Factors affecting the production of L-phenylacetylcarbinol by yeast: a case study.

L-Phenylacetylcarbinol (L-PAC) is the precursor for L-ephedrine and D-pseudoephedrine, alkaloids possessing alpha- and beta-adrenergic activity. The most commonly used method for production of L-PAC is a biological method whereby the enzyme pyruvate decarboxylase (PDC) decarboxylates pyruvate and then condenses the product with added benzaldehyde. The process may be undertaken by either whole cells or purified PDC. If whole cells are used, the biomass may be grown and allowed to synthesize endogenous pyruvate, or the cells may be used as a catalyst only, with both pyruvate and benzaldehyde being added. Several yeast species have been investigated with regard to L-PAC-producing potential; the most commonly used organisms are strains of Saccharomyces cerevisiae and Candida utilis. It was found that initial high production rates did not necessarily result in the highest final yields. Researchers then examined ways of improving the productivity of the process. The substrate, benzaldehyde, and the product, L-PAC, as well as the by-products, were found to be toxic to the biomass. Methods examined to reduce toxicity include modification of benzaldehyde dosing regimes, immobilization of biomass or purified enzymes, modification of benzaldehyde solubility and the use of two-phase reaction systems. Various means of modifying metabolism to enhance enzyme activity, relevant metabolic pathways and yield have been examined. Methods investigated include the use of respiratory quotient to influence pyruvate production and induce fermentative activity, reduced aeration to increase PDC activity, and carbohydrate feeding to modify glycolytic enzyme activity. The effect of temperature on L-PAC yield has been examined to identify conditions which provide the optimal balance between L-PAC and benzyl alcohol production, and L-PAC inactivation. However, relatively little work has been undertaken on the effect of medium composition on L-PAC yield.

Effect of a rhinovirus-caused upper respiratory illness on pulmonary function test and exercise responses.

Upper respiratory illness (URI) may cause more frequent acute disability among athletes than all other diseases combined. The purposes of this study were to determine the impact of a rhinovirus-caused URI on resting pulmonary function submaximal exercise responses and on maximal exercise functional capacity. Twenty-four men and 21 women (18-29 yr) of varying fitness levels were assigned to the experimental group (URI), and 10 additional individuals served as a control group (CRL). An initial serological screening was performed on all URI group subjects to exclude those with the rhinovirus 16 (HRV16) antibody. All subjects completed both a baseline pulmonary function test and a graded exercise test to volitional fatigue. URI subjects were inoculated with HRV 16 on two consecutive days within 10 d of completing these tests. The day following the second inoculation (peak of illness), post-inoculation pulmonary function and graded exercise tests were performed. A noninfected control group completed these same pulmonary and exercise tests 1 wk apart. ANOVA identified no significant differences (P < 0.05) at minutes 2, 5, and 8 for the physiological responses measured between the pre- and post-exercise tests for both the URI and CRL, groups. Furthermore, there were no significant differences between maximal exercise performance between running trials for either group. There was also no significant interaction between treatment (pre/post URI) and group for any of the pulmonary function measures obtained. In conclusion, physiological responses to pulmonary function testing and submaximal and maximal exercise do not appear to be altered by an URI.

A novel spectrophotometric method for the enzymatic determination of NAD+ and NADH.

The theory and practice of a novel spectrophotometric method for the enzymatic determination of NAD+ and NADH is described. The method can not discriminate between NAD+ and NADH, but determines the concentration of the sum of both nucleotides. The method is based on the bleaching of p-nitroso-N,N-dimethylaniline (NMDA) (epsilon 440 nm = 35400 M-1cm-1) with NADH, in the presence of ethanol and yeast alcohol dehydrogenase, under the conditions of enzymatic cycling (ethanol > NDNA > NAD/H). The initial rates of -NDMA bleaching are proportional to the concentration of NAD+ or NADH, in a broad range from 10 nM to 100 microM.

Nucleotide sequence of high-passage hepatitis A virus strain HM175: comparison with wild-type and cell culture-adapted strains.

The nucleotide sequence of cDNA from a high-passage, cell culture-adapted variant of hepatitis A virus strain HM175 was compared with the previously determined sequences of wild-type virus and two other cell culture-adapted variants. A total of 42 nucleotide changes were detected when the sequence was compared with wild-type virus. Five of these changes were common to all cell culture-adapted strains and a further two changes were shared by the strains that had experienced the greatest number of cell culture passages. The mutations were distributed throughout the genome coding for amino acid substitutions in regions 2B, 2C and 3D with silent changes in 1C and the 5' non-coding region. The possible relevance of these mutations to cell culture adaptation and attenuation is discussed.

Atypical campylobacters associated with gastroenteritis.

Nine strains of Campylobacter species other than Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis were isolated from patients with acute diarrhea. All nine strains showed preferred growth at 37 degrees C under microaerophilic conditions. Conventional microbiological tests and DNA-DNA dot blotting were used to identify these strains. Three of the nine Campylobacter strains hydrolyzed hippurate, reduced nitrate, produced catalase, were resistant to cephalothin, and were shown to be highly related to C. jejuni type strains. Two strains had negative or weak catalase activity and were hippurate negative. Three other strains had characteristics similar to those of Campylobacter cinaedi. The ninth strain, isolated from a homosexual man with antibodies to human immunodeficiency virus (human T-cell lymphotropic virus type III), showed unique features different from those of all the known campylobacters used in this study. This strain grew well at 25 and 37 degrees C and was catalase and nitrate positive, hippurate negative, and resistant to cephalothin.

Randomized clinical investigation of Ro 15-1788, a benzodiazepine antagonist, in reversing the central effects of flunitrazepam.

In a double-blind, randomized study the efficacy of Ro 15-1788 as a benzodiazepine antagonist was evaluated in patients anaesthetized with flunitrazepam. The effects were compared with a comparable group of patients who did not receive the antagonist. Ro 15-1788 significantly reversed the sedative and hypnotic effects of flunitrazepam and reduced the degree of anterograde amnesia. No side-effects were attributable to the drug.

Further characterization of 41 isolates of adenovirus types 19/37 by serum neutralization and DNA restriction enzyme analysis.

Forty-one strains of adenovirus type 19/37 (Ad19/37) mainly isolated from patients with keratoconjunctivitis or conjunctivitis between 1974 and 1984 were re-evaluated by serum neutralization (SN), haemagglutination inhibition (HI) and DNA restriction analysis. Of 19 isolates which were neutralized to high titre by antiserum prepared against prototype Ad19, 5 showed cross-reactivity with 32-64 units of Ad37 antiserum, while of 22 strains neutralized by high titre by Ad37 antiserum, 3 showed cross-reactivity with 32 units of Ad19 antiserum. By DNA restriction analysis, all Ad19 isolates were identical to each other and to Ad19A virus. Using endonuclease Bgl 1, three variants were observed among the Ad37 isolates.

Molecular cloning of cDNA from hepatitis A virus strain HM-175 after multiple passages in vivo and in vitro.

Hepatitis A virus (HAV) strain HM-175 was passaged six times in marmosets, 59 times in cell culture and purified from infected cell culture supernatant fluid. The viral RNA was extracted, copied into cDNA and the cDNA:RNA hybrids were cloned into the PstI site of plasmid pBR322. The cDNA clones were authenticated by hybridization to RNA extracted from HAV-infected cells and clones representing the 3' end of the genome were identified using a previously authenticated cDNA clone. The clones represented all but 29 bases of the HAV genome. They were compared to HAV strain HM-175 cDNA cloned from viral RNA after three passages in marmosets on the basis of restriction endonuclease mapping and DNA sequencing. No differences were found in either the presence or absence of restriction endonuclease sites using 33 different restriction enzymes. Sequencing of cDNA representing bases 29 to 1002 of the HAV genome revealed eight base changes all of which were within the 5' noncoding region.

Jaundice and eosinophilia associated with amitriptyline.

Eosinophilia and jaundice occured in a depressed patient treated with amitriptyline, an association which is previously unreported. These complications cleared with withdrawal of the drug. This clinical picture should be added to the known hepatic complications of amitriptyline.