The accessibility of DNA to dimethylsulfate in complexes with recA protein.

recA protein coats DNA co-operatively to form filaments approximately 100 A thick, which in the presence of ATP, and more stably so in the presence of the non-hydrolyzable analog ATP gamma S, have a helical appearance with a deep cleft in the protein coat. This protein helix follows the DNA helix, to which it imparts a new helicity of 18.5 bp per turn of 97 A pitch. Here we test the accessibility of the DNA in the complex to modification by dimethylsulfate, and find that the complexed DNA is approximately 2-fold more reactive on the major groove side than it was in B-DNA (methylation of guanine N7), while it is protected approximately 2-fold on the minor groove side (methylation of adenine N3), suggesting that the protein coats the DNA along the minor groove. Furthermore, N3 of cytosine, a residue involved in base pairing, is found exposed in complexes with single strands as it is in naked single-stranded DNA, while it remains inaccessible in complexes with double strands, suggesting that the latter is not melted at this stage of the strand exchange reaction.     

Bulb mite,Rhizoglyphus robini (Astigmata: Acaridae) as an experimental animal

Rhizoglyphus robini Claparède (Acari: Astigmata: Acaridae) is proposed as a model laboratory animal for biological, ecological, physiological and toxicological studies. The mite is easy and inexpensive to rear, quite fecund, convenient to manipulate, and may rapidly be raised to gram quantities. Examples are presented of its use in soil pest ecology and control studies, and in physiological, biochemical and toxicological investigations. Efforts to explore the induction of detoxification systems by various chemicals, and a demonstration of its control by solarization, are also described.     

Twenty-year outcome analysis of genetic screening programs for Tay-Sachs and beta-thalassemia disease carriers in high schools.

Programs for education, screening, and counseling of senior-high-school students, in populations at high risk for Tay-Sachs and beta-thalassemia diseases, have existed for >20 years in Montreal. Four process and outcome variables are reported here: (i) voluntary participation rates in the high-school cohort; (ii) uptake rates for the screening test; (iii) origin of carrier couples seeking the prenatal diagnosis option in the programs; and (iv) change in incidence of the two diseases. Between 1972 and 1992, we screened 14,844 Ashkenazi-Jewish students, identified 521 HexA-deficient carriers (frequency 1:28), reached 89% of the demographic cohort in the educational component of the program, and achieved 67% voluntary participation in the subsequent screening phase. The corresponding data for the beta-thalassemia program are 25,274 students (mainly of Mediterranean origin) representing 67% of the cohort with 61% voluntary participation in the screening phase (693 carriers; frequency 1:36). From demographic data, we deduce that virtually all the carriers identified in the high-school screening program remembered their status, had their partner tested if they did not already know they were a carrier couple, and took up the options for reproductive counseling/prenatal diagnosis. In Montreal, the current origin of all couples using prenatal diagnosis for Tay-Sachs and beta-thalassemia diseases is the corresponding genetic screening/testing program, whereas, at the beginning of the programs, it was always because there was a history of an affected person in the family. Incidence of the two diseases has fallen by 90%-95% over 20 years; the rare new cases are born (with two exceptions) outside the target communities or to nonscreened couples.     

Elongation of duplex DNA by recA protein

recA protein, which is essential for the recombination process in Escherichia coli, was incubated in the presence of 5′-γ-thiotriphosphate with circular plasmid pBR_βG containing small single-stranded gaps. Stable complexes were formed which appear in the electron microscope as fibres with a diameter about five times that of naked DNA. Complex formation appears to be a co-operative process whereby the average rise per base-pair with respect to the fibre axis increases from 3·39 ± 0·08 Å to 5·20 ± 0·18 Å. The elongation of DNA by about 50% is compatible with an unwinding of the double helix and an intercalating mode of binding of recA and/or 5′-γ-thiotriphosphate to DNA.     

The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.     

Synthesis,biological evaluation and molecular modeling of novel selective COX-2 inhibitors: sulfide,sulfoxide, and sulfone derivatives of 1,5-diarylpyrrol-3-substituted scaffold

A novel series of 1,5-diarylpyrrol-3-sulfur derivatives (10–12) was synthesized and characterized by NMR and mass spectroscopy and x-ray diffraction. The biological activity of these compounds was evaluated in in vitro and in vivo tests to assess their COX-2 inhibitory activity along with anti-inflammatory and antinociceptive effect.Results showed that the bioisosteric transformation of previously reported alkoxyethyl ethers (9a-c) into the corresponding alkyl thioethers (10a-c) still leads to selective and active compounds being the COX-2 inhibitory activity for most of them in the low nanomolar range. The oxidation products of 10a,b were also investigated and both couple of sulfoxides (11a,b) and sulfones (12a,b) showed an appreciable COX-2 inhibitory activity. Molecular modeling studies were performed to investigate the binding mode of the representative compounds 10b, 11b, and 12b into COX-2 enzyme and to explore the potential site of metabolism of 10a and 10b due to the different in vivo efficacy. Among the developed compounds, compound 10b showed a significant in vivo anti-inflammatory and antinociceptive activity paving the way to develop novel anti-inflammatory drugs.     

A synthetic peptide reproducing the mitochondrial targeting motif of AKAP121: a conformational study

The conformational features of a peptide derived by the 10-30 sequence of the mitochondrial domain of AKAP121 [Ac-1XKKPLALPGMLALLGWWWFFSRKKX25-NH2 (X=beta-Ala)] in water and in a water/trifluoroethanol (TFE) mixture at 298 K have been determined by NMR and CD spectroscopy. Backbone clustering analysis of NMR-derived structures led to the identification of a single representative structure in water/TFE. The structure of the peptide consists mainly of an alpha-helix, whose core is the region 7-23, with a less ordered N-terminal part. These data are confirmed by CD analysis. It is noteworthy that the high hydrophobic Trp16-Phe20 segment, that might also mediate interaction with tubulin, is organized in an alpha-helical wheel. Our conformational data can be the starting point for the development of highly selective peptides that interfere with the biological function of the Protein Kinase A scaffold protein AKAP121.