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Biomedical subjects

W Davis

Publications and source records attributed to W Davis.

At least 19 recordsLinked to original sources
PubMed2006

Prophylactic administration of ponazuril reduces clinical signs and delays seroconversion in horses challenged with Sarcocystis neurona.

The ability of ponazuril to prevent or limit clinical signs of equine protozoal myeloencephalitis (EPM) after infection with Sarcocystis neurona was evaluated. Eighteen horses were assigned to 1 of 3 groups: no treatment, 2.5 mg/kg ponazuril, or 5.0 mg/kg ponazuril. Horses were administered ponazuril, once per day, beginning 7 days before infection (study day 0) and continuing for 28 days postinfection. On day 0, horses were stressed by transport and challenged with 1 million S. neurona sporocysts per horse. Sequential neurologic examinations were performed, and serum and cerebrospinal fluid were collected and assayed for antibodies to S. neurona. All horses in the control group developed neurologic signs, whereas only 71 and 40% of horses in the 2.5 and 5.0 mg/kg ponazuril groups, respectively, developed neurologic abnormalities. This was significant at P = 0.034 by using Fisher exact test. In addition, seroconversion was decreased in the 5.0 mg/kg group compared with the control horses (100 vs. 40%; P = 0.028). Horses with neurologic signs were killed, and a post-mortem examination was performed. Mild-to-moderate, multifocal signs of neuroinflammation were observed. These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but does not eliminate, infection and clinical signs of EPM in horses.

Animals↗
PubMed2003

US physicians' attitudes toward genetic testing for cancer susceptibility.

Genetic testing for an inherited susceptibility to cancer is an emerging technology in medical practice. Little information is currently available about physicians' attitudes toward these tests. To assess US physicians' opinions on unresolved issues surrounding genetic testing, a 15-min survey was administered to a stratified random sample of 1,251 physicians from 8 specialties, selected from a file of all licensed physicians in the US (response rate = 71.0%). Dependent measures included physicians' attitudes toward genetic counseling and testing qualifications, availability of guidelines, patient confidentiality and insurance discrimination issues, and clinical utility of genetic tests. More than 89% of physicians reported a need for physician guidelines, 81% thought that patients with positive genetic test results are at risk for insurance discrimination, and more than 53% thought that it was difficult to ensure the confidentiality of test results. Almost 25% indicated that genetic tests for cancer susceptibility have too many inaccurate or ambiguous results; nearly 75% thought that clear guidelines are not available for managing patients with positive test results. Only 29% of physicians reported feeling qualified to provide genetic counseling to their patients. More than 84% of oncologists considered themselves qualified to recommend genetic testing to their patients compared with 40% of primary care physicians (PCPs), and 57% of tertiary care physicians (TCPs). US physicians expressed great uncertainty about issues surrounding genetic testing for cancer susceptibility. Results of this national survey underscore the need to provide physicians with clear guidelines on the use of genetic cancer susceptibility tests and effective medical training on their appropriate implementation.

Attitude of Health Personnel↗
PubMed2001

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion.

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of human adenosine 5'-triphosphate-binding cassette, sub-family A, transporter 2 (ABCA2).

We have isolated the full-length cDNA for human ATP-binding cassette, sub-family A, member 2 transporter (ABCA2). The ORF of this cDNA encodes a protein consisting of 2436 amino acids with apparent molecular weight of M(r) 270,000. Accordingly, ABCA2 is the largest known mammalian ABC transporter described thus far. Analysis of mRNA expression levels indicated that ABCA2 is highest in human brain and has a broad expression pattern in a panel of tumor cell lines. Using specific antibodies to ABCA2 and various organelle marker proteins, ABCA2 was found to colocalize with the lysosomal/endosomal marker LAMP1, forming discrete, punctate intracellular vesicles. In ABCA2-transfected cells, the transporter also colocalized with a fluorescently labeled steroid analogue, estramustine. The sequestration of the steroid into the lysosomal/endosomal compartment indicates a potential substrate specificity for ABCA2. Furthermore, the presence of a lipocalin signature motif in the ABCA2 sequence suggests a possible broad role for this protein in the transport of steroids, lipids, and related molecules.

ATP-Binding Cassette Transporters↗
PubMed2001

Analysis of mitogen-activated protein kinase pathways used by interleukin 1 in tissues in vivo: activation of hepatic c-Jun N-terminal kinases 1 and 2, and mitogen-activated protein kinase kinases 4 and 7.

The effects of interleukin 1 (IL-1) are mediated by the activation of protein kinase signalling pathways, which have been well characterized in cultured cells. We have investigated the activation of these pathways in rabbit liver and other tissues after the systemic administration of IL-1alpha. In liver there was 30-40-fold activation of c-Jun N-terminal kinase (JNK) and 5-fold activation of both JNK kinases, mitogen-activated protein kinase (MAPK) kinase (MKK)4 and MKK7. IL-1alpha also caused 2-3-fold activation of p38 MAPK and degradation of the inhibitor of nuclear factor kappaB ('IkappaB'), although no activation of extracellular signal-regulated protein kinase (ERK) (p42/44 MAPK) was observed. The use of antibodies against specific JNK isoforms showed that, in liver, short (p46) JNK1 and long (p54) JNK2 are the predominant forms activated, with smaller amounts of long JNK1 and short JNK2. No active JNK3 was detected. A similar pattern of JNK activation was seen in lung, spleen, skeletal muscle and kidney. Significant JNK3 activity was detectable only in the brain, although little activation of the JNK pathway in response to IL-1alpha was observed in this tissue. This distribution of active JNK isoforms probably results from a different expression of JNKs within the tissues, rather than from a selective activation of isoforms. We conclude that IL-1alpha might activate a more restricted set of signalling pathways in tissues in vivo than it does in cultured cells, where ERK and JNK3 activation are often observed. Cultured cells might represent a 'repair' phenotype that undergoes a broader set of responses to the cytokine.

Animals↗
PubMed2001

Characterization of the murine epidermal 12/15-lipoxygenase.

The murine lipoxygenase (LO) family consists of at least seven members classified according to the HETE (hydroxyeicosatetraenoic acid) metabolite generated during arachidonic acid metabolism and the site of tissue expression. At present there are four 12-lipoxygenases that are functionally distinct, vary in cell and tissue distribution, catalytic activity and each are products of separate, linked genes. They are "platelet-type" 12-LO (P-12LO), "leukocyte-type" 12-LO (L-12LO), "epidermal-type" 12-LO (e-12LO) and the most recently discovered 12(R)-LO. In this report we characterize e-12LO, which was overexpressed in the baculovirus/insect cell expression system. The enzyme functions as a dual specificity 12/15-lipoxygenase with a 12-HETE/15-HETE product ratio of approximately 6:1 with arachidonic acid as substrate. Several other polyunsaturated fatty acids served as substrates for e-12LO such as gamma-linolenic, dihomo-gamma-linolenic and eicosapentaenoic acids. A green fluorescent protein/e-12LO fusion protein was localized to the cytosol of transfected HEK 293 cells. The e-12LO gene was expressed in mouse oocytes and early embryos. Western blot analysis revealed high level expression in postnatal day 3 mouse epidermal lysates. Together these data suggest that e-12LO plays a role in normal epidermal function and as yet an undiscovered role in early development.

Amino Acid Sequence↗
PubMed2001

Inability to obtain formal informed consent in the face of a standard surgical indication.

A thirty-eight-year-old intoxicated man was admitted to the surgical trauma service following a single motor-vehicle accident. He had a severe closed head injury, bilateral pulmonary contusions, a fracture-dislocation of the right acetabulum, and an open injury of the right knee joint. The acetabular fracture pattern was an associated both-column fracture with the femoral head dislocated into a widely displaced posterior-column fracture line. The treating physicians agreed that it would be in the patient's best interest to take him to the operating room for emergent debridement and irrigation of his knee wound. At surgery, the patient also underwent attempted closed reduction of the acetabular fracture and placement of a skeletal traction pin. Radiographs obtained with the patient in traction showed reduction of the femoral head beneath a displaced superior dome fragment, but there remained a 12-mm gap in the posterior column, greater than 3 mm of step incongruity, and a large articular fragment entrapped in the anterior aspect of the hip joint. The patient remained intubated and sedated for several days. Upon weaning from the ventilator, it became evident that his head injury would prevent him from being able to give informed consent in the foreseeable future. The patient's family members refused to become involved with his care or medical decision-making, as he had become completely estranged from them as a result of his chronic alcohol abuse. Further delay in surgical treatment for the acetabular fracture would be associated with greater difficulty in obtaining an anatomic reduction, the potential for additional articular damage to the femoral head, and an increased risk of surgical complications. The question that arises is whether it is in the patient's best interest for the surgeon to proceed with open reduction and internal fixation of the acetabular fracture without having had the opportunity to fully inform him of the treatment options or the risks associated with an extensive surgical exposure.

Acetabulum↗
PubMed2001

Cellular thiols and reactive oxygen species in drug-induced apoptosis.

In higher eukaryotes, reactive oxygen species (ROS) are generated during respiration in mitochondria in the course of reduction of molecular oxygen as well as by distinct enzyme systems. ROS have been implicated in the regulation of diverse cellular functions including defense against pathogens, intracellular signaling, transcriptional activation, proliferation, and apoptosis. The reduction-oxidation (redox) state of the cell is primarily a consequence of the precise balance between the levels of ROS and endogenous thiol buffers present in the cell, such as glutathione and thioredoxin, which protect cells from oxidative damage. Dramatic elevation of ROS, exceeding compensatory changes in the level of the endogenous thiol buffers, may result in the sustained activation of signaling pathways and expression of genes that induce apoptosis in affected cells. Many cytotoxic drugs function selectively to kill cancer cells by the abrogation of proliferative signals, leading to cell death, and numerous reports have demonstrated that ROS are generated following treatment with these drugs. In this review, we will summarize recent contributions to our understanding of the importance of cytotoxic drug-induced modulation of cellular redox status for signaling and transcription leading to activation of apoptotic effector mechanisms.

Animals↗
PubMed2000

Sex differences in the activation of tamoxifen to DNA binding species in rat liver in vivo and in rat hepatocytes in vitro: role of sulfotransferase induction.

Previous work has indicated that metabolic activation of tamoxifen in rat liver cells involves cytochrome P450-mediated alpha-hydroxylation, followed by sulfate ester formation, mediated by hydroxysteroid sulfotransferase a (rHSTa), a member of the SULT2A subfamily, which efficiently metabolizes dehydroepiandrosterone. Because it is known that the expression of rHSTa and other SULT2A forms is substantially higher in female rats than in males, it might be predicted that tamoxifen would be a more potent liver carcinogen in females than in males. Yet tamoxifen has been shown to be equipotent in both sexes. To investigate this paradox, primary cultures of hepatocytes were prepared from Fischer F-344 rats and treated with tamoxifen (10 microM) or alpha-hydroxytamoxifen (1 microM). Rats were also treated with tamoxifen daily by gavage (0.12 mmol/kg/day) for up to 14 days. DNA was isolated from hepatocytes and liver and analyzed by 32P-postlabeling. Liver cytosol fractions were prepared and analyzed for dehydroepiandrosterone sulfotransferase activity and SULT2A protein levels. In tamoxifen-treated hepatocytes and after a single dose of tamoxifen in vivo, DNA adduct formation in male cells was significantly lower than in female cells, 11- and 6-fold, respectively. However, with increasing daily doses of rats with tamoxifen, the adduct level in males increased to a level 89% of that in females by 14 days. Dehydroepiandrosterone sulfotransferase activity in male rat liver cytosols was only 17% of the activity of female cytosols after one dose of tamoxifen but 64% after 14 days of exposure to the compound. This increase in activity correlated with increases in the levels of SULT2A protein, detected by Western blotting. Western blotting did not allow the unambiguous identification of the induced SULT2A form(s). However, by using a specific reverse transcriptase/PCR technique, it was found that it was primarily rHSTa that was induced. Thus, after prolonged exposure to tamoxifen, DNA adduct formation and rHSTa expression in males are significantly closer to the levels in females than they are after initial exposure. These changes explain the similar susceptibility of male and female rats to tamoxifen carcinogenesis.

Animals↗
PubMed2000

Developmental change in TATA-box utilization during preimplantation mouse development.

Activation of the embryonic genome during preimplantation mouse development is characterized by a marked reprogramming of gene expression that is essential for further development. Expression of the protein translation initiation factor eIF-1A gene is driven by a proximal TATA-containing promoter and a distal TATA-less promoter. Using specific amplification of cDNA ends that resolves transcripts derived from the TATA-less and TATA-containing promoters, we find that 70% of the eIF-1A transcripts are derived from the TATA-containing promoter in the fully-grown oocyte. Activation of the embryonic genome during the two-cell stage is accompanied by a change in promoter utilization such that only 25% of the transcripts are now derived from the TATA-containing promoter, i.e., 75% are derived from the TATA-less promoter. When one-cell embryos are cultured to the two-cell stage in the presence of alpha-amanitin, this change in transcript abundance is not observed, i.e., the distribution of transcripts is similar to that observed in the oocyte. By the blastocyst stage only 5% of the transcripts are generated from the TATA-containing promoter. If the change in TATA-box utilization for the eIF-1A reflects an underlying global change in TATA-box utilization, a dramatic change in promoter utilization may occur during preimplantation development such that TATA-less promoters are more efficiently utilized. Such a change in promoter utilization could contribute significantly to the reprogramming of gene expression that occurs during the maternal-to-zygotic transition.

Animals↗
PubMed2000

Drosophila D-titin is required for myoblast fusion and skeletal muscle striation.

An ethylmethane sulfonate (EMS) mutagenesis of Drosophila melanogaster aimed at discovering novel genes essential for neuromuscular development identified six embryonic lethal alleles of one genetic locus on the third chromosome at 62C. Two additional lethal P element insertion lines, l(3)S02001 and l(3)j1D7, failed to complement each other and each of the six EMS alleles. Analysis of genomic sequence bracketing the two insertion sites predicted a protein of 16,215 amino acid residues, encoded by a 70 kb genomic region. This sequence includes the recently characterized kettin, and includes all known partial D-Titin sequences. We call the genetic locus, which encodes both D-Titin and kettin, D-Titin. D-Titin has 53 repeats of the immunoglobulin C2 domain, 6 repeats of the fibronectin type III domain and two large PEVK domains. Kettin appears to be the NH2-terminal one third of D-Titin, presumably expressed via alternative splicing. Phenotype assays on the allelic series of D-Titin mutants demonstrated that D-Titin plays an essential role in muscle development. First, D-Titin has an unsuspected function in myoblast fusion during myogenesis and, second, D-Titin later serves to organize myofilaments into the highly ordered arrays underlying skeletal muscle striation. We propose that D-Titin is instrumental in the development of the two defining features of striated muscle: the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays.

Actin Cytoskeleton↗
PubMed2000

Rational dosages of nutrients have a prolonged effect on learning disabilities.

CONTEXT: Reports that administration of nutrients has increased the academic performance of learning-disabled children exist in the literature. OBJECTIVE: To document the effects of nutrients on learning-disabled children in a controlled study. DESIGN: A randomized, double-blind, placebo-controlled crossover trial, which followed 1 year of open-label nutrients. Children who improved in the open-label trial were eligible to enter the controlled phase of the study. SETTING: Subjects were enrolled from the general community through advertisements. PATIENTS OR OTHER PARTICIPANTS: Twenty children met the criteria for being learning disabled. INTERVENTION: Each child was tried out on some (but not necessarily all) of the B vitamins and minerals used in this study. These were administered semi-blinded for the first year; double-blinded in crossover rotations during the second year; and open-label in the ensuing years. MAIN OUTCOME MEASURES: At various time points, school-certified psychologists administered psychoeducational tests. School report cards were evaluated at baseline and for all subsequent periods. RESULTS: Twenty learning-disabled children entered the study, but 1 dropped out because of nausea. The remaining 19 children showed significant academic and behavioral improvements within a few weeks or months of open-label treatment with nutrient supplements. Some children gained 3 to 5 years in reading comprehension within the first year of treatment; and all children in special education classes became mainstreamed, and their grades rose significantly. Twelve of the children completed the 1-year double-blind phase, after which approximately half of the children chose to remain on the nutrients for at least 2 additional years. For those who discontinued, it took at least 1 year to begin to see the first indications of decline in academic performance, and another year for their grades to drop significantly. In contrast, for children who remained on nutrients, the gains continued the upward trend; at the end of year 4, the difference in scores between the 2 groups had reached statistical significance (P < .01). CONCLUSION: The overall results of this study tentatively support the concept that learning disabilities may in some cases be a nutrient-responsive disorder.

Adolescent↗
PubMed1999

Reprogramming of gene expression during preimplantation development.

A fundamental problem in early mammalian development is the transformation of the highly differentiated oocyte into the totipotent blastomeres by the 2-cell stage. This remarkable transformation probably entails reprogramming the pattern of gene expression. The maternal-to-zygotic transition occurs during the 1-cell stage, i.e., 1-cell embryo is transcriptionally active. DNA replication is a likely locus of regulation, since DNA replication, with consequent nucleosome displacement, would serve to facilitate the access of maternally derived transcription factors to their cis-acting DNA-binding sequences. In fact, the first round of DNA replication is essential for the expression of two genes, eIF-1A and the transcription-requiring complex (TRC), as well as global transcription. A growing body of evidence suggests that a transcriptionally repressive state develops during the 2-cell stage. Interestingly, inhibiting the second round of DNA replication inhibits the decrease in expression of both the TRC and eIF-1A, as well as total endogenous gene expression. This repression may be linked to a change in chromatin structure, since treatment of 2-cell embryos with histone deacetylase inhibitors prevents the decrease in expression of the TRC and eIF-1A, and also inhibits the development of the transcriptionally repressive state for global gene expression. The findings that histone deacetylases can be targeted to chromatin provide a mechanism to link histone deacetylation with repression of gene expression. J. Exp. Zool. (Mol. Dev. Evol.) 285:276-282, 1999.

Animals↗
PubMed1999

Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase.

Ligation of the B cell antigen receptor (BCR) induces a cascade of signaling pathways that lead to clonal expansion, differentiation, or abortive activation-induced apoptosis of B lymphocytes. BCR-mediated cross-linking induces the rapid phosphorylation of protein tyrosine kinases. However, the pathways leading to the activation of downstream serine/threonine kinases such as mitogen-activated protein kinase, p90(Rsk), and p70S6 kinase (p70(S6k)) that mediate reorganization of the actin cytoskeleton, cell cycle progression, gene transcription, and protein synthesis have not been delineated. We recently demonstrated that cross-linking of BCR leads to activation of p70(S6k) in B lymphocytes. In this report, we demonstrate that multiple protein tyrosine kinase-dependent signal transduction pathways induced by BCR lead to the activation of p70(S6k). These distinct pathways exhibit different thresholds with respect to the extent of receptor cross-linking required for their activation. Activation of p70(S6k) by suboptimal doses of anti-Ig is Syk-dependent and is mediated by protein kinase C and phosphoinositol 3-kinase. Moreover, the activation of p70(S6k) results in phosphorylation of S6 protein which is important for ribosomal protein synthesis and may be coupled to BCR-induced protein and DNA synthesis in primary murine B cells.

Animals↗
PubMed1999

Synergistic activation of JNK/SAPK by interleukin-1 and platelet-derived growth factor is independent of Rac and Cdc42.

The c-Jun N-terminal kinases (JNKs) are activated strongly by inflammatory cytokines and environmental stresses, but only weakly by growth factors. Here we show that platelet-derived growth factor (PDGF) strongly potentiates activation of JNK by interleukin 1 (IL-1) in human fibroblasts and a pig aortic endothelial (PAE) cell line. This synergistic activation of JNK by IL-1 and PDGF was unaffected by bacterial toxins that inactivate Rho proteins and Ras. Since Rho proteins have been implicated in JNK activation, their possible involvement was investigated further using stably expressed, inducible N17 or V12 mutants in PAE cell lines. N17 Rac non-selectively reduced JNK activity by 30% in resting or stimulated cells (IL-1 alone, or with PDGF). N17 Cdc42 had no effect. V12 Rac weakly activated JNK and synergized with IL-1, but not with PDGF. V12 Cdc42 weakly activated JNK, but synergized with PDGF and not IL-1. Our results imply that Rho GTPases are not directly involved in mediating IL-1-induced JNK activation, or in the potentiation of this activation by PDGF.

Bacterial Toxins↗
PubMed1999

4-Hydroxytamoxifen gives DNA adducts by chemical activation, but not in rat liver cells.

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumors in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and in rat hepatocytes in culture. These arise primarily from its metabolite alpha-hydroxytamoxifen, and may also arise, in part, from another metabolite, 4-hydroxytamoxifen. We have prepared two model compounds for the potential reactive metabolite formed from 4-hydroxytamoxifen in rat liver. One of these was its alpha-acetoxy ester. This was much more reactive than that from tamoxifen, and could not be isolated in pure form. It reacted with DNA in the same way that alpha-acetoxytamoxifen did, to give adducts which were isolated by hydrolysis and chromatography, and identified as alkyldeoxyguanosines. The second derivative was alpha, beta-dehydro-4-hydroxytamoxifen. This also reacts with DNA in vitro, to give the same products as those from alpha-acetoxy-4-hydroxytamoxifen. Reaction probably proceeds through the same resonance-stabilized carbocation in either case. However, when primary cultures of rat hepatocytes were treated with either 4-hydroxytamoxifen, 4,alpha-dihydroxytamoxifen, or alpha, beta-dehydro-4-hydroxytamoxifen at a concentration of 10 microM, no adducts could be detected in their DNA by the 32P-postlabeling technique. Similarly, no adducts could be found in the liver DNA of female Fischer F344 rats treated orally (at 0.12 mmol kg-1) with the same substances. If 4-hydroxytamoxifen is metabolized to 4, alpha-dihydroxytamoxifen in rat liver, then either this substance is not converted to reactive esters or they are rapidly detoxified.

Animals↗