Effects of plant-derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food

The ability of 3 plant flavonoids (morin, myricetin and quercetin) and 4 polyphenolic acids (caffeic acid, chlorogenic acid, ellagic acid and ferulic acid) to inhibit the genotoxic effects of a number of cooked-food mutagens (IQ, MeIQ, MeIQx, Trp-P-1 and Trp-P-2), was investigated in a bacterial mutation assay using Salmonella typhimurium TA98 as indicator and hepatic S9 mixes from either SWR mice or Syrian hamster as metabolic activating systems. Although the polyphenolic acids failed to have an effect, the flavonoids generally inhibited IQ, MeIQ, MeIQx and Trp-P-1 induced mutagenesis in a dose-dependent manner, irrespective of the source of S9. This was not the case with Trp-P-2 where the flavonoids were only observed to inhibit when SWR mouse S9 but not Syrian hamster S9 was used. Of the 3 compounds, myricetin and quercetin were superior to morin in their inhibitory capacity.     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production.

The availability of a technique for site-directed mutagenesis by gene replacement provides a powerful tool for genetic analysis in any bacterial species. We report here a general technique for gene replacement in Pseudomonas aeruginosa. Genes on fragments of cloned P. aeruginosa DNA, altered by transposon mutagenesis, can be transduced into a recipient strain and can replace homologous genes in the P. aeruginosa genome. In this study we applied this technique to the construction of recA mutants of P. aeruginosa. A cloned segment of P. aeruginosa FRD1 DNA was isolated which encoded a protein analogous to the recA gene product of Escherichia coli. The P. aeruginosa recA gene was able to complement several defects associated with recA mutation in E. coli. Transposon Tn1 and Tn501 insertions in the cloned recA gene of P. aeruginosa were used to generate chromosomal recA mutants by gene replacement. These recA strains of P. aeruginosa were more sensitive to UV irradiation and methyl methane sulfonate and showed reduced recombination proficiency compared with the wild type. Also examined was the effect of recA mutations on the expression of alginate, a virulence trait. Alginate is a capsulelike polysaccharide associated with certain pulmonary infections, and its expression is typically unstable. The genetic mechanism responsible for the instability of alginate biosynthesis was shown to be recA independent.     

A note on the survival of Legionella pneumophila in stagnant tap water

Tap water, from an experimental hot water system, containing a known virulent strain of Legionella pneumophila was stored in screw-capped bottles for 14 months. Viable counts showed survival of L. pneumophila and at least three other bacterial species. This reinforces the view that L. pneumophila can survive in stagnant water foatively long periods of time.     

The persistence of Legionella pneumophila in non-sterile, sterile and artificial hard waters and their growth pattern on tap washer fittings

Simultaneous experiments were performed with sterilized and non-sterile water and an artificial hard water. After seeding with an environmental isolate of Legionella pneumophila numbers in the sterile land hard water decreased rapidly and colonization of various tap washer fittings failed to take place. Adhesion and growth of an environmental isolate of L. pneumophila to washers in non-sterile tap water was followed over a 4-month period with fluorescein-labelled antibody and by scanning electron microscopy. After adherence the individual cells appeared to divide to form chains which spread over the surfaces. Organisms other than legionellas were also present and a complex colonization matt was formed which was embedded in a protective coat of slime and debris. The numbers of L. pneumophila recovered from the water were highest between 4 and 7 weeks but they could still be cultivated after 4 months.     

Improvements in exercise performance: effects of carbohydrate feedings and diet

In an effort to determine the effects of carbohydrate (CHO) feedings immediately before exercise in both the fasted and fed state, 10 well-trained male cyclists [maximum O2 consumption (VO2 max), 4.35 +/- 0.11 l/min)] performed 45 min of cycling at 77% VO2 max followed by a 15-min performance ride on an isokinetic cycle ergometer. After a 12-h fast, subjects ingested 45 g of liquid carbohydrate (LCHO), solid carbohydrate confectionery bar (SCHO), or placebo (P) 5 min before exercise. An additional trial was performed in which a high-CHO meal (200 g) taken 4 h before exercise was combined with a confectionery bar feeding (M + SCHO) immediately before the activity. At 10 min of exercise, serum glucose values were elevated by 18 and 24% during SCHO and LCHO, respectively, compared with P. At 0 and 45 min no significant differences were observed in muscle glycogen concentration or total use between the four trials. Total work produced during the final 15 min of exercise was significantly greater (P less than 0.05) during M + SCHO (194,735 +/- 9,448 N X m), compared with all other trials and significantly greater (P less than 0.05) during LCHO and SCHO (175,204 +/- 11,780 and 176,013 +/- 10,465 N X m, respectively) than trial P (159,143 +/- 11,407 N X m). These results suggest that, under conditions when CHO stores are less than optimal, exercise performance is enhanced with the ingestion of 45 g of CHO 5 min before 1 h of intense cycling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance

Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuberosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuberosum parent was a late blight differential possessing the R4 gene which confers resistance to race 0. The S. brevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed.     

Bacillus thuringiensis and control of plant pests

Summary The feasibility ofB. thuringiensis as an economic insect control agent is dependent upon various factors. Consideration of three situations. whereB. thuringiensis could be used illustrates the interaction of these factors, and their contribution to determining the success or failure of the organism as an insecticide. Apple crops suffer from attack by a complex of insect pests, many of which are very susceptible to theB. thuringiensis toxin. However chemical control methods are preferred by growers in order to satisfy the consumers' demand for top quality blemish-free fruit. By contrast cotton growers are beset with problems of pest resistance to chemical insecticides. This problem is tackled by using pest management strategies which orchestrate all possible methods of control. Such a situation is ideally suited toB. thuringiensis products, but as yet they have proven to be of inadequate efficacy. The softwood forest industry is an example whereB. thuringiensis is both needed and effective. The pest complex is relatively simple, and there is public concern about the health risks of chemical sprays. Recent developments of high performance formulations means thatB. thuringiensis is an economic, cost-effective, biological alternative to chemical control for forestry pests.

---

Resumen La posibilidad de utilizarB. thuringiensis para el control de insectos de una forma económica depende de varios factores. La consideración de tres situaciones distintas en las queB. thuringiensis puede ser utilizado ilustra la interacción de estos factores y su contribución al éxito o fracaso del organismo como insecticida. El cultivo de manzanas sufre el ataque de una serie de plagas de insectos, muchas de las cuales son susceptibles a la toxina deB. thuringiensis. Sin embargo, los agricultores prefieren utilizar métodos de control químico a fin de satisfacer la demanda del consumidor por un fruto de primera calidad sin marcas ni señales. Por el contrario, los cultivadores de algodon estan sensibilizados a los problemas de la aparición de plagas resistentes a los insecticidas químicos. Este problema se ha abordado utilizando sistemas de control integrado que aglutinan todos los posibles métodos de control. Esta situación parece la ideal para el uso de productos a base deB. thuringiensis, pero hasta ahora estos han resultado ser poco eficaces. La industria de la madera blanda constituye en ejemplo en el cualB. thuringiensis es a la vez necesario y efectivo. Las plagas en cuestión forman un sistema relativamente simple y los efectos de los sprays químicos sobre la salud pública son causa de preocupación. Los desarrollos recientes en formulaciones de elevada eficacia convierten aB. thuringiensis en una alternativa biológica económicamente factible frente al control químico de plagas forestales.

---

Résumé La possibilité d'utiliser économiquementB. thuringiensis dans la lutte contre les insectes nuisibles dépend de facteurs divers. L'examen de trois situations dans lesquellesB. thuringiensis peut être utilisé illustre l'interaction de ces facteurs et leur contribution dans le succès ou l'échec de cet organisme comme insecticide. Les récoltes de pommes sont menacées par les attaques d'un mélange complexe d'insectes, dont beaucoup sont sensibles à la toxine deB. thuringiensis. Cependant, les horticulteurs préfèrent les méthodes chimiques à cause de l'exigence des consommateurs pour des fruits de haute qualité et sans aucune tache. Par contre, les cultivateurs de coton sont obsédés par les problèmes de résistance aux insecticides chimiques. Ce problème est affronté par des stratégies faisant appel à tous les moyens de lutte possibles. C'est une situation convenant bien aux produits qui contiennentB. thuringiensis, mais jusqu'ici ces produits se sont montrés inefficaces. L'industrie du bois tendre forestier est toutefois un exemple de cas oùB. thuringiensis est à la fois nécessaire et efficace. En effet, le complexe des insectes agressifs est relativement simple et il existe en outre une prévention du public contre les risques sanitaires des pulvérisations de produits chimiques. La mise au point récente de formulations à haute performance démontre queB. thuringiensis est, pour la lutte contre les insectes nuisibles des forêts, une alternative économique et bon-marché.

---

---

Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsiki, 12–16 August 1985. Session 8     

Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase

During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.     

On the relationship of ultrastructural and cytochemical features to color in mammalian skeletal muscle

Summary The semitendinosus muscle of the albino rat is divided grossly into two clearly distinguishable parallel longitudinal bands, one red (anterior) and the other white (posterior). By using mitochondrial content as a criterion for distinguishing fiber types, it is demonstrated that the red portion of the muscle is composed predominantly of red (52%) and intermediate (40%) fibers, while the white portion consists primarily of white fibers (82%). Red fibers have the smallest and white fibers have the largest average diameter. Ultrastructural characteristics of the three fiber types resemble closely those previously described for the rat diaphragm. Red fibers are rich in large mitochondria with abundant cristae, and possess the widest Z lines. In red fibers, the H-band region of the sarcoplasmic reticulum consists of an elaborate network of narrow tubules. In white fibers, mitochondria are smaller, less numerous, and have fewer cristae; Z lines are about half as wide as in red fibers. In the H-band region of the sarcoplasmic reticulum there is a more compact arrangement of broad more or less parallel tubules. Intermediate fibers are similar to red fibers except that their diameters are larger; mitochondria are somewhat smaller and cristae are less abundant; the width of the Z lines is close to that of white fibers. The consistent difference in Z line width establishes this dimension as an important criterion for distinguishing fiber types and facilitates ultrastructural identification, especially of the intermediate fiber.The clear relationship between color of the semitendinosus and cytological features of its component fibers supports the use of the terms red, white, and intermediate as simple and valid designations for fiber types in mammalian skeletal muscle. Measurement of the cross-sectional area contributed by each fiber type to the total area indicates that both red and intermediate fibers may contribute to redness in mammalian skeletal muscle.An early portion of this work was carried out with MissSharon Whelan (Mrs.Bernard Weiss). The author acknowledges the important contribution of Mr.Richard Stearns through his skillful work on the photographic illustrations and the technical assistance of MissAnn Campbell and Mrs.Joan Normington. — This study was supported by Grant No. HD 01026-04 from the United States Public Health Service.