Induction of aflatoxin biosynthesis inAspergillus parasiticus by ascorbic acid-mediated lipid peroxidation

The effect ofl-ascorbic acid on the biosynthesis of aflatoxin inAspergillus parasiticus was studied. Ascorbic acid at lower concentrations did not inhibit the growth of fungus but markedly induced aflatoxin biosynthesis. At a concentration of 1000 ppm of ascorbic acid, 4.8-fold higher levels of aflatoxin were detected. Copper did not enhance the induction of toxin synthesis by ascorbic acid when added to the growth medium. Ascorbic acid at 1000 ppm was also found to induce aflatoxin synthesis in resting mycelia. Chloroform (1% vol/vol) was found to induce aflatoxin synthesis under similar conditions. Ascorbic acid in the presence of ferrous ion can cause lipid peroxidation, which in turn is responsible for the induction of aflatoxin synthesis. During the induction of aflatoxin synthesis by ascorbic acid, the uptake of carbon source (acetate) was not affected. This observation suggests that on ascorbic acid treatment a precursor or an intermediate of aflatoxin biosynthesis is synthesized in vivo and is responsible for the higher levels of toxin without increasing the uptake of acetate.     

Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5''-[beta-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction.

(1) We [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637 and Davies, Muir & Offord (1986) Biochem. J. 240, 609-612] have previously identified a major product in the degradation of insulin by insulin proteinase (the N-terminal fragment produced by cleavage between residues LeuA13 and TyrA14, SerB9 and HisB10) together with evidence for a minor cleavage site between HisB10 and LeuB11 or between LeuB11 and ValB12. (2) We now present evidence for minor sites of cleavage between TyrA14 and GlnA15, GluB13 and AlaB14 as well as HisB10 and LeuB11.     

Ascorbic acid enhancement of organogenesis in tobacco callus

The effect of ascorbic acid on growth and shoot formation in callus cultures of tobacco (Nicotiana tabacum L.) was investigated, using young (4–12 subcultures) and old (more than 30 subcultures) tissue. It was found that ascorbate, at levels of 4–8×10^-4M, enhanced shoot formation in both young and old callus. Treatment with ascorbate also speeded up the shoot-forming process. In addition, ascorbate completely reversed the inhibition of shoot formation by gibberellic acid in young callus, but was less effective in old callus.     

Pyrimidine.pyrimidine base-pair mismatches in DNA. A nuclear magnetic resonance study of T.T pairing at neutral pH and C.C pairing at acidic pH in dodecanucleotide duplexes

Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.     

Purification and biochemical characterisation of the EcoR124 type I modification methylase.

Large scale purification of the type I modification methylase EcoR124 has been achieved from an over-expressing strain by a two step procedure using ion-exchange and heparin chromatography. Pure methylase is obtained at a yield of 30 mg per gm of cell paste. Measurements of the molecular weight and subunit stoichiometry show that the enzyme is a trimeric complex of 162 kDa consisting of two subunits of HsdM (58 kDa) and one subunit of HsdS (46 kDa). The purified enzyme can methylate a DNA fragment bearing its cognate recognition sequence. Binding of the methylase to synthetic DNA fragments containing either the EcoR124 recognition sequence GAAN6RTCG, or the recognition sequence GAAN7RTCG of the related enzyme EcoR124/3, was followed by fluorescence competition assays and by gel retardation analysis. The results show that the methylase binds to its correct sequence with an affinity of the order 10(8) M-1 forming a 1:1 complex with the DNA. The affinity for the incorrect sequence, differing by an additional base pair in the non-specific spacer, is almost two orders of magnitude lower.     

Multipoint linkage analysis of the short arm of chromosome 11 in non-insulin dependent diabetes including maturity onset diabetes of youth

Summary Members of three families with maturity onset diabetes of youth (MODY) and seven with common type 2 diabetes were typed for six DNA markers (H-RAS, INS, HBBC, PTH, CALC1, CAT) on the short arm of chromosome 11. Using conventional pairwise linkage analysis, close linkage in the MODY families was excluded for all six markers. By multipoint analysis and a genetic map of the short arm of chromosome 11, MODY was excluded from a region of at least 35 and up to 60 centiMorgans (cM) on the short arm of chromosome 11. Multipoint analysis in the type 2 families also excludes linkage to the INS, H-RAS region of at least 3 and up to 30 cM. This study using multipoint linkage analysis in non-insulin dependent diabetes provides strong evidence against a role for mutations in or around the insulin gene in the causation of MODY or type 2 diabetes in the families studied.     

Somatic cell hybrids, sequence-tagged sites, simple repeat polymorphisms, and yeast artificial chromosomes for physical and genetic mapping of proximal 17p.

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.     

Stereospecific microbial reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl-1H-1)-benzazepin+ ++-2-o ne.

A key intermediate, (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluorome thyl)- 2H-1-benzazepin-2-one (compound II or SQ32191), with high optical purity was made by the stereoselective microbial reduction of the parent ketone 1. Several strains of bacterial and yeast cultures were screened for the ability to catalyse the stereoselective reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl)-1H-1-benzazepin++ +-2,3-dione [compound I or SQ32425]. Microorganisms from the genera Nocardia, Rhodococcus, Alkaligenes, Corynebacterium, Arthrobacter, Hansenula, and Candida reduced compound I to compound II with 60-70% conversion yield. In contrast, microorganisms from the genera Pseudomonas and Acinetobacter reduced compound I stereospecifically to (trans)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromet hyl-2H- 1-benzazepin-2-one (compound III or SQ32408). Among various cultures evaluated, N. salmonicolor SC6310 effectively catalysed the transformation of compound I to compound II with 96% conversion yield at 1.5-2.0 gl-1 concentration. Compound II was isolated and identified by NMR analysis, mass spectrometry, and comparison to an authentic sample. Preparative scale fermentation process and transformation process were developed using cell suspensions of N. salmonicolor SC6310 to catalyse the transformation of compound I to compound II. The isolated compound II had a melting point of 222 degrees C (reference 221-223 degrees C), optical rotation of +130.4 (reference +128 degrees C), and optical purity of greater than 99.9% as analyzed by NMR and chiral HPLC.     

Comparative testing of disinfectant and antiseptic products using proposed European suspension testing methods

The development of standard suspension test methods for disinfectants and antiseptics for adoption in Europe is described. Evaluation of a range of disinfectant agents and products currently used in the UK under conditions as proposed for these tests indicates that the majority of products diluted in water of standard hardness showed satisfactory activity producing a 4·5–5 log reduction in viable count within 5 min against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Proteus mirabilis and Candida albicans in the absence and presence of 1% albumin. All the products, when diluted with distilled water, produced greater than 5 log reduction in 60 min.