Comparison of nucleic acid hybridization and nucleic acid amplification using conserved sequences from the 5' noncoding region for detection of bovine viral diarrhea virus

Primers and probes derived from conserved sequences located in the 5' noncoding region of pestiviruses were evaluated for detection of bovine viral diarrhea virus. With these reagents, hybridization and polymerase chain reaction tests detected 62 of 90 and 90 of 90 bovine viral diarrhea virus isolates, respectively. A quick lysis method for preparing RNA for use in polymerase chain reaction amplification also was evaluated.     

Role of the thrombin receptor's cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-triggered versus tonic internalization and intracellular localization

The G protein-coupled thrombin receptor is activated by an irreversible proteolytic mechanism and, perhaps as a result, exhibits an unusual trafficking pattern in the cell. Naive receptors tonically cycle between the cell surface and a protected intracellular pool, whereas receptors cleaved and activated at the cell surface internalize and move to lysosomes. Toward understanding how these trafficking events are regulated, we examined a series of receptor mutants. A receptor with alanine substitutions at all potential phosphorylation sites in the cytoplasmic tail failed to display agonist-triggered internalization but, like wild type receptor, displayed robust signaling, tonic cycling, and localization to both the cell surface and an intracellular pool. A truncation mutant that lacked most of the cytoplasmic tail also signaled robustly, lacked phosphorylation, and was defective in agonist-triggered internalization. However, in contrast to the specific phosphorylation site mutant, the truncation mutant did not display tonic cycling and localized exclusively to the cell surface. An analysis of a series of truncation mutants localized residues important for receptor trafficking to a 10-amino acid stretch in its cytoplasmic tail. These data suggest that phosphorylation may trigger internalization of activated thrombin receptors but that a second phosphorylation-independent signal mediates tonic internalization of naive receptors. They further suggest that maintenance of the intracellular pool of naive thrombin receptors requires tonic receptor internalization.     

Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer

Cells possess within their epigenetic repertoire the ability to undergo an active process of cellular suicide termed programmed (or apoptotic) cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy-dependent cascade of biochemical and morphologic changes (also termed apoptosis) within the cell, resulting in its death and elimination. Although the final steps (i.e., DNA and cellular fragmentation) are common to cells undergoing programmed cell death, the activation of this death process is initiated either by sufficient injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones and growth/survival factors). Within the prostate, androgens are capable of both stimulating proliferation as well as inhibiting the rate of the glandular epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen ablation; however, they do retain the cellular machinery necessary to activate the programmed cell death cascade when sufficiently damaged by exogenous agents. In the normal prostate epithelium, cell proliferation is balanced by a equal rate of programmed cell death, such that neither involution nor overgrowth normal occurs. In prostatic cancer, however, this balance is lost, such that there is greater proliferation than death producing continuous net growth. Thus, an imbalance in programmed cell death must occur during prostatic cancer progression. The goal of effective therapy for prostatic cancer, therefore, is to correct this imbalance. Unfortunately, this has not been achieved and metastatic prostatic cancer is still a lethal disease for which no curative therapy is currently available. In order to develop such effective therapy, an understanding of the programmed death pathway, and what controls it, is critical. Thus, a review of the present state of knowledge concerning programmed cell death of normal and malignant prostatic cells will be presented.     

Synthesis of 3 beta-aryl-8-azabicyclo[3.2.1]octanes with high binding affinities and selectivities for the serotonin transporter site

A novel entry to tropane analogs of cocaine was developed based on the reaction of rhodium-stabilized vinylcarbenoids with pyrroles. These analogs were tested in binding to dopamine, serotonin (5-HT), and norepinephrine transporters in membranes from rat striatum and frontal cortex. In all the analogs, the aryl group at the 3 position was directly bound to the tropane ring and an ethyl ketone moiety was present at the 2 position. By appropriate modification of the aryl and nitrogen substituents, highly potent and 5-HT selective tropanes were prepared. The most potent and selective compound was 3 beta-[4-(1-methylethenyl)phenyl]-2 beta-propanoyl-8-azabicyclo[3.2.1]octane (13b) which had a Ki of 0.1 nM at 5-HT transporters and was 150 times more potent at 5-HT vs dopamine transporters and almost 1000 times more potent at 5-HT vs norepinephrine transporters.     

Acute and chronic effects of insulin on leptin production in humans: Studies in vivo and in vitro

This study was undertaken to investigate the changes in obesity (OB) gene expression and production of leptin in response to insulin in vitro and in vivo under euglycemic and hyperglycemic conditions in humans. Three protocols were used: 1) euglycemic clamp with insulin infusion rates at 40, 120, 300, and 1,200 mU / m / min carried out for up to 5 h performed in 16 normal lean individuals, 30 obese individuals, and 31 patients with NIDDM; 2) 64-to 72-h hyperglycemic (glucose 12.6 mmol/l) clamp performed on 5 lean individuals; 3) long-term (96-h) primary culture of isolated abdominal adipocytes in the presence and absence of 100 nmol/l insulin. Short-term hyperinsulinemia in the range of 80 to > 10,000 microU/ml had no effect on circulating levels of leptin. During the prolonged hyperglycemic clamp, a rise in leptin was observed during the last 24 h of the study (P < 0.001). In the presence of insulin in vitro, OB gene expression increased at 72 h (P < 0.01), followed by an increase in leptin released to the medium (P < 0.001). In summary, insulin does not stimulate leptin production acutely; however, a long-term effect of insulin on leptin production could be demonstrated both in vivo and in vitro. These data suggest that insulin regulates OB gene expression and leptin production indirectly, probably through its trophic effect on adipocytes.     

Isolation and refined regional mapping of expressed sequences from human chromosome 21

To increase candidate genes from human chromosome 21 for the analysis of Down syndrome and other genetic diseases localized on this chromosome, we have isolated and studied 9 cDNA clones encoded by chromosome 21. For isolating cDNAs, single-copy microclones from a chromosome 21 microdissection library were used in direct screening of various cDNA libraries. Seven of the cDNA clones have been regionally mapped on chromosome 21 using a comprehensive hybrid mapping panel comprising 24 cell hybrids that divide the chromosome into 33 subregions. These cDNA clones with refined mapping positions should be useful for identification and cloning of genes responsible for the specific component phenotypes of Down syndrome and other diseases on chromosome 21, including progressive myoclonus epilepsy in 21q22.3.     

Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales

Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.     

Vertebral artery location in relation to the vertebral body as determined by two-dimensional computed tomography evaluation

STUDY DESIGN: This study analyzed the precise two-dimensional location of the vertebral artery within cervical vertebrae as determined by measurements obtained from axial computed tomographic images of the cervical spine. OBJECTIVE: To determine the margin of safety necessary to avoid vertebral artery laceration during central decompression and lateral nerve root decompression for cervical spinal stenosis. SUMMARY OF BACKGROUND DATA: Laceration of the vertebral artery is a rare but potentially catastrophic complication of anterior decompressive surgery of the cervical spine. METHODS: The mean, standard deviation, and 95% confidence interval of the mean of measurements localizing the vertebral artery within the vertebral body were calculated from 50 transaxial computed tomography images of each of the second through sixth cervical vertebrae. RESULTS: Both the mean interforaminal distance (from 25.90 +/- 1.89 mm at C3 to 29.30 +/- 2.70 mm at C6) and the average distance of the posterior border of the foramen transversarium from the ventral border of the spinal canal (from 2.16 +/- 1.18 mm at C3 to 3.53 +/- 1.56 mm at C6) increased from C3 to C6. CONCLUSIONS: According to our measurements, the risk of vertebral artery laceration is greater at more cephalad vertebrae during lateral extension of central decompressive procedures and lateral nerve root decompression. Because of the variability of these parameters between individuals, accurate individual preoperative localization of the vertebral arteries is recommended.