Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa.

Patients with retinitis pigmentosa (RP) typically develop night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors are the mainstay of their vision; however, over years or decades, these cones slowly degenerate, leading to blindness. We created transgenic pigs that express a mutated rhodopsin gene (Pro347Leu). Like RP patients with the same mutation, these pigs have early and severe rod loss; initially their cones are relatively spared, but these surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. Given the strong similarities in phenotype to that of RP patients, these transgenic pigs will provide a large animal model for study of the protracted phase of cone degeneration found in RP and for preclinical treatment trials.

Production of 2,3-butylene glycol from whey by Klebsiella pneumoniae and Enterobacter aerogenes.

Production of 2,3-butylene glycol from whey with Klebsiella pneumoniae and Enterobacter aerogenes was studied. Sterilization of the whey was unnecessary. Acid whey required neutralization, but sweet whey did not. Butylene glycol production was most efficient at 33 degrees C for Klebsiella pneumoniae and at 37 degrees C for Enterobacter aerogenes. Aeration significantly improved yields. Klebsiella pneumoniae produced more butylene glycol than did Enterobacter aerogenes in unsupplemented whey. The addition of 50 mM sodium acetate to whey increased the production of butylene glycol and acetoin by Enterobacter aerogenes; it also increased the production of glycol by Klebsiella pneumoniae, but the increase in this case was offset by a decrease of production of acetoin. Maximal yields of the glycol plus acetoin in whey were obtained in 48 to 64 h, but Enterobacter aerogenes required about 160 h for complete utilization of the lactose. Highest yields were about .3 M butylene glycol plus acetoin, which corresponds to the production of about 10 kg of glycol from 380 liters of whey.

Frequencies of Bacteriophage-Resistant and Slow Acid-Producing Variants of Streptococcus cremoris.

The frequencies of bacteriophage-resistant and slow acid-producing variants in 10 purified strains of Streptococcus cremoris were studied. There were considerable differences among the strains in the occurrence of both slow acid-producing and phage-resistant mutants. Nevertheless, the spontaneous rates of mutation to slow acid production were three to five orders of magnitude greater than the corresponding rates of mutation to phage resistance, suggesting that slow acid production and phage resistance are not genetically linked, although they appear in cultures concomitantly. The frequencies of slow acid-producing variants among resistant and sensitive isolates from the same parent culture were similar and appeared to be strain dependent. All phage-resistant mutants tested were found to be deficient in adsorption of the homologous bacteriophage.

Specificity of the Westerfeld adaptation of the Voges-Proskauer test.

Aliphatic chain compounds at least four carbons long with vicinal carbonyl groups in the 2,3 positions were detected by the Westerfeld test. Acetoin, which has one carbonyl group and an adjacent hydroxyl group, gave positive results, but methyl action (3-hydroxy-3-methyl-2-butanone) was negative, and subsequent tests supported the conclusion that acetoin is oxidized to diacetyl by alpha-naphthol during the Westerfeld test in the absence or presence of air. 2,3-Pentanedione and 2,3-heptanedione gave positive results, but equimolar concentrations of these compounds gave maximal absorbancy readings that were only 35% (2,3-pentanedione) and 31% (2,3-heptanedione) of those obtained with diacetyl or acetoin. Negative results were obtained with pyruvic acid, 2,3-butylene glycol, and carbon ring compounds (1,2-cyclohexanedione, alloxan, and 3,4-dihydroxy-3-cyclobutene-1,2-dione). alpha-Naphtho could not be replaced in the test by beta-naphthol, 1,2,3,4,-tetrahydroxy-1-naphthol, or 5,6,7,8-tetrahydroxy-1-naphthol. Creatine could not be replaced by arginine, guanidine . HCl, or guanidinoacetic acid.

Microbial production of 2,3-butylene glycol from cheese whey.

Six microorganisms that produced acetoin or diacetyl or both from glucose were tested for the production of 2,3-butylene glycol from lactose. Bacillus polymyxa and Streptococcus faecalis gave positive results and were tested in unmodified wheys. Cottage cheese whey was unsatisfactory, but B. polymyxa produced large amounts of the glycol in sweet whey, about 60 mmol of glycol per 100 mmol of lactose utilized. Aeration and an increased ratio of surface area to volume of whey enhanced the production of glycol. 2,3-Butylene was separated from the spent whey and from acetoin and diacetyl with a Sephadex G-10 column.

Inhibition of Candida albicans by Lactobacillus acidophilus.

Candida albicans grew at pH 4.6 or above in nutrient broth containing 5% glucose but was retarded at pH 7.7 by filtrates of Lactobacillus acidophilus grown in casitone broth. Vaginal implantation of nonfermented acidophilus milk, yogurt, or low-fat milk for preventing recurrence of monilia vaginitis subsequent to treatment with Nystatin was studied with 30 women. Reinfections within 3 mo according to product received were: no milk product, 3; yogurt, 1; nonfermented acidophilus milk, 1; and low-fat milk, 0.

Production of Hydrogen peroxide by Lactobacillus acidophilus.

Hydrogen peroxide production in reconstituted skim milk (10%) and low-fat milk by four strains lf Lactobacillus acidophilus was studied at 37 and 4 C. Strains A and B produced little, but strains C and D produced larger amounts, especially if agitated continuously during growth at 37 C or storage at 4 C. Continuous shaking was required at 4 C for C or D (4.0 X 10(8)/ml) to produce sufficient hydrogen peroxide to retard growth of Pseudomonas fragi. Flavin adenine dinucleotide stimulated the oxidation of reduced nicotinamide adenine dinucleotide by dialyzed cell-free extracts of C and D, which indicated that the reduced nicotinamide adenine dinucleotide oxidases of these strains produce hydrogen peroxide as an end product.

Enumeration of Lactobacillus acidophilus with the Agar Plate Count.

Higher plate counts on MRS agar were obtained under anaerobic conditions for three of four strains of Lactobacillus acidophilus and commercially prepared nonfermented acidophilus milks (products A and B) made with two of the strains. Average values for aerobic counts of products A and B were 87 and 60%, respectively, of those for corresponding anaerobic counts. Incubation of plates poured with MRS agar for 72 ± 3 h at 37 C was sufficient for maximal counts. Two strains and the nonfermented acidophilus milks gave highest counts on Standard Methods Agar (SMA). Greatest numbers of bile-resistant colonies were indicated by MRS agar (with 0.2% oxgall). The average of plate counts for products A and B determined on MRS agar with oxgall was 65% of that for corresponding plate counts determined on MRS without oxgall. Buffered distilled water, 0.9% NaCl, and 1% peptone each served satisfactorily as diluent. Overlaying MRS agar in poured plates with additional medium was not advantageous. Plate counts of samples that had been frozen and stored at -26 C or in dry ice were as high as those of duplicate samples that had been stored at 1.7 C.

End products and fermentation balances for lactic streptococci grown aerobically on low concentrations of glucose.

Maximum acetate produced aerobically by Streptococcus diacetilactis and Streptococcus cremoris was 14% of 1 to 7 mumol of glucose/ml in a partially defined medium that contained lipoic acid. Y (glucose) values were 35.3 (S. diacetilactis) and 31.4 (S. cremoris) with low concentrations (1 to 7 mumol/ml) of glucose in the medium and 21 (S. diacetilactis) with higher concentrations (6 to 15 mumol/ml). Y (adenosine 5'-triphosphate) values for the bacteria, determined by taking into account the end products produced, were 15.6 and 13.9 for S. diacetilactis and S. cremoris, respectively, in the partially defined medium containing 1 to 7 mumol of glucose/ml and higher (21.5 and 18.9, respectively) in a complex medium that contained 2 mumol of glucose/ml. Addition of citrate in addition to glucose did not result in higher molar growth yields.

Microorganisms and characteristics of laban.

Laban had a titratable acidity of about 1.0%, a pH of 4.25, an ethanol content of 1.25%, and contained 4.2 mug acetaldehyde and 34 mug acetoin/ml. There was no diacetyl. Five microorganisms, classified as Streptococcus thermophilus, Lactobacillus acidophilus, Leuconostoc lactis, Kluyveromyces fragilis, and Saccharomyces cerevisiae, were responsible for the fermentation. Streptococcus thermophilus and L. acidophilus were responsible for acid production with S. thermophilus producing acid more rapidly. Most of the acetaldehyde was produced by K. fragilis, little ethanol was found in absence of S. cerevisiae, and the acetoin was producted by S. thermophilus.

Incorporation of radioactive acetate into diacetyl by Streptococcus diacetilactis.

Streptococcus diacetilactis was grown in a partially defined, lipoic acid-free medium containing radioactive acetate with and without addition of 0.1% unlabeled sodium pyruvate. Labeled carbon was incorporated into diacetyl, but neither the amount of diacetyl produced nor its specific activity was influenced by addition of pyruvate. Acetoin had low specific activity, indicating that it was a mixture of radioactive and nonradioactive acetoin. The specific activity of acetoin was lower when pyruvate, a precursor of unlabeled acetoin, was added to the medium, which indicated that the radioactive acetoin was produced from radioactive diacetyl by diacetyl reductase. Results substantiate condensation of acetyl-coenzyme A with hydroxyethylthiamine pyrophosphate as the in vivo mechanism for synthesis of diacetyl.

Isolation and identification of psychrophilic species of Clostridium from milk.

Four of 48 raw milk samples contained catalase-negative, gram-positive, motile, sporeforming, rod-shaped bacteria that grew optimally at 22 to 30 C and slowly at low temperatures. Isolates from two samples had a minimal growth temperature of 4 C, were anaerobic, and had characteristics similar to Clostridium hastiforme; those from the other two samples had a minimal growth temperature of 0 +/- 1 C, were anaerobic, aerotolerant, and had characteristics similar to C. carnis. Specific growth rates, doubling times, ability to grow in pasteurized milk stored in commercial cartons, and resistance of spores to heating were determined for one strain of C. hastiforme.