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Biomedical subjects

M M Oosthuizen

Publications and source records attributed to M M Oosthuizen.

At least 19 recordsLinked to original sources
PubMed2001

Hydroxyl radical generation: the effect of bicarbonate, dioxygen and buffer concentration on pH-dependent chemiluminescence.

Enhanced chemiluminescence (CL) induced by HO* was detected using the primary enhancers luminol (Lum), isoluminol (ILum) and lucigenin (Luc), in the absence of HCO3-, at pH 9.5, 10.0 and 10.5 but not between pH 4.0 and 9.0. This was confirmed using nine different HO* generators. FeCl3/NTA/H2O2 was the only HO* generator that was able to generate singular HO* which was obtained entirely from the Fenton reaction. However, this was so only at pH 10.0 since at all other pHs multiple ROS were produced. This was confirmed by the chemical detection of the fluorescent hydroxylated product of terephthalic acid in the absence of O2. No HO* Lum-CL pH optima coincided with the O2*- mediated Lum-CL optima found at pH 8.0 and 9.0. Bicarbonate had an enhancing effect on Lum-CL which was 70-2700% at pH 10.0 for the different HO* generators. This was due to the conversion of the radical-electron from HO* to CO3*-, making CL detection more efficient since less HO* were lost initially before detection. Methyl-cypridine-luciferin analogue (MCLA) elicited CL in the pH range 4.0-10.0 with the same set of generators in the absence of HCO3-. The iron-containing generators had their different MCLA-CL optima at pH 4.5, 5.0 or 6.0, excluding those overlapping with the O2(*-)-mediated CL optima. The two copper-containing generators had optima at the same pHs, viz, 7.0 and 10.5. Again, FeCl3/NTA/H2O2 was the only HO* generator able to produce singular HO* by the Fenton reaction. However, whereas Lum-CL was able to detect singular HO* only at pH 10.0, MCLA-CL detected it at pH 5.0 and 5.5. Therefore, MCLA is the most suitable CL enhancer for physiological assessments since it is the most sensitive enhancer and has HO* CL optima nearer to physiological pH than the other probes. The HCO3- enhancement of MCLA-CL was even greater than that of Lum-CL, since increases of 114-fold and 37-fold, respectively, were obtained at these HO*-specific pH optima for FeCl3/NTA/H2O2. Therefore, bicarbonate concentration is as important a parameter as pH when the enhanced CL of a non-cellular system is determined. Hydrogen peroxide was not able to elicit CL directly but, due to trace metal contamination, it produced artifactual CL due to HO* formation. High H2O2 levels, which prevent spontaneous O2*- dismutation, helped to establish the overlapping pH optima of CL mediated by O2*- and HO* which were artifactually produced either by O2*- via H2O2 and trace metals or by perferryl intermediates, respectively. Due to spontaneous dismutation to H2O2, only 22% of the O2*- produced by HX/XO could be detected by enhanced CL.

Acridines↗
PubMed1999

Glutathione-linked enzymes in benign and malignant oesophageal tissue.

Oxyradicals are involved in multiple mutational events and can contribute to the conversion of healthy cells to cancer cells. Glutathione (GSH) and the GSH-replenishing enzymes keep the antioxidant status of normal cells at a level where they can avert oxyradical derived mutations. The aim of this study was to determine whether in cancer cells the GSH-replenishing, GSH antioxidant and GSH-depleting enzymes were not at appropriate levels and therefore not able to protect cancer cells adequately against oxyradical-induced mutations. Cancer of the oesophagus was chosen since it is the most common gastrointestinal malignancy in South African Blacks. Biopsies and blood from 31 patients with cancer of the oesophagus and 29 non-cancer patients were assessed for these enzymes. The mean activity of the antioxidant and depleting enzyme GSH-peroxidase was elevated significantly by twofold in the cancer tissue compared to normal tissue. However, the activity of the replenishing enzyme GSSG-reductase and the level of the depleting enzyme GSH-s-transferase P1-isoenzyme were significantly reduced by 23% and 33% respectively. As in a previous paper we found that GSH was depleted and gamma-glutamine transpeptidase was diminished in oesophageal cancer. There can be two reasons for GSH depletion. Firstly, elevated GSH-peroxidase will use more GSH in an attempt to cope with the excessive production of oxyradicals as revealed by elevated lipid peroxidation; this was, as shown by us before, elevated sixfold in oesophageal cancer. Secondly, if little replenishment of GSH occurred the level of GSH would become lower. This was confirmed by our findings that the activities of the replenishing enzymes were significantly diminished in oesophageal cancer tissue. Contrary to what was expected, the other depleting enzyme GSH-s-transferase P1 was not elevated in cancer tissue but was significantly lower. However, in the blood of the same patients it was significantly elevated. An explanation for this phenomenon is that, although the production of GST-P1 was enhanced in cancer, it did not show because it was rapidly extruded into the blood by an unknown mechanism operational only in cancer cells.

Esophageal Neoplasms↗
PubMed1999

Antioxidants suitable for use with chemiluminescence to identify oxyradical species.

From a panel of 24 alleged antioxidants the most suitable antioxidants (AO) for use with chemiluminescence (CL) experiments were determined. Superoxide dismutase (SOD), using luminol as the chemiluminescence probe (Lum-CL), was inhibitory only towards O2*- and not HO* or (1)O2. SOD was thus a suitable antioxidant for O2*-, as was tiron. Tiron had advantages, however, since SOD acted as a pro-oxidant in the presence of H2O2 or H2O2/HO* generators. The two most suitable antioxidants for (1)O2 were diphenylisobenzofuran (DBF) and tryptophan, for both Lum and Lucigenin-CL (Luc-CL). Desferrioxamine, with both Lum and Luc-CL, was a very effective scavenger for HO*, but appeared to be an even more effective scavenger for (1)O2. Cysteamine showed the best discrimination between IC50s when the two (1)O2 generators NaOCl/H2O2 and NDPO2 were compared. Cysteamine was, therefore, the only scavenger that was appropriate for studies with hypochlorite. Melatonin, with Lum-CL, was found to be the most suitable scavenger for HO*. Mannitol, the classical AO for HO*, was not suitable when used with CL since it acted as a pro-oxidant. Some of the AOs revealed either calyx- or bell-shaped CL inhibition profiles and presumably, therefore, may act as both pro- or antioxidants at different concentrations. Antioxidants showing these kinds of dual activities should be used with caution in CL studies.

Antioxidants↗
PubMed1997

Lipid peroxidation in eclampsia.

In order to investigate the extent of lipid peroxidation in hypertensive disorders of pregnancy, a case-control study was designed. Eight eclamptic women were matched with women with severe pre-eclampsia (n = 8) and healthy pregnant controls (n = 8). Lipid peroxidation was measured by the malondialdehyde thiobarbituric acid reactive assay (MDA-TBAR) and expressed as nmol/ml. Both severe pre-eclampsia and eclampsia groups had significantly higher MDA-TBAR levels than healthy pregnant women. Eclamptic women before delivery had highest MDA-TBAR levels which decreased after delivery.

Journal Article↗
PubMed1994

Amino acid sequence and properties of vasopressin-associated elephant neurophysin.

The primary structure of an elephant neurophysin, homologous to vasopressin-associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact with residues 77 and 78 of the monomer-monomer interface. This Tyr residue therefore serves as a potential reporter of the path involved in the long-range linkage between peptide binding and dimerization in this system. NMR studies of the protein in unliganded and liganded states demonstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elevated pKa of 11.1 assigned to Tyr-75 by UV spectrophotometric titration, the NMR signals from the 3,5 and 2,6 ring protons of Tyr-75 were shifted 0.3 and 0.2 ppm upfield, respectively, relative to their positions in small peptides, indicating significant shielding and/or hydrogen bonding. The Tyr-75 ring proton signals narrowed slightly, with no discernible change in chemical shift, on conversion from dimer to monomer in the unliganded state. Ring protons of Tyr-49, distant from the monomer-monomer interface, but adjacent to the peptide-binding site, were markedly perturbed by dimerization, in accord with their behavior in bovine neurophysins. The results suggest that the secondary and tertiary structure of the region 75-78 is largely unchanged by dimerization, and argue against an important role for this region in dimerization-mediated conformational changes that alter the binding site in the unliganded state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Effect of verapamil on hepatic ischemia/reperfusion injury.

Alterations in cellular calcium homeostasis are a critical factor in the pathogenesis of hepatic ischemic damage and may mediate oxygen free radical injury during the reperfusion period. We investigated the effect of the calcium channel blocker verapamil on hepatic ischemia/reperfusion injury in normal rats and rats sensitized to oxidative injury by chemical depletion of the endogenous antioxidant glutathione. Forty-five minutes of complete hepatic ischemia followed by reperfusion caused an increase in the serum glutamic pyruvic transaminase (SGPT) level and a decline in the endogenous hepatic glutathione level but no increase in hepatic lipid peroxidation products. Chemical depletion of hepatic glutathione with diethylmaleate did not induce hepatocellular injury but augmented hepatic ischemia/reperfusion-induced SGPT release and promoted lipid peroxidation. Pretreatment with the calcium entry blocker verapamil protected against the ischemia/reperfusion-induced drop in hepatic glutathione but did not reduce SGPT release in normal rats. In rats sensitized to oxidative injury by chemical depletion of endogenous glutathione, the calcium channel blocker verapamil protected against ischemia/reperfusion-induced lipid peroxidation and reduced the release of SGPT. These findings indicate that the rat liver is protected against oxidative injury after short periods of total ischemia by its rich supply of endogenous glutathione. A beneficial effect of verapamil occurs only in rats sensitized to oxidative injury, suggesting that the calcium channel blocker protects against oxygen radical attack.

Alanine Transaminase↗
PubMed1992

The effect of hypovolaemia on healing of the right and the left colon. An experimental study.

An experimental study was carried out to investigate the effect of a period of hypovolaemia prior to surgery on healing of the right and left colon of the baboon. Colonic healing was assessed in three ways: in terms of (i) anastomotic leakage, (ii) hydroxyproline concentrations and (iii) breaking strength. The results suggest that hypovolaemia before surgery affects the right and left colon similarly.

Anastomosis, Surgical↗
PubMed1991

A steady-state kinetic analysis of the reaction between arginine esterase E-I from Bitis gabonica venom and synthetic arginine substrates and the influence of pH, temperature and solvent deuterium isotope.

1. Using synthetic arginines as substrates, steady-state kinetic studies showed a deviation from Michaelis-Menten kinetics for esterase E-I purified from the venom of Bitis gabonica. Graphical analysis indicated a rate equation of at least a degree of 3:3. 2. pH variation of the kinetic parameters indicated the involvement of groups with pK values of approximately 7 and approximately 9 which had to be in the ionic form for activity. 3. Solvent isotope studies suggested transition states where proton transfer or reorganization was the rate-limiting step of proteolytic catalysis. A single protogenic site was postulated. 4. Temperature effects on the enzymic reaction showed a significant reduction in entropy loss upon formation of the transition state with both esters and extended tail polypeptide-anilides in comparison with the activation entropy for benzoyl-L-arginine p-nitroanilide.

Arginine↗
PubMed1991

Inactivation of arginine esterase E-I of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone and isatoic anhydride.

1. Esterase E-I from Bitis gabonica was inactivated with irreversible inhibitors which included studies with a water-soluble carbodiimide, an affinity labelling peptide and a mechanism-based inactivator. 2. The reaction with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide was biphasic and the dominant part followed saturation kinetics. At pH 5.5 a rate constant of 0.4 min-1 for inactive enzyme formation was calculated and a dissociation constant (Ki) of 0.2 M for the enzyme-inhibitor complex. 3. Inactivation with D-Phe-Pro-Arg-chloromethyl ketone indicated a two-step mechanism, for which the reaction parameters at pH 8.0 were determined. The Ki value was 0.2 microM and the inactivation rate was 2.5 min-1. 4. With isatoic anhydride pseudo-first-order kinetics was observed. At pH 8.0 a rate constant of 0.9 min-1 and a Ki of 2.0 mM were obtained. The inactivation of the enzyme was found to be governed by a group in the enzyme showing a pK value of 7.3.

Amino Acid Chloromethyl Ketones↗
PubMed1991

Properties of the isoenzyme forms A-1, A-2 and B of N-acetyl-beta-D-glucosaminidase purified from baboon kidneys.

Three forms of N-acetyl-beta-D-glucosaminidase (NAG: A, B and I) were separated from baboon kidney using Con A-Sepharose and DEAE-Trisacryl chromatography. 2. The A form was further purified into two forms A-1 and A-2 using hydroxylapatite chromatography and anodic PAGE. Both were homogeneous on SDS-PAGE and anodic PAGE but microheterogeneous on PAG-IEF, which could be eliminated by prior treatment with endoglycosidase H or glycopeptidase F. 3. The carbohydrate content accounted for some of this microheterogeneity since it varied from 31 for A-1 to 17% for A-2 and the sialic acid was 6 and 1%. Deamidation may also contribute since the acidic amino acids (29 mol%) and ammonia were high following acid hydrolysis. 4. The mol. wt for A-1, determined by SDS-PAGE, was 52.1 K. 5. The pH optimum was 4.55 and the pI4.97. 6. The optimum temperature for NAG A and B was 50 degrees and 42 degrees C, but B retained more activity above 55 degrees C. 7. The Km for N-acetyl-beta-D-glucosamine and -galactosamine for both isoforms was 0.497 and 0.627 mM respectively. 8. Several ions were found to be uncompetitive inhibitors. Ag+ and Pb2+ were the most potent having Ki values of 3.6 and 8.5 mM respectively. Acetate acted as a competitive inhibitor.

Acetylglucosaminidase↗
PubMed1991

Oxygen free radicals and glutathione in hepatic ischemia/reperfusion injury.

Oxygen free radicals have been implicated as mediators of ischemia/reperfusion injury in a variety of organs. We investigated the role of oxidative injury and endogenous hepatic glutathione (GSH) in liver cell injury associated with complete hepatic ischemia and reperfusion. Forty-five minutes of complete hepatic ischemia followed by reperfusion caused an increase in serum GPT and a fall in hepatic GSH but no increase in hepatic lipid peroxidation products. Chemical depletion of hepatic GSH with diethyl maleate did not cause hepatocellular injury but augmented hepatic ischemia/reperfusion-induced SGPT release and promoted lipid peroxidation. Pretreatment with the selective, membrane-permeable oxygen radical scavenger dimethyl sulfoxide protected against the ischemia/reperfusion-induced drop in hepatic GSH but did not reduce SGPT release in normal rats. In rats sensitized to oxidative injury by depletion of endogenous GSH with diethyl maleate the oxygen radical scavenger protected against ischemia/reperfusion-induced lipid peroxidation and reduced the release of SGPT. These findings suggest that the rich hepatic supply with endogenous GSH has a crucial role in the protection against oxygen radical injury following short periods of total hepatic ischemia. Oxygen radical injury only occurs after depletion of these endogenous GSH stores.

Alanine Transaminase↗
PubMed1990

Elephant pituitary gonadotropins.

We describe for the first time the purification and some properties of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) isolated from anterior pituitary tissue of the African elephant (Loxodonta africana). Methodology previously applied to equine and donkey pituitaries was used to obtain purified preparations of elephant LH and FSH in yields of 8.8 and 0.48 mg, respectively, per 10 g pituitary powder. The preparations were characterized by HPLC gel filtration and amino acid analysis, both of which showed the elephant LH and FSH to be very similar to ovine LH and FSH. The preparations were also characterized by radioimmunoassays and bioassays for LH and FSH and a radioreceptor assay for FSH. Results showed virtually no cross-contamination of hormonal activities in the elephant LH and FSH preparations. Elephant LH potencies ranged from 50 to 66% of highly purified ovine LH and elephant FSH potencies ranged from 21 to 52% of highly purified ovine FSH in the various assays employed. No evidence was found for any demonstrable intrinsic FSH activity in elephant LH. The assays employed suggest possible usage for making physiological measurements of gonadotropins in the elephant.

Amino Acids↗
PubMed1990

Gastric mucosal injury caused by hemorrhagic shock and reperfusion: protective role of the antioxidant glutathione.

Oxygen free radicals have been implicated as mediators of gastric mucosal injury caused by ischemia/reperfusion. We investigated the role of exogenous and endogenous glutathione (reduced glutathione, GSH) in gastric mucosal injury associated with hemorrhagic shock and reperfusion. Mucosal GSH content was found to be consistently higher in the antrum than in the corpus. Ischemia (hemorrhage to 25 to 30 mm Hg) followed by retransfusion of shed blood, but not ischemia alone, caused a marked drop in gastric mucosal GSH and gross mucosal injury, which was confined to the corpus and spared the antrum. Chemical depletion of gastric mucosal GSH with diethylmaleate or inhibition of GSH synthesis with buthionine sulfoximine increased mucosal injury in the corpus and also rendered the antral mucosa susceptible to ischemia/reperfusion injury. Pretreatment with exogenous GSH provided marked protection against gross mucosal ischemia/reperfusion injury and prevented the ischemia/reperfusion-induced drop in mucosal GSH. These data suggest that the mucosal availability of the antioxidant GSH is an important protective factor against the development of gastric mucosal ischemia/reperfusion injury and supports a major role of oxygen radical release in the pathogenesis of gastric ischemia/reperfusion injury.

Animals↗
PubMed1989

An explanation of the noncorrespondence between assessment methods of cyclosporin.

Various methods of determining cyclosporin (CyA) levels in patients after kidney transplantation were compared. These included polyclonal antibody (pcAb-), specific and nonspecific monoclonal antibody (S- and NmcAb-) radioimmunoassays (RIA), and high performance liquid chromatography (HPLC). The results obtained by the various methods when compared showed some correlation but did not correspond. A probable explanation for part of this noncorrespondence is the presence of monoclonally crossreactive metabolites (CyA-M). Another reason was that the concentration of CyA in the standards supplied with the RIA kits was found to be higher than stated.

Antibodies, Monoclonal↗
PubMed1989

Primary structure of elephant pituitary prolactin.

Tryptic digests of elephant pituitary prolactin (elePRL) were separated by reverse phase high performance liquid chromatography (HPLC) and paper electrophoresis. From the amino acid composition, the amino acid sequencing of selected peptides, and from their alignment with expected tryptic peptides from ovine prolactin (oPRL), the primary sequence of elePRL is proposed.

Amino Acid Sequence↗