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Biomedical subjects

W W Nichols

Publications and source records attributed to W W Nichols.

At least 19 recordsLinked to original sources
PubMed2002

Two diarylurea electron transport inhibitors reduce Staphylococcus aureus hemolytic activity and protect cultured endothelial cells from lysis.

Reduction in electron transport is associated with decreased production in alpha-toxin despite the fact that Staphylococcus aureus is able to grow from 1 CFU to >10(7) CFU. Similarly, under anaerobic conditions, S. aureus does not produce alpha-toxin. Although the pathways that connect oxidative metabolism and toxin production are unknown, agents are available that exhibit greater inhibition of plant versus mammalian electron transport. Herbicides block electron transport in plants by inhibiting the formation of phosphoquinol (QH(2)) in plants. Commercial use in farming is possible because these compounds are much less active against the quinones found mammalian mitochondria. Because bacterial electron transport systems are closer to plant than mammalian systems, we hypothesized that inhibitors of respiration might be able to reduce S. aureus electron transport and block the production of alpha-toxin. We studied two compounds and found that the effective dose for the inhibition of bacterial respiration was 50 to >3,500 times lower than the concentration required to cause similar inhibition of rat mitochondrial respiration. Compounds I and II also reduced toxin production in S. aureus without causing overt toxicity to cultured endothelial cells. Finally, the compounds reduced the damage caused by S. aureus when cocultured with the endothelial cells. This raises the possibility that compounds that inhibit bacterial respiration might be prove valuable for the prevention of toxin production in S. aureus.

Animals↗
PubMed2001

Arterial elastance and wave reflection augmentation of systolic blood pressure: deleterious effects and implications for therapy.

Systolic and pulse blood pressures are stronger predictors of stroke, coronary heart disease, myocardial infarction, heart failure, end-stage renal disease, and cardiovascular mortality than diastolic pressure. Furthermore, diastolic pressure is inversely related to coronary heart disease and cardiovascular mortality. Increased elastance (or stiffness, inverse of compliance) of the central elastic arteries is the primary cause of increased systolic and pulse pressure with advancing age and in patients with cardiovascular disease, including hypertension, and is due to degeneration and hyperplasia of the arterial wall; diastolic pressure decreases as arterial elastance increases. As elastance increases, transmission velocity of both forward and backward (or reflected) traveling waves increases, which causes the reflected wave to arrive earlier in the central aorta and augments pressure in late systole. These changes in arterial wall properties cause an increase in left ventricular afterload and myocardial oxygen consumption and a decrease in myocardial perfusion pressure, which may induce an imbalance in the supply-demand ratio, especially in hypertrophied hearts with coronary artery disease. Also, an increase in systolic pressure increases arterial wall circumferential stress, which promotes fatigue and development of atherosclerosis. Vasodilator drugs have little direct active effect on large elastic arteries but can markedly reduce wave reflection amplitude and augmentation index by decreasing elastance of the muscular arteries and reducing pulse wave velocity of the reflected wave from the periphery to the heart. This decrease in intensity (or amplitude) and increase in travel time (or delay) of the reflected wave causes a generalized decrease in systolic pressure and arterial wall stress and an increase in ascending aortic flow during the deceleration phase. The decrease in systolic pressure brought about by this mechanism is grossly underestimated when systolic pressure is measured in the brachial artery.

Adult↗
PubMed2000

Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice.

The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/microg DNA (representing approximately 150,000 diploid cells). Using this assay, we carried out integration studies of three different plasmid DNA vaccines, containing either the influenza hemagglutinin, influenza matrix or HIV gag gene. Six weeks after intramuscular injection, free plasmid was detected in treated muscle at levels ranging from approximately 1,000 to 4,000 copies/microg DNA. At 6 months, the plasmid levels ranged between 200 and 800 copies/microg DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was extrachromosomal. If integration had occurred, the frequency was </= 1-8 integrations per 150,000 diploid cells, which would be at least three orders of magnitude below the spontaneous mutation rate. Our results suggest that the risk of mutation due to integration of plasmid DNA vaccines following intramuscular injection is negligible.

Animals↗
PubMed2000

Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA.

A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro. Six weeks following intramuscular injection, the vast majority of the plasmid was detected in the muscle and skin near the injection site; lower levels of plasmid were also detected in the draining lymph nodes. At early time points (1-7 days) after injection, a low level of systemic exposure could be detected. Occasionally, plasmid was detected in gonads, but it dissipated rapidly and was extrachromosomal - indicating a low risk of germline transmission. Aluminum phosphate adjuvant had no effect on the tissue distribution and did not result in a detectable increase in integration frequency. Biojector delivery, compared with needle injection, greatly increased the uptake of plasmid (particularly in skin at the injection site), but did not result in a detectable increase in integration frequency. Finally, injection of a plasmid DNA vaccine containing the human papilloma virus type 16 E7 gene, known to increase integration in vitro, did not result in detectable integration in mice. These results suggest that the risk of integration following intramuscular injection of plasmid DNA is low under a variety of experimental conditions.

Adjuvants, Immunologic↗
PubMed2000

Plasmid DNA vaccines: assay for integration into host genomic DNA.

The primary safety concern for DNA vaccines is their potential to integrate into host cellular DNA. We describe a sensitive and quantitative assay for investigating the tissue distribution and integration of plasmid DNA vaccines. By including gonadal tissues in the analysis, the potential for germline transmission is also assessed. At various time points after injection, total DNA is isolated from a variety of tissues and assayed by PCR for the presence of plasmid. To test for integration, genomic DNA is first purified away from free plasmid using a series of different gel electrophoresis procedures. The gel-purified genomic DNA is then assayed for integrated plasmid using PCR. Stringent methods are used to prevent contamination. The assay, validated using a variety of positive and negative controls, is capable of detecting one copy of plasmid per ug DNA (approximately 150,000 diploid cells). Using this assay, we have carried out intramuscular studies in mice or guinea pigs for four different DNA vaccine plasmids. There was no evidence of integration to a sensitivity of about one copy/microg DNA, which is at least three orders of magnitude below the spontaneous mutation frequency.

Animals↗
PubMed1999

Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan.

Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes, and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2.6 x 10(7) s(-1) M(-1) by monitoring the onset of inhibition during assays started by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 A resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.

Anti-Infective Agents, Local↗
PubMed1999

The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation.

Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesin (PIA), which is composed of linear beta-1,6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica) locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Sequence comparison with the S. epidermidis ica genes revealed 59 to 78% amino acid identity. Deletion of the ica locus results in a loss of the ability to form biofilms, produce PIA, or mediate N-acetylglucosaminyltransferase activity in vitro. Cross-species hybridization experiments revealed the presence of icaA in several other Staphylococcus species, suggesting that cell-cell adhesion and the potential to form biofilms is conserved within this genus.

Bacterial Adhesion↗
PubMed1999

Stenosis of the main artery supplying an organ: effect of end-organ vascular resistance on the poststenotic peak systolic velocity in an in vitro hydraulic model at Doppler US.

PURPOSE: To test the hypothesis that increased end-organ vascular resistance reduces blood flow to the kidney, thus reducing the mean velocity in the renal artery and secondarily lowering the peak systolic velocity (PSV). MATERIALS AND METHODS: An in vitro hydraulic model with a pulsatile pump, blood-mimicking fluid, interchangeable stenoses, and variable compliance and resistance was used to investigate the relationship between end-organ vascular resistance and poststenotic PSV. RESULTS: Poststenotic PSV was mildly dependent on end-organ vascular resistance and decreased with increasing resistance. CONCLUSION: The results help explain some of the reported variability from using poststenotic PSV to detect hemodynamically significant renal arterial stenoses, but the effect is not great enough to completely explain the variability. Other factors not investigated in this study must be at work as well.

Blood Flow Velocity↗
PubMed1999

Inhibition of arterial thrombus formation by ApoA1 Milano.

The mutant form of human apoA1, known as apoA1 Milano, is formed as a result of arginine 173 to cysteine substitution and inhibits experimental atherosclerosis in cholesterol-fed animals. This study was designed to determine if apoA1 Milano would modify arterial thrombogenesis. Sprague Dawley rats were intravenously administered the carrier alone (n=8) or apoA1 Milano (20 mg. kg-1. d-1 for 4 to 10 days, n=17). The abdominal cavity was opened, and the abdominal aorta was isolated. Whatman paper impregnated with 35% FeCl3 was wrapped around the surface of the aorta, and aortic flow was recorded continuously. In carrier-treated rats, an occlusive platelet-fibrin-rich thrombus was formed in 21.2+/-4.1 (mean+/-SD) minutes. Treatment of rats with apoA1 Milano markedly delayed time to thrombus formation (38.8+/-11.9 versus 21.2+/-4.1 minutes, P<0. 01), inhibited platelet aggregation (25+/-7% versus 50+/-11%, P<0. 01), and reduced weight of the thrombus (18.5+/-1.8 versus 23.7+/-2. 3 mg/cm, P<0.01). Total cholesterol and HDL levels remained similar in both groups of rats, but plasma apoA1 Milano levels were elevated in apoA1 Milano-treated rats. In in vitro studies, incubation of platelets with apoA1 Milano reduced ADP-induced platelet aggregation by about 50%, but apoA1 Milano had no direct effect on vasoreactivity. This study provides further evidence for critical role of platelets in thrombosis. Use of apoA1 Milano offers a novel approach to inhibit arterial thrombosis.

Animals↗
PubMed1998

DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens.

Positive results in the in vitro assay for chromosome aberrations sometimes occur with test chemicals that apparently do not react with DNA, being negative in tests for mutation in bacteria, for DNA strand breaks, and for covalent binding to DNA. These chromosome aberrations typically occur over a narrow concentration range at toxic doses, and with mitotic inhibition. Indirect mechanisms, including oxidative damage, cytotoxicity and inhibition of DNA synthesis induced by chemical exposure, may be involved. Understanding when such mechanisms are operating is important in evaluating potential mutagenic hazards, since the effects may occur only above a certain threshold dose. Here, we used two-parameter flow cytometry to assess DNA synthesis inhibition (uptake of bromodeoxyuridine [BrdUrd]) associated with the induction of aberrations in CHO cells by DNA-reactive and non-reactive chemicals, and to follow cell cycle progression. Aphidicolin (APC), a DNA polymerase inhibitor, induces aberrations without reacting with DNA; 50 microM APC suppressed BrdUrd uptake during a 3-h treatment to <10% of control levels. Several new drug candidates induced aberrations concomitant with marked reductions in cell counts at 20 h (to 50-60% of controls) and suppression of BrdUrd uptake (<15% of control). Several non-mutagenic chemicals and a metabolic poison, which induce DNA double strand breaks and chromosome aberrations at toxic dose levels, also suppressed DNA synthesis. In contrast, the alkylating agents 4-nitroquinoline-1-oxide, mitomycin C, methylnitrosourea, ethylnitrosourea, methylmethane sulfonate and ethylmethane sulfonate, and a topoisomerase II inhibitor, etoposide, produced many aberrations at concentrations that were less toxic (cell counts >/=73% of controls) and gave little inhibition of DNA synthesis during treatment (BrdUrd uptake >/=85% of controls), although cell cycle delay was seen following the 3-h treatment. Thus, inhibition of DNA synthesis at the time of treatment is supporting evidence for an indirect mechanism of aberrations, when there is no direct DNA reactivity.

Animals↗
PubMed1998

From famine to feast: the role of methylglyoxal production in Escherichia coli.

The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

Carbon-Oxygen Lyases↗
PubMed1998

Melagatran, an oral active-site inhibitor of thrombin, prevents or delays formation of electrically induced occlusive thrombus in the canine coronary artery.

Intravenous administration of thrombin inhibitors, such as hirudin, has been shown to decrease the frequency of coronary artery reocclusion after thrombolysis. However, recent findings in large clinical trials in patients with unstable angina and myocardial infarction have failed to demonstrate a sustained antithrombotic effect after cessation of drug treatment. These findings indicate a need for a prolonged antithrombotic regimen, preferably an orally active thrombin inhibitor. To test the hypothesis that a regimen consisting of oral thrombin inhibitor will delay or prevent the formation of occlusive clot, anesthetized dogs were given saline (n = 9) or a single dose of a novel active site low-molecular-weight thrombin inhibitor melagatran by nasogastric tube (1.5 mg/kg, n = 6; 2.5 mg/kg, n = 6), and 15 min later, a potent thrombogenic stimulus in the form of anodal current (100 microA) was applied to the intimal surface of the narrowed left anterior descending coronary artery (LAD). All saline-treated dogs developed stable thrombus, indicated by zero flow at 34 +/- 7 min after initiation of direct current. On the other hand, one of the six dogs given high-dose melagatran did not develop thrombotic occlusion of the LAD during the entire 4 h of observation. Mean time to occlusive thrombus formation in 11 other dogs was prolonged 4-5 times as compared with that in the saline-treated dogs (p < 0.001). Spontaneous thrombolysis was observed in three of 11 dogs after initial clot formation. Overall, the coronary artery was patent for 68% (low dose) and 75% (high dose) of the observation period in melagatran-treated dogs (vs. 14% of observation period in saline-treated dogs). Peak plasma concentration was 0.87 +/- 0.22 microM in dogs given low-dose and 1.38 +/- 0.30 microM in dogs given high-dose melagatran. The activated partial thromboplastin time (aPTT) increased 1.5-fold at peak plasma concentration of melagatran. These observations imply (a) thrombin generation plays a critical role in thrombus formation in narrowed coronary arteries, (b) oral melagatran prevents or delays thrombus formation, whereas the aPTT is only modestly prolonged, and (c) the thrombus formed in the presence of melagatran is prone to spontaneous lysis in this canine model of coronary thrombosis.

Administration, Oral↗
PubMed1997

Calculating vascular resistances.

Vascular resistance calculations often affect decisions regarding therapeutic options encountered by physicians and their patients. However, many of the terms, units, and methods used when calculating vascular resistances are ambiguous. This report attempts to clarify some of these ambiguities and suggests methods for predicting normal vascular resistances.

Blood Flow Velocity↗
PubMed1997

Transition to post-operative epidural or patient-controlled intravenous analgesia following total intravenous anaesthesia with remifentanil and propofol for abdominal surgery.

Remifentanil is an ultrashort acting mu opioid, well suited to total intravenous (i.v.) anaesthesia. Pain immediately following emergence from anaesthesia is a potential problem because of the rapid offset. This study investigated the transition from remifentanil/propofol total intravenous anaesthesia to post-operative analgesia with epidural or patient controlled analgesia morphine in 22 patients undergoing major abdominal surgery. A remifentanil post-operative infusion initiated during emergence was titrated in the recovery room for 30 min, at which time 14% of patients had a pain score of 2 and 86% had pain scores of 0 or 1 (0 = no pain; 1 = mild pain; 2 = moderate pain; 3 = severe pain), at a mean infusion rate of 0.086 microgram kg-1 min-1. A smooth transition was then made to either epidural analgesia or patient controlled analgesia with morphine; pain scores were not significantly changed during the transition. Nausea occurred in 16 of the 22 patients, but only following administration of morphine. Epidural analgesia produced significantly lower pain scores on the surgical ward compared with patient controlled analgesia.

Abdomen↗
PubMed1996

Growth regulation by peroxisome proliferators: opposing activities in early and late G1.

Peroxisome proliferators (PPs) are a diverse group of nongenotoxic rodent liver carcinogens. One potential mechanism for the carcinogenicity of PPs is epigenetic modulation of growth-regulatory signal transduction pathways. We investigated the effects of PPs on growth-regulatory gene expression and cell proliferation in immortalized mouse liver cells, comparing PPs with other growth regulators and tumor promoters of known activity. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, and ciprofibrate ethyl-ester were found to be potent inducers of immediate-early gene expression (including c-fos, c-jun, junB, egr-1, NUP475, and to a lesser extent fosB, JE, and KC, with maximal expression seen 1 h after treatment of serum-deprived quiescent cells. The gene induction was potently inhibited by protein kinase inhibitor H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride] but not by H8 [N-¿2-(methyl-amino)ethyl¿-5-isoquinolinesulfonamide dihydrochloride], indomethacin, or nordihydroguaiaretic acid. Compared with other growth regulators, the profile of PP-induced gene expression was most similar to that induced by arachidonic acid and eicosatetraynoic acid. The induction of immediate-early gene expression by PPs was followed by enhanced progression into S phase (DNA synthesis) when quiescent cells were treated with the PPs for only 1 h, washed, and then incubated without PPs. However, no stimulation of DNA synthesis was seen when the PPs were continually present. Furthermore, the PPs inhibited serum-induced DNA synthesis, even when they were added 6 h after serum stimulation (in late G1). Dehydroepiandrosterone-sulfate, a unique PP in being a steroid, had no detectable effect on immediate-early gene expression, did not stimulate DNA synthesis when applied for only 1 h, but did inhibit serum-induced DNA synthesis. Thapsigargin and A23187 mimicked this mitoinhibitory activity of PPs, suggesting that calcium mobilization by PPs might be involved. Our results demonstrate that PPs can modulate cell proliferation either by a stimulatory activity that functions in early G1, associated with activation of immediate-early gene expression, or by an inhibitory activity that functions in late G1; both activities could potentially play a role in tumor promotion by PPs.

Animals↗