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Background: Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome. Methodolgy/Principal Findings: This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogonous. Genetic markers were identified that could discriminate between the two clades and cluster 1. Conclusions/Significance: Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here. © 2008 Paauw et al. Chemicals / CAS: Bacterial Proteins; DNA, Bacterial

A total of 20 Bifidobacterium strains were isolated from fecal samples of 4 breast- and bottle-fed infants and all were characterized as Bifidobacterium breve based on 16S rRNA gene sequence and metabolic analysis. These isolates were further characterized and compared to the type strains of B. breve and 7 other Bifidobacterium spp. by comparative genome hybridization. For this purpose, we constructed and used a DNA-based microarray containing over 2000 randomly cloned DNA fragments from B. breve type strain LMG13208. This molecular analysis revealed a high degree of genomic variation between the isolated strains and allowed the vast majority to be grouped into 4 clusters. One cluster contained a single isolate that was virtually indistinguishable from the B. breve type strain. The 3 other clusters included 19 B. breve strains that differed considerably from all type strains. Remarkably, each of the 4 clusters included strains that were isolated from a single infant, indicating that a niche adaptation may contribute to variation within the B. breve species. Based on genomic hybridization data, the new B. breve isolates were estimated to contain approximately 60-90% of the genes of the B. breve type strain, attesting to the existence of various subspecies within the species B. breve. Further bioinformatic analysis identified several hundred diagnostic clones specific to the genomic clustering of the B. breve isolates. Molecular analysis of representatives of these revealed that annotated genes from the conserved B. breve core encoded mainly housekeeping functions, while the strain-specific genes were predicted to code for functions related to life style, such as carbohydrate metabolism and transport. This is compatible with genetic adaptation of the strains to their niche, a combination of infants and diet. © 2011 Institut Pasteur.

Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a wheat-based liquid medium. Hybridization of A. oryzae RNAs to a macroarray bearing cDNAs isolated from a library representing at least 16% of the total number of A. niger genes identified 14 differentially expressed cDNA clones, showing that heterologous macroarray analysis with an A. niger cDNA library can be used to identify regulated gene transcripts in the related species A. oryzae. Moreover, Northern analysis with a selection of eight probes for A. niger genes encoding proteins involved in morphological development and cell wall biosynthesis identified five more differentially expressed genes. A suppression subtractive hybridization procedure revealed another 12 differentially expressed genes. The results presented show that, of the 29 identified genes which are expressed at higher levels during growth on wheat kernels, six encode proteins that are functionally related to polarized growth, four encode products known to be involved in morphogenesis, three code for proteins related to cell wall composition, and nine of the cDNA clones encode novel proteins. These findings pinpoint genes associated with the changes in cellular morphogenesis seen in A. oryzae grown on wheat kernels as opposed to wheat-based liquid medium. © Springer-Verlag 2005.

The rapid identification of spoilage microorganisms is of eminent importance to the food industry. It provides the food industry with the opportunity to reduce economical losses by designing adequate intervention measures. The use of identification systems based on biochemical and physiological characteristics resulted often in disappointing identification results and misidentifications. This will inevitably lead to inappropriate strategies to prevent spoilage. This review discusses the potential of the DNA based identification technology including the polymerase chain reaction (PCR) for the identification and specific detection of microorganisms. Fingerprinting methods based on the DNA-probe technology enable a clear insight in the identity of microorganisms on different levels, varying from genus to strain level depending on the systems used. Discrimination between subspecies and strain level is shown to be helpful for investigating routes and sources of contamination. Differentiation at the species level is demonstrated to be essential in order to design a highly specific detection system enabling to signalize a microorganism that belongs to a particular species. Also indicated in this review is the necessity and the technical approach to detect microorganisms that display a particular undesirable trait.

A genomic DNA-based microarray was constructed containing over 6000 randomly cloned genomic fragments of approximately 1-2 kb from six mammalian intestinal Bifidobacterium spp. including B. adolescentis, B. animalis, B. bifidum, B. catenulatum, B. longum and B. pseudolongum. This Bifidobacterium Mixed-Species (BMS) microarray was used to differentiate between type strains and isolates belonging to a set of nine Bifidobacterium spp. Hierarchical clustering of genomic hybridization data confirmed the grouping of the Bifidobacterium spp. according to the 16S rRNA-based phylogenetic clusters. In addition, these genomic hybridization experiments revealed high homology between the type-strain B. animalis subsp. lactis LMG18314 and B. animalis subsp. animalis LMG10508 (79%) as well as between the type strains B. longum biotype longum LMG13197 and B. longum biotype infantis LMG8811 (72%) - nevertheless, discrimination between these species was possible due to the high resolution output of the BMS-array. In addition, it was shown that the BMS-array could be used for assigning unknown Bifidobacterium isolates to a species group. Finally, a set of 54 diagnostic clones for Bifidobacterium identification was selected and sequenced to advance the understanding of the species-related differences. Remarkably, a large fraction (31%) of these was predicted to encode proteins that belong to the bifidobacterial glycobiome and another 11% had functional homology with genes involved in the protection against foreign DNA. Overall, the BMS-microarray is a high-resolution diagnostic tool that is able to facilitate the detection of strain- and species-specific characteristics of bifidobacteria. © 2008 Elsevier B.V. All rights reserved.

Thermophilic Campylobacter species are the most common cause of gastroenteritis in developed countries. Campylobacter spp. are ubiquitous in nature and widespread in livestock. Infections occur sporadically, and are believed to occur through consumption of contaminated meat products and from environmental sources. DNA-based techniques are essential for tracing origins of pathogens, and thereby for control over infection. This chapter provides an overview of existing technologies for characterizing Campylobacter isolates, in comparison to the use of pan-genomic microarrays. It is clear that microarray tools allow insight in the genetic factors important for host specificity, virulence, adaptation and antibiotic resistance. ɠ2011 Woodhead Publishing Limited All rights reserved

Enterococcus faecium, an ubiquous colonizer of humans and animals, has evolved in the last 15 years from an avirulent commensal to the third most frequently isolated nosocomial pathogen among intensive care unit patients in the United States. E. faecium combines multidrug resistance with the potential of horizontal resistance gene transfer to even more pathogenic bacteria. Little is known about the evolution and virulence of E. faecium, and genomic studies are hampered by the absence of a completely annotated genome sequence. To further unravel its evolution, we used a mixed whole-genome microarray and hybridized 97 E. faecium isolates from different backgrounds (hospital outbreaks (n = 18), documented infections (n = 34) and asymptomatic carriage of hospitalized patients (n = 15), and healthy persons (n = 15) and animals (n = 21)). Supported by Bayesian posterior probabilities (PP = 1.0), a specific clade containing all outbreak-associated strains and 63% of clinical isolates was identified. Sequencing of 146 of 437 cladespecific inserts revealed mobile elements (n = 74), including insertion sequence (IS) elements (n = 42), phage genes (n = 6) and plasmid sequences (n = 26), hypothetical (n = 58) and membrane proteins (n = 10), and antibiotic resistance (n = 9) and regulatory genes (n = 11), mainly located on two contigs of the unfinished E. faecium DO genome. Split decomposition analysis, varying guanine cytosine content, and aberrant codon adaptation indices all supported acquisition of these genes through horizontal gene transfer with IS16 as the predicted most prominent insert (98% sensitive, 100% specific). These findings suggest that acquisition of IS elements has facilitated niche adaptation of a distinct E. faecium subpopulation by increasing its genome plasticity. Increased genome plasticity was supported by higher diversity indices (ratio of average genetic similarities of pulsed-field gel electrophoresis and multi locus sequence typing) for clade-specific isolates. Interestingly, the previously described multi locus sequence typing-based clonal complex 17 largely overlapped with this clade. The present data imply that the global emergence of E. faecium, as observed since 1990, represents the evolution of a subspecies with a presumably better adaptation than other E. faecium isolates to the constraints of a hospital environment. © 2007 Leavis et al.

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)+ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals. Chemicals/CAS: DNA, 9007-49-2; RNA, 63231-63-0; Amino Acids; DNA, 9007-49-2; RNA, Messenger; Serum Albumin

In the present study, the expression of the epidermal growth factor receptor (EGFR) was investigated in putative preneoplastic and neoplastic acinar cell lesions induced in the rat pancreas by azaserine, using Northern blotting, in situ hybridisation (ISH) and immunohistochemistry. EGFR protein levels were decreased in putative preneoplastic eosinophilic acinar cell lesions (atypical acinar cell nodules, AACN) in comparison with normal acinar cells of the pancreas. However, EGFR mRNA expression correlated positively with the volume of AACN in pancreatic homogenates and ISH showed equal or stronger EGFR mRNA expression in AACN than in the surrounding normal acinar cells. Neither EGFR protein nor EGFR mRNA was detected in more advanced lesions such as acinar adenocarcinomas (in situ). Moreover, EGFR protein expression showed an inverse relationship with the mitotic rate of the acinar cells. These findings suggest that down-regulation of EGFR at the protein level may abrogate negative constraints on cell growth, which may stimulate the development of putative preneoplastic AACN to more advanced lesions and, ultimately, acinar adenocarcinomas.

Monosaccharides transporters play important roles in assimilate supply for sink tissue development. In this study, a new monosaccharide transporter gene OsMST6 was identified from rice (Oryza sativa L.). The predicted OsMST6 protein shows typical features of sugar transporters and shares 79.6% identity with the rice monosaccharide transporter OsMST3. Heterologous expression in yeast (Saccharomyces cerevisiae) demonstrated that OsMST6 is a broad-spectrum monosaccharide transporter, with a K m of 266.1 μΜ for glucose. OsMST6-green fluorescent protein fusion protein is localized to the plasma membrane in plant. Semi-quantitative RT-PCR analysis exhibited that OsMST6 is expressed in all tested organs/tissues. In developing seeds, OsMST6 expression level is high at the early and middle grain filling stages and gradually declines later. Further analysis detected its expression in both maternal and filial tissues. RNA in situ hybridization analysis indicated that OsMST6 is predominantly expressed in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm of young seeds, in mesophyll cells of source leaf blades, and in pollens and the connective vein of anthers. In addition, OsMST6 expression is up-regulated by salt stress and sugars. The physiological role of OsMST6 for seed development and its roles in other sink and source tissues are discussed. © 2008 Springer-Verlag.

The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint.The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale. Copyright (C) 2000 Elsevier Science B.V.

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291T), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291T. Pseudomonas monteilii (DSM14164) clustered well with P. putida strains. © 2007 Springer-Verlag.

We investigated the lobular localization and molecular level of expression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7α-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7α-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7α-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways. Chemicals/CAS: 3 hydroxy 3 methylglutaryl coenzyme A, 1553-55-5; alanine aminotransferase, 9000-86-6, 9014-30-6; cholesterol 7alpha monooxygenase, 9037-53-0; colestipol, 25085-17-0, 37296-80-3, 50925-79-6; glutamate ammonia ligase, 9023-70-5; glyceraldehyde 3 phosphate dehydrogenase, 9001-50-7; pyruvate kinase, 9001-59-6; sterol 27 hydroxylase, 134712-57-5; Biological Markers; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Colestipol, 50925-79-6; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-

Messenger RNA levels change on a minutes scale due to both degradation and de novo transcription. Consequently, alterations in the transcript profiles that are not representative for the condition of interest are easily introduced during sample harvesting and work-up. In order to avoid these unwanted changes we have validated a - 45°C methanol-based quenching method for obtaining reliable and reproducible 'snapshot' samples of Lactobacillus plantarum cells for transcriptome analyses. Transcript profiles of cells harvested with the quenching method were compared with transcript profiles of cells that were harvested according to two different commonly applied protocols. Significant differences between the transcript profiles of cells harvested by the different methods from the same steady-state culture were observed. In total, 42 genes or operons were identified from which the transcript levels were altered when the cells were not immediately quenched upon harvesting. Among these, several have previously been associated with cold-shock response. Furthermore, the reproducibility of transcript profiles improved, as indicated by the fact that the variation in the data sets obtained from the quenched cells was smaller than in the data sets obtained from the cells that were harvested under non-quenched conditions. © 2005 Elsevier B.V. All rights reserved.

DNA fragments containing genetic information for five secretion-related small GTPases of Aspergillus niger (srgA-E) were isolated and identified as members of different Rab/Ypt subfamilies. This isolation and the search for similar sequences in fungal genomic and EST databases showed that, in contrast to Saccharomyces cerevisiae, filamentous fungi also possess homologues of mammalian Rab2 GTPases. Multiple transcripts with unusually long 5′ and 3′ untranslated regions were found for all srg genes. Their level of expression was independent of the type of carbon source used for growth. Although the transcripts of srgA and srgB were abundant to the same extent throughout the cultivation, that of the other genes peaked during the early growth phase and then declined. Two genes, srgA and srgB, were characterized further. The protein encoded by srgA exhibited relatively low identity (58%) to its closest S. cerevisiae homologue SEC4, whereas the protein encoded by srgB showed 73% identity with S. cerevisiae YPT1. In contrast to other SEC4 homologues, srgA was unable to complement an S. cerevisiae sec4 mutant, and its disruption was not lethal in A. niger. SrgA mutants displayed a twofold increase in their hyphal diameter, unusual apical branching and strongly reduced protein secretion during growth on glucose. Molecular Sequence Numbers: A44334, AJ224685, AJ278657, AJ278658, AJ278659, AJ278660, AJ278661, AJ278662, AJ404733, AJ404734, P07560, S32965, X52099, X52100, X52469, X52475, X59598, X72833, X72834, X76173, X76174, X76175, Z22220, Z98598; Chemicals/CAS: Fungal Proteins; Glucose, 50-99-7; maltodextrin, 9050-36-6; Polysaccharides; rab GTP-Binding Proteins, EC 3.6.1.-; Saccharomyces cerevisiae Proteins; SEC4 protein, S cerevisiae, EC 3.6.1.-.

Despite the fact that pigs are increasingly used in pharmacological and toxicological studies, knowledge on the enzymes which metabolize xenobiotics, in particular cytochrome P450 (CYP) enzymes, in pigs is still very limited. Primary cultures of pig hepatocytes were used to characterize CYP enzymes. The characterization was performed at the level of enzymatic activities, apoprotein and mRNA analyses. Enzyme inducers investigated were β- naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) and rifampicin (RIF). After 48 hr of BNF treatment, CYP1A protein and mRNA levels were increased, and ethoxyresorufin O-deethylation and caffeine 3-demethylation were strongly induced. PB and RIF increased the levels of CYP3A apoprotein and mRNA, whereas BNF down-regulated CYP3A and related activities. PB and RIF treatment resulted in increased ethylmorphine N-demethylation and testosterone hydroxylation, which appears to be the result of CYP3A induction. Hybridization of pig RNA with a human CYP2C9 cDNA probe showed a PB and RIF inducible CYP, which was down-regulated by BNF. Similar inducing effects were observed for tolbutamide, a marker substrate for CYP2C. DEX was not a potent inducer, although some induction of CYP3A mRNA was observed. The present results indicate the absence of CYP2B and probably CYP2D enzymes and activities in pig liver. Despite some dissimilarities, the results indicate that pigs, apart from their very human-like physiology, might represent a more appropriate model species for oxidative drug metabolism in humans than rats.

Chemicals/CAS: tryptophan, 6912-86-3, 73-22-3; RNA, Bacterial; RNA, Viral; Tryptophan, 73-22-3; Tryptophan-tRNA Ligase, EC 6.1.1.2

To improve the predictability of the zebrafish embryotoxicity test (ZET) for developmental (neuro)toxicity screening, we used a multiple-endpoints strategy, including morphology, motor activity (MA), histopathology and kinetics. The model compounds used were antiepileptic drugs (AEDs): valproic acid (VPA), carbamazepine (CBZ), ethosuximide (ETH) and levetiracetam (LEV). For VPA, histopathology was the most sensitive parameter, showing effects already at 60. μM. For CBZ, morphology and MA were the most sensitive parameters, showing effects at 180. μM. For ETH, all endpoints showed similar sensitivity (6.6. mM), whereas MA was the most sensitive parameter for LEV (40. mM). Inclusion of kinetics did not alter the absolute ranking of the compounds, but the relative potency was changed considerably. Taking all together, this demo-case study showed that inclusion of multiple-endpoints in ZET may increase the sensitivity of the assay, contribute to the elucidation of the mode of toxic action and to a better definition of the applicability domain of ZET. © 2014 Elsevier Inc. Molecular Sequence Numbers: GENBANK: NM_131146, NM_131850; Chemicals/CAS: carbamazepine, 298-46-4, 8047-84-5; ethosuximide, 77-67-8; etiracetam, 102767-28-2, 33996-58-6; valproic acid, 1069-66-5, 99-66-1 Manufacturers: Sigma Aldrich, United States

Monosaccharide transporters mediate the membrane transport of a variable range of monosaccharides, which plays a crucial role in sugar distribution throughout the plant. To investigate the significance of monosaccharide transporters for rice (Oryza sativa L.) seed development, cDNA of a new putative monosaccharide transporter gene OsMST4 was isolated. The deduced OsMST4 protein shows typical features of monosaccharide transporters, and shares high homology with other plant homologues. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that OsMST4 is a functional monosaccharide transporter capable of transporting glucose, fructose, mannose and galactose. Transcriptional analysis revealed that OsMST4 is expressed in all tested organs/tissues. In developing caryopses, its expression is high at the early and middle grain filling stages, and declines gradually to low levels after that. Further analysis revealed that it is expressed in both the maternal tissue and the filial tissue, with its highest expression in embryo. Cellular location in young caryopses through RNA in situ hybridization showed that OsMST4 mRNA mainly accumulates in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm. The expression pattern of OsMST4 was further confirmed by histochemical analysis of the OsMST4-promoter-β-glucuronidase (GUS) transgenic rice plants. These data indicate that OsMST4 is actively involved in monosaccharides supply for seed development during the course of grain filling. In addition, the cell type-specific expression patterns of OsMST4 in other sink and source tissues were also investigated, and its corresponding physiological roles were discussed. © 2007 Springer Science+Business Media B.V.