Intracerebral gadolinium deposition following blood-brain barrier disturbance in two different mouse models.

To evaluate the influence of the blood-brain barrier on neuronal gadolinium deposition in a mouse model after multiple intravenous applications of the linear contrast agent gadodiamide. The prospective study held 54 mice divided into three groups: healthy mice (A), mice with iatrogenic induced disturbance of the blood-brain barrier by glioblastoma (B) or cerebral infarction (C). In each group 9 animals received 10 iv-injections of gadodiamide (1.2 mmol/kg) every 48 h followed by plain T1-weighted brain MRI. A final MRI was performed 5 days after the last contrast injection. Remaining mice underwent MRI in the same time intervals without contrast application (control group). Signal intensities of thalamus, pallidum, pons, dentate nucleus, and globus pallidus-to-thalamus and dentate nucleus-to-pons ratios, were determined. Gadodiamide complex and total gadolinium amount were quantified after the last MR examination via LC-MS/MS and ICP-MS. Dentate nucleus-to-pons and globus pallidus-to-thalamus SI ratios showed no significant increase over time within all mice groups receiving gadodiamide, as well as compared to the control groups at last MR examination. Comparing healthy mice with group B and C after repetitive contrast administration, a significant SI increase could only be detected for glioblastoma mice in globus pallidus-to-thalamus ratio (p = 0.033), infarction mice showed no significant SI alteration. Tissue analysis revealed significantly higher gadolinium levels in glioblastoma group compared to healthy (p = 0.013) and infarction mice (p = 0.029). Multiple application of the linear contrast agent gadodiamide leads to cerebral gadolinium deposition without imaging correlate in MRI.

Alterations of oral microbiota and impact on the gut microbiome in type 1 diabetes mellitus revealed by integrated multi-omic analyses.

BACKGROUND: Alterations to the gut microbiome have been linked to multiple chronic diseases. However, the drivers of such changes remain largely unknown. The oral cavity acts as a major route of exposure to exogenous factors including pathogens, and processes therein may affect the communities in the subsequent compartments of the gastrointestinal tract. Here, we perform strain-resolved, integrated meta-genomic, transcriptomic, and proteomic analyses of paired saliva and stool samples collected from 35 individuals from eight families with multiple cases of type 1 diabetes mellitus (T1DM). RESULTS: We identified distinct oral microbiota mostly reflecting competition between streptococcal species. More specifically, we found a decreased abundance of the commensal Streptococcus salivarius in the oral cavity of T1DM individuals, which is linked to its apparent competition with the pathobiont Streptococcus mutans. The decrease in S. salivarius in the oral cavity was also associated with its decrease in the gut as well as higher abundances in facultative anaerobes including Enterobacteria. In addition, we found evidence of gut inflammation in T1DM as reflected in the expression profiles of the Enterobacteria as well as in the human gut proteome. Finally, we were able to follow transmitted strain-variants from the oral cavity to the gut at the individual omic levels, highlighting not only the transfer, but also the activity of the transmitted taxa along the gastrointestinal tract. CONCLUSIONS: Alterations of the oral microbiome in the context of T1DM impact the microbial communities in the lower gut, in particular through the reduction of "mouth-to-gut" transfer of Streptococcus salivarius. Our results indicate that the observed oral-cavity-driven gut microbiome changes may contribute towards the inflammatory processes involved in T1DM. Through the integration of multi-omic analyses, we resolve strain-variant "mouth-to-gut" transfer in a disease context. Video Abstract.

Individual Physiological Adaptations Enable Selected Bacterial Taxa To Prevail during Long-Term Incubations.

Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incubation itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were combined to evaluate bacterial communities derived from marine, mesohaline, and oligohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic carbon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the manipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter In the oligohaline enclosures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously described grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of external recalcitrant carbon appeared to be more important (hgc-I). Enclosure experiments with complex natural microbial communities are important tools to investigate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures.IMPORTANCE In microbial ecology, enclosure studies are often used to investigate the effect of single environmental factors on complex bacterial communities. However, in addition to the manipulation, unintended effects ("bottle effect") may occur due to the enclosure itself. In this study, we analyzed the bacterial communities that originated from three different salinities of the Baltic Sea, comparing their compositions and physiological activities both at the early stage and after 55 days of incubation. Our results suggested that internal carbon storage strategies impact the success of certain bacterial species, independent of the experimental manipulation. Thus, while enclosure experiments remain valid tools in environmental research, microbial community composition shifts must be critically followed. This investigation of the metaproteome during long-term batch enclosures expanded our current understanding of the so-called "bottle effect," which is well known to occur during enclosure experiments.

An Easy and Fast Protocol for Affinity Bead-Based Protein Enrichment and Storage of Proteome Samples.

Analysis of dilute protein samples is a challenging task for scientific and industrial labs all over the world. Although there are different methods available that allow for protein enrichment from various biological sources, all of them have serious limitations apart from their advantages. In order to perform highly reproducible and sensitive protein analysis of lowest concentrated samples, we optimized a method to enrich proteins on affinity beads (StrataClean) recently. This chapter describes the general protocol of this strategy, thereby discussing the power as well as the limits of this technique for qualitative and quantitative proteomic studies. Moreover, additional application and protocol variants will be discussed, expanding the number of compatible up- and downstream processing techniques compared to the originally published method. Hence, we evaluated the reduction of time for sample preparation by use of preprimed affinity beads and shorter incubation durations as well as the influence of high concentration of salts or urea in the sample buffer.

Activity control of the ClpC adaptor McsB in Bacillus subtilis.

Controlled protein degradation is an important cellular reaction for the fast and efficient adaptation of bacteria to ever-changing environmental conditions. In the low-GC, Gram-positive model organism Bacillus subtilis, the AAA+ protein ClpC requires specific adaptor proteins not only for substrate recognition but also for chaperone activity. The McsB adaptor is activated particularly during heat stress, allowing the controlled degradation of the CtsR repressor by the ClpCP protease. Here we report how the McsB adaptor becomes activated by autophosphorylation on specific arginine residues during heat stress. In nonstressed cells McsB activity is inhibited by ClpC as well as YwlE.

The bphC gene-encoded 2,3-dihydroxybiphenyl-1,2-dioxygenase is involved in complete degradation of dibenzofuran by the biphenyl-degrading bacterium Ralstonia sp. SBUG 290.

AIMS: Biphenyl-degrading bacteria are able to metabolize dibenzofuran via lateral dioxygenation and meta-cleavage of the dihydroxylated dibenzofuran produced. This degradation was considered to be incomplete because accumulation of a yellow-orange ring-cleavage product was observed. In this study, we want to characterize the 1,2-dihydroxydibenzofuran cleaving enzyme which is involved in dibenzofuran degradation in the bacterium Ralstonia sp. SBUG 290. METHODS AND RESULTS: In this strain, complete degradation of dibenzofuran was observed after cultivation on biphenyl. The enzyme shows a wide substrate utilization spectrum, including 1,2-dihydroxydibenzofuran, 2,3-dihydroxybiphenyl, 1,2-dihydroxynaphthalene, 3- and 4-methylcatechol and catechol. MALDI-TOF analysis of the protein revealed a strong homology to the bphC gene products. We therefore cloned a 3.2 kb DNA fragment containing the bphC gene of Ralstonia sp. SBUG 290. The deduced amino acid sequence of bphC is identical to that of the corresponding gene in Pseudomonas sp. KKS102. The bphC gene was expressed in Escherichia coli and the meta-fission activity was detected using either 2,3-dihydroxybiphenyl or 1,2-dihydroxydibenzofuran as substrate. CONCLUSIONS: These results demonstrate that complete degradation of dibenzofuran by biphenyl degraders can occur after initial oxidation steps catalysed by gene products encoded by the bph-operon. The ring fission of 1,2-dihydroxydibenzofuran is catalysed by BphC. Differences found in the metabolism of the ring fission product of dibenzofuran among biphenyl degrading bacteria are assumed to be caused by different substrate specificities of BphD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time that the gene products of the bph-operon are involved in the mineralization of dibenzofuran in biphenyl degrading bacteria.

Cometabolic ring fission of dibenzofuran by Gram-negative and Gram-positive biphenyl-utilizing bacteria.

Thirty-five strains of soil bacteria were grown with biphenyl (BP) and tested for their capacity to cooxidize dibenzofuran (DBF). During metabolism of DBF, the culture medium of 17 strains changed from colorless to orange, indicating a meta-cleavage pathway of DBF degradation. The ring cleavage product of these isolates was shown to be 2-hydroxy-4-(3'-oxo-3' H-benzofuran-2'-yliden)but-2-enoic acid (HOBB). The strain SBUG 271, studied in detail and identified as Rhodococcus erythropolis, degraded DBF via 1,2-dihydroxydibenzofuran. The ensuing meta-cleavage yielded HOBB and salicylic acid. In addition, the four monohydroxylated monomers of DBF and two metabolites, which were not further characterized, were detected. Thus, our results demonstrate that the metabolic mechanism involves lateral dioxygenation of DBF followed by meta-cleavage and occurs in Gram-negative as well as in Gram-positive BP-degrading bacteria.

Cometabolic degradation of dibenzofuran by biphenyl-cultivated Ralstonia sp. strain SBUG 290.

Cells of the gram-negative bacterium Ralstonia sp. strain SBUG 290 grown in the presence of biphenyl are able to cooxidize dibenzofuran which has been 1,2-hydroxylated. Meta cleavage of the 1, 2-dihydroxydibenzofuran between carbon atoms 1 and 9b produced 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid, which was degraded completely via salicylic acid. The presence of these intermediates indicates a degradation mechanism for dibenzofuran via lateral dioxygenation by Ralstonia sp. strain SBUG 290.

A mutant for the yeast scERV1 gene displays a new defect in mitochondrial morphology and distribution.

The yeast scERV1 gene is the best characterized representative of a new gene family found in different lower and higher eukaryotes. The gene product is essential for the yeast cell and has a complex influence on different aspects of mitochondrial biogenesis. The homologous mammalian ALR(Augmenter of Liver Regeneration) genes from man, mouse and rat are important at different developmental stages of the organism as, for example, in spermatogenesis and liver regeneration. In this study the influence of scERV1 on the morphology of mitochondria and its submitochondrial localization are investigated. A temperature-sensitive mutant of the gene was stained with a mitochondria-specific dye and fluorescence was inspected at the permissive and restrictive temperature. A new phenotype for morphological defects of mitochondria was identified. Already at the permissive temperature mitochondrial vesicles accumulate at defined positions in the cell. After shift to the restrictive temperature, morphological changes, and finally complete loss of mitochondrial structures, are observed. Ultrastructural studies confirm these findings and demonstrate the loss of the mitochondrial inner membrane and at the final stage a drastic reduction or complete absence of mitochondria from the cell. GFP fusion experiments with the scERV1 gene and subcellular localization by fractionation experiments identify the gene product inside mitoplasts and the cytosol. Re-investigation of the mutant phenotype demonstrates that after longer incubation of the mutant at the restrictive temperature an irreversible defect of the cells, even on glucose complete medium, is found that is in accordance with a complete loss or irreversible damage of mitochondria.

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach.

In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

Azf1p is a nuclear-localized zinc-finger protein that is preferentially expressed under non-fermentative growth conditions in Saccharomyces cerevisiae.

In previous studies the AZF1 gene has been identified as a second high-copy number suppressor for a special mutant of the gene for the mitochondrial core enzyme of RNA polymerase. The first high-copy number suppressor of this mutant turned out to be the specificity factor MTF1 for mitochondrial transcription. Up to now, the influence of AZF1 on mitochondrial transcription, its precise localization in the cell and the regulation of its expression has not been determined. The putative protein contains a long stretch of poly-asparagine amino acids and a typical zinc-finger domain for DNA binding. These characteristic structural features were used to create the abbreviation AZF1 (Asparagine-rich Zinc Finger protein). An initial computer analysis of the sequence gave no conclusive results for the presence of a mitochondrial import sequence or a typical nuclear-targeting sequence. A recent more-detailed analysis identified a possible nuclear localization signal in the middle of the protein. Disruption of the gene shows no effect on plates with glucose-rich medium or glycerol. In this report a specific polyclonal antibody against Azf1p was prepared and used in cell-fractionation experiments and in electron-microscopic studies. Both of these clearly demonstrate that the AZF1 protein is localized exclusively in the nucleus of the yeast cell. Northern analysis for the expression of the AZF1 messenger RNA under different growth conditions was therefore performed to obtain new insights into the regulation of this gene. Together with the respective protein-expression analysis these data demonstrate that Azf1p is preferentially synthezised in higher amounts under non-fermentable growth conditions. Over-expression of Azf1p in the yeast cell does not influence the expression level of the mitochondrial transcription factor Mtf1p, indicating that the influence of Azf1p on the suppression of the special mitochondrial RNA polymerase mutant is an indirect one. Subcellular investigation of the deletion mutant by electron microscopy identifies specific ultrastructural cell-division defects in comparison to the wild-type.

Oxygenation cascade in conversion of n-alkanes to alpha,omega-dioic acids catalyzed by cytochrome P450 52A3.

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1, 16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1, 16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high Vmax values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min-1, respectively), whereas the oxidation of hexadecanoic acid, 1, 16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min-1, respectively). 1-Hexadecanol was found to be the main branch point between mono- and diterminal oxidation. Taken together with data on the incorporation of 18O2-derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono- and diterminal monooxygenation reactions from n-alkanes to alpha, omega-dioic acids with high regioselectivity.

Effect of selected environmental factors on degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost.

By varying selected environmental factors, the degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost were investigated. An optimized soil moisture and enhanced bioavailability by using the nonionic surfactant Tween 80 were of great importance for the degradation rates of biaryl compounds like biphenyl and 4-chlorobiphenyl by cells of Ralstonia picketti SBUG 290 inoculated into soil. Additionally, degradation of these compounds by the investigated strain in soil was strongly dependent upon the medium of precultivation. Also the influence of temperature and soil pH-value was tested. In contrast to the used soil, the autochthonous flora of the compost seemed to have a higher physiological activity. All investigated compounds (biphenyl, 4-chlorobiphenyl and dibenzofuran) were degraded quickly in compost. Inoculation with the investigated bacterium did not enhance the degradation rates significantly.

Chromosome polymorphisms close to the cm-ADE1 locus of candida maltosa.

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succino-carboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE1; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it residues.

Molecular analysis of a leu2-mutant of Candida maltosa demonstrates the presence of multiple alleles.

Three different alleles of the beta-isopropylmalate dehydrogenase gene were cloned and sequenced from a leucine auxotrophic mutant, G587, of Candida maltosa. The cloning of functionally-intact wild-type genes from this mutant strain suggests the presence of silent gene copies. An interallelic-divergence comparison has provided evidence for new regulatory mechanisms. Sequence data and karyotype analysis argue for a highly-aneuploid genome of C. maltosa. An interpretation for the spontaneous auxotrophy-prototrophy-auxotrophy sequence of mutations in C. maltosa is suggested.

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.

Correlation of biochemical blocks and genetic lesions in leucine auxotrophic strains of the imperfect yeast Candida maltosa.

The four enzymatic steps in the conversion of alpha-ketoisovaleriate to leucine were examined in the wild type and in 13 leucine auxotrophic strains of Candida maltosa. The genetic lesions in the auxotrophs, involve at least five different loci and are correlated with three enzymatic steps. This was confirmed by gene cloning, protoplast fusion, and enzyme assays. The pathway for leucine biosynthesis in C. maltosa shows general similarity to that of other lower eukaryotes but there are individual differences in the numbers of genes responsible for single enzymatic steps. A disomic state of the chromosomes carrying genes coding for alpha-isopropylmalate synthase and beta-isopropyl-malate dehydrogenase was elucidated.

[Gene transfer using fusion of isolated cell nuclei with protoplasts of the yeast Saccharomyces cerevisiae]. / Perenos genov s pomoshch'iu sliianiia izolirovannykh kletochnykh iader s protoplastami drozhzhei Saccharomyces cerevisiae.

Hybrid clones of Saccharomyces cerevisiae with different genotypes have been obtained by polyethyleneglycol induced fusion of isolated cellular nuclei with protoplasts. The genetic instability of complete nuclei after fusion results in formation of different genotypes.