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Biomedical subjects

M J Rhodes

Publications and source records attributed to M J Rhodes.

At least 37 records · Page 2Linked to original sources
PubMed1996

Dietary quercetin glycosides: antioxidant activity and induction of the anticarcinogenic phase II marker enzyme quinone reductase in Hepalclc7 cells.

It has recently been shown by Hollman et al. (Am. J. Clin. Nutr., 62, 1276-1282) that flavonoid glycosides are preferentially absorbed from dietary onions compared to the flavonoid aglycone. In the light of this, we have compared the bioactivities of the two most abundant flavonoid glycosides that we have purified from onions (quercetin-3,4'-diglucoside and quercetin-4'-glucoside) to the quercetin aglycone, and also to the more commonly studied commercially-available flavonoid glycosides, rutin (quercetin-3-rutinoside) and isoquercitrin (quercetin-3-glucoside). Quercetin aglycone was the most effective inducer of the anticarcinogenic phase II marker enzyme, quinone reductase (QR), in mouse Hepalclc7 cells. Of the glycosides, only quercetin-4'-glucoside was able to induce QR activity in this assay. Inhibition of NADPH/iron- and ascorbate/iron-induced lipid peroxidation of human liver microsomes, and the Trolox C-equivalent antioxidant capacity (TEAC), were also measured. The 4'-glycosylation dramatically decreased activity in the 'antioxidant' assays, whereas 3-substitutions produced much smaller changes. These results show that the preferentially-absorbed quercetin glycosides in onions have markedly different biological properties compared with the aglycone.

Animals↗
PubMed1995

Isolation and expression pattern of a cDNA encoding a cathepsin B-like protease from Nicotiana rustica.

Sequence analysis of a 1.33 kb clone from a root cDNA library of Nicotiana rustica revealed an open reading frame encoding a protein of 356 amino acids. The deduced protein has high levels of homology to human cathepsin B protease and a cathepsin B-like cysteine protease from wheat but much lower levels of homology with other plant cysteine proteinases. Southern blotting experiments suggest a limited number of cathepsin B-like genes are present in the genome of N. rustica and also that of N. tabacum. RNA analysis involving a range of tissues, harvested from both Nicotiana species 4-5 h after the beginning of a 16 h photoperiod, revealed the cathepsin B-like gene was being expressed strongly in roots, stem and developing flowers but weakly in mature leaves. Further analysis of RNA extracted from leaf tissue of N. tabacum revealed the gene showed rhythmic expression and also that its expression increased in response to wounding. Analysis of leaf tissues harvested during the latter part of a 16 h photoperiod (11 and 16 h after illumination commenced) showed that transcript levels were two three times higher than in leaf tissue harvested either towards the end of the dark period or 5 h after illumination commenced. When leaf tissue was wounded at 11:00 (5 h after plants were illuminated), and harvested for RNA extraction 6 h later, the level of cathepsin B-like transcript in mesophyll tissue was found to be increased ca. 2-fold relative to the level detected in unwounded controls.

Amino Acid Sequence↗
PubMed1995

Monophosphoryl lipid A as a prophylactic for sepsis and septic shock.

The ability of monophosphoryl lipid A (MLA) to provide prophylactic protection against septic shock was evaluated in a mouse model of induced endotoxin hypersensitivity. Treatments of hypersensitized animals with low doses of MLA attenuated endotoxin lethality and endotoxin-mediated liver damage. These effects were related to the ability of MLA to suppress accumulation of TNF-alpha and IFN-gamma in the bloodstream of animals. MLA treatments had only a modest effect in suppressing the accumulation of nitrate in the bloodstream. This implied that MLA did not suppress induction of macrophage and hepatocyte nitric oxide synthetases that contribute to antimicrobial defense and protect against endotoxin-mediated liver damage. The MLA treatments did not appear to compromise inflammatory defenses against local infection since locally recruited leukocytes remained responsive to endotoxin after hypersensitivity had been attenuated. In agreement with these findings, other studies have shown that the induction of endotoxin tolerance by MLA parallels the induction of resistance of animals to lethal challenges with either Gram negative or Gram positive bacteria. As predicted from preclinical studies, human trials of the clinical form of MLA (MPL-immunostimulant) have confirmed that MLA could attenuate systemic responses to endotoxin in normal volunteers, including the attenuation of blood cytokine accumulation and attenuation of symptomatic responses.

Adjuvants, Immunologic↗
PubMed1994

Effects of tumor necrosis factor and dexamethasone on the regulation of interferon-gamma induction by monophosphoryl lipid A.

Monophosphoryl lipid A (MLA), derived from lipopolysaccharide (LPS) of Salmonella minnesota strain R595, induced rapid accumulation of interferon (IFN)-gamma in mice. Tumor necrosis factor (TNF)-alpha appeared to be a cofactor for IFN-gamma induction by MLA. With low doses of MLA (< 5 micrograms), IFN-gamma induction was dependent upon exogenous TNF-alpha administered either in advance of or with MLA. A 25 micrograms dose of MLA induced significant IFN-gamma accumulation in the absence of exogenous TNF-alpha. In this case, endogenous TNF-alpha appeared to be a cofactor in the response, since suppression of TNF-alpha production with dexamethasone inhibited IFN-gamma induction, and this inhibition was overcome by administration of exogenous TNF-alpha with MLA. Treatment of animals with MLA tolerized them against LPS. Tolerant animals did not produce IFN-gamma when challenged with LPS, and this tolerance was not abrogated by supplementing mice with exogenous TNF-alpha during the challenge. Although dexamethasone inhibited IFN-gamma induction by MLA, it did not inhibit tolerance induction by MLA.

Animals↗
PubMed1992

A rationale for the prophylactic use of monophosphoryl lipid A in sepsis and septic shock.

Monophosphoryl lipid A (MLA), a substructure of bacterial lipopolysaccharide (LPS), is being developed as a prophylactic for sepsis and septic shock. In the present study it was shown that MLA induced a rapid accumulation of IFN-gamma in mice that correlated with an in vivo priming of macrophages. Primed macrophages could be induced in vitro to synthesize nitric oxide, a key mediator of macrophage cytotoxicity. Due to its rapid clearance, MLA was not present in circulation at the time when IFN-gamma accumulated, suggesting that MLA could not synergize with IFN-gamma to systemically activate macrophages in vivo. MLA treatment tolerized mice against the IFN-gamma response--ie., treatment of mice with MLA on day 1 blocked LPS from inducing IFN-gamma on days 2-4. The significance of these results in relation to MLA's ability to enhance non-specific resistance and block LPS lethality in animals is discussed.

Animals↗
PubMed1991

The formation of 3 alpha- and 3 beta-acetoxytropanes by Datura stramonium transformed root cultures involves two acetyl-CoA-dependent acyltransferases.

Tropine (tropan-3 alpha-ol) is an intermediate in the formation of hyoscyamine. An acyltransferase activity that can acetylate tropine using acetylcoenzyme A as cosubstrate has been found in transformed root cultures of Datura stramonium. A further acyltransferase activity that acetylates pseudotropine (tropan-3 beta-ol) with acetyl-coenzyme A is also present. These two activities can be partially resolved by anion-exchange chromatography, some fractions containing only the pseudotropine-utilizing activity. The basic properties of these two enzymes are reported and their roles in forming the observed alkaloid spectrum of D. stramonium roots discussed.

Acetyltransferases↗
PubMed1991

Strategies for the genetic manipulation of alkaloid-producing pathways in plants.

Increasingly, as a result of recent biochemical work, there exists a realistic possibility of taking a molecular genetic approach to the manipulation of alkaloid-producing pathways in plant tissue cultures. In the pathways forming indole alkaloids in CATHARANTHUS ROSEUS, tropane alkaloids in DATURA and HYOSCYAMUS species, and nicotine in NICOTIANA species, recent studies have identified a number of key enzymes and at least some of the factors that regulate their levels of activity. Such knowledge contributes the basis upon which it has become feasible to design a strategy by which the flux in these pathways may be enhanced at the genomic level. This review presents a summary of the state-of-the-art pertaining to these pathways and discusses the strategy to be adopted for a molecular approach to their manipulation, together with some of the pitfalls that may arise when trying to alter their natural regulation.

Journal Article↗
PubMed1990

Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation.

Gene Expression↗
PubMed1990

Growth and hyoscyamine production of 'hairy root' cultures of Datura stramonium in a modified stirred tank reactor.

The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg.g-1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g-1 wet weight). Growth at 30 degrees C was initially faster than at either 25 degrees C or 35 degrees C and by day 12, the drained weight of roots in the fermentor at 30 degrees C was about fourfold greater than at 25 degrees C and twice that at 35 degrees C. The ultimate hyoscyamine levels attained (approximately 0.5 mg.g-1 wet weight) were similar at both 25 degrees C and 30 degrees C but some 40% lower at 35 degrees C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25 degrees C and the highest production rate of 8.2 mg hyoscyamine 1(-1) per day was obtained for roots grown at 30 degrees C. In continuous fermentation at 25 degrees C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30 degrees C or 35 degrees C.

Atropine↗
PubMed1989

Leukotriene metabolism and hypersensitivity in man and mouse.

The relative rates of cysteinyl-leukotriene metabolism were analyzed in fresh human and mouse serum. Human serum contained higher gamma-glutamyl-transpeptidase activity than mouse serum, and a higher percentage of the metabolized leukotriene C4 was recovered as leukotriene D4 in the human serum than in the mouse serum. The results suggest that the patterns of metabolism of the cysteinyl-leukotrienes could be an important factor in determining the relative sensitivity of an animal to the development of hypersensitivity reactions.

Animals↗
PubMed1987

A spectrophotometric assay for strictosidine synthase.

A spectrophotometric assay for strictosidine synthase is described. Strictosidine is extracted with ethyl acetate and, where high substrate concentrations are used, the organic extract is washed with dilute ammonia to remove coextracted secologanin; after evaporation of the solvent, the residue is heated with 5 M H2SO4 for 45 min and the A348 value is measured. Strictosidine production is calculated from the response of similarly treated standards. A minimum production of 10-25 nmol of strictosidine may be determined. The assay is demonstrated using extracts of cultured Cinchona ledgeriana cells.

Carbon-Nitrogen Lyases↗
PubMed1984

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay.

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.

Ascorbic Acid↗
PubMed1984

An enzyme-linked immunosorbent assay for quassin and closely related metabolites.

A microtitration plate enzyme-linked immunosorbent assay has been developed for quassin and closely related seco-triterpenes. The method is much more sensitive than conventional chromatographic techniques and enables the routine analysis of large numbers of samples with a detection limit of 5 ng quassin. Antibody production was elicited by injecting a conjugate of iso-quassinic acid linked to bovine serum albumin into rabbits. Important features of the technique are (a) the use of a double-antibody, noncompetitive enzyme-linked immunosorbent assay for a low-molecular-weight, nonantigenic compound using microtitration plates; and (b) that the initial incubation of samples with primary antiserum can be carried out in buffer containing up to 20% (v/v) methanol with minimal loss of sensitivity. The structural requirements for recognition of the hapten by the antiserum are considered based on the levels of cross-reaction found with a wide range of other quassinoids.

Chemical Phenomena↗
PubMed1982

Trichobilharzia physellae (Digenea: Schistosomatidae) from endemic waterfowl on the high plains of Texas.

Adult Trichobilharzia physellae were recovered from the liver and mesenteric veins of green-winged teal, Anas creca, blue-winged teal, A. discors, and pintail, A. acuta, breeding on the High Plains of Texas. Wild ducks naturally-infected with these schistosomes were emaciated with mild to severe atrophy of the breast musculature. The liver was considerably enlarged, pale and somewhat friable. Numerous viable and dead T. physellae were recovered from the cut surface. On histologic examination, viable adult schistosomes were observed in the portal veins and dead, partly calcified T. physellae were found in the interlobular bile ducts. The normal liver architecture was well preserved but there was mild to extensive fibroplasia of most portal triads. An intense inflammatory response consisting mainly of eosinophils and fibroblasts with a few histiocytes, epithelioid cells, plasma cells, and lymphocytes surrounded the infected triads. There was pressure atrophy of adjacent hepatic parenchyma, hyperplasia or atrophy of the bile duct epithelium, bile stasis in smaller interlobular ducts, ectasia of larger portal ducts, focal areas of inflammatory cells throughout the liver, and periphlebitis of portal veins. The basic lesion presented as an obstructive fibrosis of the portal triads. These lesions are compared with those reported in infections by other schistosome species in their respective hosts. The oculate, apharyngeate, furcocercous cercariae of T. physellae were recovered from the snail first intermediate host, Physa anatina. Attempts to infect domestic mallard ducks via skin penetration by these cercariae were unsuccessful. This is the first record of a cercarial dermatitis-producing schistosome species from the Texas Panhandle and the first detailed description of such extensive lesions produced by adult schistosomes in the liver of a vertebrate host.

Animals↗