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Biomedical subjects

W A Smith

Publications and source records attributed to W A Smith.

At least 19 recordsLinked to original sources
PubMed1997

Tissue distribution of DNA adducts in rats treated by intramammillary injection with dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene.

Dibenzo[a,l]pyrene (DBP) has recently emerged as a potent environmental carcinogen having greater carcinogenicity in the rat mammary epithelial glands than 7,12-dimethylbenz[a]anthracene (DMBA), previously considered to be the most potent mammary carcinogen and benzo[a]pyrene (BP), a ubiquitous environmental carcinogen. Previous studies on the tumor-initiating potential of DBP, DMBA, and BP demonstrated that DBP was 2.5 times more potent in inducing the tumors in mouse skin and rat mammary glands than DMBA; BP was a weak mammary carcinogen in these animals. The present study was designed to investigate if the significantly increased mammary carcinogenicity of DBP over DMBA and BP was related to increased DNA adduction at the target site. Female Sprague-Dawley rats were treated by intramammillary injection with an equimolar dose of 0.25 micromol/gland of DBP, DMBA, and BP at the 3rd, 4th and 5th mammary glands on both sides. 32P-Postlabeling analysis of mammary epithelial DNA of rats treated with DBP produced two major (nos. 3 and 6) and at least 5 minor adducts. DMBA treatment resulted in one major and 4 minor DNA adducts while BP produced one major and two minor adducts. Quantitation of the adduct radioactivity revealed that DNA adduction was 6- and 9-fold greater in DBP-treated animals than in BP- and DMBA-treated animals, respectively. The adduct levels per 10(9) nucleotides in mammary epithelial cells for DBP, BP and DMBA were in the following descending order: 1828 +/- 378, 300 +/- 45 and 207 +/- 72, respectively. Tissue distribution of DNA adducts in non-target organs following DBP treatment showed similar adduct pattern as found in the mammary epithelial cells except the liver, which resulted in 4 additional adduct spots; vehicle-treated tissue DNA processed in parallel did not show any detectable adducts. DMBA- and BP-DNA adduct patterns in various tissues were similar to that found in mammary epithelial cells, however, significant quantitative differences were found; BP-DNA adducts were undetectable in the pancreas and bladder. Quantitation of adduct radioactivity showed a 15- to 60-fold lower DBP-DNA adduction in these tissues than the levels found in the mammary tissue; similarly 5-20 and 30-100 times lower DNA adduction was found following treatment with DMBA and BP, respectively. The significantly increased binding of DBP to the mammary epithelial DNA over BP and DMBA is in concordance with its known higher mutagenicity and tumorigenicity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Insulin receptor-like tyrosine kinase in the tobacco hornworm, Manduca sexta.

Phosphotyrosine-containing proteins are present in the prothoracic glands, muscle, and fat body of Manduca sexta, as determined by immunoprecipitation followed by kinase assay, and by Western blotting. One such protein (M(r) 178,000) can also be immunoprecipitated using antibodies directed against the human insulin receptor and insulin receptor substrate. The 178 kD protein appears to be expressed more strongly in prothoracic glands removed just prior to wandering (days 3-4) and prior to pupation (days 7-9), and phosphorylation of the protein is enhanced by an M. sexta brain factor. The results suggest that a tyrosine-kinase-linked molecule similar to the insulin receptor may play a regulatory role in M. sexta.

Animals↗
PubMed1996

Involvement of microtubules in prothoracicotropic hormone-stimulated ecdysteroidogenesis by insect (Manduca sexta) prothoracic glands.

Secretion of ecdysteroid molting hormones by insect prothoracic glands is stimulated by neuropeptide prothoracicotropic hormones (PTTH). Studies reported here were conducted to assess the effects of microfilament and microtubule inhibitors on in vitro ecdysteroidogenesis by prothoracic glands of Manduca sexta. Microfilament inhibitors (cytochalasins B and D) had no effect on basal or big PTTH-stimulated ecdysteroidogenesis. Microtubule inhibitors (colchicine, podophyllotoxin, nocodazole) had no effect on basal ecdysteroid secretion, but suppressed PTTH-stimulated secretion in a concentration-dependent manner. The effect of nocodazole was partially reversible, suggesting it was not due to nonspecific toxicity. Colchicine had no effect on glandular ecdysteroid levels, indicating that inhibition was not due solely to blockage of secretion. The combined results are consistent with the hypothesis that microtubule-mediated transport of ecdysteroid precursors plays a critical role in stimulation of ecdysteroidogenesis by PTTH.

Actin Cytoskeleton↗
PubMed1996

Cleveland clinic rotodynamic pump.

BACKGROUND: It is now accepted that 70% to 80% of patients with end-stage heart failure would benefit from a permanent implanted left ventricular assist device. Previously there was little consideration of the use of nonpulsatile pumps for this function. METHODS: An extensive 5-year engineering research and development program to develop a permanent implanted nonpulsatile blood pump has been undertaken. RESULTS: We have developed a continuous-flow blood pump of small size (207 g) and low power requirement (6.5 watts) producing 5 L/min flow with low hemolysis. CONCLUSIONS: This pump has the potential to be the basis of an innovative ventricular assist system.

Animals↗
PubMed1996

Cyclic AMP is a requisite messenger in the action of big PTTH in the prothoracic glands of pupal Manduca sexta.

Prothoracicotropic hormone (PTTH), a peptide produced by the insect brain, stimulates the prothoracic glands to secrete ecdysteroids. The big form of this peptide (25.5 kDa) has been postulated to act through cyclic AMP in larval Manduca sexta, but the role of the cyclic nucleotide in the action of PTTH in pupal glands has been less clear. Results of the present study indicate that PTTH-stimulated ecdysteroid secretion and protein phosphorylation by glands removed from pupal Manduca sexta are blocked by two inhibitors of cAMP-dependent protein kinase: Rp-cAMPS, an antagonist of cAMP binding to the regulatory subunit of the kinase, and H-89, an inhibitor of the catalytic subunit of the kinase. Further, PTTH stimulates significant accumulation of cAMP in pupal glands, although less than that previously seen in PTTH-stimulated larval glands. Cyclic AMP-dependent protein kinase is found in cytoplasmic and membrane-associated glandular subfractions, as measured by incorporation of [32P]8-N3cAMP into the regulatory subunit of the kinase. PTTH enhances cytoplasmic cAMP content and appears to increase the amount of cAMP bound to a cytoplasmic type II regulatory subunit of cAMP-dependent protein kinase. The results indicate that cAMP plays a requisite role in PTTH action in pupal glands, thus arguing in favor of a uniform mechanism of action for the peptide during Manduca development.

Animals↗
PubMed1996

Investigation of presumptive mobilization pathways for calcium in the steroidogenic action of big prothoracicotropic hormone.

Ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm Manduca sexta is stimulated by the cerebral neuropeptide prothoracicotropic hormone (PTTH). PTTH-stimulated cAMP synthesis and ecdysone secretion are dependent on the presence of extracellular calcium, suggesting that PTTH enhances calcium entry into the cytosol. Such entry into the cytosol might involve the opening of a plasma membrane calcium channel, or a mechanism dependent upon prior inositol triphosphate (IP3)-mediated release of intracellularly stored calcium. In pupal prothoracic glands, PTTH does not increase IP3 or other inositol phosphates over-times ranging from seconds up to 30 min, even in the presence of lithium. However, the L-type calcium channel antagonist nitrendipine completely prevents PTTH-stimulated ecdysone synthesis. A 41 kDa G-protein in prothoracic glands is ADP-ribosylated by pertussis toxin. However, PTTH-stimulated ecdysone synthesis is unaffected by prior exposure to pertussis toxin, indicating that the 41 kDa protein is not involved in the acute stimulation of steroidogenesis. By contrast, cholera toxin has a stimulatory effect on ecdysone secretion suggesting the involvement of a Gs-like protein. Based on the absence of PTTH-stimulated inositol phosphate formation in pupal prothoracic glands, it is suggested that calcium mobilization may occur through the opening of a calcium channel, possibly regulated by Gs.

Animals↗
PubMed1996

Use of a microsome-mediated test system to assess efficacy and mechanisms of cancer chemopreventive agents.

There is a growing need for short-term assays which can assess the mechanisms and efficacy of cancer chemopreventive agents. In the present study we have employed a microsome-mediated test system concomitantly with DNA adduct detection to assess the efficacy of five chemopreventive agents, N-acetylcysteine, butylated hydroxytoluene (BHT), curcumin, oltipraz, and ellagic acid. 32P-Postlabeling analysis of DNA incubated with benzo[a]pyrene (BP) in the presence of Aroclor 1254-induced microsomes produced two major adducts: one derived from the interaction of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) with deoxyguanosine (dG) and the other from further activation of 9-OH-BP (309 and 34 adducts/10(7) nucleotides, respectively). With the exception of N-acetylcysteine, all test agents significantly altered BP-DNA adduct levels: Intervention with ellagic acid and oltipraz substantially (64-94%) inhibited both BPDE-dG and 9-OH-BP adducts, while intervention with curcumin and BHT inhibited the BPDE-dG adduct (57% and 38%, respectively) and enhanced the 9-OH-BP adduct (230% and 650%, respectively). Furthermore, ellagic acid was the only test agent observed to inhibit the anti BPDE-dG adduct in the absence of microsomal enzymes, which is consistent with the known conjugation of ellagic acid with BPDE. These results suggest that oltipraz may be acting as an inhibitor of P4501A1, the isozyme involved in activation of BP to BPDE, or by conjugation of the electrophilic species by a metabolite of oltipraz. A plausible mechanism for inhibition of the BPDE-dG adduct and enhancement of the 9-OH-BP adduct by curcumin and BHT includes inhibition of epoxide hydrolase. Our results also indicate that N-acetylcysteine does not act as an electrophilic trapping agent of BP metabolites but may exert its protective effect in vivo by various other means, including modulation of detoxification enzymes and altering DNA repair processes. These data suggest that this cell-free system in conjunction with the sensitive 32P-postlabeling DNA adduct analysis may prove a viable test system for assessing the mechanisms and efficacy of chemopreventive agents.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Feasibility of an air motor-driven centrifugal blood-pumping system.

The use of cardiopulmonary bypass (CPB) is extending out of the cardiac surgery operating room into new venues. The long-term goal of this project is the development of a completely disposable temporary-use CPB system that could be economically distributed to all of the units where it might be needed. Centrifugal blood pumps have demonstrated successful and widespread use. However, they are not as widely available as might be desired because they require a large and expensive console. An inexpensive, small, lightweight, disposable unit, in contrast, could be widely distributed for emergency care of patients and would be logistically practical for patient transportation between the presenting institution and a major cardiac care facility equipped for definitive treatment. An air motor might be an approach to such a device. The current research project underway at the University of Akron in conjunction with the Cleveland Clinic Foundation has focused on the following key feasibility issues: air consumption, air motor noise, and sealing the rotating shaft. Prototypes have been constructed from commercially available vane and turbine motors. Early studies have demonstrated favorable results with regard to air consumption and shaft sealing and directions for handling air motor noise.

Cardiopulmonary Bypass↗
PubMed1996

Comparative analysis of the genes encoding group 3 allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae.

Group 3 allergens of the genus Dermatophagoides represent one of the major groups of house dust mite allergens. The cDNA sequence data for Der p 3, in combination with both N-terminal amino acid sequences and substrate affinity data, have confirmed that the group 3 allergens are trypsin-like proteases. Using the information from the Der p 3 P3WS1 cDNA clone, genes encoding both Der f3 and Der p 3 have now been amplified by the polymerase chain reaction and analysed. Two Der f3 clones and three Der p 3 genomic clones were sequenced. Each of the clones contained a single small intron and encoded a mature protein of 233 amino acids. The nucleotide sequence was identical for both Der f3 clones. There was 81% identity between the Der f3 sequence and the original Der p 3 P3WS1 clone. The calculated molecular weight of Der f3 was 25.27 kDa compared to 24.98 kDa for Der p 3. All the amino acid residues required for the catalytic activity and the substrate specificity were conserved between the two homologues. The coding sequences of two of the three Der p 3 genomic clones were identical to the original Der p 3 P3WS1 clone with the third having nucleotide changes resulting in four non-conservative amino acid substitutions in the mature protein. These substitutions resulted in a molecule with a slightly larger molecular weight and a more acidic pI value than the original Der p 3 clone. This third Der p 3 genomic clone is, therefore, an isoform of the Der p 3 P3WS1 clone and is classified as an isovariant of the allergen. The nucleotide sequence data presented are the first reported for Der f 3. The Der f 3 gene, like the Der p 3 gene, encoded a trypsin-like protease, but with a slightly larger molecular weight.

Allergens↗
PubMed1996

Sequence polymorphisms of the Der p 3 house dust mite allergen.

BACKGROUND: The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectric-focusing studies with the natural Der p 3 protein have indicated that several isoforms exist. OBJECTIVE: To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen. METHODS: Five cDNA clones of Der p 3 have been isolated from a lambda gt10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco R1 digested genomic DNA was performed. RESULTS: Southern blot analysis of Eco R1 digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones differed only in their 3' untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen. CONCLUSIONS: These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group 1 and group 2 house dust mite allergens.

Allergens↗
PubMed1996

The Cleveland Clinic rotodynamic pump program.

The Cleveland Clinic Foundation has developed a unique rotodynamic blood pump for future use as a permanent implant. This pump is small (2.5 x 2.5 inches) and requires an electric input power of 7 watts to produce 5 L/min of blood flow against 100 mm Hg at 3,000 rpm. Initial in vivo testing has confirmed in vitro function and shown low hemolysis. Endurance bench testing has exceeded 12 months of continuous function. This pump is the basis of an innovative ventricular assist system in which power is supplied by a tranocutaneous electrical transmission system, and pacer technology is used for both control logic and telemetry functions. The resulting system will be completed and tested under an NHLBI contract during the next 5 years.

Cardiac Pacing, Artificial↗
PubMed1995

Regulation and consequences of cellular changes in the prothoracic glands of Manduca sexta during the last larval instar: a review.

The prothoracic glands of the tobacco hornworm, Manduca sexta, respond to prothoracicotropic hormone (PTTH) by a regulatory pathway involving cAMP, protein phosphorylation, protein synthesis, and enhanced secretion of ecdysteroids including ecdysone and 3-dehydroecdysone. Recent investigations have revealed that PTTH acts by this general mechanism throughout the fifth larval instar, i.e., during the transition from larva to pupa. However, the glands undergo developmental changes in size, steroidogenic capacity, and in elements of the signalling pathway associated with synthesis, degradation, and intracellular action of cAMP. The present review describes such changes, and their possible regulation and consequences, in the general context of endocrine events underlying larval-pupal metamorphosis during the fifth larval stage.

Animals↗
PubMed1995

Prescribing benzodiazepines for noninstitutionalized elderly.

OBJECTIVE: To describe benzodiazepine prescribing for elderly people living in the community in British Columbia, and to compare such prescribing with an indicator of current guidelines. DESIGN: Descriptive analysis of pharmacy billing data. SETTING: Province of British Columbia. PARTICIPANTS: All elderly persons (age 65 and older) dispensed benzodiazepines by community pharmacies in British Columbia during 1990. MAIN OUTCOME MEASURE: Potentially inappropriate prescriptions were defined by a maximum 2-month limit of 20 diazepam equivalents daily, as determined by the BC Drug Usage Review Program in consultation with experts in the field. Physicians' rates of potentially inappropriate prescribing were determined per 100 benzodiazepine prescriptions written. RESULTS: Almost 24% of elderly people in British Columbia were prescribed benzodiazepines at least once during 1990. Of these, 17.1% were given potentially inappropriate prescriptions. Physicians who prescribed benzodiazepines most frequently had the highest rates of potentially inappropriate prescriptions. CONCLUSION: Prescribing practice does not correspond with our indicator of current guidelines.

Age Factors↗
PubMed1994

Cloning and sequencing of the Dermatophagoides pteronyssinus group III allergen, Der p III.

House dust mites are widely recognized as major factors involved in the triggering of allergic diseases such as asthma. It is now apparent that the group III allergens of the Dermatophagoides mite species may play a significant role in a number of house dust mite allergic cases. Natural Der p III was isolated by gel filtration of salt precipitated Dermatophagoides pteronyssinus extract and as reported previously ran as a doublet of Mr 28 and 30 K on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Natural Der fIII was isolated by affinity purification with the 5A12 monoclonal antibody. Amino acid sequence data was generated for both these proteins which was used to construct DNA probes to screen a Dermatophagoides pteronyssinus cDNA library by hybridization and resulted in the isolation of a recombinant Der p III cDNA clone, P3WS1. The 1059 bp cDNA fragment included a 786 bp open reading frame which encodes a pre-pro region of 29 amino acids and a mature protein of 232 amino acids with a calculated Mr 24,985. A search of the BLAST protein database has confirmed that the Der pIII P3WS1 clone is approximately 50% homologous with other trypsin proteins. We have confirmed with both our natural protein sequence and the P3WS1 amino acid sequence data that the group III allergens are trypsin-like proteins.

Allergens↗
PubMed1994

An assessment of the toxicity of parenteral treatment with copper EDTA and copper heptonate in sheep.

The toxicity of 2 parenteral copper (Cu) supplements was investigated. Di-sodium copper ethylene diamino tetra acetate (Cu EDTA) and Cu heptonate were administered to sheep (n = 9) by a single subcutaneous injection at a concentration of 0,2, 1 and 2 mg Cu/kg each (Trial 1.) Nine sheep were untreated and served as controls. The same treatments were applied to 2 sheep each (Trial 2) with the addition of 3 mg Cu/kg live body mass as Cu heptonate, and Cu heptonate administered intravenously at rates of 0,2, 0,4 and 0,6 mg Cu/kg live body mass. In Trial 1, 67% of the sheep treated with Cu EDTA at 2 mg Cu/kg live body mass died within 3 to 17 d after treatment, while no mortalities occurred in sheep where Cu heptonate was administered at the same dosage rate and even at 3 mg Cu/kg live body mass (P < or = 0,01). Post-mortem examination suggested acute Cu toxicity in all cases. Liver Cu concentrations were markedly increased (P < or = 0,05) by both supplements in groups of 3 treated sheep slaughtered over a 3-month period compared to control animals. The liver Cu concentrations of sheep that succumbed to Cu toxicity were within the normal range of 100 to 450 mg/kg DM. Results from Trial 2 suggested that the 2 sheep treated with 2 mg Cu/kg live body mass as Cu EDTA, experienced a haemolytic crisis between 5 and 11 d after treatment, resulting in the death of one of these sheep. The haemolytic crisis was characterised by a severe decrease in haemoglobin concentration and haematocrit.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Chronic nonpulsatile blood flow. I. Cerebral autoregulation in chronic nonpulsatile biventricular bypass: carotid blood flow response to hypercapnia.

To investigate the response of the carotid blood flow and general circulation to hypercapnia in chronic nonpulsatile blood flow, we performed 18 carbon dioxide gas inhalation studies on three calves undergoing a centrifugal biventricular bypass with ventricular fibrillation. An ultrasonic flow probe was put on the carotid artery during biventricular bypass pump implantation, and pump flows were maintained at 90, 100, and 120 ml/kg per minute for 1 week each. The carbon dioxide inhalation studies were performed twice a week. Hypercapnia was induced by administering pure carbon dioxide gas through a nasal tube at flow rates of 0, 5, 7.5, 10, 12.5, and 15 L/min for 5 minutes each at three different nominal pump flow rates, and the resultant arterial blood gas and hemodynamic changes were recorded. No significant correlation existed between the carotid blood flow and mean aortic pressure, which varied from 70 to 140 mm Hg, but the carotid blood flow correlated significantly (p < 0.01) with the systemic pump flow rate. A significant (p < 0.01) linear relationship was found between the carotid blood flow and arterial carbon dioxide tension. For each 1 mm Hg change in arterial carbon dioxide tension, there was a 2.8 % change in the carotid blood flow. The percent changes in the carotid blood flow in response to arterial carbon dioxide tension were calculated as 2.9%, 3.7%, and 2.5% for each 1 mm Hg change in arterial carbon dioxide tension at pump flows of 90, 100 and 120 ml/kg per minute. No significant differences in the carotid blood flow response to hypercapnia were detected among the three systemic pump flow rates. These results thus suggested that chronic nonpulsatile blood flow had no detrimental effects on cerebral autoregulation.

Animals↗
PubMed1993

Developmental changes in cyclic AMP-dependent protein kinase associated with increased secretory capacity of Manduca sexta prothoracic glands.

In Manduca sexta, basal and PTTH-stimulated secretion of ecdysteroids by prothoracic glands in vitro increases from days 1 to 4 of the fifth larval stage. Glandular content of cAMP-dependent protein kinase was analyzed to determine if the enzyme changes in concert with increased secretory response. Photoaffinity labeling with [32P]8-N3 cAMP revealed a 55-kDa cAMP-binding protein characteristic of the regulatory subunit of type-II cAMP-dependent protein kinase (RII). It appears that RII is one of a limited number of cellular proteins that is phosphorylated in the presence of [gamma-35S]ATP; the thiophosphorylated protein and the photoaffinity-labeled regulatory subunit possess the same M(r) and pI, and thiophosphorylation is blocked by mammalian cAMP-dependent protein kinase inhibitor. From days 1 to 4 of the fifth instar, glandular content of RII increases in conjunction with increased ecdysteroid secretory capacity. Application of JH analog on day 1 significantly inhibits the observed increase in RII. Catalytic subunit activity does not change from days 1 to 4 of the fifth instar, nor does cellular content of a 34-kDa protein previously shown to be phosphorylated in response to PTTH. While it is unlikely that increased content of RII is solely responsible for enhanced ecdysteroid secretion by the prothoracic glands, it may serve as a convenient marker for investigating the mechanism by which steroidogenic capacity is regulated.

Animals↗