Procedures in the biochemical estimation of age of vertebrates

The most accurate method known to data for estimating age of wild vertebrates involves biochemical assay of eye lens proteins. Laboratory procedures that may be unfamiliar to many ecologists are described in detail. The method is based on precise changes in the amount of insoluble lens protein, and consists of two procedures: obtaining the appropriate lens fraction, and quantitatively analyzing its protein composition. Lenses are homogenized, and the insoluble fraction isolated by centrifugation. Protein content is measured colorimetrically by using the Lowry test. Instructions are given for single and double-beam spectrophotometers.     

Species of Pseudomonas obtained at 7 degrees C and 30 degrees C during aerobic storage of lamb carcasses.

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30 degrees C were Ps. fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7 degrees C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% SSM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group (Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (> 74% SSM) and Ps. lundensis (> 80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.     

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1-3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2-3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.