Effect of arsenite on swimming motility delays surface colonization in Herminiimonas arsenicoxydans.

Herminiimonas arsenicoxydans is a Gram-negative bacterium able to detoxify arsenic-contaminated environments by oxidizing arsenite [As(III)] to arsenate [As(V)] and by scavenging arsenic ions in an extracellular matrix. Its motility and colonization behaviour have been previously suggested to be influenced by arsenite. Using time-course confocal laser scanning microscopy, we investigated its biofilm development in the absence and presence of arsenite. Arsenite was shown to delay biofilm initiation in the wild-type strain; this was partly explained by its toxicity, which caused an increased growth lag time. However, this delayed adhesion step in the presence of arsenite was not observed in either a swimming motility defective fliL mutant or an arsenite oxidase defective aoxB mutant; both strains displayed the wild-type surface properties and growth capacities. We propose that during the biofilm formation process arsenite acts on swimming motility as a result of the arsenite oxidase activity, preventing the switch between planktonic and sessile lifestyles. Our study therefore highlights the existence, under arsenite exposure, of a competition between swimming motility, resulting from arsenite oxidation, and biofilm initiation.

Yeast regulatory gene PPR1. I. Nucleotide sequence, restriction map and codon usage.

The PPR1 gene of Saccharomyces cerevisiae controls the transcription of two unlinked structural genes URA1 and URA3. The primary structure of this eukaryotic regulatory gene and its flanking regions has been established by the dideoxynucleotide chain termination method. Our data show an open reading frame of 2712 nucleotides, corresponding to 904 amino acid residues. The 3' untranslated messenger RNA region presents consensus yeast termination and polyadenylation sequences. The pattern of codon usage in the gene is clearly random. This result is discussed in relation to protein abundance and is compared with the codon usage in 20 yeast structural and regulatory genes and with that found for Escherichia coli genes.

Yeast regulatory gene PPR1. II. Chromosomal localization, meiotic map, suppressibility, dominance/recessivity and dosage effect.

The Saccharomyces cerevisiae gene PPR1 encodes a positive regulator of the expression of the two unlinked structural genes URA1 and URA3. The gene has been mapped to a position 6.5 cM from the centromere of chromosome XII. Uninducible alleles have been selected and used to establish a meiotic map. Suppressible alleles have been identified. The sequencing of a suppressible allele confirms the nonsense nature of the mutation as well as the reading frame deduced from the nucleotide sequence. No evidence of intracistronic complementation was found, and enzymatic analysis of leaky mutants did not reveal any mutations dissociating regulation of URA1 from that of URA3. Three in vitro-constructed deletions of PPR1 have been integrated at the chromosomal locus, giving strains with a completely negative phenotype. These deletion mutants display the wild-type basal level of URA1 and URA3 expression and show a semi-dominant phenotype in heteroallelic ppr1+/ppr1-delta diploids. Amplifying PPR1 by introduction into yeast on a multicopy vector increases the induction factor of URA1 and URA3 expression. These results show that the extent of regulation of the two structural genes is dependent on the concentration of the active PPR1 protein.

Chromatographic, capillary electrophoretic and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of urinary modified nucleosides as tumor markers.

Modified nucleosides are formed posttranscriptionally in RNA. During RNA turnover free modified nucleosides are formed which circulate in the blood stream and are excreted in the urine. Their levels are increased in a number of malignant diseases, and they can be used in clinical chemistry as tumor markers. The analysis includes the isolation of the nucleosides from urine with phenylboronate gel and their separation and quantitation by HPLC on C18 columns or by capillary electrophoresis on uncoated columns applying a sodium dodecyl sulfate-borate-phosphate buffer. Identification of the nucleosides is performed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry including post source decay spectra. In two clinical studies the diagnostic value of urinary modified nucleosides is investigated, in a study on children with leukemia and other malignant diseases and a study on women with breast cancer. Candidate markers are pseudouridine, 1-methylguanosine, N2-methylguanosine, 3-methyluridine and 1-methyl-inosine.

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography.

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b(0) + b(1) (1/x) with the regression coefficients b(0) and b(1,) the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1-2 months, when they reach their absolute maximum for all age periods, and then decrease.

Structure and organisation of the pyrimidine biosynthesis pathway genes in Lactobacillus plantarum: a PCR strategy for sequencing without cloning.

This report describes the sequence and structural organisation of the pyrimidine biosynthesis pathway genes of Lactobacillus plantarum CCM 1904. It also describes an in vitro technique based on PCR for sequencing without cloning. This new technique was developed because it was impossible to clone certain parts of the L. plantarum genomic DNA in the Escherichia coli host. L. plantarum pyr genes are organised as a 9.8-kb operon with the following order: pyrR, pyrB, pyrC, pyrAA, pyrAB, pyrD, pyrF and pyrE. There are two major differences from the pyrimidine operons of Bacillus subtilis (Quinn et al., J. Bacteriol. 266 (1991) 9113-9127; Turner et al., J. Bacteriol, 176 (1994) 3708-3722) and Bacillus caldolyticus (Ghim et al., Microbiology 140 (1994) 479-491): the absence of pyrP encoding for uracil permease, and the absence of an open reading frame named orf2, whose function is unknown. Two mutually exclusive stem-loop structures were predicted at the 5'-end of L. plantarum pyr mRNA; this operon could be regulated by transcriptional attenuation under the control of PyrR. Complementation of E. coli pyrD, pyrF and pyrE mutants was obtained with a L. plantarum genomic DNA library. Alignment of the L. plantarum Pyr proteins with other known procaryotic Pyr proteins indicates that they display highly conserved regions in Gram-positive and Gram-negative bacteria.

Effect of hypotension on the results of kidney storage and the use of dopamine under these conditions.

The influence of the length and severity of hypotension on the results of kidney preservation was examined in dogs. Successful 24-hr hypothermic kidney storage was possible, if the donor animal was subjected to hypovolemic hypotension (mean blood pressure 60 mm Hg) for a duration 1 to 4 hr. If the blood pressure was lowered to 50 mm Hg, successful kidney preservation could not be obtained. It was concluded that the level of hypotension was of more importance than its duration. After 24 hr of cold ischemia, the function of kidneys from hypotensive donors could be improved significantly if dopamine was given to the recipient. The preservation injury itself could not be counteracted by dopamine because dopamine did not improve the function of kidneys which were removed from normotensive donors but were stored for 24 hr under hypothermia.

Determination of chromosome size and number of rrn loci in Lactobacillus plantarum by pulsed-field gel electrophoresis.

The size of the Lactobacillus plantarum CCM 1904 chromosome was determined by pulse-field gel electrophoresis. It was found to be 3.3-3.4 Mb using SfiI or AscI restriction endonucleases, compared to 3-4 Mb found for the other L. plantarum strains tested. L. plantarum CCM 1904 5S rDNA was clonedl by polymerase chain reaction, sequenced, and used as a probe to characterize strains. At least five rrn loci were found. The pulsed-field gel electrophoresis macrorestriction patterns were strain-specific, while the rDNA restriction hybridization patterns were species-specific.

Cloning and structure of the pyrE gene of Lactobacillus plantarum CCM 1904.

The pyrE gene of Lactobacillus plantarum CCM 1904, coding for the orotate phosphoribosyl transferase involved in the pyrimidine biosynthetic pathway, was cloned in Escherichia coli and sequenced. The predicted polypeptide sequence extending over 212 amino acids (MW 22,690) was compared to those of E. coli and to those of lower eukaryotes (Saccharomyces cerevisiae, Podospora anserina, Sordaria macrospora, Dictyostelium discoideum). Important conserved stretches were revealed, implying that these proteins are closely related.

In vitro metabolite identification of ML3403, a 4-pyridinylimidazole-type p38 MAP kinase inhibitor by LC-Qq-TOF-MS and LC-SPE-cryo-NMR/MS.

Prediction of the metabolic profile of a potential new drug is recommended at an early stage in industrial drug discovery process to determine whether or not any potentially reactive or toxic metabolites are formed. In the present study, we investigated the in vitro metabolism of ML3403 ({4- [5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl -(1-phenylethyl)-amine), a potent and selective p38 MAP kinase inhibitor using mouse liver microsomes. The combination of LC-ESI-Qq-TOF (tandem quadrupole time-of-flight)-MS (mass spectrometer) and LC-SPE (solid phase extraction)-cryo-NMR (nuclear magnetic resonance)/MS at 600 MHz has been applied for comprehensive and straightforward structural elucidation of ML3403 metabolites. It was possible to determine the metabolic profile of ML3403, revealing eight different metabolites formed by N-desalkylation, S-mono- and di-oxidation, aliphatic hydroxylation and pyridine-N-oxidation. The ESI-Qq-TOF-MS data yielded elemental compositions of all metabolites and their fragments by evaluation of the accurate mass and isotopic pattern information using the sigma-fit algorithm. Evaluation of 2D NMR spectra obtained from pure ML3403 an its major metabolite ML3603 allowed the unequivocal assignment of the resonances in 1D NMR spectra obtained directly from the microsomal incubation by LC-SPE-cryo-NMR/MS. The presented method significantly decreases the time required for a complete structural assignment of metabolites from microsomal in vitro assays.

Intelligence and phenylketonuria: effects of diet termination.

Trends in intellectual functioning before and after diet termination were examined in 30 children with PKU treated before 6 weeks of age and on a liberal diet for a mean of three years since the mean age of 59 months. Comparisons of trends in pre- and posttermination IQ scores with the linear spline technique revealed no significant differences. Comparisons of pretermination IQ scores with posttermination scores and scores from the most recent evaluation also yielded no significant differences. These data fail to support a relationship between termination of diet and impairment of intellectual functioning, at least during this period of observation.

Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged protein by a single metal-affinity chromatography step.

The mature part of the chloroplast triose phosphate-phosphate translocator was cloned into the yeast expression vector pEVP11. This construct was used to transform cells from both Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. The chloroplast translocator protein was functionally expressed in the transformed yeast cells and represented about 1-2% of the Sch. pombe cell membrane protein. It was localized to mitochondrial membranes and/or membranes of the rough endoplasmic reticulum. In order to purify the recombinant translocator protein, a sequence encoding a C-terminal tag of six histidine residues was introduced into the corresponding cDNA. The expressed histidine-tagged translocator protein was purified from the transformed yeast cells under nondenaturing conditions to apparent homogeneity by a single-step affinity chromatography using a Ni2+. nitrilotriacetic acid resin. Both the expressed triose phosphate translocator and the recombinant histidine-tagged protein possess substrate specificities identical to those of the authentic chloroplast protein, providing definitive evidence for its identity as the triose phosphate translocator and further disproving its assignment as the receptor for chloroplast protein import. The yeast expression system in combination with the Ni2+. nitrilotriacetic acid chromatography thus provides a valuable tool for the production of purified membrane proteins in a functional state.

Neurophysiological, psychological, and nutritional investigations during discontinuation of the phenylalanine-restricted diet in children with classic phenylketonuria.

Assessment of cortical sensory and psychological functions in children with classic phenylketonuria during the time period of diet discontinuation did not uncover any significant changes when pre-diet and post-diet termination data were compared. Analysis of relationships among the study variables found that children who had adequate dietary control and who were of average or above average intelligence displayed normal somatosensory evoked potentials (SEP) whereas those children with less adequate dietary control and lower IQ scores had atypical SEP waveforms.

Genetic polymorphisms of cytochrome P450 2C9 causing reduced phenprocoumon (S)-7-hydroxylation in vitro and in vivo.

The effect of cytochrome P450 (CYP) 2C9 polymorphisms on the stereoselective biotransformation of the oral anticoagulant phenprocoumon (PPC) to inactive, monohydroxylated metabolites was studied in vitro and in vivo. In human liver microsomes, the (S)-7-hydroxylation--being the major metabolic pathway--was significantly compromised in a gene-dose-dependent manner in samples expressing the CYP2C9*2 or CYP2C9*3 allele. The CYP2C9*3/*3 genotype corresponded to an almost fourfold lower (S)-7-hydroxylation rate than CYP2C9*1/*1 (wild-type). The intrinsic clearance of human recombinant CYP2C9*2 and CYP2C9*3 for the (S)-7-hydroxylation was 28.9 and 50.9% lower than of CYP2C9*1, respectively. The area under the plasma concentration-time curve (AUC) of PPC metabolites after oral intake of 12 mg racemic PPC was significantly lower in volunteers expressing the CYP2C9*2 or CYP2C9*3 allele. Increasing plasma AUC metabolic ratios (parent compound/metabolite) in CYP2C9*2 and CYP2C9*3 variant allele carriers were found for each hydroxylation reaction and the CYP2C9*3/*3 genotype corresponded to an about 10-fold higher metabolic ratio of PPC (S)-7-hydroxylation relative to CYP2C9*1/*1. CYP2C9 polymorphisms cause a markedly compromised PPC (S)-7-hydroxylation. However, PPC metabolism appears overall less influenced by CYP2C9 genotype compared with other oral anticoagulants and it may thus be a valuable alternative for therapeutic anticoagulation of patients expressing CYP2C9 variant alleles.

Complete sequence of a eukaryotic regulatory gene.

Dihydroorotase, the third enzymatic activity of the pyrimidine pathway, is encoded in Saccharomyces cerevisiae by a single gene URA4, which is induced at the transcriptional level by accumulation of ureidosuccinic acid. A regulatory gene PPR2 (pyrimidine pathway regulatory 2) acting specifically on this step, has been characterized, cloned and sequenced. The main open reading frame is 384 nucleotides long and potentially codes for a basic protein, favoring a molecular mechanism involving direct binding of a regulatory protein to DNA. The short length of the PPR2 polypeptide chain and the presence of seven cysteine residues suggest that the active form of the protein is an oligomer assembled through disulphide bonds. An uninducible allele has been cloned and sequenced. The mutation corresponds to an A leads to T transversion changing a lysine triplet into an ochre codon. The uninducible phenotype of this mutant is completely suppressed by an ochre suppressor, strengthening the hypothesis that PPR2 acts on URA4 transcription through the synthesis of a regulatory protein.

Molecular characterization of a carbon transporter in plastids from heterotrophic tissues: the glucose 6-phosphate/phosphate antiporter.

Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only approximately 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates.

Levels of transforming growth factor beta and transforming growth factor beta receptors in rat liver during growth, regression by apoptosis and neoplasia.

Transforming growth factor beta1 (TGF-beta1) has been implicated as inhibitor of cell proliferation and a potent inducer of apoptosis in vitro and in vivo after the administration of high doses. To assess the role of endogenous TGF-beta1, we quantitated the cytokine and its receptors in rat liver during regenerative and hyperplastic growth, regression by apoptosis, and in hepatocellular carcinoma (HCC). This was accomplished by Northern blot analysis and by RNase protection assay of the messenger RNA (mRNA) of TGF-beta1 and TGF-beta receptors (TbetaR) types I to III and by an activity bioassay of the TGF-beta proteins. Untreated rat livers were found to contain 15.6 +/- 4.8 ng TGF-beta1 protein/g tissue; TGF-beta2 protein was not detected. To induce toxic cell death and subsequent regenerative DNA synthesis in the liver, rats were treated with a necrogenic dose of carbon tetrachloride (CCl4). After 24 and 48 hours, there was an upregulation of TGF-beta1 (mRNA, up to tenfold; protein, about twofold) and of TbetaRs (mRNA: two- to fourfold); that indicates an overall enhanced production of and sensitivity to TGF-beta1, which may serve to confine the regenerative response. Hyperplastic liver growth and regression of the hyperplasia were induced by treatment with cyproterone acetate (CPA) or nafenopin (NAF) followed by withdrawal; neither mRNAs of TGF-beta1 and TbetaR types I to III nor TGF-beta1 protein exhibited significant changes during the growth phase or during regression by apoptosis. We also studied neoplastic growth. HCC, obtained after long-term treatment with NAF, exhibited high rates of cell replication and apoptosis. The majority of lesions contained mRNA and protein of TGF-beta1 and mRNA of TbetaR types I to III at concentrations similar to those of the surrounding tissue. In conclusion, during liver regeneration there is a pronounced upregulation of expression of both TGF-beta1 and TbetaRs I to III, but not during mitogen-induced liver growth or regression. It appears that apoptosis is induced via altered local concentration of TGF-beta1, in a paracrine and/or autocrine way. By this mechanism the lethal effects of TGF-beta1 may be locally confined, and overshoots of apoptosis in the liver may be prevented.

A new class of plastidic phosphate translocators: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/phosphate antiporter.

We have purified a plastidic phosphate transport protein from maize endosperm membranes and cloned and sequenced the corresponding cDNAs from maize endosperm, maize roots, cauliflower buds, tobacco leaves, and Arabidopsis leaves. All of these cDNAs exhibit high homology to each other but only approximately 30% identity to the known chloroplast triose phosphate/phosphate translocators. The corresponding genes are expressed in both photosynthetically active tissues and in nongreen tissues, although transcripts were more abundant in nongreen tissues. Expression of the coding region in transformed yeast cells and subsequent transport measurements of the purified recombinant translocator showed that the protein mediates transport of inorganic phosphate in exchange with C3 compounds phosphorylated at C-atom 2, particularly phosphoenolpyruvate, which is required inside the plastids for the synthesis of, for example, aromatic amino acids. This plastidic phosphate transporter is thus different in structure and function from the known triose phosphate/phosphate translocator. We propose that plastids contain various phosphate translocators with overlapping substrate specificities to ensure an efficient supply of plastids with a single substrate, even in the presence of other phosphorylated metabolites.

Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa.

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.