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T J Smith

Publications and source records attributed to T J Smith.

At least 145 records · Page 8Linked to original sources
PubMed1998

Identification of viral mutants by mass spectrometry.

A method to identify mutations of virus proteins by using protein mass mapping is described. Comparative mass mapping was applied to a structural protein of the human rhinovirus Cys1199 --> Tyr mutant and to genetically engineered mutants of tobacco mosaic virus. The information generated from this approach can rapidly identify the peptide or protein containing the mutation and, in cases when nucleic acid sequencing is required, significantly narrows the region of the genome that must be sequenced. High-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry were used to identify amino acid substitutions. This method provides valuable information for those analyzing viral variants and, in some cases, offers a rapid and accurate alternative to nucleotide sequencing.

Amino Acid Sequence↗
PubMed1998

Leukoregulin induction of protein expression in human orbital fibroblasts: evidence for anatomical site-restricted cytokine-target cell interactions.

The molecular basis for the profound inflammatory response and the accumulation of hyaluronan in orbital connective tissues seen in thyroid-associated ophthalmopathy is unknown. Moreover, the link between the orbital manifestations of Graves' disease and those in the pretibial skin, localized dermopathy, has yet to be established. We have reported recently that leukoregulin, an activated T lymphocyte-derived cytokine, dramatically induces hyaluronan synthesis and prostaglandin-endoperoxide H synthase 2 in human orbital fibroblasts in culture. In the current studies, utilizing giant two-dimensional gel electrophoresis, we find that orbital fibroblasts express constitutively a protein profile that distinguishes them from skin fibroblasts derived from the abdominal wall and from the pretibium. We further demonstrate that leukoregulin, when present in culture medium for 16 hr, up-regulates a set of orbital fibroblast proteins not present in untreated cultures or in fibroblasts from the abdominal wall. However, some of the same protein inductions are present in the pretibial fibroblasts. These leukoregulin-induced changes in protein expression are completely blocked by dexamethasone (10 nM). Our findings are the first to identify proteins that appear to be expressed and differentially regulated in an anatomical site-restricted manner in orbital and pretibial fibroblasts and seem to establish a molecular link between fibroblasts from the orbit and those in pretibial skin.

Binding Sites↗
PubMed1998

Antiviral agent blocks breathing of the common cold virus.

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a "breathing" model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

Antiviral Agents↗
PubMed1998

CD40 engagement up-regulates cyclooxygenase-2 expression and prostaglandin E2 production in human lung fibroblasts.

A newly emerging view of fibroblasts is that they are vital for initiating inflammation and respond to and direct the activities of leukocytes. Human fibroblasts can express CD40, an activation Ag the ligand of which is displayed by activated leukocytes. We demonstrate here that CD40 engagement on human lung fibroblasts dramatically increases proinflammatory PGE2 synthesis. This up-regulation is mediated through an induction of cyclooxygenase-2 (Cox-2) since Cox-2-selective inhibitors block the up-regulation. Western and Northern blot analyses demonstrated that Cox-2 protein and mRNA are dramatically increased in fibroblasts following CD40 engagement. We conclude that CD40 is a major pathway in human fibroblasts for the induction of Cox-2. There is intense interest in devising strategies for disruption of the CD40-CD40 ligand system to blunt inflammation. Such an intervention would be expected to attenuate the up-regulation of fibroblast Cox-2 and PGE2 production at the site of tissue injury.

CD40 Antigens↗
PubMed1998

Development of walking, swimming and neuronal connections after complete spinal cord transection in the neonatal opossum, Monodelphis domestica.

Development of coordinated movements was quantitatively assessed in adult opossums (Monodelphis domestica) with thoracic spinal cords transected by (1) crushing 7-8 d after birth [postnatal days 7-8 (P7-P8)]; at 2-3 years of age, systematic behavioral tests (e.g., climbing, footprint analysis, and swimming) showed only minor differences between control (n = 5) and operated (n = 10) animals; and (2) cutting on P4-P6; at 1 month these opossums exhibited coordinated walking movements but were unable to right themselves from a supine position, unlike controls (n = 6). When tested at 2 or 6 months, they could right themselves and showed remarkable coordination, albeit with more differences from controls than after a crush. No animals with spinal cords that were crushed at P14-18 survived because of cannibalism by the mother. Morphological studies (n = 10) 3 months-3 years after crush at 1 week showed restoration of structural continuity and normal appearance at the lesion site. Animals with cut rather than crushed cords showed continuity but greater morphological deficits. That lesions were complete was demonstrated by examining morphology and nerve impulse conduction immediately after crushing or cutting the spinal cord in controls. After lumbar spinal cord injection of 10 kDa dextran amine, retrogradely labeled cells were found rostral to the lesion in hindbrain and midbrain nuclei. Conduction was restored across the site of the lesion. Thus complete spinal cord transection in neonatal Monodelphis was followed by development of coordinated movements and repair of the spinal cord, a process that included development of functional connections by axons that crossed the lesion.

Animals↗
PubMed1998

Mortality studies of metalworking fluid exposure in the automobile industry: VI. A case-control study of esophageal cancer.

BACKGROUND: Results are reported from a nested case-control study of 60 esophageal cancer deaths among 46,384 automobile manufacturing workers potentially exposed to metalworking fluids (MWF) in machining and grinding operations. METHODS: By using incidence-density sampling, controls were selected with a sampling ratio of 20:1 from among co-workers who remained at risk by the age of death of the case, matched on race, gender, plant, and year of birth. Conditional logistic regression was used to evaluate the risk associated with cumulative exposure (mg/m3-years) to each of three types of metalworking fluid (straight, soluble, and synthetic MWF), as well as with years of exposure to selected components of MWF, including nitrosamines, sulfur, biocides, and several metals. RESULTS: Esophageal cancer was found to be significantly associated with exposure to both soluble and synthetic MWF in grinding operations. The odds ratios (ORs) for grinding with soluble MWF were elevated at 2.5 or greater in all categories of cumulative exposure, although the exposure-response trend was statistically significant only when exposure was measured as duration. Those with 12 or more years exposure to soluble MWF in grinding operations experienced a 9.3-fold relative risk of esophageal cancer mortality (95% CI = 2.1-42.1). The OR for ever grinding with synthetic MWF was 4.1 (95% CI = 1.1-15.0). Elevated risk was also associated with two agents found in both synthetic and soluble fluids, nitrosamines, and biocides. For exposure to nitrosamines, the OR was 5.4 (95% CI = 1.5-19.9); for biocides the OR was 3.8 (95% CI = 0.8-18.9). However, because the same workers were exposed to grinding with synthetics, nitrosamines and biocides, it was not possible to separate the specific risks associated with these components.

Automobiles↗
PubMed1998

Actions of the pyrethroid insecticides cismethrin and cypermethrin on house fly Vssc1 sodium channels expressed in Xenopus oocytes.

Voltage-sensitive sodium channels encoded by the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1/tipE sodium channels expressed in oocytes were highly sensitive to tetrodotoxin; half-maximal inhibition of sodium currents by tetrodotoxin was obtained at a concentration of 2.4 nM. Cismethrin, a pyrethroid that produces Type I effects on intact nerve, slowed the inactivation of sodium currents carried by Vssc1/tipE channels during a depolarizing pulse and induced a tail current after repolarization that decayed with a first-order time constant of approximately 650 ms. The voltage dependence of activation and steady-state inactivation of cismethrin-modified channels were shifted to more negative potentials. Cypermethrin, a pyrethroid with Type II effects on intact nerve, also prolonged the inactivation of Vssc1/tipE sodium channels and induced a tail current. However, the cypermethrin-induced tail current was extremely persistent, decaying with a first-order time constant of approximately 42 s. Unlike cismethrin, the effect of cypermethrin was use dependent, requiring repeated depolarizing pulses for the full development of modified sodium currents. The divergent effects of cismethrin and cypermethrin on Vssc1/tipE sodium channels expressed in oocytes are consistent with the actions of these and related compounds on sodium channels in invertebrate and vertebrate nerve preparations and provide insight into the mechanisms underlying the production of Type I and II effects on neuronal excitability.

Animals↗
PubMed1998

Health service studies in the terminally ill cancer patient.

The number of high quality health service research studies in care of the terminally ill patient is very limited. For some areas of care, such as coordination of care for the dying, the clinical benefit is not clear, but the cost-effectiveness evidence seems compelling enough to provide services. For others, such as the use of advanced directives or hospice care, the ethical and medical rationale is compelling, but the evidence of clinical benefit or better cost-effectiveness is limited.

Health Services Research↗
PubMed1998

Cyclooxygenases as the principal targets for the actions of NSAIDs.

The recent identification, cloning, and characterization of two cyclooxygenases has provided insight into how nonsteroidal anti-inflammatory drugs can beneficially inhibit prostaglandin production in inflammation but also produce side-effects in the gut and kidney. The subtle differences in the sites in which these drugs bind the enzymes has allowed development of inhibitors that exhibit selectivity for the inflammatory cyclooxygenase and spare the housekeeping enzyme. This selectivity in theory should enhance the therapeutic potential of these new drugs.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

The American Society of Clinical Oncology program to improve end-of-life care.

Too few cancer patients have optimal care at the end of life, as measured by unrelieved pain, death in a setting other than home, and uncoordinated care. The American Society of Clinical Oncology (ASCO), as the largest professional society whose members care for most cancer patients in the United States, has begun an initiative to improve end-of-life care. Educational programs for ASCO members have been started at the national level. A Task Force on End of Life Care was created to study the current state of end-of-life care, barriers to improved end-of-life care, and what ASCO can do to improve end-of-life care. A member-wide survey has been completed to assess, from the professional's perspective, the current state of and barriers to end-of-life care. Specific sections of the Task Force will address hospice care, physician- assisted suicide, clinical barriers, economic barriers, research initiatives, educational deficiencies, and quality of care at the end of life. Improvements in end-of-life care can be made if the current deficiencies are noted, barriers ascertained, and specific solutions found. Professional societies have both the interest and responsibility to improve end-of-life care.

Journal Article↗
PubMed1998

Characterization of xenobiotic-metabolizing enzymes and nitrosamine metabolism in the human esophagus.

Esophageal cancer has been associated with tobacco smoking, and nitrosamines are possible causative agents for this cancer. The present study investigated the metabolism of the tobacco carcinogens N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosodimethylamine (NDMA), as well as the presence of xenobiotic-metabolizing enzymes in human esophageal tissues from individuals in the United States and Huixian, Henan Province, China (a high-risk area for esophageal cancer). All esophageal microsomal samples activated NNN and the metabolic rate was 2-fold higher in the esophageal samples from China than the USA. All microsomal samples activated NDMA. However, most of the microsomal samples did not activate NNK. Troleandomycin (an inhibitor of cytochrome P450 3A) decreased the formation of NNN-derived keto acid by 20-26% in the esophageal microsomes. The activities for NADPH: cytochrome c reductase, ethoxycoumarin O-deethylase, NAD(P)H: quinone oxidoreductase and glutathione S-transferase were present in the esophageal samples. Coumarin 7-hydroxylase (a representative activity for P450 2A6) activity was not detected in the esophageal microsomal samples. The activities for nitrosamine metabolism and xenobiotic-metabolizing enzymes were decreased (by 30-50%) in the squamous cell carcinomas compared with their corresponding non-cancerous mucosa. The presence of activation and detoxification enzymes in the esophagus may play an important role in determining the susceptibility of the esophagus to the carcinogenic effect of nitrosamines. Our results suggest that P450s 3A4 and 2E1 are involved in the activation of NNN and NDMA, respectively, in the human esophagus.

Adult↗
PubMed1998

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis by dietary olive oil and squalene.

Epidemiological studies have suggested that frequent olive oil consumption may be a protective factor against lung cancer formation. Squalene, a characteristic compound in olive oil, is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and has been proposed to inhibit the farnesylation of ras oncoproteins. The present study investigated the effect of dietary olive oil and squalene in a mouse lung tumorigenesis model. Female A/J mice were fed AIN-76A diets containing 5% corn oil (control), 19.6% olive oil, or 2% squalene starting at 3 weeks before a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (103 mg/kg, i.p.). Animals were maintained on their respective diets throughout the study. At 16 weeks after NNK administration, 100% of the mice in the control group had lung tumors with a tumor multiplicity of 16 tumors per mouse. The olive oil and squalene diets significantly (P < 0.05) decreased the lung tumor multiplicity by 46 and 58%, respectively. The squalene diet significantly (P < 0.05) decreased lung hyperplasia by 70%. In mice fed a diet containing 2% squalene for 3 weeks, the activation of NNK was increased by 1.4- and 2.0-fold in lung and liver microsomes, respectively, but its relationship to the inhibition of carcinogenesis is not clear. These results demonstrate that dietary olive oil and squalene can effectively inhibit NNK-induced lung tumorigenesis.

Animals↗
PubMed1998

A comparison of three additive tree algorithms that rely on a least-squares loss criterion.

The performances of three additive tree algorithms which seek to minimize a least-squares loss criterion were compared. The algorithms included the penalty-function approach of De Soete (1983), the iterative projection strategy of Hubert & Arabie (1995) and the two-stage ADDTREE algorithm, (Corter, 1982; Sattath & Tversky, 1977). Model fit, comparability of structure, processing time and metric recovery were assessed. Results indicated that the iterative projection strategy consistently located the best-fitting tree, but also displayed a wider range and larger number of local optima.

Algorithms↗
PubMed1998

Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate.

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 microg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 microg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50 of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of >/=0.5 microg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of >/=1, 600, as measured by ELISA, survived, but only 6 of 42 mice with titers of </=100 survived. This work shows that the purified BoNT/A(Hc) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.

Animals↗