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Biomedical subjects

N M Steele

Publications and source records attributed to N M Steele.

15 recordsLinked to original sources
PubMed2001

Ten isoenzymes of xyloglucan endotransglycosylase from plant cell walls select and cleave the donor substrate stochastically.

To map the preferred cleavage sites of xyloglucan endotransglycosylases (XETs; EC 2.4.1.207) along the donor substrate chain, we incubated the enzymes with tamarind (Tamarindus indica) xyloglucan (donor substrate; approximately 205 kDa; 21 microM) plus the nonasaccharide [(3)H]XLLGol (Gal(2).Xyl(3).Glc(3). [(3)H]glucitol; acceptor substrate; 0.6 microM). After short incubation times, to minimize multiple cleavages, the size of the (3)H-labelled transglycosylation products (determined by gel-permeation chromatography) indicated the positions of the cleavage sites relative to the non-reducing terminus of the donor. There was very little difference between the size profiles of the products formed by any of ten XETs tested [one native XET purified from cauliflower (Brassica oleracea) florets, four native XET isoenzymes purified from etiolated mung-bean (Phaseolus aureus) shoots, native XETs purified from lentil (Lens culinaris) and nasturtium (Tropaeolum majus) seeds, and three insect-cell-produced thale-cress (Arabidopsis thaliana) XETs (EXGT, TCH4 and MERI-5)]. All such product profiles showed a good fit to a model in which the enzyme chooses its donor substrate independently of size and attacks it, once only, at a randomly selected cleavage site. The results therefore do not support the hypothesis that different XET isoenzymes are adapted to produce longer or shorter products such as might favour either the efficient integration of new xyloglucan into the cell wall or the re-structuring of old xyloglucan within an expanding wall.

Cell Wall↗
PubMed2000

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET).

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan-enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0-6.5), Km for the nonasaccharide XLLGol (Gal2.Xyl3.Glc3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher Km values for XLLGol (70-130 microM) than the four mung bean isoenzymes (16-35 microM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.

Catalysis↗
PubMed2000

Effect of exercise on blood pressure in pregnant women with a high risk of gestational hypertensive disorders.

OBJECTIVE: To prospectively determine whether moderate exercise during pregnancy lowers blood pressure. STUDY DESIGN: A randomized, controlled trial with one test group and one control group. All subjects have a history of mild hypertension, gestational hypertensive disorders or a family history of hypertensive disorders. Subjects were recruited before 14 weeks' gestation. After four weeks of observation, the subjects were randomly assigned to either the exercise or control group. The exercise group visited the laboratory three times a week for 10 weeks (18-28 gestation weeks) to perform 30 minutes of exercise at Rating of Perceived Exertion level 13. RESULTS: A total of 16 pregnant women (mean age, 30 years) participated. The mean metabolic equivalent during exercise sessions was 4.7 (SD = 0.8). Blood pressure measurements were compared before and after the 10-week exercise period in the two groups. Systolic blood pressures did not change significantly, but diastolic blood pressure (DBP) in the exercise group decreased by 3.5 mm Hg, while that in the control group increased by 1.1 mm Hg. Thus, the pre-post change in DBP differed by 4.6 mm Hg between groups. Exercise treatment reduced the diastolic blood pressure to a near-significant level in the exercise group (t = 2.34, df = 7, P = .052). Percent body fat did not differ between the exercise and control groups either before or after exercise treatment. ANOVA revealed that pregnancy had a significant effect (F(1, 14) = 5.7, P = .03) on increasing the percentage of fat, but exercise treatment did not (F(1, 14) = .18, P = .68). Estimated energy expenditure in overall daily physical activities during the intervention did not differ between the two groups despite the inclusion of exercise. CONCLUSION: This study detected a strong trend that 10 weeks of moderate exercise lowered the diastolic blood pressure among pregnant women at risk of hypertensive disorders. The reductions were probably due to the effect of exercise itself, not to weight or overall daily physical activity levels.

Adult↗
PubMed1999

Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex.

We describe a novel and general, mechanism-based, method for purification of xyloglucan endotransglycosylases (XETs) from crude plant extracts. Putative isoforms, obtained by step-wise precipitation with (NH4)2SO4, were incubated with tamarind xyloglucan (approximately 1 MDa) to form stable xyloglucan-XET complexes with apparent molecular masses >500 kDa on gel-permeation chromatography (GPC). Subsequent addition of xyloglucan-derived oligosaccharides (a mixture of XET acceptor substrates) caused a shift in the GPC elution volume of the activity back to that expected of a approximately 32 kDa protein, presumably by completing the transglycosylation reaction and so freeing the enzyme from the xyloglucan (donor substrate). This simple two-step method enabled the isolation of each XET activity attempted [various (NH4)2SO4 cuts from extracts of cauliflower florets and mung bean seedlings], in pure form as judged by SDS/PAGE.

Ammonium Sulfate↗
PubMed1998

Xyloglucan endotransglycosylase: evidence for the existence of a relatively stable glycosyl-enzyme intermediate.

Xyloglucan endotransglycosylases (XETs) catalyse the breakdown of xyloglucan molecules predominantly by transglycosylation. In this process, fragments of cleaved polysaccharide are preferentially transferred to other xyloglucan molecules or their oligosaccharide subunits, with overall retention of the anomeric configuration of the glycosidic bond. In accordance with the theory, we propose that the cleavage and re-formation of the glycosidic bond in xyloglucan involves the formation of a glycosyl-enzyme intermediate which decomposes by transfer of the glycosyl moiety to a suitable carbohydrate acceptor. XETs from nasturtium seed cotyledons, mung bean hypocotyls and cauliflower florets interacted with xyloglucan to form complexes of high Mr as judged by gel-permeation chromatography. The nasturtium enzyme also showed evidence of XET-xyloglucan complex-formation according to anion-exchange chromatography and adsorption of the complex to filter paper on the basis of affinity of its xyloglucan moiety for cellulose. The XET-xyloglucan complex was stable in water, 6 M urea and acidic and alkaline buffers (pH 2.5-9.5), but readily decomposed by transferring its glycosyl moiety to xyloglucan-derived oligosaccharides or by incubation with the strong nucleophile imidazole at pH 3.8-9.6. These results strongly support the assumption that XET forms a relatively stable covalently linked glycosyl-enzyme intermediate.

Cell Line↗
PubMed1987

Hepatic bile formation in the rat. Addition of vasoactive intestinal peptide to the equation.

While changes in gastric, pancreatic, and intestinal secretion in response to more recently identified gastrointestinal peptides have been characterized, there has been less investigation into effects of these hormones on hepatic bile production. The isolated perfused rat liver model has been used to examine effects of vasoactive intestinal peptide (VIP), somatostatin, bombesin, and thyrotropin-releasing hormone (TRH) on bile flow and bile acid transport. No changes were seen following bolus administration of bombesin (3 X 10(-8)-1.5 X 10(-6) M) or TRH (3 X 10(-7)-3 X 10(-6) M), while somatostatin (6 X 10(-6) M) produced a small decrease in bile flow without any change in bile acid output. VIP (3 X 10(-7) M) caused a highly significant increase in both volume of bile flow (0.85 +/- 0.8 to 1.11 +/- 0.09 microliter/min/g liver, P less than 0.001) and bile acid output (31.6 +/- 1.5 to 43.2 +/- 1.7 nmol/min/g liver, P less than 0.001). Elimination of Ca2+ from liver perfusate did not prevent VIP-induced increases in bile flow and bile acid output, and no synergistic effect of concomitant theophylline administration was observed. While effects of VIP on bile flow appear to be due to alterations in hepatic transport of bile acids, the exact mechanism(s) producing these changes remains to be elucidated.

Animals↗
PubMed1987

Influence of gastrointestinal peptides on bile acid transport by isolated rat hepatocytes.

While numerous effects of gut peptides on gastric, pancreatic, and intestinal secretion have been described, there has been little investigation of the influence of these peptides on hepatic function. In the present studies, effects of vasoactive intestinal peptide (VIP), somatostatin, thyrotropin-releasing hormone (TRH), and bombesin on taurocholate transport by isolated rat hepatocytes have been examined. Somatostatin, TRH, and bombesin in incubation media produced no change from control incubations with regard to either uptake of taurocholate by hepatocytes or efflux of bile acid from preloaded cells. However, incubation of hepatocytes with VIP produced a significant decrease in taurocholate uptake (1.34 +/- 0.13 versus 1.73 +/- 0.16 nmole.min-1.10(6) cells-1, P less than 0.001). Studies with verapamil, a calcium-channel blocking agent, and theophylline, an inhibitor of cAMP catabolism, failed to provide evidence for transmembrane Ca2+ flux or alteration in intracellular levels of cAMP, respectively, as mechanisms for the observed inhibition of hepatocyte taurocholate uptake by VIP. These data, coupled with both clinical and other basic observations, suggest that VIP may play a significant role in the regulation of hepatic bile secretion.

Animals↗
PubMed1984

Effects of parenteral and enteral hyperalimentation on hepatic drug metabolism in the rat.

Whereas patients receiving parenteral hyperalimentation frequently have abnormalities in serum liver enzymes, the influence of i.v. administration of hypertonic glucose-protein solutions on hepatic function has received little attention. Recent data from this laboratory indicated that in vivo clearance of pentobarbital was significantly decreased in rats receiving total parenteral hyperalimentation (TPN) vs. animals receiving the same hyperalimentation solution enterally (total enteral hyperalimentation; TEN). To determine the cause of this decreased clearance, we have analyzed mixed-function oxidase activity in hepatic microsomes prepared from livers of rats receiving 7 day continuous i.v. or i.g. infusions of hypertonic glucose (25%) combined with fibrin hydrolysate or crystalline amino acids. Hepatic microsomal cytochrome P-450 and capacity for demethylation of meperidine and hydroxylation of pentobarbital were significantly reduced in TPN animals as compared to TEN and ad libitum chow-fed control rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of microsomal protein revealed increased staining of a 46,500 molecular weight band and decreased intensity of a 52,700 molecular weight band in TPN experiments as compared to TEN studies. Increasing amino acid concentration in infusions to 4.25% from the standard 2.75% resulted in marked reductions in hepatic microsomal drug metabolism in enterally hyperalimented animals. These studies show that the route of alimentation delivery has an important influence on hepatic drug metabolic function in rats and suggest that quantitative and/or qualitative differences in hepatic delivery of protein may be responsible for the quantitative and qualitative differences in hepatic microsomal mixed-function oxidases observed between TPN and TEN animals. If similar changes occur in humans receiving TPN, major alterations in drug regimens may be required.

Amino Acids↗
PubMed1983

Prevention of acetaminophen and cocaine hepatotoxicity in mice by cimetidine treatment.

Hepatotoxicity occurs in animals after administration of large doses of acetaminophen and cocaine and is thought to result from production of reactive metabolites of these parent drugs by cytochrome P450. Because cimetidine binds to cytochrome P450 and inhibits hepatic drug metabolism in both humans and animals, we determined the effects of cimetidine coadministration on acetaminophen and cocaine hepatotoxicity in mice. Marked elevations of serum glutamic pyruvic transaminase and severe pericentral hepatocellular necrosis occurred in animals receiving intraperitoneal doses of 350 mg/kg acetaminophen or 35 mg/kg cocaine, while minimal serum glutamic pyruvic transaminase elevations and liver necrosis were seen in animals who also received 100 mg/kg cimetidine 1 h before and 1 h after administration of either acetaminophen or cocaine. Consistent with the hypothesis that these in vivo protective effects resulted from interaction with cytochrome P450, cimetidine inhibited in vitro hepatic microsomal metabolism of cocaine. However, despite its protective effect against acetaminophen-induced hepatic injury, concomitant administration of cimetidine did not significantly affect plasma pharmacokinetics of acetaminophen, prevent depletion of hepatic glutathione after acetaminophen administration, or alter in vivo covalent binding of [3H]acetaminophen to hepatic proteins. These studies suggest that current theories regarding production of acetaminophen-induced liver damage require reexamination. The possibility that cimetidine treatment might be useful in preventing hepatic damage due to acetaminophen and other hepatotoxins in humans is intriguing and also warrants consideration.

Acetaminophen↗
PubMed1982

Drug metabolism by rat and human hepatic microsomes in response to interaction with H2-receptor antagonists.

Cimetidine has been reported to decrease plasma clearance of drugs in humans and animals. This reduction in hepatic drug metabolism could be due to cimetidine's intrinsic H2-receptor blocking activity. Alternatively, the imidazole ring structure of cimetidine could explain these observations because imidazole derivatives have been reported to be potent inhibitors of hepatic microsomal drug metabolism. Rat and human hepatic microsomal drug metabolism in the presence of cimetidine and ranitidine, a nonimidazole H2-receptor antagonist, have been studied. High binding affinity of cimetidine for cytochrome P 450(Ks = 31 micro M) was seen, while no evidence for ranitidine binding to cytochrome P450- was observed. Cimetidine inhibited meperidine and pentobarbital metabolism by both rat and human hepatic microsomes while ranitidine did not affect these two cytochrome P450-medicated biotransformation reactions. Conjugation of morphine, a reaction not mediated by cytochrome P450, was unaffected by either cimetidine or ranitidine. The imidazole structure of cimetidine rather than its H2-receptor blocking activity is primarily responsible for cimetidine-induced inhibition of hepatic drug metabolism.

Animals↗
PubMed1982

Effects of liver congestion on hepatic drug metabolism in the rat.

Pharmacokinetic alterations in drug disposition have been demonstrated for a variety of hepatic disease states, but there is little information concerning the effects of elevated hepatic venous pressure on hepatic drug metabolism. A rat model of hepatic congestion which was characterized by significantly elevated hepatic venous pressure, marked prolongation of prothrombin time, reduced total hepatic blood flow and histological changes of marked pericentral fibrosis and central vein dilation was used to study the effects of liver congestion on hepatic microsomal biotransformation and in vivo disposition of morphine and pentobarbital. No significant differences in levels of microsomal cytochrome P-450 or NADPH-dependent cytochrome c reductase were seen between the two groups. Total hepatic microsomal capacity for glucuronidation of morphine and hydroxylation of pentobarbital was not altered by elevated hepatic vein pressure and no change in in vivo systemic clearance was seen for either drug in response to hepatic venous congestion. These animal data demonstrate that hepatic congestion produces minimal alterations in hepatic metabolism of morphine and pentobarbital and may not have the severe detrimental effects on drug biotransformation and disposition which would be predicted.

Animals↗
PubMed1976

Inhibition of potentially pathogenic yeastlike fungi by clotrimazole in combination with 5-fluorocytosine or amphotericin B.

Clotrimazole (CTM) has a doubtful future with respect to use in treatment of the systemic mycoses. To assess the potential of CTM in combined drug regimens, antifungal effects of CTM together with 5-fluorocytosine (5-FC) or amphotericin B (AMB) were tested in a synthetic liquid medium against Candida albicans, Candida tropicalis, and Torulopsis glabrata. Viable counts were monitored over a 48-h incubation period. Weak inhibitory concentrations of CTM were tested in combination with levels of 5-FC or AMB that alone produced transient antifungal effects followed by rapid recovery of proliferative capacity. Results were similar for each of the organisms studied. Between 24 and 48 h, when cultures containing 5-FC or AMB alone were in the recovery phase, CTM plus 5-FC and CTM plus AMB continued to markedly suppress cell multiplication. It would appear that weak inhibitory concentrations of CTM can act together with 5-FC or AMB to produce antifungal effects greater than that obtained with either of the latter two drugs alone.

Amphotericin B↗
PubMedDate not supplied

Effect of acupressure by Sea-Bands on nausea and vomiting of pregnancy.

OBJECTIVE: To determine the effect of continuous acupressure at P6 applied by Sea-Bands with acupressure buttons on the frequency and severity of nausea and vomiting of pregnancy during the 1 st trimester. DESIGN: A two-group, quasi-experimental, posttest-only and posttest-repeated measure. SETTING: Seventeen medical clinics or offices in southern Michigan. PARTICIPANTS: Convenience sample of English-speaking, healthy pregnant women in their 1 st trimester, who had at least one episode of nausea, vomiting, or both before their prenatal clinic/office visit where they were recruited. After being accepted for the study, the women were randomly assigned to treatment or placebo groups. INTERVENTION: Treatment group 1 applied SeaBands with acupressure buttons to both wrists for 4 days and removed the Sea-Bands for 3 subsequent days. Placebo group 2 applied the Sea-Bands without acupressure buttons to both wrists on the same time schedule as group 1. MAIN OUTCOME MEASURE: Self-report daily diaries of the number of times per day that participants experienced nausea, the severity of nausea, the number of vomiting episodes per day, and the severity of vomiting. RESULTS: Mann-Whitney U procedures revealed that the treatment group had significantly less frequency and severity of nausea and vomiting of pregnancy while wearing the Sea-Bands than did the placebo group. The treatment group also had significantly less frequency and severity of nausea and vomiting of pregnancy while wearing the SeaBands than when not wearing the Sea-Bands. CONCLUSIONS: Sea-Bands with acupressure buttons are a noninvasive, inexpensive, safe, and effective treatment for the nausea and vomiting of pregnancy.

Acupressure↗