Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

[Range of substrates and steroid bioconversion reactions performed by recombinant microorganisms Saccharomyces cerevisiae and Yarrowia lipolytica expressing cytochrome P450c17].

The relationship between 17alpha-hydroxylation and 20-oxidation-reduction of progesterone and some of its derivatives was studied in yeast strains Saccharomyces cerevisiae YEp51alpha, Yarrowia lipolytica E129A15, and expressing cytochrome P450c17. The key metabolites were found to be 17alpha-hydroxyprogesterone and 17alpha,20(alpha,beta)-dihydroxypregn-4-ene-3-ones. The bioconversion pathways of pregn-4-ene-20(alpha,beta)-ol-3-ones were determined. They included cycles of 20-oxidation, 17alpha-hydroxylation, and stereospecific 20-reduction. The efficiency and kinetic parameters of steroid bioconversion by the recombinant strains were determined. The role of yeast analogs of mammalian steroid dehydrogenases is discussed. It was found that any of the desired derivatives, 17alpha-hydroxyprogesterone or progesterone 17alpha,20(alpha,beta)-diols, could be obtained from progesterone.

[Effect of steroid biosynthesis modificators on the progesterone biotransformation by a recombinant yeasts expressing cytochrome P450c17].

Using the recombinant microorganisms S. cerevisiae GRF18 YEp5117alpha, expressing cytochrome P450c17 from bovine adrenal cortex, we investigated the influence of the various modificators of steroids biosynthesis on the relationship between the 17alpha-hydroxylation of progesterone and 20alpha-reduction. Dexamethasone and metirapon had no effect on the reaction of progesterone 17alpha-hydroxylation and on the reaction of 17alpha-hydroxyprogesterone 20alpha-reduction. Mifepriston and danazol did not covalently modify amino acid residues of the cytochrome P450c17 or its heme group under the conditions of the biotransformation of progesterone by recombinant yeasts. Ketokonazol, mifepriston and danazol acted as low-affinity competitive inhibitors, but the 20-dihydro derivatives of progesterone were mixed type inhibitors for the cytochrome P450c17. All modifiers that we used did not influence the functional properties of the yeast analog of 20alpha-hydroxysteroid dehydrogenase. According to the influence on the catalytic parameters of the cytochrome P450c17, the modifiers used can be arranged in the following order: 20beta-dihydroprogesterone (maximum effect) > mifepriston = ketokonazol > 20alpha-dihydroprogesteron > danazol > dexamethasone, metirapon (without effect).

Distribution of caveolin-1 and connexin43 in normal and injured alveolar epithelial R3/1 cells.

Using the new alveolar epithelial type I-like cell line R3/1 derived from fetal rat lung, we studied the distribution of connexin43 and caveolin-1 under conditions of bleomycin-induced injury in vitro. We show that under normal as well as under conditions of injury, endogenous connexin43 does not directly interact with endogenous caveolin-1 as revealed by immunofluorescence, glutathione S-transferase/caveolin-1 "pull down" assay, and co-immunoprecipitation experiments. The assessment of Triton X-100 solubility revealed that caveolin-1 was abundant in detergent-resistant membrane fractions. This is consistent with the localization of caveolin-1 in the lipid rafts/caveolae. Similarly, phosphorylated connexin43 was preferably detected in the Triton-insoluble fraction. Using a sucrose gradient we demonstrated that the majority of phosphorylated connexin43 colocalizes with caveolin-1 in lipid rafts, whereas all other forms of connexin43 remain in the bulk of cellular membranes and cytosolic proteins. Triton solubility assessment of bleomycin-treated cells revealed no differences in the caveolin-1 and connexin43 distribution. A further interesting outcome of our study is the shift of caveolin-1 from the lipid raft/caveolae fractions to the non-caveolar fractions after bleomycin treatment indicating an intracellular retention of caveolin-1. This result suggests the possibility that the translocation of caveolin-1 could be an important event regulating the metabolism of alveolar epithelial lung cells after injury.

Cytochrome P450 2D6 deficiency and its clinical relevance in a patient treated with risperidone.

In contrast to several authors who found hepatic cytochrome P 450 2D6 (CYP2D6) metabolising status to be clinically unimportant in treatment with the CYP2D6 substrate, risperidone, we report on a 17-year-old schizophrenic patient who suffered from severe extrapyramidal side effects (EPS) while being treated with risperidone at 4 mg per day. He was genotyped as a CYP2D6 poor metaboliser (PM). The active moiety of risperidone (sum of risperidone and 9-hydroxyrisperidone) was elevated and increased even further under co-medication with haloperidol and biperiden. We conclude that the PM phenotype for CYP2D6 of this patient had major clinical importance in treatment with risperidone. Most likely metabolic pathways other than CYP2D6 were also involved that are probably inhibited by haloperidol.

Insertional mutagenesis in the n-alkane-assimilating yeast Yarrowia lipolytica: generation of tagged mutations in genes involved in hydrophobic substrate utilization.

Tagged mutants affected in the degradation of hydrophobic compounds (HC) were generated by insertion of a zeta-URA3 mutagenesis cassette (MTC) into the genome of a zeta-free and ura3 deletion-containing strain of Yarrowia lipolytica. MTC integration occurred predominantly at random by nonhomologous recombination. A total of 8,600 Ura(+) transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain lengths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethanol as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas). A total of 257 mutants were obtained, of which about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk(-)) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants. Among Alk(-) mutants, growth defects depending on the alkane chain length were observed (alkAa to alkAc). Furthermore, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated. Flanking borders of the integrated MTC were sequenced to identify the disrupted genes. Sequence analysis indicated that the MTC was integrated in the LEU1 locus in N083, a leucine-auxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gene (PEX14) in N078 (alkD). This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in Y. lipolytica.

[Clinical and histopathological characteristics of early Leser-Trélat syndrome]. / Klinische und histopathologische Charakteristika des initialen Leser-Trélat-Syndroms.

Leser-Trélat-syndrome is characterized as the eruptive appearance of multiple seborrheic keratoses in association with underlying malignant disease. A 73 year old female patient with a metastatic adenocarcinoma of the colon presented with this paraneoplastic change. The rapid appearance of solitary seborrheic keratoses with associated inflammation, seen clinically and histologically, may be an early sign of Leser-Trélat-syndrome. The recognition of this inflammatory component as an early sign may contribute to the prompt diagnosis of this paraneoplasia, even before the eruption of numerous seborrheic keratoses.

Food intake of patients with atopic dermatitis.

There is only restricted information about the nutritional behavior of adult patients with atopic dermatitis (AD). Our purpose was to evaluate the food intake in a series of patients with AD with particular consideration of self-reported food intolerance. Particular attention was paid to the risks of nutrient deficiencies. We examined the intake of 28 food items in 116 AD patients with a food-frequency questionnaire (FFQ). For each food item the cohort was divided in two groups according to whether symptoms were reported or not (symptomatic vs. asymptomatic). We found in a series of food items a significant lower food intake among symptomatic patients. Significantly lower intakes were reported by symptomatic patients for dairy products, fish, egg, pork, oranges, non-specified fruits, apples, kiwis, green or red peppers, peanuts and hazelnuts. We concluded that in symptomatic AD patients supplementation with specific nutrients might become mandatory. This is particularly pertinent for calcium, iodine, vitamin C and n-3 fatty acids.

Vectors for gene expression and amplification in the yeast Yarrowia lipolytica.

New vector systems were developed for gene expression in Y. lipolytica. These plasmids contain: (a) as integration target sequences, either a rDNA region or the long terminal repeat zeta of the Y. lipolytica retrotransposon Ylt1; (b) the YlURA3 gene as selection marker for Y. lipolytica, either as the non-defective ura3d1 allele for single integration or the promotor truncated ura3d4 allele for multiple integration; (c) the inducible ICL1 or XPR2 promoters for gene expression; and (d) unique restriction sites for gene insertion. Multiple plasmid integration occurred as inserted tandem-repeats, which are present at 3-39 copies per cell. A correlation between gene copy number and the expressed enzyme activity was demonstrated with Escherichia coli lacZ as reporter gene under the control of the regulated ICL1 promoter. Increases in copy numbers from 5 to 13 for the lacZ expression cassettes resulted in an up to 10-11-fold linear increase of the beta-galactosidase activity in multicopy transformants during their growth on ethanol or glucose, compared with the low-copy replicative plasmid transformants (1.6 plasmid copies). These new tools will enhance the interest in Y. lipolytica as an alternative host for heterologous protein production.

trans-dominant mutations in the GPR1 gene cause high sensitivity to acetic acid and ethanol in the yeast Yarrowia lipolytica.

Acetate non-utilizing strains harbouring trans-dominant mutations in the GPR1 gene (GPR1(d)) of the dimorphic yeast Yarrowia lipolytica have been selected and characterized. These mutants are highly sensitive to low concentrations of acetic acid and ethanol, even in presence of glucose. The toxic effect of acetic acid is pH-dependent and has the strongest effect at low pH. In contrast, the action of ethanol is pH-independent. One GPR1(d) mutant has been detected that was highly sensitive to acetic acid but could still grow on ethanol, which indicates putative differences in the function of the GPR1 gene product in the sensitivity to acetic acid and ethanol. The GPR1(d) mutants exhibit a complex pleiotropic phenotype. The mutations cause changed colony morphology as well as dimorphism of cells, and induce early cell death during growth on glucose, even without the presence of dicarbon compounds. Composition of intracellular membranes, as well as morphology of vacuole and mitochondria, were strongly changed. Back-crosses with wild-type strains and analysis of recombinant strains have shown that the expression of the pleiotropic phenotype depends on the site of mutation in the GPR1 gene, as well as on the genetic background of the strain harbouring the responsive mutation. Our data suggest that Gpr1p is involved in a general response of cells to the toxic action of dicarbon compounds like acetic acid and ethanol.

Selective peroxisome degradation in Yarrowia lipolytica after a shift of cells from acetate/oleate/ethylamine into glucose/ammonium sulfate-containing media.

We have shown that peroxisomes of the yeast Yarrowia lipolytica are subject to specific degradation after exposure of acetate/oleate-grown cells to glucose excess conditions. Electron microscopic analysis has revealed that the peroxisomes were degraded by uptake in the vacuole. Our results suggest that peroxisomes are taken up by macroautophagic processes, because sequestration of individual peroxisomes, which occurs typically at the beginning of microautophagy, was never observed. The observation that a peroxisomal amine oxidase activity is specifically induced by ethylamine was used for the development of a plate assay screening procedure to isolate peroxisome degradation-defective mutants.

[Blood tests for PCB in teachers at a heavily PCB-polluted school]. / Blutuntersuchungen auf PCB bei Lehrerinnen und Lehrern einer stark PCB-belasteten Schule.

The levels of PCB 28, 52, 101, 153, 138 and 180 were determined in blood samples of subjects (mainly teachers; n = 18) who had worked in a PCB-contaminated school-building for many years (average: 14.2 years). The PCB concentrations in indoor air ranged up to 13,500 ng/m3. For comparison PCB blood levels were determined in 18 teachers working in schools not contaminated by PCB. The subjects of the reference group were matched with the exposed subjects with respect to age and gender. PCB 28, 52 and 101 were not detectable in the blood samples (detection limit: 0.1 microgram/l). In the exposed subjects blood levels of PCB 153 and 138 were, on average, slightly higher and the levels of PCB 180 slightly lower when compared with the reference subjects, but the differences were not statistically significant. The levels of PCB 153, 138 and 180 increased with age. With the exception of two subjects the levels of these congeners were below the reference values proposed by Kappos et al. The results show that inhalative exposure to PCB 153, 138 and 180 is very low in comparison to PCB background exposure via food. Due to rapid metabolisation and elimination the blood levels of PCB 28, 52 and 101 are usually very low.

[Interspecies comparison of healing standardazed bone defects with and without autogenous bone transplantation]. / Interspeziesvergleich zur Heilung standardisierter Knochendefekte mit und ohne autogene Knochentransplantation.

This study tries to establish a basis for comparison of animal studies regarding bone defect healing. Pigs, sheep and rabbits were operated on according to a standardized scheme where each received bilateral defects of the femoral condylus. One of the defects was filled with cancellous autograft, the other remained empty. Bone defect healing was followed with several different methods of investigation, the results were put into perspective with the help of a standardized score-scheme.

Physiology and genetics of the dimorphic fungus Yarrowia lipolytica.

The ascomycetous yeast Yarrowia lipolytica (formerly Candida, Endomycopsis, or Saccharomyces lipolytica) is one of the more intensively studied 'non-conventional' yeast species. This yeast is quite different from the well-studied yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its phylogenetic evolution, physiology, genetics, and molecular biology. However, Y. lipolytica is not only of interest for fundamental research, but also for biotechnological applications. It secretes several metabolites in large amounts (i.e. organic acids, extracellular proteins) and the tools are available for overproduction and secretion of foreign proteins. This review presents a comprehensive overview on the available data on physiology, cell biology, molecular biology and genetics of Y. lipolytica.

Neuronal protection by intraischemic brain perfusion: an electron microscopy study in the rat.

Organ perfusion with bloodless solutions is an established clinical method for protecting the heart against ischemic damage. In our study, we evaluated the effects of intraischemic bloodless brain perfusion on postischemic ultrastructural neuronal changes in a model of severe incomplete forebrain ischemia produced by hemorrhagic hypotension combined with temporary carotid occlusion in the rat. Four groups of rats were compared. During an ischemic insult of 30 min, the brains of two groups were perfused via both external carotids with either a normosmolar normothermic magnesium-enriched perfusate (MgSO4, 30 mM; NaCl, 37 mM; mannitol, 180 mM; n = 10) or a normothermic normal saline solution (n = 9) at a rate of 6 ml/h. Two other groups (ischemia without perfusion, n = 8; no ischemia and no perfusion, n = 7) served as controls. After 30 min of ischemia, withdrawn blood for hemorrhagic hypotension was reinfused, the carotid arteries reopened, and the brains reperfused for 2 h. After perfusion-fixation, qualitative and quantitative evaluation of postischemic cell changes of hippocampal CA1 neurons was performed by electron microscopy. Brain perfusion with the magnesium-containing solution significantly protected neurons against ischemic cell changes and provided an ultrastructural pattern similar to that seen in the nonischemic control group. In contrast, brain perfusion with normal saline solution did not result in neuronal protection. We conclude that intraischemic intracarotid brain perfusion with magnesium-enriched perfusate protects hippocampal neurons significantly against ischemic cell changes in the early reperfusion period after transient severe forebrain ischemia.

Ylt1, a highly repetitive retrotransposon in the genome of the dimorphic fungus Yarrowia lipolytica.

A highly repetitive composite element, Ylt1, was detected in the genome of the dimorphic fungus Yarrowia lipolytica. Ylt1 resembles retrotransposons found in other eukaryotes. It is about 9.4 kb long and can transpose in the genome. The Ylt1 element is bounded by a long terminal repeat (LTR), the zeta element. Several copies of zeta were isolated and sequenced. The sequence of this element is well conserved. It is 714 bp long and is bounded by nucleotides 5'-TG...CA-3', which are part of a short inverted repeat, a feature conserved in the LTRs of retroviruses and retrotransposons. Sequence analysis revealed motifs commonly found in LTR elements, like signals for the start and termination of transcription. The zeta element exists as part of retrotransposon Ylt1, as well as a solo element in the genome. Ylt1 and solo zeta elements are flanked by a 4-bp directly repeated genomic sequence. The copy numbers of Ylt1 and solo zeta are dependent on the strain examined, but at least 35 copies of the composite Ylt1 element and more than 30 copies of the solo zeta element per haploid genome have been observed.

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein.

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 bp long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Saccharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.

Cerebral malformation, seizures, hypertrichosis, distinct face, claw hands, and overlapping fingers in sibs of both sexes.

We report on sibs of both sexes with a multiple malformation syndrome of cerebral malformation, seizures, hypertrichosis, distinct face, claw hands, and overlapping fingers. The older boy died in a tonic extension spasm at age 4 months. When discharged, the younger girl was 3.5 months old. This appears to be an autosomal recessive syndrome which to our knowledge has not been described before.

Primary and secondary erythema migrans in central Wisconsin.

BACKGROUND AND DESIGN: We report a series of 28 consecutive patients with erythema migrans (EM) who underwent skin biopsies for culture of Borrelia burgdorferi. Culture results, Lyme serologic findings, and clinical features in patients with only primary EM are compared with those in patients with secondary EM. RESULTS: Culture preparations of skin specimens obtained from six of 12 patients with only primary EM, and from 14 of 16 patients with secondary EM were positive for B burgdorferi. Seven patients with only primary EM were initially seronegative, and only one patient had an annular lesion. A central crusted punctum was present in five of six primary EM lesions that were not culture positive, but in none of nine culture-positive primary EM lesions. Patients with secondary EM were all seropositive and had less cutaneous and more constitutional symptoms than patients with only primary EM. Three patients with secondary EM had abnormal liver enzyme profiles, one patient had complete heart block, and one patient had myocarditis simulating infarction. Less than one third of all patients recalled a tick bite. An isomorphic nonresponse was seen in skin previously involved with secondary EM in one patient who had a drug exanthem from amoxicillin. CONCLUSIONS: Borrelia burgdorferi can be reliably cultured from skin biopsy specimens of secondary EM. Culture preparation aids definitive diagnosis of Lyme disease in patients with only primary EM who often lack constitutional symptoms, have nondiagnostic lesions, or are seronegative.