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PubMed1978

Studies on the negative circular dichroic bands around 297 nm of ribosomes from bacterial cells.

The small negative CD bands around 297 nm of isolated 30-S and 50-S ribosomal subunits were precisely measured for three bacteria, Bacillus stearothermophilus, Bacillus subtilis and Escherichia coli Q 13. The intensities of the negative CD bands of 30-S subunits were always much greater than those of 50-S subunits irrespective of the bacterial strains, which may be related to the difference in comformations of rRNAs and proteins in the complexes between these subribosomal particles. The dissociation of 70-S ribosomes into two subunits by lowering Mg2+ concentration caused evident enhancement of intensity of the 297 nm CD band, which was completely reversed by the association of the two subunits into 70-S particles. The melting profiles of CD spectra 3 B. stearothermophilus and E. coli were compared and both subunits of the former were found to be more heat stable than those of the latter. It was found that 5 M urea and 0.5% sodium dodecyl sulfate (SDS) treatment caused considerable reduction of the negative CD intensity around 297 nm of 30-S subunits but no significant change of 50-S subunits, while no significant change was observed for the CD spectra of isolated 16-S and 23-S rRNAs by the same treatment. Effects of EDTA treatment and then addition of Mg2+ on the CD spectra and fluorescence emission spectra of the subunits were also observed and the contribution by the interaction between rRNA s and proteins in ribosomes to the small negative band around 297 nm was discussed.

Bacillus subtilis↗
PubMed2006

Rapid quantitative profiling of complex microbial populations.

Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities--a 'census'--is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10,462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples--the current 'gold standard' method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.

Adult↗
PubMed2005

The 18S rDNA sequence of Synchytrium endobioticum and its utility in microarrays for the simultaneous detection of fungal and viral pathogens of potato.

Resting spores extracted from wart (Synchytrium endobioticum)-infected potato tubers were used for DNA extraction and amplification of 18S rDNA. Analysis of the cloned, sequenced fragment revealed high similarity to members of the Chytridiomycota. Using this information, specific oligonucleotide probes were designed and arrayed onto glass slides for detection of the pathogen. Viral sequence information available in the databank was retrieved, or new viral sequences were generated, and used to design probes for specific detection of important quarantine viruses of potato. To determine the sensitivity and specificity of the oligonucleotide probes, total RNA from infected plants was reverse transcribed, labelled with Cyanine 5, and hybridised with the microarray. A significant number of the oligonucleotide probes exhibited high specificity to S. endobioticum, Andean potato latent virus, Andean potato mottle virus, Potato black ringspot virus, and Potato spindle tuber viroid. Hybridisation signals of sub-arrays within slides were reproducible (r = 0.79) with a high correlation coefficient of hybridisation repetitions (0.73). Our results demonstrate the potential of microarray-based hybridisation for identification of multiple pathogen targets, which will find application in quarantine laboratories, where parallel testing for diverse pathogens is essential.

Chytridiomycota↗
PubMed1984

Physiological concentrations of GTP stimulate fusion of the endoplasmic reticulum and the nuclear envelope.

Incubation of highly purified nuclei with rough microsomes stripped of associated ribosomes and physiological concentrations of guanosine triphosphate (GTP) led to the fusion of outer membranes of nuclei with microsomes to form large irregular membrane extensions. Measurement of membrane profiles in electron micrographs revealed that the outer membranes of nuclei incubated under these conditions increased significantly in length compared with that of outer membranes of unincubated or control incubated nuclei. This morphometric assay for fusion was used to check membrane and tissue specificity. It was found that GTP did not stimulate fusion between other intracellular membranes (e.g. mitochondrial or Golgi) or between such membranes and nuclear envelopes. GTP did, however, stimulate fusion between stripped rough microsomes from rat liver and outer membranes of nuclei from rat brain. These studies have revealed that membranes of the rough endoplasmic reticulum and nuclear envelope possess unique recognition and fusion properties and as such constitute the first demonstration of membrane interaction specificity at the intracellular level.

Adenosine Triphosphate↗
PubMed2005

Development of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance.

A group-specific primer pair was designed to amplify the 16S rRNA gene of representative reference strains from environmentally sourced, mesophilic aerobic spore-forming Bacillus taxa. The PCR generated a 1114 bp amplicon but did not do so with DNA extracted from 16 other Eubacterial species. When amplicons were digested with restriction enzymes AluI or TaqI, different profiles containing between 2 and 5 fragments ranging in size from 76 to 804 base pairs were seen with different Bacillus species. This procedure, known otherwise as amplified ribosomal DNA restriction analysis or ARDRA, produced unique and distinguishable patterns to differentiate between 15 ATCC reference strains (10 Bacillus, 3 Paenibacillus and 2 Brevibacillus member species) as well as 3 misidentified Bacillus probiotic strains in a commercial collection. Our simplified PCR-ARDRA protocol provides a facile method for the identification of most environmentally important species of Bacillus.

Animals↗
PubMed2003

Microbial diversity in a thermophilic aerobic biofilm process: analysis by length heterogeneity PCR (LH-PCR).

A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site treatment of pulp and paper mill whitewater lining. The microbial diversity in this process was analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA. The primer pair selected for PCR amplification was first evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups. The fragment contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length. The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier elements was found to be diverse in both stages of the SCBP. During normal operating conditions, sequences belonging to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most prominent LH-PCR peak. Samples from the suspended biomass consisted of completely different bacterial populations, which were, however, similar in the serial reactors. The pilot process experienced alkaline shocks, after which Bacillus-like sequences were detected in both the biofilm and suspended biomass. However, when the conditions were reversed, the normal microbial population in the biofilm recovered rapidly without further biomass inoculations. This study shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater processes.

Bacteria↗
PubMed2003

Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes.

In order to determine the global effects of oncogenic Ras and Akt signaling pathways on translational efficiencies, we compared the gene expression profiles of total cellular mRNA and mRNA associated with polysomes. We found that the immediate effect of Ras and Akt signaling blockade on transcription was relatively modest; however, the profile of mRNA associated with polysomes was substantially altered. These observations indicate that the immediate effect of Ras and Akt signaling regulates the recruitment of specific mRNAs to ribosomes to a far greater extent than they regulate the production of mRNAs by transcriptional effects. The mRNAs most affected are those encoding proteins that regulate growth, transcription regulation, cell to cell interactions, and morphology. These data support a model whereby Ras and Akt signaling primarily lead to cellular transformation by altering the transcriptome and producing a radical shift in the composition of mRNAs associated with actively translating polysomes.

Animals↗
PubMed1996

Molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans Candida species.

The reported incidence of fungal infections associated with non-albicans species from the Candida genus is increasing. Most of these infections occur in immunocompromised patients, particularly those infected with HIV. The role of molecular genetic techniques alongside the existing techniques for the identification and typing of these organisms is discussed. Species-specific genomic DNA fragments cloned from C. tropicalis and C. krusei have been developed for identification and strain typing. Analysis of tRNA profiles has been shown to be effective for the identification of C. glabrata, C. guilliermondii, C. parapsilosis and C. tropicalis. A PCR method employing primers complimentary to large ribosomal subunit genes and the lanosterol-alpha-demethylase gene has been applied for several species, including C. glabrata, C. krusei and C. tropicalis. Strain typing by comparison of genomic DNA fingerprints has been demonstrated for C. tropicalis and C. krusei following hybridisation analysis with species-specific probes. Synthetic oligonucleotide probes--which do not have to be species-specific and which can detect minor polymorphisms--have also been used for strain typing of isolates of several non-albicans species. Random amplification of polymorphic DNA (RAPD) has also been used for analysis of C. glabrata, C. lusitaniae and C. tropicalis isolates. The potential for the application of these and other techniques to Candida spp. taxonomy--and the example of a recently discovered novel species, C. dubliniensis--is discussed.

Candida↗
PubMed2005

Global gene expression responses to cadmium toxicity in Escherichia coli.

Genome-wide analysis of temporal gene expression profiles in Escherichia coli following exposure to cadmium revealed a shift to anaerobic metabolism and induction of several stress response systems. Disruption in the transcription of genes encoding ribosomal proteins and zinc-binding proteins may partially explain the molecular mechanisms of cadmium toxicity.

Adaptation, Physiological↗
PubMed2002

The immunosuppressant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation.

RAFT1/FRAP/mTOR is a key regulator of cell growth and division and the mammalian target of rapamycin, an immunosuppressive and anticancer drug. Rapamycin deprivation and nutrient deprivation have similar effects on the activity of S6 kinase 1 (S6K1) and 4E-BP1, two downstream effectors of RAFT1, but the relationship between nutrient- and rapamycin-sensitive pathways is unknown. Using transcriptional profiling, we show that, in human BJAB B-lymphoma cells and murine CTLL-2 T lymphocytes, rapamycin treatment affects the expression of many genes involved in nutrient and protein metabolism. The rapamycin-induced transcriptional profile is distinct from those induced by glucose, glutamine, or leucine deprivation but is most similar to that induced by amino acid deprivation. In particular, rapamycin treatment and amino acid deprivation up-regulate genes involved in nutrient catabolism and energy production and down-regulate genes participating in lipid and nucleotide synthesis and in protein synthesis, turnover, and folding. Surprisingly, however, rapamycin had effects opposite from those of amino acid starvation on the expression of a large group of genes involved in the synthesis, transport, and use of amino acids. Supported by measurements of nutrient use, the data suggest that RAFT1 is an energy and nutrient sensor and that rapamycin mimics a signal generated by the starvation of amino acids but that the signal is unlikely to be the absence of amino acids themselves. These observations underscore the importance of metabolism in controlling lymphocyte proliferation and offer a novel explanation for immunosuppression by rapamycin.

Amino Acids↗
PubMed2003

Influence of histochemical stains on quantitative gene expression analysis after laser-assisted microdissection.

Laser-assisted microdissection (LAM) allows isolation of specific cell populations for molecular studies. The combination of LAM and of real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) enables generation of quantitative cell-specific gene expression data. Histochemical stains used to identify cells desired for LAM should provide acceptable morphology and not interfere with RNA or with subsequent molecular analysis techniques. To determine a reliable stain for analysing RNA, using the housekeeping gene, RPL13A, we performed quantitative gene expression analysis of laser microdissected cells from prostatic frozen tissues. The frozen sections were histochemically stained with hematoxylin, methyl green, toluidine blue O and May-Grunwald. After laser microdissection real-time quantitative RT-PCR was performed. Methyl green yielded more RT-PCR product than did the other dyes. The lowest yield of amplification was obtained after May-Grunwald staining. Therefore we recommend methyl green for general use in gene expression analysis, especially when handling small amounts of RNA.

Ethidium↗
PubMed1998

Streptomycin bactericidal action is dependent on polyamine endogenous levels in E. coli.

E. coli polyamine-supplemented and depleted cultures showed an important difference in survival to streptomycin; the bactericidal effect of the antibiotic was remarkably higher in cells with normal levels of polyamines. Similar results were observed with kanamycin. Analysis of the polyamine-containing cells pulse-labelled with 35S-methionine during streptomycin action indicated that the amounts of newly-synthesized peptides in various subcellular fractions was different from the amounts formed in the untreated controls; the most dramatic change was found in the residual particulate fraction where the antibiotic treatment caused a 3-fold increase of radioactive proteins. On the contrary, equivalent amounts of labelled peptides were detected in the different fractions prepared from polyamine-depleted bacteria incubated with or without antibiotic. In this case the corresponding residual fraction was only slightly increased. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the different fractions showed some changes elicited by streptomycin in the protein patterns of polyamine-containing bacteria, especially in the residual fractions. The electrophoretic profile corresponding to deprived cells was very similar in all cases. The role of polyamines in the conformation of the outer membrane and in the correct assembly of ribosomes is discussed on account of the enhancing effect of these polycations on the bactericidal action of streptomycin.

Anti-Bacterial Agents↗
PubMed1997

A low molecular weight artificial RNA of unique size with multiple probe target regions.

Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E. coli. These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring. Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs. This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence. This aRNA is 160 nucleotides in length and again accumulates to high levels in the E. coli cytoplasm without incorporating into ribosomes. The 3xPen aRNA illustrates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs. Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization. These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

Base Sequence↗
PubMed2002

Comparative analysis of multiple genome-scale data sets.

The ongoing analyses of published genome-scale data sets is evidence that different approaches are required to completely mine this data. We report the use of novel tools for both visualization and data set comparison to analyze yeast gene-expression (cell cycle and exit from stationary phase/G(0)) and protein-interaction studies. This analysis led to new insights about each data set. For example, G(1)-regulated genes are not co-regulated during exit from stationary phase, indicating that the cells are not synchronized. The tight clustering of other genes during exit from stationary-phase data set further indicates the physiological responses during G(0) exit are separable from cell-cycle events. Comparison of the two data sets showed that ribosomal-protein genes cluster tightly during exit from stationary phase, but are found in three significantly different clusters in the cell-cycle data set. Two protein-interaction data sets were also compared with the gene-expression data. Visual analysis of the complete data sets showed no clear correlation between co-expression of genes and protein interactions, in contrast to published reports examining subsets of the protein-interaction data. Neither two-hybrid study identified a large number of interactions between ribosomal proteins, consistent with recent structural data, indicating that for both data sets, the identification of false-positive interactions may be lower than previously thought.

Cell Cycle↗
PubMed2004

Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathways.

Transformation progression of epithelial cells involves alterations in their morphology, polarity, and adhesive characteristics, all of which are associated with the loss and/or reorganization of actin structures. To identify the underlying mechanism of formation of the adhesion-dependent, circumferential actin network, the expression and localization of the actin binding and regulating proteins (ABPs), vinculin, VASP, and profilin were evaluated. Experimental depolarization of epithelial cells results in the loss of normal F-actin structures and the transient upregulation of vinculin, VASP, and profilin. This response is due to the loss of cell-cell, and not cell-substrate interactions, since cells that no longer express focal adhesions or stress fibers are still sensitive to changes in adhesion and manifest this in the altered profile of expression of these ABPs. Transient upregulation is dependent upon de novo protein synthesis, and protein kinase-, but not phosphatase-sensitive signal transduction pathway(s). Inhibition of the synthesis of these proteins is accompanied by dephosphorylation of the ribosomal S6 protein, but does not involve inhibition of the PI3-kinase-Akt-mTOR pathway. Constitutive expression of VASP results in altered cell morphology and adhesion and F-actin and vinculin structures. V12rac1 expressing epithelial cells are constitutively nonadhesive, malignantly transformed, and constitutively express high levels of these ABPs, with altered subcellular localizations. Transformation suppression is accompanied by the restoration of normal levels of the three ABPs, actin structures, adhesion, and epithelial morphology. Thus, vinculin, VASP, and profilin are coordinately regulated by signal transduction pathways that effect a translational response. Additionally, their expression profile maybe indicative of the adhesion and transformation status of epithelial cells.

Actins↗
PubMed2026

Comparative toxicoproteomics reveals distinct proteostasis and ribosomal stress signatures of CMIT and PHMG.

Methylchloroisothiazolinone (CMIT) and polyhexamethylene guanidine (PHMG) are antimicrobial biocides associated with pulmonary toxicity, although their comparative cellular stress mechanisms remain unclear. Here, we investigated how CMIT and PHMG differentially alter the proteome of human alveolar epithelial A549 cells under subcytotoxic conditions. Cells were exposed to CMIT or PHMG, and global proteomic profiling was performed using label-free liquid chromatography-tandem mass spectrometry. Differentially expressed proteins (DEPs) were identified at a 1% false discovery rate with an absolute log2 fold change &#x2265;1. Functional analyses were conducted using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Ingenuity Pathway Analysis, and selected proteins were validated by western blotting. Comparative toxicoproteomics revealed distinct stress-response signatures induced by the two biocides. CMIT preferentially altered proteins associated with proteostasis, oxidative stress, and protein quality control, whereas PHMG was characterized by coordinated depletion of ribosome-associated and translation-related proteins. A total of 73 and 155 DEPs were identified in CMIT- and PHMG-treated cells, respectively, with 22 proteins shared between treatments. Western blotting confirmed PSMD3, TUBB2A, and GLRX1 as CMIT-responsive proteins and THRAP3, DHX15, and RPL4 as PHMG-responsive markers. These findings provide comparative mechanistic insight into how CMIT and PHMG induce distinct epithelial stress responses and identify candidate protein markers that may support future in vitro assessment of biocide-induced pulmonary toxicity.

CMIT↗
PubMed1987

The human muscle-tendon junction. A morphological study during normal growth and at maturity.

The myotendon junction of human paravertebral skeletal muscle was studied by light and electron microscopy. Transverse and longitudinal sections of myotendinous regions of normal multifidus muscles were examined at three chronological stages from birth to maturity. Variations in the appearance of surface extensions at the terminal ends of muscle fibers consisted of brush-like evaginations at birth and villous-like projections in the adult. Regardless of age, they were invariably covered by a prominent external lamina, and mutually interdigitated with connective-tissue elements in the adjacent tendon. Various stages of myofibrillar assembly and sarcomere alignment were evident in the muscle fiber terminus at birth. With advancing age, splitting of terminal sarcomeres at Z bands commonly gave rise to diverging myofilament bundles that attached to electron-dense patches under the sarcolemma. In these regions, leptomeric organelles were also encountered in neonatal and adolescent myotendons. At all stages, the ends of muscle fibers possessed cytological features consistent with active synthesis and secretion. Densely-packed sarcoplasmic organelles including multiple Golgi complexes, clusters of ribosomes, mitochondria, cytoplasmic vesicles, and elements of rough- and smooth-surfaced endoplasmic reticulum were prevalent. Peripheral and centrally-placed heterochromatic nuclei with prominent nucleoli were arranged singly or in groups at the ends of muscle fibers. Satellite cell profiles and unmyelinated axons in the subjacent tendon were also identified at these sites in the adult. Fibroblasts in growing tendon were plentiful, and at all stages, possessed morphological features indicative of high metabolic and secretory activities.

Adolescent↗