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PubMed1982

Glucosyldiphosphoundecaprenol, the mannose acceptor in the synthesis of the O9 antigen of Escherichia coli. Biosynthesis and characterization.

We describe the biosynthesis in vitro of the mannose acceptor of the O9 mannan synthesis by Escherichia coli membranes and its analysis with chemical, enzymatic and physical means. Membranes from E. coli 1357 (O9:K29-:H-his,pmi,rfe) were incubated with 10 mM UDP-glucose and 20 mM magnesium chloride in large scale. The incubation mixtures were extracted with butan-1-ol and the extract was fractionated by ion-exchange chromatography on DEAE-cellulose. The presence of the mannose acceptor was detected in the column effluent by using aliquots of the fractions in membrane-reconstitution experiments. The purified mannose acceptor was hydrolyzed for 10 min in 0.1 M hydrochloric acid at 100 degrees C and the hydrolyzate was extracted with light petroleum. Mass spectrometric analysis of the material from the organic phase showed it to be undecaprenol. The aqueous phase contained phosphate and glucose (as determined with glucose oxidase peroxidase) in the ratio of 1.9, alpha-Galactosyldiphosphoundecaprenol and beta-glucosylphosphoundecaprenol were prepared for comparison in these experiments. The results obtained showed that the mannose acceptor in the synthesis of the O9 mannan of E. coli is alpha-glucosyldiphosphoundecaprenol.

Antigens, Bacterial↗
PubMed1989

Influence of drugs on albumin and bilirubin interaction.

Twenty-eight drugs, including cephem antibiotics and anti-inflammatory agents currently used or considered potentially useful in neonatal intensive care nurseries in Japan, were examined to determine their influence on albumin and bilirubin interaction by means of a glucose oxidase - peroxidase method, using an automated analyzer (Arrows) for unbound bilirubin (U.B.). The apparent binding constant for drugs to the high-affinity site on albumin (KD) was determined. Of cephem antibiotics, latamoxef sodium (LMOX) and cefazolin sodium (CEZ) were found to displace bilirubin from albumin (KD = 5.9 x 10(3) M-1 and 4.5 x 10(3) M-1, respectively) as strongly as Na salicylate (KD = 6.8 x 10(3) M-1). Mephenamate and indomethacin, which are used for medical closure of patent ductus arteriosus in premature infants, were also found to be stronger bilirubin displacers (KD = 1.3 x 10(5) M-1 and 1.2 x 10(5) M-1, respectively) than sulfisoxazole (KD = 1.6 x 10(4) M-1). Maximal displacement factors (MDF's) were also estimated in reference to protein binding (%) and effective serum concentration (M) of each drug in human adults. Of these drugs, mephenamate showed a higher risk of bilirubin displacement (MDF = 3.79) than sulfisoxazole (MDF = 2.58) and LMOX had a higher risk of displacement (MDF = 1.97) than Na salicylate (MDF = 1.85). On the other hand, indomethacin and CEZ showed minimal effects on displacement at therapeutic levels (MDF = 1.03 and 1.00, respectively). At therapeutic serum levels, mephenamate and LMOX may possess the potential for displacing bilirubin from albumin and increasing the risk of bilirubin encephalopathy, in a manner similar to sulfisoxazole.

Albumins↗
PubMed2007

Potential antioxidant activity of celecoxib and amtolmetin guacyl: in vitro studies.

1. In vitro studies of the potential antioxidant activity of the selective cyclo-oxygenase-2 inhibitor celecoxib and the non-steroid anti-inflammatory drug amtolmetin guacyl (AMG) were carried out. The study included experiments on the ability of these drugs to affect some indices of the oxidative stress [lipid peroxidation (LP), activity of antioxidant enzymes, glutathione (GSH) level] in rat stomach and colon mucosa and in liver. 2. Celecoxib and AMG did not change the activity of the enzymes GSH-peroxidase, oxidased glutathione (GSSG)-reductase and glucose-6-phosphate-dehydrogenase, as well as the GSH level in all tissue preparations. An increased superoxide dismutase (SOD) activity and a tendency to a decreased Fe/ascorbic acid-induced LP in stomach and colon mucosa were found, but only in the presence of AMG. 3. In the liver, both celecoxib and AMG decreased spontaneous and Fe/ascorbic acid-induced LP. SOD activity was enhanced only in the presence of AMG. 4. Experiments aimed at studying celecoxib and AMG in free oxygen radical-generating systems were also carried out. AMG and tolmetin (the main metabolite of AMG) inhibited OH*-provoked deoxyribose degradation in a Fenton system. Celecoxib had no effect on free radicals when tested in the same system. 5. In conclusion, the results of the present in vitro studies suggest that AMG and celecoxib possess antioxidant and metal-chelating abilities, which might contribute to their beneficial effects.

Animals↗
PubMed1990

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.

Aldose-Ketose Isomerases↗
PubMed1980

Relevance of colour vision and diabetic retinopathy to self-monitoring of blood glucose.

A study was performed to determine the effect of colour vision defects and diabetic retinopathy on diabetic patients' ability to use a visual method of measuring their own blood glucose concentrations. Forty-eight diabetics whose colour vision and retinal status was assessed by an ophthalmologist carried out 311 blood glucose estimations using oxidase-peroxidase test strips which were then compared with laboratory values. There was a trend towards poor performance with advancing age but neither colour vision nor diabetic retinopathy had a significant effect on patients' ability to use this visual method of estimating blood glucose concentrations. The vast majority of diabetics who will benefit from being able to monitor their own blood glucose control should have no difficulty in using a visual method of testing, even if they do have defects of colour vision.

Adolescent↗
PubMed1977

Influence of fosfomycin on intestinal disaccharidases in rats.

The influence of orally administered fosfomycin on intestinal disaccharidases in albino rats was studied. The enzymes were evaluated in a homogenate of the intestinal mucosa, using the method of SOLS and DE LA FUENTE which is based on the colored reaction of the glucose that is freed by the disaccharidases from disaccharides with a glucose-oxidase-peroxidase reagent and a chromogen (Glucostat). The results obtained in animals treated with a daily dose of 1 g/kg fosfomycin for 6 consecutive days are compared with the corresponding results of animals that received 1 ml/day of saline solution during the same period. Our conclusion was that fosfomycin has no effect on the intestinal disaccharidases in rats.

Administration, Oral↗
PubMed1976

Cytochemical demonstration of extraperoxisomal catalase. I. Sheep liver.

In sheep hepatocytes catalase activity was demonstrated both within peroxisomes and within the cytosol. In the cytosol the catalase reaction product is contiguous to the plasma membrane and surrounds the nuclei, rough endoplasmic reticulum, cisternae, mitochondria and Golgi apparatus. This is the first cytochemical demonstration of guine extraperoxisomal catalase. No catalase reaction product was seen in the cytosol of nonparenchymal cells. To demonstrate catalase, both glutaraldehyde and formaldehyde fixation were used, followed by a diaminobenzidine technique modified from Novikoff and Goldfischer. Control reactions were performed to distinguish catalase reaction product from adsorption of oxidized diaminobenzidine and from precipitate due to oxidase-, peroxidase- or heat-stable peroxidatic activities. The results were evaluated in the light and electron microscopes.

Animals↗
PubMed2005

Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine.

Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins. Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. Here, we describe two rapid, specific, and sensitive enzymatic measurements for both phospholipids. Plasma was incubated with bacterial sphingomyelinase (for SM measurement) or bacterial PC-specific phospholipase D (for PC measurement), alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, and 4-aminoantipyrine for 45 min. A blue dye, with an optimal absorption at 595 nm, was generated. PC levels did not influence SM measurement and vice versa. The linear range for the SM measurement was 0.5-5 microg, and that for PC was 2.5-20 microg. The mean percentage recovery was 98.0 +/- 5.2% for SM and 96.6 +/- 3.8% for PC. The interassay coefficient of variation of the assay was 1.7 +/- 0.05% for SM and 3.1 +/- 0.13% for PC. These two new methods are amenable to automation and can be adapted for large-scale, high-throughput assays.

Dose-Response Relationship, Drug↗
PubMed2005

The coexistence of the oxidative and reductive systems in roots: the role of plasma membranes.

Different components of the plasma membrane bound and associated redox system, which participate in the energy transfer from the predominantly reducing intercellular environment to the extracellular oxidizing environment, are reviewed. Special attention is given to plant root cells. An analysis of the plasma membrane-associated redox components, such as the cytochromes, quinones, and different types of oxidoreductases (dehydrogenases, oxidases, peroxidases, and superoxide dismutases), is made, as well as their coupling with naturally occurring extracellular substrates, such as oxygen and its reactive forms, phenols, ascorbate, nitrate, ferric ion, and organic acids. The participation of different free radical species in most of the plasma membrane-bound redox reactions is documented.

Cell Membrane↗
PubMed1985

Biochemical mechanism of oxidative damage by redox-cycling drugs.

Biochemical mechanisms of production of redox intermediates of redox-cycling drugs include: photochemical events, either photoionization process or electron transfer from photoexcited states; electron exchange of reduced form of a drug with the oxy state of oxygen-binding hemoproteins; oxidation by catalytic metal centers (oxidases, peroxidases, oxygenases) of the reduced forms of drugs; or electron transfer to the oxidized form of a drug from activated intracellular electron transfer chain (mitochondria, microsomes, etc.). Further reaction of these drug free radicals can lead to oxidative damage by either direct attack of biological macromolecules or via oxygen reduction, giving O2-, H2O2, and OH. The reaction pathway depends on the presence of metal ions, natural scavengers, enzymes that control relative concentrations of reactive species, and availability of oxygen in the environment.

Animals↗
PubMed1995

Probable immune system mediation of the antitumor activity of the 2,6-dimethoxybenzo-p-semiquinone radical.

We recently reported that the antitumor activity of an immobilized oxidase-peroxidase system, which can produce radical species as intermediates, is dependent on the presence of an intact and functioning immune system in the host. A number of recent investigations have indicated that the bioactive form of many quinone-type anticancer agents may be the semiquinone-type radical. To investigate if the antitumor activity of some of the quinone-type anticancer agents may also be dependent on a fully functioning immune system in the host, we used as a simple, well-defined model the 2,6-dimethoxybenzo-p-semiquinone radical. This free radical has a potent antitumor activity against Ehrlich ascites tumors in mice, as first described by Szent-Gyorgyi's group. In the present study, we investigated its antitumor activity in immunocompromised BALB/c mice. We found that the antitumor activity of the semiquinone radical is completely abolished in mice that had previously been subjected to whole body gamma irradiation, indicating a possible involvement of one or more cells of the host's defense system. The antitumor activity was not abolished in mice with dysfunctional macrophages induced by a severe selenium deficiency. However, the radical was inactive in mice whose immune system was suppressed with cyclosporin A and in nude (nu/nu) mice. These results suggest that the dimethoxybenzo-semiquinone radical, rather than acting primarily as a cytotoxic agent as previously reported, exerts its antitumor activity mainly via a more complex mechanism in which T lymphocytes appear to play a major role.

Animals↗
PubMed2004

Incidence and risk factors for neonatal hypoglycaemia in Kerala, India.

OBJECTIVES: To detect the incidence and risk factors that predicted the occurrence of neonatal hypoglycaemia and to identify neonates who require mandatory blood glucose screening during the first 48 h of life. DESIGN: Hospital based prospective study in a maternity centre in south India. SETTING: Labour room, postnatal wards and newborn nursery of the Institute of Maternal and Child Health, Medical College, Kozhikode, India. PATIENTS: Six hundred and four neonates were enrolled in the study by a systematic random sampling method from 1 August to 1 November 2002. INTERVENTION/MEASUREMENT: Random blood glucose levels were estimated by the standard glucose oxidase--peroxidase method on two occasions 24 h apart, during the first 2 days of life. Nineteen clinical characteristics of the mother-baby pair were analysed statistically in relation to the occurrence of hypoglycaemia. RESULTS: The incidence of neonatal hypoglycaemia in the present study group was 41/1000 live births. Eight variables strongly and independently predicted the risk of neonatal hypoglycaemia, at least one being present in 89.1% of the hypoglycaemic neonates. They included prematurity, low birthweight, maternal diabetes mellitus, delay in initiation of breastfeeding for more than 2 h postnatally, maternal pre-eclampsia and eclampsia, birth asphyxia, cold stress or hypothermia, and maternal oligohydramnios. CONCLUSIONS: Hypoglycaemia was a common problem in apparently normal asymptomatic babies. Apart from the classic 'text book risk factors', maternal oligohydramnios and a breastfeeding delay of more than 2 h after delivery predicted the risk of neonatal hypoglycaemia in this group. Mandatory blood glucose screening in babies with any one of the above mentioned risk factors serves as an easy and cost effective measure for the prompt identification of this condition.

Blood Glucose↗
PubMed1999

Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase.

BACKGROUND: Various methods are available to measure serum cholesterol concentrations. Of these, the cholesterol ester hydrolase (CEH)-cholesterol oxidase-peroxidase chromogenic method is widely used. However, this method has the disadvantage of interference by reducing substances. We developed and evaluated an endpoint assay for serum cholesterol, based on a CEH-cholesterol dehydrogenase (CDH)-ultraviolet method. METHODS: Cholesterol esters are first hydrolyzed to free cholesterol by CEH. The free cholesterol is then reduced by CDH to cholest-4-ene-3-one with the simultaneous production of beta-NADH from beta-NAD(+). At equilibrium, the CDH reaction gives incomplete conversion of cholesterol to cholest-4-ene-3-one. To overcome this disadvantage, we added hydrazine monohydrate to the reaction mixture to remove cholest-4-ene-3-one, which allowed the reaction to proceed to completion and gave stoichiometric production of beta-NADH from the reaction of beta-NAD(+) with cholesterol. RESULTS: We tested whether the amount of cholesterol added was equivalent to the absorbance change of NADH at 340 nm with six aqueous samples. Recoveries were 97.1-100.3%. The reaction was linear up to 20.28 mmol/L. The mean within-day (n = 20) and between-day (n = 10) imprecision (CV) was 0. 29-0.43% and 0.22-0.61%, respectively. No interference by bilirubin, hemoglobin, ascorbic acid, and other reducing agents was observed. The equation obtained in comparison with the modified Abell-Levy-Brodie-Kendall method was: y = 0.992x - 0.0058 mmol/L; r = 0.997; S(y|x) = 0.117 mmol/L; n = 50. CONCLUSION: This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.

Cholesterol↗
PubMed2000

Semiquantitative determination of urinary glucose: comparison of home-made strip and routine tests.

Glucose oxidase paper strips for semiquantitative determination of glucose in urine are commercially available but details of their preparation are not published. The purpose of this study was to produce such strips, using a coupled enzyme glucose oxidase-peroxidase reaction which would yield purple color with ortho-tolidine, and safranin upon dipping into urine containing glucose. In this present study, without any special equipment, special humidity and temperature control, the new reagent strip named R-strip could be successfully prepared in the atmospheric conditions of Thailand. R-strips were evaluated against random urine added with various amounts of glucose in comparison with a commercial strip (T-strip), Benedict's test, and a commercial tablet (C-tablet), routinely used in laboratories. The developed strips were found to be as specific as T-strip and more sensitive than other tests.

Diagnostic Tests, Routine↗
PubMed2002

Highly sensitive cholesterol assay with enzymatic cycling applied to measurement of remnant lipoprotein-cholesterol in serum.

BACKGROUND: Remnant lipoprotein-cholesterol (RLP-C) concentrations in sera of healthy individuals are very low (0.080-0.437 mmol/L), making conventional cholesterol methods poorly suited to this purpose. We have developed a highly sensitive cholesterol assay (CD method) and applied it to the RLP-C assay. METHODS: The CD shuttled cholesterol reversibly between reduced and oxidized forms in the presence of thio-NAD and NADH. The production rate of thio-NADH correlated with the cholesterol concentration and was measured by the absorbance at 404/500 nm. This CD method was combined with an immunoaffinity separation procedure with specific monoclonal antibodies to apolipoprotein (apo) A1 and apo B-100 and used for RLP-C assay. Results were compared with a RLP-C method that uses cholesterol oxidase, peroxidase, and chromogenic substrate. RESULTS: The CD method could detect 0.10 x 10(-3) mmol/L cholesterol and was at least 5 times more sensitive than the conventional enzymatic method. Within- and between-day imprecision (as CVs) of the RLP-C assay with the CD method was <4%. Regression analysis of RLP-C assays with the new (y) and conventional (x) cholesterol methods yielded: y = 1.02x - 0.008 mmol/L (S(y/x) = 0.0065 mmol/L; r = 0.997; n = 297). CONCLUSIONS: Serum RLP-C can be measured by the CD method. The CD method may be useful for other assays that require sensitive cholesterol measurements in biological materials.

Cholesterol↗
PubMedDate not supplied

A semi-micromethod for determination of oxalate in human plasma.

An enzymatic semi-micromethod for oxalate determination in human plasma was elaborated. The principle of the method depends on the oxalate isolation from deproteinized plasma, following determination by the calorimetric oxalate oxidase-peroxidase-indamine system. This method protects against internal oxalate losses and excludes an interference of contaminations. Results, obtained by this method, were reliable and ideally suited for use as real normal values (less than or equal to 6 microM) of oxalate content in the plasma of healthy individuals. The elaborated method, which can assay plasma oxalate accurately in normal individuals as well as in hyperoxalemic conditions is superior to those previously used. The procedure of semi-micromethod does not require expensive equipments and apparatus: it is simple and easy to perform in every laboratory and takes little time.

Algorithms↗
PubMed1992

Simultaneous patient-side measurement of hemoglobin, glucose, and cholesterol in finger-stick blood.

We describe a multianalyte assay system for patient-side use comprising single-use plastic cartridges and a small monitor. Hemoglobin, glucose, and cholesterol can be simultaneously measured in 3 min in an unmeasured volume of blood. The sample is drawn by capillary action into four channels for delivery to assay-specific stacks containing a set of closely apposed layers. The distal layer is a membrane that acts as the optical surface for reflectance optics. For glucose and cholesterol assays, erythrocytes are removed by a fibrous filter layer and oxidase-peroxidase chemical reactions contained in the optical membrane generate a colored product. For hemoglobin measurement, blood is lysed by detergent contained in a porous disk. The amount of color reaching the optical membrane is measured by fiber optics. To ensure fail-safe operation, sensors verify sample sufficiency and degree of hemolysis. The assays perform comparably with laboratory methods.

Blood Glucose↗
PubMed2004

[Earthworm feces-induced systemic resistance of cucumber against anthracnose].

Pot experiment showed that mixing earthworm feces into soil induced a significantly lower (P < 0.05) cucumber anthracnose than mixing peat, but had no significant difference (P > 0.05) with the treatment of system acquired resistance. The activities of polyphenol oxidase, peroxidase and phenylalanine ammonia-lyase in cucumber leaves treated with and without pathogen were increased in different degrees by mixing earthworm feces, compared with by mixing peat. All these suggested that mixing with earthworm feces could induce the system resistance of cucumber and suppress the plant disease via starting up the recovery enzyme system in plant.

Animals↗