Activity of mushroom polyphenol oxidase in organic medium

A kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p-cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p-substituted phenolic substrates, yielding o-quinone products. Michaelis-Menten kinetics were used to obtain apparent K(M) and V(max) values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o-quinones were measured by a novel method: (1)H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water-sensitive, unstable o-quinones. (c) 1993 John Wiley & Sons, Inc.     

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus–oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.     

Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.     

Potential of fungi as biocontrol agents of polyphagotarsonemus latus (Acari: Tarsonemidae)

Infection of broad mites, Polyphagotarsonemus latus, by conidia of Beauveria bassiana, Hirsutella thompsonii, and Paecilomyces fumosoroseus conidia was investigated in the laboratory under controlled temperature and moisture conditions. Infection of P. latus by the fungus (LC₅₀) was related positively to the 1.16 × 10⁶ B. bassiana conidia per ml, 2.39 × 10³ H. thompsonii conidia per ml, and 1.29 × 10⁵ P. fumosoroseus conidia per ml. Mortality caused by B. bassiana occurred the fastest among densities fluctuating between 65 and 125 mites per leaf. The pathogens B. bassiana and P. fumosoroseus, adjuvants (oil and molasses), and the acaricide ABG6364, a microbial insecticide containing the Beta-exotoxin of Bacillus thuringiensis were evaluated for efficacy against P. latus in a greenhouse test. The B. bassiana treated plants had the greatest and most persistent effect on percentage mortality of total broad mites present per leaf (88 %) followed by the acaricide ABG6364. Use of adjuvants (oil and molasses) did not increase infection of P. latus.     

Effect on lipoprotein profile of replacing butter with margarine in a low fat diet: randomised crossover study with hypercholesterolaemic subjects.

OBJECTIVE--To examine the effect on lipid and lipoprotein concentrations when butter or an unsaturated margarine is used for cooking or spreading in a reduced fat diet. DESIGN--Randomised crossover study with two intervention periods of six weeks'' duration separated by a five week washout. SETTING--Community setting in New Zealand. SUBJECTS--49 volunteers with polygenic hypercholesterolaemia and baseline total cholesterol concentration in the range 5.5-7.9 mmol/l. MAIN OUTCOME MEASURES--Concentrations of total and low density lipoprotein, Lp(a) lipoprotein, high density lipoprotein, apolipoprotein B 100, and apolipoprotein A I. RESULTS--Concentrations of low density lipoprotein cholesterol and apolipoprotein B were about 10% lower with margarine than with butter. Lp(a) lipoprotein and high density lipoprotein cholesterol concentrations were similar with the two diets. CONCLUSION--Despite concerns about adverse effects on lipoproteins of trans fatty acids in margarines, the use of unsaturated margarine rather than butter by hypercholesterolaemic people is associated with a lipoprotein profile that would be expected to reduce cardiovascular risk.     

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.)

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H₂O₂. The cells recover and start producing ATP immediately upon the cessation of H₂O₂ production. This indicates that the increased O₂ uptake is primarily incorporated into active O₂ species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M_r 40 000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.