Acid production by oral strains of Candida albicans and lactobacilli.

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42-66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important.

From structure and functions of steroidogenic enzymes to new technologies of gene engineering.

Abstract (3/4) This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.

Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization.

In order to get deeper insights into oxidative degradation of the hydrophobic substrates (HS) triglycerides and alkanes by yeasts, tagged mutants affected in these pathways were generated by random insertion of a mutagenesis cassette MTC into the genome of Yarrowia lipolytica. About 9.600 Ura+ transformants were screened in plate tests for utilization of alkanes (C10, C16), oleic acid and tributyrin. HS degradation mutants were recovered as unable to grow on alkane or on intermediates of the pathway (AlkA-AlkE phenotype classes). To identify the disrupted genes, insertion points of the MTC were sequenced using convergent and divergent PCR. Sequence analysis evidenced both known and new genes required for HS utilization, e.g. for AlkD/E mutants MTC insertion had occurred in genes of thioredoxin reductase, peroxines PEX14 and PEX20, succinate-fumarate carrier SFC1, and isocitrate lyase ICL1. Several mutants were affected in alkane utilization depending on chain length. Mutant Z110 (AlkAb: C10- C16+) was shown to be disrupted for ANT1 encoding a peroxisomal membrane localized adenine nucleotide transporter protein, providing ATP for the activation of short-chain fatty acids by acyl-CoA synthetase II in peroxisomes. Mutants N046 and B095 (AlkAc: C10+ C16-) were disrupted for the ABC transporter encoded by ABC1 gene, thus providing first evidence for its participation in chain length dependent alkane transport processes.

[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).

Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications.

The alkane-assimilating yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n-alkanes, fatty acids, fats and oils for which it has specific metabolic pathways. An overview of the oxidative degradation pathways for alkanes and triglycerides in Y. lipolytica is given, with new insights arising from the recent genome sequencing of this yeast. This includes the interaction of hydrophobic substrates with yeast cells, their uptake and transport, the primary alkane oxidation to the corresponding fatty alcohols and then by different enzymes to fatty acids, and the subsequent degradation in peroxisomal beta-oxidation or storage into lipid bodies. Several enzymes involved in hydrophobic substrate utilisation belong to multigene families, such as lipases/esterases (LIP genes), cytochromes P450 (ALK genes) and peroxisomal acyl-CoA oxidases (POX genes). Examples are presented demonstrating that wild-type and genetically engineered strains of Y. lipolytica can be used for alkane and fatty-acid bioconversion, such as aroma production, for production of SCP and SCO, for citric acid production, in bioremediation, in fine chemistry, for steroid biotransformation, and in food industry. These examples demonstrate distinct advantages of Y. lipolytica for their use in bioconversion reactions of biotechnologically interesting hydrophobic substrates.

Pelvic magnetic resonance imaging after bone marrow harvest--a retrospective study in 50 unrelated marrow donors.

A total of 50 unrelated marrow donors were examined by pelvic magnetic resonance imaging (MRI) to investigate the morphological sequelae of bone marrow harvesting (BMH). Signal increase in T2-weighted sequences and contrast media enhancement in T1 sequences at the operative sites were found as typical MRI morphology 4 weeks after harvest (group A, n=16), corresponding to edema, hyperemia and proliferative activity. Although tissue repair was completed in the majority of donors 1 year after BMH, about 36% of donors in group B (n=16) had abnormal findings. These included a persistence of the 'acute injury' signal pattern (2/16, 12%), and signal alterations due to fatty marrow conversion (4/16, 24%). The proportion of MRI abnormalities increased to over 70% in two-time donors (group C, n=11), which might indicate a cumulation of tissue damage after repetitive harvests. If donors had experienced prolonged discomfort after BMH (group D, n=7), MRI revealed pathological signals in 86%. In conclusion, the MRI morphology reflects the pathophysiological reactions after BMH, including inflammation and tissue repair. A further prospective evaluation in a larger number of donors is necessary to confirm these results and to identify the factors which influence the extent and duration of tissue damage.

[Oxidation of 17alpha,20beta- and 17alpha,20alpha-dihydroxysipregn-4-en-ones--side products of biotransformation of progesterone by recombinant microorganisms expressing cytochrome P45017alpha]. / Okislenie 17alpha,20beta- i 17alpha,20alpha-digidroksipregn-4-en-3-onov--pobochnykh produktov biotransformatsii progesterona rekombinantnymi mikroorganizmami, ékspressiruiushchimi tsitokhorm P45017alpha.

Progesterone biotransformation with recombinant yeast Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 alpha expressing bovine adrenocortical cytochrome P45017 alpha yielded 17 alpha-hydroxyprogesterone and two diols, 17 alpha, 20 beta- and 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17-20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20 beta-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17 alpha-hydroxylation and 20 alpha- and 20 beta-reduction. The results widen the possibilities for enzymatic and chemical modifications of steroids. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017alpha.

The cDNA encoding cytochrome P-45017alpha from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactose-induced yeast cells showed expression of about 240 nmol of P-45017alpha per liter of the culture. Binding of progesterone to the cytochrome P-45017alpha was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17alpha-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017alpha and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17alpha-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017alpha-dependent hydroxylation reaction. The reduction of the 20-keto group of 17alpha-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017alpha expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11beta- > 11alpha- > 19-hydroxyprogesterone. In conclusion, the present results show that the host-vector system used is suitable for high-level functional expression of P-45017alpha and further application of enzymatic properties of this protein to perform specific steroid biotransformations.

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.

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.

[The Dresden Cord Blood Bank. Experiences of the Cord Blood Bank in Dresden, promoted by the German bone Marrow Donor Registry]. / Dresdner Nabelschnurblutbank. Erfahrungen der Nabelschnurblutbank in Dresden, unterstützt durch die Deutsche Knochenmarkspenderdatei.

BACKGROUND: Allogeneic bone marrow and peripheral blood stem cell transplantation is the treatment of choice for a number of malignant hematological diseases, marrow failure syndromes and severe congenital immunodeficiency states. As a new, valuable source of hematopoietic stem cells, cord blood has become increasingly attractive to the medical community. More than 1500 related and unrelated cord blood transplantations have already been performed worldwide. Cord blood can be a particularly good alternative source of stem cells for pediatric patients, if no HLA-identical donor can be found. MATERIAL AND METHODS: In August 1997 the Cord Blood Bank at the University Hospital of Dresden initiated the collection, processing and cryopreservation of placental blood. This Cord Blood bank is promoted by the German bone marrow donor registry DKMS in Tübingen/Germany collaborating with 8 gynecological clinics in Dresden, Bautzen and Erlabrunn. Before cryopreservation, volume reduction of cord blood units is routinely performed by centrifugation and by separation of the buffy coat. RESULTS: As of March 2000, more than 2200 cord blood units have been collected. 60% of the samples had to be discarded because of insufficient quality (low volume and/or cell count, bacterial contamination, positive infectious disease markers). However, more than 800 cord blood units met all quality control criteria and were cryopreserved. CONCLUSION: These data from the Cord Blood Bank at the University Hospital of Dresden are comparable with results from other cord blood banks. Efforts directed toward the cryopreservation and banking of increased numbers of cord blood units are being continued worldwide and should be supported by the general public.

Experiences of the Dresdner Cord Blood Bank, supported by the Deutsche Knochenmarkspenderdatei.

Allogeneic bone marrow and peripheral blood stem cell transplantation is the treatment of choice for some malignant hematologic diseases, marrow failure syndromes, and severe congenital immunodeficiency states. Since Gluckman et al reported in 1988 the first successful human leukocyte antigen (HLA)-matched sibling umbilical cord blood stem cell transplantation, it has been known that cord blood is a valuable source of hematopoietic stem cells. The Cord Blood Bank at the University Hospital of Dresden was founded in 1997 and started collecting, processing, and cryoconserving umbilical cord blood in August 1997. The cord blood bank is supported by the largest German donor registry: Deutsche Knochenmarkspenderdatei (DKMS) in Tubingen, Germany. With the informed consent of the mothers, the collection is performed in collaboration with six hospitals in Dresden, Berlin, and Bautzen. We routinely perform a volume reduction by centrifuging the blood bag and expressing the leukocyte-rich supernatant. Routinely, sterility, total nucleated cells (TNC), CD34+ cell count, HLA class I and II, ABO/Rh blood group, and colony-forming units are evaluated. The maternal blood is screened for anti-immunodeficiency virus (anti-HIV), anti-hepatitis C virus (anti-HCV), anti-hepatitis B surface antigen (HBsAg), anti-hepatitis B surface (anti-HBs), anti-hepatitis B core (anti-HBc), anticytomegalovirus (anti-CMV), and toxoplasmosis and with Treponema pallidum hemagglutination assay (TPHA). More than 1,000 cord blood units could be collected. Because of the required volume and cell count and because of sterility, 50% of the collected units had to be discharged. Our results are comparable with data of other cord blood banks: mean volume 79 mL; cell count after volume reduction-TNC, 7.16 x 10(8); mononucleated cells (MNC), 3.75 x 10(8); CD34+ cells, 1.95 x 10(6); colony-forming units (CFU), 67.1 x 10(4). To increase the pool of potential umbilical cord blood units and in order to evaluate the possibility for unrelated transplants, cryopreservation and banking of large numbers of cord bloods are necessary.

Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum.

cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2. The amino acid sequences deduced were about 55% identical. Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences. A massive proliferation of the endoplasmic reticulum was observed in the S. cerevisiae cells which produced P-450. Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm. As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.

Molecular cloning and characterization of the primary structure of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa.

A cDNA library was established starting from poly(A) RNA of n-alkane-grown Candida maltosa cells and cDNA clones were isolated containing the entire coding sequence for the alkane hydroxylating cytochrome P-450. The deduced protein consists of 521 amino acids, contains two putative transmembrane segments in the N-terminal region and has a characteristic heme-binding sequence in the C-terminal part. Sequence alignments with members of 11 reported cytochrome P-450 families revealed a strong homology to an alkane-inducible cytochrome P-450 from Candida tropicalis.

Oxygen limitation induces indirectly the synthesis of cytochrome P-450 mRNA in alkane-growing Candida maltosa.

Candida maltosa cells grown on hexadecane under oxygen limitation have an up to 6-fold higher cytochrome P-450 content in comparison to cells cultivated at oxygen saturation. We show by mRNA quantification using an in vitro translation system and subsequent specific immunoprecipitation that the cytochrome P-450 induction occurs mainly on the transcriptional level. The signal for induction may be the enhanced intracellular hexadecane concentration owing to a reduced hydroxylation capacity of the cytochrome P-450 at oxygen limitation.

The induction of cytochrome P-450 in Lodderomyces elongisporus.

In the alkane-utilizing yeast strain Lodderomyces elongisporus cytochrome P-450 is induced by aliphatic hydrocarbons and to a lesser degree also by some of their derivatives. Cycloheximide and glucose inhibit the induction process, the former by inhibition of cytoplasmic translation, the latter presumably by catabolite repression. Among the nearly 40 checked compounds tetradecane and 1-tetradecene are the most effective inducers. The branching of the alkyl chain as well as the terminal introduction of electrophilic atoms decrease the induction effect.

[Cytochrome P-450 content in yeast cells during growth on hexadecane]. / Soderzhanie tsitokhroma P-450 v kletkakh drozhzhei pri roste na geksadekane.

The content of cytochrome P-450 was studied in the cells of alkane oxidizing yeasts Candida guilliermondii, C. tropicalis and C. lipolytica. The cells of all the studied yeast strains growing on hexadecane were found to contain cytochrome P-450. The cytochrome was not detected when the yeast strains grew on glucose. The concentration of cytochrome P-450 remained constant at the exponential growth phase, but decreased at the beginning of the stationary growth phase. Cytochrome P-450 was shown to be synthesized de novo in the course of physiological adaptation of the cells to hexadecane.

[Cytochrome P-450 induction during yeast organism growth on various substrates]. / Induktsiia tsitokhroma P-450 pri roste drozhzhevykh organizmov na razlichnykh substratakh.

The induction of cytochrome P-450 was studied in Torulopsis candida and other yeast species in the process of their growth on glucose, hexadecane, ethanol, hexadecanol and palmitate. Cytochrome P-450 was induced in all of the studied alkane oxidizing yeast strains during their growth on hexadecane or on the intermediates of its oxidation, viz. hexadecanol and palmitate. However, the maximal content of P-450 in the cells was 3-4 times lower during the growth on hexadecanol and palmitate as compared to the growth on hexadecane. In Candida utilis incapable of growing on n-alkanes but growing on hexadecanol and palmitate, cytochrome P-450 was not detected upon the yeast growth on these substrates. Cytochrome P-450 was not induced in any of the strains during their growth on ethanol. In the course of growth on glucose, cytochrome P-450 was found only in T. candida; it was induced in its cells just prior to the stationary phase caused by the exhaustion of the carbon source.