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Biomedical subjects

D M Walker

Publications and source records attributed to D M Walker.

At least 37 records · Page 2Linked to original sources
PubMed1999

Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway.

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.

Aldehyde Oxidoreductases↗
PubMed1999

Mutagenicity of 1,3-butadiene at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

The species specific response to 1,3-butadiene (BD), an important industrial chemical, was investigated by determining the influence of exposure duration and exposure concentration on the mutagenicity of BD in mice and rats and by defining the spectra of mutations in the Hprt gene T-cell mutants from control and BD-exposed mice. Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed by inhalation to 0, 20, 62.5, or 625 ppm of BD for up to 4 weeks (6 h/day, 5 days/week). Groups of control and exposed animals (n=4-12/group) were necropsied at multiple time points after exposure and the T-cell cloning assay was used to measure Hprt mutant frequencies in lymphocytes isolated from spleen. Mutant clones collected from control and BD-exposed mice were propagated and analyzed by RT-PCR to produce Hprt cDNA for sequencing. In animals necropsied 4 weeks after 2 or 4 weeks of BD exposure (0 or 625 ppm), the rate of accumulation of mutations was greater in mice than in rats. Supra-linear dose-response curves were observed in BD-exposed mice, indicating a higher efficiency of mutant induction at lower concentrations of BD. The mutagenic potency estimates (represented by the differences in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals) in mice were 11 and 61 following 4 weeks of exposures to 62.5 and 625 ppm of BD, respectively, while mutant frequencies (Mfs) in rats were significantly increased only at 625 ppm BD (mutagenic potency of 7). Molecular analysis of Hprt cDNA from expanded T-cell clones from control and BD-exposed mice demonstrated an increased frequency of mutants in exposed animals that likely contain large deletions in the Hprt gene (P=0.016). These data indicate that both exposure duration and exposure concentration are important in determining the magnitude of mutagenic response to BD, and that mutagenic and carcinogenic properties of BD in mice may be related more to the ability of its metabolites to cause chromosomal deletions than to produce point mutations.

Administration, Inhalation↗
PubMed1999

Mutagenicity of the racemic mixtures of butadiene monoepoxide and butadiene diepoxide at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

The purpose of this study was to determine if Hprt mutant frequency (Mf) data from rodents exposed directly to individual epoxy metabolites of 1,3-butadiene (BD) can be used to identify the relative significance of each intermediate in the mutagenicity of BD in mice vs. rats. To this end, the relative contributions of the racemic mixtures of BD monoepoxide (BDO) and BD diepoxide (BDO(2)) to BD-induced mutagenicity was investigated by exposing mice and rats to selected concentrations of BDO and BDO(2) (i.e., 2.5 and 4.0 ppm, respectively) and comparing the mutagenic potency of each intermediate to that of BD (at 62.5 ppm) when comparable blood levels of metabolites are achieved (in the mouse). Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed to rac-BDO (0, 2.5, or 25 ppm) or (+/-)-BDO(2) (0, 2, 4 ppm) by inhalation for 4 weeks (6 h/day, 5 days/week), and then groups of control and exposed animals (n=3-12/group) were necropsied at multiple time points post-exposure for measuring Hprt Mfs in splenic lymphocytes (via the T-cell cloning assay) and estimating mutagenic potencies (represented by the difference in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals). The resulting Mf data, along with the extant metabolism data, suggest that at lower BD exposures (</=62.5 ppm) (+/-)-BDO(2) is a major contributor to the mutagenicity of BD in mice, whereas other metabolites and stereochemical configurations are responsible for mutations in BD-exposed rats and for the incremental mutagenic effects at higher BD exposures in mice. These studies indicate that additional work is needed to determine more definitively the relative contributions of these and other metabolites and stereochemical forms to BD-induced mutagenicity. Also, the novel approach of measuring mutagenic potencies as the change in Hprt Mfs over time in T-cells of exposed vs. control animals, as used in this study, can be valuable for predicting the potential role of these intermediates in each species.

Administration, Inhalation↗
PubMed1999

Butenolide endothelin antagonists with improved aqueous solubility.

Continued development around our ETA-selective endothelin (ET) antagonist 1 (CI-1020) has led to the synthesis of analogues with improved aqueous solubility profiles. Poor solubility characteristics displayed by 1 required a complex buffered formulation in order to conduct iv studies. To overcome the use of specific iv formulations for preclinical studies on additional drug candidates, analogues with improved aqueous solubility were desired. Several analogues were synthesized with substitution patterns that allowed for the formation of either acid or base addition salts. These derivatives had dramatically improved aqueous solubility. In addition, these analogues retained equivalent or improved ETA receptor selectivity and antagonist potency, versus 1, both in vitro and in vivo. Compound 29, which contains as a substituent the sodium salt of a sulfonic acid, has an ETA IC50 = 0.38 nM, ETA selectivity of 4200-fold, and ETA functional activity of KB = 7.8, all of which are similar or superior to those of 1. Compound 29 also has vastly superior aqueous solubility and solubility duration, compared to 1. Furthermore, 29 after iv infusion displays improved activity to 1 in preventing acute hypoxia-induced pulmonary hypertension in rats with an ED50 = 0.3 microg/kg/h.

Animals↗
PubMed1999

Massive osteolysis of the mandible: report of a case with multifocal bone loss.

Osteolysis of the jaws has been reported in association with infection, cysts, neoplasia, and metabolic, endocrine, or hematologic abnormalities. Rare cases of idiopathic osteolysis have also been recorded. We report the case of a 10-year-old girl with mandibular basal and alveolar bone resorption that has continued over a period of 9 years. The patient has subsequently developed bilateral resorption of the ascending rami and condyles. The maxilla is uninvolved. Investigations included radiology, computerized tomography, scintigraphy, hematology, serum chemistry, endocrinology, histopathology, microbiology, and immunology. Neutrophil chemotaxis, chemiluminescence, and random migration values were low but within the normal range. These findings are interpreted as indicating an unusual variant of massive osteolysis.

Child↗
PubMed1999

Phase space reconstruction using input-output time series data.

In this paper we suggest that an extension of a procedure recently proposed by Wayland et al. [Phys. Rev. Lett. 70, 580 (1993)] for recognizing determinism in an autonomous time series can also be used as a diagnostic for determining an appropriate embedding dimension for driven ("input-output") systems. We compare the results of this extension to the results produced by the extensions to the method of false nearest neighbors put forward by Rhodes and Morari [Proceedings of the American Control Conference, Seattle, edited by The American Automatic Control Council (IEEE, Piscataway, 1995)] and the method of averaged false nearest neighbors by Cao et al. [Int. J. Bifurcation Chaos 8, 1491 (1998)].

Journal Article↗
PubMed1999

Detection of cyclophosphamide-induced mutations at the Hprt but not the lacI locus in splenic lymphocytes of exposed mice.

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6-7 weeks- of-age with the indirect-acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 x 10(-5) using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 x 10(-5) in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP-induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC((R)) bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 +/- 0.09 x 10(-5); 25 mg CP/kg: 0.39 +/- 0.05 x 10(-5)) versus controls (0.10 +/- 0.01 x 10(-5)); however, the Mfs in lacI of lymphocytes from the same CP-treated animals were not significantly different from controls (100 mg CP/kg: 9.4 +/- 1.1 x 10(-5); 25 mg CP/kg: 6.7 +/- 0. 8 x 10(-5); control: 7.7 +/- 0.7 x 10(-5)). Hprt mutational spectra data in CP-treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI of lymphocytes from Big Blue((R)) transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP-induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacI transgene of this nontarget tissue for CP-induced cancer.

Animals↗
PubMed1998

Preliminary study of microsurgical repairs of the inferior alveolar nerve in rats using primary suturing and laser weld techniques.

A comparison of primary suturing and a new laser weld technique is described for the microsurgical repair of the inferior alveolar nerve in Wistar rats. A reliable method of exposure of the inferior alveolar nerve has been developed in order to allow intraosseous repairs of the nerve involving suturing with 10:0 nylon and a laser weld technique using an albumin-based solder, containing indocynine cardiogreen, plus an infrared (810 nm wavelength) diode laser. Seven cases of microsuture and laser weld repairs were performed with a 29.4% reduction in total operating time in the laser weld group. Histochemical analysis showed comparable mean neuron counts and mean tracer uptake by neurons for the microsuture and laser weld groups. Giant cell reactions were identified in two of the primary suture cases and axon deflection in three cases demonstrating possible advantages of the laser weld technique which showed no adverse reactions by axons or epineurium to the coagulative repair with the solder. The technique of laser weld repair, on initial analysis, therefore appears comparable with traditional suture repairs and indeed may possess several advantages. Further studies are recommended.

Animals↗
PubMed1998

Fatal Staphylococcus aureus infective endocarditis: the dental implications.

Infective endocarditis remains an important and life-threatening infection despite improvements in diagnosis and management. There is currently a greater role for nosocomial acquisition of organisms and immunosuppression in the pathogenesis of this disease and emergence of a broader spectrum of infective organisms including those not commonly isolated from the mouth such as staphylococci. We report a case of infective endocarditis caused by Staphylococcus aureus in which the patient developed disseminated intravascular coagulation and multiple septic infarcts resulting in a frontal lobe brain abscess. Multiple dental extractions were complicated by delayed postextraction hemorrhage and the immediate cause of death was abdominal hemorrhage. The dental management in infective endocarditis should be planned in consultation with the attending physician, and should take into account both the causative organism and the presence of complications. When the oral cavity cannot be proven as the bacterial source for infective endocarditis, the immediate dental management should be directed toward improving the patient's oral hygiene and providing pain relief. Definitive long-term treatment, including any extractions, is ideally delayed until the patient has fully recovered from the infective endocarditis and its attendant complications.

Brain Abscess↗
PubMed1998

Epithelial p53 gene expression and mutational analysis, combined with growth fraction assessment, in oral lichen planus.

The immunohistochemical detection of epithelial p53 protein expression in oral lichen planus (OLP) biopsies was supplemented with molecular analysis for mutations of the p53 gene using the polymerase chain reaction - single stranded conformational polymorphism (PCR-SSCP) technique. p53 protein expression, in the basal epithelial cell layer, as detected by the DO7 and 1801 antibodies, was significantly more frequent in OLP compared with other oral keratoses and normal mucosa, as was the growth fraction. The 10 OLP biopsies that had the most frequent p53 staining (plus a case of OLP found in continuity with a SCC) were screened by the PCR-SSCP technique, but no mutations were detected in the p53 gene (exons 5 9). The p53 overexpression in the OLP samples may be a physiological response to the hyper-proliferative state, as revealed by the growth fraction determination. This may usefully serve to protect against mutagenesis, and so be a factor in the low incidence of carcinoma associated with OLP.

Adult↗
PubMed1998

Cervical lymph node involvement in head and neck cancer detectable as expression of a spliced transcript of type II keratin K5.

Metastatic spread to cervical lymph nodes is a major determinant of outcome in head and neck cancer. One hundred and ninety-six lymph nodes dissected from fresh surgical specimens from 24 patients with primary head and neck squamous cell carcinoma (SCC) were bisected. Messenger RNA (mRNA) extracted from one half and from a segment of the primary tumour was amplified by reverse transcriptase (RT)-polymerase chain reaction (PCR) with primers flanking the fifth intron of human type II keratin K5. DNA bands resolved by agarose gel electrophoresis were confirmed as specific transcripts by sequencing. The other half of each node was fixed in formalin for histology and, in selected nodes, for immunohistology for cytokeratins. Of 153 nodes suitable for analysis, 14 nodes contained metastatic tumour detected by light microscopy and also tested positive for K5 mRNA by RT-PCR. Fifty-six nodes were histologically negative for tumour but positive for K5 mRNA, and 83 nodes were negative for both histology and K5 mRNA. Extracts of the primary tumour always reacted positively for K5 by RT-PCR, whereas lymph nodes from patients without malignancies, and blood lymphocytes from a healthy volunteer reacted negatively. RT-PCR designed to detect the presence of processed transcripts of type II keratin K5 in stratified squamous epithelial cells may be a sensitive technique to detect the presence of viable and potentially metastatic carcinoma cells in lymph nodes draining head and neck SCC.

Biomarkers, Tumor↗
PubMed1998

Culture and propagation of Hprt mutant T-lymphocytes isolated from mouse spleen.

The optimization of the mouse lymphocyte Hprt mutation assay has been impeded by the relatively poor growth potential of mouse T-cells in vitro, which leads to low cloning efficiencies (CEs) and limited expansion of Hprt mutant clones for molecular analysis of mutations occurring in control and treated mice. In this study, the addition and manipulation of concanavalin A (Con A), mouse interleukin-2 (IL-2), and a commercially available culture supplement, rat T-STIM with Con A, were used to identify growth conditions producing relatively high CEs for mouse T-cells. Supplementation of medium with 10% rat T-STIM, along with appropriate amounts of Con A for priming and exogenous IL-2 for cloning, resulted in average CEs of 15-16% in lymphocytes isolated from spleens of control mice (n = 32) or mice exposed to 1,3-butadiene (n = 27). In addition, several reagents were assessed for their potential to stimulate long-term growth of Hprt mutant clones; these T-cell stimulatory agents included Con A, phytohemagglutinin, and a calcium ionophore ionomycin combined with a tumor promoter phorbol 12-myristate 13-acetate. In a pilot study, stimulation with Con A proved to be the most effective means for propagating mouse T-cell clones under the various conditions tested. In follow-up experiments, transfer of mutant clones to 24-well plates and repeated stimulation with Con A in IL-2 and rat T-STIM supplemented medium was found to expand 76% of 536 mutant clones to about 400,000 to several million cells per clone. These data indicate that rat T-STIM-supplemented medium enhances the initial outgrowth of mouse T-cells, and that repeated mitogenic stimulation with Con A in the presence of IL-2 and rat T-STIM provides a means for propagating mouse T-cell clones for mutation analyses by a variety of methods.

Animals↗
PubMed1997

Design of a potent combined pseudopeptide endothelin-A/endothelin-B receptor antagonist, Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of its pharmacokinetic and spectral properties.

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

Amino Acid Sequence↗
PubMed1997

Intraperitoneal administration of coumarin causes tissue-selective depletion of cytochromes P450 and cytotoxicity in the olfactory mucosa.

Coumarin is a naturally occurring fragrant compound widely used in consumer products and also as a therapeutic agent. The effects of intraperitoneal (ip) and oral administration of coumarin on cytochrome P450 (P450) expression in olfactory mucosa were examined. A single ip injection of coumarin at 50 mg/kg resulted in a significant reduction of levels of CYP2A and CYP2G in the olfactory mucosa of Wistar rats and C57BL/6 mice at 48 hr following injection. Dose-response analysis of coumarin effects indicated that Wistar rats were more sensitive than C57BL/6 mice. A significant suppression of nasal CYP2A levels was observed at 25 mg/kg in rats, but not in mice. Depletion of P450 content was not observed in liver of either rats or mice at 50 mg/kg, indicating tissue-selective effects. Decreased P450 levels were observed at 24 hr, 48 hr, and 7 days following treatment, with minimal levels seen at 48 hr. The decrease in P450 levels was accompanied by necrosis, cell loss, and basal cell metaplasia in the olfactory mucosa. Intraperitoneal injection of 7-hydroxycoumarin or 3,4-dihydrocoumarin at 50 mg/kg did not result in depletion of nasal P450, indicating that the toxicity is not mediated by P450-catalyzed coumarin 7-hydroxylation and supporting the hypothesis that the formation of coumarin 3,4-epoxide may be responsible for the toxicity. Oral treatment with coumarin in drinking water led to a small, yet significant induction of CYP2A protein and coumarin hydroxylase activity in the nasal mucosa of mice, but not rats. Thus, ip administration of coumarin causes tissue-selective depletion of P450 and cytotoxicity in the olfactory mucosa of Wistar rats and C57BL/6 mice. It remains to be determined whether similar toxicity occurs following coumarin administration by other routes.

Animals↗
PubMed1997

Establishing a normal range for mouth opening: its use in screening for oral submucous fibrosis.

The maximal mouth opening of 700 healthy Nepalese adults, age range of 18-68 years, measured by paraclinical workers was determined as there are no data available for an Asian population. The mean value of the inter-incisal distance was 47.1 mm (range 33.7-60.4 mm) and 98% of the population surveyed fell within this range. The minimum limit of normal oral opening was determined to be 34 mm. Ten out of 13 patients with histologically confirmed oral submucous fibrosis (OSF) had a maximal oral opening of less than 34 mm. It was concluded that reduced oral opening measured by paraclinical workers as a single screening test for oral submucous fibrosis, has a sensitivity of only 77% and will detect only advanced cases. However, each of the 3 subjects from the healthy population found to have restricted mandibular opening and who agreed to be examined further had significant oral conditions, namely oral submucous fibrosis (2) and pericoronitis (1). The study confirms the value of this measurement as a screening procedure for significant oral disease by paraclinical staff particularly in developing countries where trained dental and medical personnel are scarce. Because of its limited sensitivity, measurement of mouth opening is unsatisfactory as a single screening test for OSF, as it will not detect early stages of the disease.

Adolescent↗
PubMed1997

Collaboration between the Air Force, VA, and a university psychiatry department on an inpatient unit.

The David Grant Medical Center at Travis Air Force Base in California is the site of a collaboration between the U.S. Air Force, the Department of Veterans Affairs, and the School of Medicine of the University of California, Davis, which was begun in 1994 to assure optimum use of resources and provide broader educational experiences to residents and students. The three systems have been integrated most fully on an inpatient mental health unit. The authors describe the history and development of the collaboration, focusing on how the model has helped the three organizations address clinical, institutional, and educational needs.

California↗