Phenotypic and genotypic identification of yeasts from cheese.

Eighty-five years strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kregervan Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.

Phytopathogenic filamentous (Ashbya, Eremothecium) and dimorphic fungi (Holleya, Nematospora) with needle-shaped ascospores as new members within the Saccharomycetaceae.

Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J Industr. Microbiol. 14, 523-530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle-shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces. The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542-549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marxianius, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution.

Autolysis parallels activation of mu-calpain.

The kinetics of autolysis and activation of mu-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2 hydrolysis was found to be a linear function of the autolysed 76 kDa form of mu-calpain large subunit at both 10 and 300 microM Ca2+, and both straight lines intersected the origin. This finding supports the view that native mu-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, K1(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 microM Ca2+), whereas the maximal value was 1.52 s-1, at 30 degrees C. The Ca(2+)-induced activation process was then monitored by using our novel, continuous fluorimetric assay with labelled MAP2 as substrate. The first-order rate constant of activation, k1(act), was derived as the reciprocal of the lag phase ('transit time') at the initial part of the progress curve: half-maximum was at pCa2+ = 3.8 (158 microM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of mu-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of mu-calpain. Pre-activated mu-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 microM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the mu-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.

Analysis of coenzyme Q systems, monosaccharide patterns of purified cell walls, and RAPD-PCR patterns in the genus Kluyveromyces.

Analysis of the coenzyme Q system and the monosaccharide pattern of purified cell walls were used for species characterization in the genus Kluyveromyces. All the type strains of the genus possess coenzyme Q-6 and the mannose-glucose ('Saccharomyces type') cell wall sugar pattern. With the help of Random Amplified Polymorphic DNA-Polymerase Chain Reaction analysis 17 species were separated: K. aestuarii, K. africanus, K. Bacillisporus, K. blattae, K. delphensis, K. dobzhanski, K. lactis (anamorph Candida sphaerica), K. lodderae, K. marxianus (syn. K. fragilis, K. bulgaricus, K. cicerisporus, anamorphs Candida macedoniensis, C. pseudotropicalis, C. kefyr), K. phaffii, K. piceae, K. polysporus, K. sinensis, K. thermotolerans (syn. K. veronae, anamorph Candida dattila), K. waltii, K. wickerhamii, K. yarrowii (anamorph Candida tannotolerans). A strain of K. drosophilarum showed with the type strain of K. lactis only 63% similarity. The strain originally described as the type strain of K. cellobiovorus nom. nud. was excluded from the genus (Q-9), and found to be conspecific with the type strain of Candida intermedia.

Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia and Sterigmatomyces species.

The qualitative and quantitative monosaccharide spectra of purified yeast cell walls revealed that there are three phylogenetically distinct lineages of sterigma-forming basidiomycetous yeasts: (i) Kurtzmanomyces and Sterigmatomyces species, which contain high levels of mannose; (ii) Tilletiopsis species, which contain glucose, galactose, and small amounts of mannose; and (iii) Fellomyces, Kockovaella, Sterigmatosporidium, and Tsuchiyaea species, which appear to be closely related on the basis of their high levels of glucose and the presence of xylose. The yeast cell wall neutral sugars of Sporobolomyces antarcticus and Sterigmatomyces aphidis were similar to those of members of the genus Tilletiopsis. However, the possibility that these taxa are conspecific was eliminated by the results of a random amplified polymorphic DNA (RAPD) analysis. The conspecificity of Mrakia frigida and Mrakia nivalis, the conspecificity of Mrakia gelida and Mrakia stokesii, and the conspecificity of Sterigmatomyces halophilus and Sterigmatomyces indicus were confirmed by RAPD analysis results. RAPD analysis was found to be a simple and highly sensitive method which can be used to differentiate species at the DNA level; it can replace nuclear DNA-nuclear DNA hybridization experiments for species identification, characterization, and delimitation.

[Effects of various quantities of dietary iodine on total serum T3 and T4 in gilts during various phases of the reproductive cycle]. / Uticaj razlicitih kolicina joda u ishrani nazimica na koncentraciju ukupnog serumskog T3 i T4 u razlicitim fazama reprodukcijskog ciklusa.

The effect of different dietary iodine levels (1 p.p.m.-control, 2 p.p.m. and 3 p.p.m.) on the serum levels of thyroid hormones (T3 and T4) has been studied in gilts at various stages of reproductive cycle, as well as on the their reproductive performances. The gilts were the crossbred of Swedish Landrace x Big Yorkshire x German Landrace, selected for reproduction. The serum concentrations of T3 and T4 in four month old gilts (at the beginning of experiment) were normal: 1.48 +/- 0.28 and 46.57 +/- 11.37, respectively. Approximately the same levels of serum thyroid hormones were found in all 3 experimental groups of the gilts at the first oestrus (about 6.5 months) when they were artificially inseminated, at the end of the first third of the pregnancy, at the end of lactation and on the day of first postlactational oestrus. Significantly lower concentrations of serum T4 were only at 1-3 days before parturition, 6-12 hours after parturition and during lactation in all groups of gilts. There were no significant differences in T3 and T4 concentrations as well as in reproductive performances between gilts on the different iodine diet. The authors concluded that 1 p.p.m. of dietary iodine, which is regularly applied in this pig farm, is sufficient to provide normal levels thyroid hormones and normal reproduction. There is not economical and physiological reasons to increase this level of iodine to 2 and 3 p.p.m., since the obtained results showed that these changes don't demonstrated neither positive or negative effects.

Alpha-fetoprotein: a marker for threatened abortion.

The role of alpha-fetoprotein (AFP) as a marker for threatened abortion was investigated in 50 patients and compared with the roles of serum human chorionic somatomammotropin (HCS) or human placental lactogen (HPL) and human chorionic gonadotropin (HCG). Of the 50 patients admitted to the hospital with threatened abortion, 30 patients aborted and 20 gave birth to live-born babies. Twenty women aborted within seven days of admission. Eighty-two percent (82%) of the 20 had abnormal AFP values: one third were above the 97.5 percentile and two thirds were below the 2.5 percentile. The AFP values were normal in almost all cases in the group who aborted after seven days. The HCG level in serum was found to be the best indicator of threatened abortion. the AFP and HCS levels were equal indicators of threatened abortion, but were not as reliable as the HCG levels The AFP values are of more diagnostic use than the HCS values, however, because both abnormally low and high AFP levels can indicate an unfavorable course in pregnancy.