Absorption and fluorescence studies of the binding of the recA gene product from E. coli to single-stranded and double-stranded DNA. Ionic strength dependence

The binding of the recA gene product from E. coli to double-stranded and single-stranded nucleic acids has been investigated by following the change in melting temperature of duplex DNA and the fluorescence of single-stranded DNA or poly(dA) modified by reaction with chloroacetaldehyde. At low ionic strength, in the absence of Mg2+ ions, RecA protein binds preferentially to duplex DNA or poly(dA-dT). This leads to an increase of the DNA melting temperature. Stabilization of duplex DNA decreases when ionic strength or pH increases. In the presence of Mg2+ ions, preferential binding to single-stranded polynucleotides is observed. Precipitation occurs when duplex DNA begins to melt in the presence of RecA protein. From competition experiments, different single-stranded and double-stranded polydeoxynucleotides can be ranked according to their ability to bind RecA protein. Structural changes induced in nucleic acids upon RecA binding are discussed together with conformational changes induced in RecA protein upon magnesium binding.     

Biochemistry and Genetics of gangliosidoses

Summary The gangliosidoses comprise an-ever increasing number of biochemically and phenotypically variant diseases. In most of them an autosomal recessive inherited deficiency of a lysosomal hydrolase results in the fatal accumulation of glycolipids (predominantly in the nervous tissue) and of oligosaccharides.The structure, substrate specificity, immunological properties of and genetic studies on the relevant glycosidases, ganglioside G_M1 -galactosidase and -hexosaminidase isoenzymes, are reviewed in this paper. Contrary to general expectation, only a poor correlation is observed between the severity of the disease and residual activity of the defective enzyme when measured with synthetic or natural substrates in the presence of detergents. For the understanding of variant diseases and for their pre- and postnatal diagnosis, the necessity of studying the substrate specificity of normal and mutated enzymes under conditions similar to the in vivo situation, e.g., with natural substrates in the presence of appropriate activator proteins, is stressed. The possibility that detergents may have adverse affects on the substrate specificity of the enzymes is discussed for the -hexosaminidases. The significance of activator proteins for the proper interaction of lipid substrates and watersoluble hydrolases is illustrated by the fatal glycolipid storage resulting from an activator protein deficiency in the AB variant of G_M2-gangliosidosis. Recent somatic complementation studies have revealed the existence of a presumably post-translational modification factor necessary for the expression of ganglioside G_M1 -galactosidase activity. This factor is deficient in a group of variants of G_M1-gangliosidosis. Among the possible reasons for the variability of enzyme activity levels in heterozygotes and patients, allelic mutations, formation of hybrid enzymes, and the existence of patients as compound heterozygotes are discussed. All these may result in the production of mutant enzymes with an altered specificity for a variety of natural substrates.Abbreviations Cer ceramide - Gal D-galactose - GalNAc 2-acetamido-2-deoxy-D-galactopyranoside - Glc D-glucose - MUF 4-methylumbelliferone - MUF--Gal 4-methylumbelliferyl--D-galactopyranoside - MUF--GalNAc 4-methylumbelliferyl-2-acetamido-2-deoxy--D-galactopyranoside - MUF--GlcNAc 4-methylumbelliferyl-2-acetamido-2-deoxy--D-glucopyranoside - NeuAc N-acetylneuraminic acid - PNP--Gal p-nitrophenyl--D-galactopyranoside. Variant B of infantile G_M2-gangliosidosis, Tay-Sachs disease; Variant 0 of infantile G_M2-gangliosidosis, Sandhoff disease, Sandhoff-Jatzkewitz disease; Variant 0 of juvenile G_M2-gangliosidosis, juvenile Sandhoff disease; Variant AB, Variant AB of infantile G_M2-gangliosidosis.     

Androgenic effects of the antiprogestagen RMI 12,936

RMI 12,936 (7alpha-methyl-17beta-hydroxy-androst-5-en-one) was tested for androgenic activity in mouse kidney and for antiprogestational activity in guinea-pig uterus. RMI 12,936 stimulated an increase in kidney weight and in the activity of the androgen-responsive renal enzymes, beta-glucuronidase, alcohol dehydrogenase and arginase. RMI 12,936 was bound by the renal androgen receptor with a relative affinity approximately one-third that of testosterone. Although RMI 12,936 did not stimulate glycogen accumulation in guinea-pig endometrium in vivo, it was active in endometrial organ culture. When RMI 12,936 was combined with progesterone, glycogen accumulation in vitro was partly inhibited. RMI 12,936 was bound by the guinea-pig uterine progesterone receptor with a relative affinity of less than 1%. It is concluded that RMI 12,936 is an androgenic steroid with antifertility actions and in-vitro antiglycogenic activity.     

Evolution of heavy metal resistant transconjugants in a soil environment with a concomitant selective pressure

Abstract Conjugal transfer between Escherichia coli and Alcaligenes eutrophus of plasmid pDN705, containing genes encoding resistance against cadmium, zinc, and cobalt ( czc genes) occurred in heavy metal polluted soil. The selective pressure from heavy metals (especially Zn²⁺) resulted in an increased number of resistant transconjugants and higher respiratory activities in sterile soil. As filter mattings showed no or even a negative effect of Zn²⁺ on plasmid transfer, the increase of the number of transconjugants in polluted was probably due to growth rather than stimulated transfer. High numbers of recipients inhibited extended growth of transconjugants in sterile unpolluted soil. This intranspecific competition was overcome in the presence of heavy metals. In non-sterile soil, such an effect of heavy metals was not always evident, and seemed to be related to the severity of the selective pressure and inversely to the overall biological competition.     

Activity of an essential oil derived from Chenopodium ambrosioides on greenhouse insect pests

This study involved both greenhouse and laboratory experiments evaluating the effect of an essential oil product (QRD 400) derived from Chenopodium ambrosioides variety nr. Ambrosioides L. (Chenopodiaceae) on greenhouse insect pests that feed on different plant parts: citrus mealybug, Planococcus citri (Risso); longtailed mealybug, Pseudococcus longispinus (Targioni Tozzetti); western flower thrips, Frankliniella occidentalis (Pergande), and fungus gnats (Bradysia spp.). Treatments were applied to coleus, Solenostemon scutellarioides plants; transvaal daisy, Gerbera jamesonii flowers; or growing medium, depending on the insect pest. The essential oil was most effective, based on adult emergence, on both the second and third instars of the fungus gnat Bradysia sp. nr. coprophila when applied as a drench to growing medium. In addition, there was a significant rate response for QRD 400 on fungus gnats. The QRD 400 treatment had the highest percentage of mortality on longtailed mealybug (55%) compared with the other treatments. However, the essential oil was less effective against citrus mealybug (3% mortality) and western flower thrips adults (18-34% mortality) compared with standard insecticides, such as acetamiprid (TriStar) and spinosad (Conserve), which are typically used by greenhouse producers. This lack of efficacy may be associated with volatility and short residual properties of the essential oil or with the essential oil taking longer to kill insect pests. Other insecticides and miticides evaluated, including sesame oil, garlic, paraffinic oil, and Bacillus thuringiensis subsp. israelensis, provided minimal control of the designated insect pests. In addition, adult rove beetle Atheta coriaria Kraatz adults were not effective in controlling the larval instars of fungus gnats when applied at a rate of five adults per container.     

Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats

The metabolism of vitamin E involves oxidation of the phytyl chain to generate the terminal metabolite 7,8-dimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (CEHC) via intermediate formation of 13'-hydroxychromanol and long-chain carboxychromanols. Conjugated (including sulfated) metabolites were reported previously but were limited to CEHCs. Here, using electrospray and inductively coupled plasma mass spectrometry, we discovered that gamma-tocopherol (gamma-T) and delta-T were metabolized to sulfated 9'-, 11'-, and 13'-carboxychromanol (9'S, 11'S, and 13'S) in human A549 cells. To further study the metabolites, we developed a HPLC assay with fluorescence detection that simultaneously analyzes sulfated and nonconjugated intermediate metabolites. Using this assay, we found that sulfated metabolites were converted to nonconjugated carboxychromanols by sulfatase digestion. In cultured cells, approximately 45% long-chain carboxychromanols from gamma-T but only 10% from delta-T were sulfated. Upon supplementation with gamma-T, rats had increased tissue levels of 9'S, 11'S, and 13'S, 13'-hydroxychromanol, 13'-carboxychromanol, and gamma-CEHC. The plasma concentrations of combined sulfated long-chain metabolites were comparable to or exceeded those of CEHCs and increased proportionally with the supplement dosages of gamma-T. Our study identifies sulfated long-chain carboxychromanols as novel vitamin E metabolites and provides evidence that sulfation may occur parallel with beta-oxidation. In addition, the HPLC fluorescence assay is a useful tool for the investigation of vitamin E metabolism.     

Redescription of Ascocotyle (Ascocotyle) felippei Travassos, 1928 (Digenea: Heterophiydae) with new synonymies

The heterophyid trematode Ascocotyle (Ascocotyle) felippei Travassos, 1928, is redescribed and new data on its life cycle are provided, based on types and metacercariae found in the heart bulb and gills of naturally infected guppies, Poecilia vivipara (new fish intermediate host), from a coastal lagoon in Rio de Janeiro, Brazil. Examination of the type and all voucher specimens of A. (A.) felippei collected by Travassos in the type host and locality in Brazil has shown that they possess only 32 (16 + 16) circumoral spines, rather than 36 (18 + 18) spines as previously reported. Based on the identical number and arrangement of circumoral spines, shape of the body, the presence of a long preoral lobe and posterior muscular prolongation of the oral sucker, short and wide ceca, a simple gonotyl lacking refractile bodies, and the site of infection of metacercariae (predominantly heart bulb), A. (A.) puertoricensis Price, 1932 and A. (A.) tenuicollis Price, 1935, are proposed as new synonyms of A. (A.) felippei.