing the a1 mating type, mitochondria from either parent were observed equally frequently. In progeny with the a2 mating type, mitochondria were almost exclusively (94%) from the a2 parent.

Crosses, Genetic↗