Restriction endonucleases for pulsed field mapping of bacterial genomes.

Fundamental to many bacterial genome mapping strategies currently under development is the need to cleave the genome into a few large DNA fragments that can be resolved by pulsed field gel electrophoresis. Identification of endonucleases that infrequently cut a genome is of key importance in this process. We show that the tetranucleotide CTAG is extremely rare in most bacterial genomes with G+C contents above 45%. As a consequence, most of the sixteen bacterial genomes we have tested are cleaved less than once every 100,000 base pairs by one or more endonucleases that have CTAG in their recognition sequences: Xba I (TCTAGA), Spe I (ACTAGT), Avr II (CCTAGG) and Nhe I (GCTAGC). Similarly, CCG and CGG are the rarest trinucleotides in many genomes with G+C content of less than 45%. Thus, Sma I (CCCGGG), Rsr II (CGGWCCG), Nae I (GCCGGC) and Sac II (CCGCGG) are often suitable endonucleases for producing fragments that average over 100,000 base pairs from such genomes. Pulsed field gel electrophoresis of the fragments that result from cleavage with endonucleases that cleave only a few times per genome should assist in the physical mapping of many prokaryotic genomes.     

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.     

The effect of feeding endophyte infected tall fescue seed on reproductive performance in female rats

Female Sprague-Dawley rats (Rattus norvegicus) were randomly assigned to diets containing mixtures of rat chow and tall fescue (Festuca arundinacea) seed with 0, 5, 10, 20 and 40% infection levels of Acremonium coenophialum to assess the effect of the diets on the reproductive potential of rats. Rats fed 40% infected seed had decreased body weight, decreased mean percent body weight of uteri, failed to maintain normal estrous cycles and were unable to become pregnant. Animals fed a diet of 20% infected fescue seed had extended estrous cycles. There were no significant differences among the 0, 5 and 10% dietary treatments.     

Manchester air disaster.

On 22 August 1985 a fire occurred on a Boeing 737 jet airliner at take off at Manchester Airport. One hundred and thirty seven passengers and crew were on board. Fifty two passengers died on the aircraft, 85 escaped. Most survivors had minor physical injuries, but 15 required admission to hospital because of smoke inhalation and two of these had severe burns. At presentation only one survivor required ventilation but within 12 hours a further five required ventilation. Although initially patients suffering from smoke inhalation may seem relatively well, lung function may deteriorate rapidly in the first 24 hours. Careful organisation and regular practice of procedures to deal with a major accident are essential to be able to respond adequately to such an event.     

Characterization of aminoalkylimidazol-4-one mutagens from liquid-reflux models

A new class of low molecular weight, aminomethylimidazol-4-one (IQ-"like") mutagens have been produced by the reaction of creatinine with the amino acid L-threonine, in liquid-reflux models, mimicking cooking, of diethylene glycol:5% distilled water (2 h at 150 degrees C). Two mutagens, 2-amino-1-methyl-5-propylideneimidazol-4-one (AMPI) and 2-amino-5-ethylidene-1-methylimidazol-4-one (AEMI) were isolated and characterized by UV absorption spectra, mass spectra, and 1H-NMR. The mutagen AEMI was identical to that obtained from the reaction of creatinine with acetaldehyde. These mutagens were positive in all IQ-sensitive Ames tester strains and were not inactivated by acidic nitrosation at pH 1.0. Products displaying mutagenicity were also obtained by refluxing creatinine with other hydroxyamino acids such as L-serine, L-homoserine, and L-4-amino-3-hydroxybutyric acid, and aldehydes such as glyoxal, methylglyoxal, glycolaldehyde, but not formaldehyde. Simple model systems such as creatinine and acetaldehyde may be useful in more clearly defining the exact mechanism of formation of IQ-type mutagens (aminomethylimidazo-quinolines and -quinoxalines) produced during cooking, as well as in screening for potential inhibitors of IQ-type mutagen formation, and elucidating the mechanism of such inhibition.     

Family physicians for underserved areas. The role of residency training.

Graduates of four rural and four urban family practice programs were interviewed to determine the nature of their practices and the factors that had influenced their practice location decisions. All programs gave residents substantial experience providing continuity of care for underserved populations. Of the 158 physicians surveyed, 58 (46%) were working in areas designated as underserved. The percentage of physicians in underserved areas was higher than that reported in other studies and was much higher than would be expected if practice sites were selected on the basis of population distribution alone. Notable differences in personal and practice characteristics were found between the physicians who chose to work in underserved areas and those who did not and between those who established practices in rural and in urban underserved areas.     

Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.     

Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation.

1,25-dihydroxyvitamin D3 is converted to calcitroic acid before being excreted in the bile. Biosynthesis of calcitroic acid has been demonstrated in two target cells of vitamin D, in the kidney and the osteoblastic cell line UMR-106. Calcitroic acid was identified by combinations of h.p.l.c., u.v. spectroscopy and mass spectrometry. Evidence is presented that calcitroate is derived from the 24-oxidation pathway, possibly through the intermediate 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3. The 24-oxidation pathway to calcitroic acid in bone cells is stimulated by 1,25-dihydroxyvitamin D3. The pathway in both bone cells and perfused kidney operates at physiological concentrations of substrate and appears to be capable of rapid clearance of the hormone.