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PubMed2004

Genotypic diversity of oral Actinomyces naeslundii genospecies 1 and 2 in caries-active preschool children.

A total of 991 isolates of Actinomyces naeslundii were obtained from sound approximal tooth sites in either caries-active (n = 35) or caries-free (n = 20) preschool children. From this group of isolates, 101 strains were chosen to study the genotypic diversity of A. naeslundii genospecies 1 (n = 30), catalase-positive (n = 30), and catalase-negative genospecies 2 (n = 41). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), with a pair of primers targeting the 16S ribosome RNA gene (16S rDNA), and MnlI digestion together with randomly amplified polymorphic DNA (RAPD) with eight arbitrary, single 10-mer primers were performed to generate genetic profiles of selected Actinomyces isolates. The hierarchic relationships of genetic profiles were finally analyzed using computerized dendrograms. There was no significant difference in the prevalence rates and proportions of either genospecies 1 or 2 between the caries-free and caries-active groups, although a higher prevalence of genospecies 2 was noted in the total population. Dendrogram analyses of the 16S rDNA PCR-RFLP profiles revealed that all strains belonging to A. naeslundii genospecies 1 could be subgrouped into three genotypes (T7, T18, and T19), with a single predominant genotype, T18 (27/30). Catalase-positive strains for genospecies 2 fell into three subtypes (T4, T7, and T17), whereas the catalase-negative counterparts were distributed amongst 16 subtypes. No specific genotype was significantly associated with caries activity. We conclude that heterogeneous subgroups of A. naeslundii genospecies 1 and 2, particularly the latter, are the constituent flora of dental plaque in children and may contribute to the pathogenesis of childhood caries.

Actinomyces↗
PubMed2004

In vivo molecular pharmacology and antitumor activity of the targeted Akt inhibitor PX-316.

Akt, a serine/threonine kinase that promotes cell survival, is activated by binding of its pleckstrin homology (PH) domain to membrane phosphatidylinositol (PtdIns)-3-phosphates formed by PtdIns-3-kinase. D-3-Deoxy-phosphatidyl-myo-inositols that cannot be phosphorylated on the 3-position of the myo-inositol group are inhibitors of the Akt PH domain. The most active compound is D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (PX-316). PX-316 administered intraperitoneally to mice at 150 mg/kg inhibits Akt activation in HT-29 human tumor xenografts up to 78% at 10 h with recovery to 34% at 48 h. Phosphorylation of GSK-3beta, a downstream target of Akt, is also inhibited. There is no decrease in PtdIns(3,4,5)-trisphosphate levels by PX-316, showing it is not an inhibitor of PtdIns-3-K in vivo. Gene expression profiling of HT-29 tumor xenografts shows many similarities between the effects of PX-316 and the PtdIns-3-K inhibitor wortmannin, with downregulation of several ribosomal-related genes, while PX-316 uniquely increases the expression of a group of mitochondrial-related genes. PX-316 has antitumor activity against early human MCF-7 breast cancer and HT-29 colon cancer xenografts in mice. PX-316 formulated in 20% hydroxypropyl-beta-cyclodextrin for intravenous administration is well tolerated in mice and rats with no hemolysis and no hematological toxicity. Thus, PX-316 is the lead compound of a new class of potential agents that inhibit Akt survival signaling.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1988

The effects of human cytomegalovirus infection on cytoskeleton-associated polysomes.

Human cytomegalovirus (HCMV) infection induces disruption of the host cell's cytoskeleton (CSK). This disruption is accompanied by three transient phases of actin depolymerization that occur at 20 min, 5 to 10 h and 48 to 72 h post infection (pi). During the 20 min peak of actin depolymerization, the level of cellular polysomes associated with the CSK was reduced, due to release of ribosomes from CSK-associated polysomes. Cellular mRNAs previously existing in these polysomes, however, remained associated with the CSK. Also during this period, nuclear to cytoplasmic transport of host cellular mRNA as well as the association of newly synthesized mRNA with the CSK was temporarily delayed. By 60 min pi, ribosomes, preexisting host cellular mRNA, and newly synthesized mRNAs (host and viral) had reestablished a distribution in the infected cell comparable to that of uninfected cells. Sedimentation profiles of soluble and CSK fractions at various times throughout the viral infection indicated that, although the amount of polysomes associated with the CSK at 20 min pi was reduced, essentially all HCMV and all host cell polysomes present were associated with the CSK. The majority of HCMV DNA hybridizable poly(A)+ RNAs were associated with the CSK throughout the viral infection. These early events appear to correlate with a transient interruption of host cellular mRNA translation early in infection and may represent a process whereby HCMV gene expression becomes competitive with that of the host cell.

Cell Fractionation↗
PubMed2002

Prevalence and characterization of macrolide resistance in clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes from North America.

Resistance to macrolides is not a new phenomenon but it deserves attention because of the widespread use of these agents and their inclusion in many clinical guidelines for respiratory tract infections. The most common mechanisms by which Streptococcus pneumoniae and Streptococcus pyogenes develop resistance to macrolides is by target site modification (erythromycin ribosome methylase, erm) and efflux of the drug out of the organisms (macrolide efflux, mef). Target site modification may be of greater concern because it confers high-level resistance to all antimicrobials in the macrolide-lincosamide-streptograminB (MLSB) group. The genotype profiles of macrolide-resistant S. pneumoniae and S. pyogenes differ somewhat across regions in the US and between the US and Canada and other countries. There is some evidence for an association between macrolide resistance and treatment failure but this must be researched more fully. S. pneumoniae and S. pyogenes isolates resistant to macrolides are generally susceptible to ketolide antimicrobials because these agents bind more strongly to the relevant domain of the ribosomal subunit (withstanding erm resistance) and are less vulnerable to efflux compared to the macrolides.

Anti-Bacterial Agents↗
PubMed2005

Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing.

It is well recognized that a dynamic biofilm develops upon plant biomass in the herbivore gastrointestinal tract, but this component of the microbiome has not previously been specifically sampled, or directly compared with the biodiversity present in the planktonic fraction of digesta. In this study, the digesta collected from four sheep fed two different diets was separated into three fractions: the planktonic phase, and the microbial populations either weakly or tightly adherent to plant biomass. The community DNA prepared from each fraction was then subjected to both ribosomal intergenic spacer analysis (RISA) and denaturing gradient gel electrophoresis (DGGE). Both types of analysis showed that dietary factors influence community structure, and that the adherent fractions produced more complex profiles. The RIS-clone libraries prepared from the planktonic and adherent populations were then subjected to restriction fragment length polymorphism (RFLP) and DNA sequence analyses, which resulted in a far greater degree of discrimination among the fractions. Although many of the sequenced clones from the adherent populations were assigned to various clusters within the low G+C Gram-positive bacteria, the clone libraries from animals consuming an all-grass diet were largely comprised of novel lineages of Clostridium, while in animals consuming the starch-containing diet, Selenomonas and Ruminococcus spp. were the dominant low G+C Gram-positive bacteria. Additionally, the libraries from hay-fed animals also contained clones most similar to asaccharolytic Clostridia, and other Gram-positive bacteria that specialize in the transformation of plant phenolic compounds and the formation of cinnamic, phenylacetic and phenylpropionic acids. These results reveal, for the first time, the phylogeny of adherent subpopulations that specialize in the transformation of plant lignins and other secondary compounds, which potentiate polysaccharide hydrolysis by other members of the biofilm.

Animals↗
PubMed2004

Identification of Carnobacterium species by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region and species-specific PCR.

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA(Ala) and tRNA(Ile), which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.

Animals↗
PubMed1985

Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms.

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.

Electrophoresis, Polyacrylamide Gel↗
PubMed2003

Adaptation to nickel spiking of bacterial communities in neocaledonian soils.

Adaptation to nickel of bacterial communities of two extreme neocaledonian soils (an ultramafic soil and an acidic soil) was investigated by nickel spiking and compared with adaptation in a non-neocaledonian soil used as reference. Soil microcosms were amended with nickel chloride (NiCl2), and bacterial community structure was analysed with the ribosomal intergenic spacer analysis (RISA) technique. Then, bacterial populations that respond to nickel stress were identified by cloning and sequencing. In the ultramafic soil, a shift occurred on day zero on the assay profiles and consisted of the emergence of a bacterial group closely related to the Ralstonia/Oxalobacter/Burkholderia group. It is hypothesized that NiCl2 had a physico-chemical impact on soil structure. Fourteen days after nickel spiking, another shift occurred in the two soils that concerned a bacterial group belonging to the Actinomycete group. Only a few changes occurred in the bacterial community structure of the neocaledonian soils compared with those of the reference soil, which is more affected by nickel spiking. These results suggest that neocaledonian soil bacteria are particularly well adapted to nickel.

Actinomycetales↗
PubMed1997

PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level.

The genus Acinetobacter is phenotypically rather homogeneous, but genotypically heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplified large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypically related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.

Acinetobacter↗
PubMed1995

Molecular characterization and identification of Saprolegnia by restriction analysis of genes coding for ribosomal RNA.

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species of Saprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except for S. hypogyna, S. delica, S. lapponica, and S. mixta. The rDNA data indicate that S. lapponica and S. lapponica and S. mixta are conspecific with S. ferax, whereas there is no support for the proposed synonymies of S. diclina with S. delica and of S. mixta with S. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in the S. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains of S. parasitica originating from the U.K. and Japan. HhaI and BsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonuclease BstUI was diagnostic for S. parasitica, generating identical fingerprints for all stains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved in S. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed that S. diclina and S. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification of Saprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.

Base Sequence↗
PubMed1998

Interaction of the 530 ribosomal site with regions of mRNA.

We evaluated the energetic stability of all possible 10-base fragments of the small subunit (SS) rRNA with a large ensemble of mRNAs. Results show that the 530 loop is the only conserved region that interacts favorably around the start codon as well as the elongation region of messengers. The interaction profile of the 530 loop around the stop codon showed that base pairing was energetically unfavorable in that region. With these results we constructed a simple scheme for the role of rRNA-mRNA interactions in the protein translation mechanism.

Base Sequence↗
PubMed1996

Pseudomonas aeruginosa ribotyping: stability and interpretation of ribosomal operon restriction patterns.

Fifty-one Pseudomonas aeruginosa isolates were differentiated into 21 types by ribotyping. Several enzyme combinations, including the best ones proposed in literature, were utilized and the highest discrimination was reached by individual digestion with PvuII, HindII, and EcoRI or BamHI. Clinical isolates from outbreaks were clonally related as identified by this molecular approach. Restriction rDNA profiles were composed of strong and weak bands. Using 6 micrograms DNA we were able to demonstrate that PvuII, HindIII, and BamHI weak bands were reproducible. These weak bands should be considered not only to accomplish the highest discrimination but also to correctly assign isolate clonality. Conversely, we found that EcoRI weak bands were not reproducible and, therefore, are not recommended for ribotype analysis. Finally, profiles differing in one single band actually represented isolates of different genotype, as confirmed by further analysis using other molecular methods. In this report on P. Aeruginosa ribotyping of clinical isolates, criteria for band pattern interpretation are established.

Bacterial Typing Techniques↗
PubMed2001

Single-strand conformation polymorphism analysis of genetic variation in Labiostrongylus longispicularis from kangaroos.

Single-strand conformation polymorphism (SSCP) analysis was employed to screen for sequence heterogeneity in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA of Labiostrongylus longispicularis, a parasitic strongylid nematode occuring in some species of kangaroo in different geographical regions of Australia. The results showed that most of the nematodes screened had different SSCP profiles, which were subsequently shown to correspond to polymorphisms and/or an indel in the ITS-2 sequence. These variable sites related mainly to unpaired regions of the predicted secondary structure of the precursor rRNA molecule. SSCP profiles could be used to distinguish L. longispicularis in Macropus robustus robustus (New South Wales) from L. longispicularis in Macropus robustus erubescens and Macropus rufus (South Australia). This difference corresponded to a transversional change in the ITS-2 sequence at alignment position 82. The study demonstrated clearly the effectiveness of SSCP analysis for future large-scale population genetic studies of L. longispicularis in order to test the hypothesis that L. longispicularis from different geographical regions represents multiple sibling species.

Animals↗
PubMed2004

Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags.

To understand the molecular mechanism associated with gastric carcinogenesis, we identified genes expressed in gastric cancer cell lines and tissues. Of 97,609 high-quality ESTs sequenced from 36 cDNA libraries, 92,545 were coalesced into 10,418 human Unigene clusters (Build 151). The gene expression profile was produced by counting the cluster frequencies in each library. Although the profiles of highly expressed genes varied greatly from library to library, those genes related to cell structure formation, heat shock proteins, the glycolysis pathway, and the signaling pathway were highly represented in human gastric cancer cell lines and in primary tumors. Conversely, the genes encoding immunoglobulins, ribosomal proteins, and digestive proteins were down-regulated in gastric cancer cell lines and tissues compared to normal tissues. The transcription levels of some of these genes were confirmed by RT-PCR. We found that genes related to cell adhesion, apoptosis, and cytoskeleton formation were particularly up-regulated in the gastric cancer cell lines established from malignant ascites compared to those from primary tumors. This comprehensive molecular profiling of human gastric cancer should be useful for elucidating the genetic events associated with human gastric cancer.

Base Sequence↗
PubMed1996

Complete growth inhibition of Escherichia coli by ribosome trapping with truncated cspA mRNA at low temperature.

BACKGROUND: CspA, the major cold-shock protein of Escherichia coli, is transiently induced upon temperature downshift and considered to play an important role in low-temperature adaptation. RESULTS: Overproduction of truncated cspA mRNAs retaining translational ability was found to completely block cell growth at low temperatures. This effect was termed 'low-temperature antibiotic effect of truncated cspA expression (LACE)'. In contrast to the significant reduction of polysomes in normal cells upon cold shock, cells under LACE maintained a high polysome profile, producing only truncated cspA products. Growth inhibition of cells under LACE was suppressed when CspA was overproduced together with the truncated cspA mRNA. CONCLUSION: LACE is caused by the overproduction of a truncated cspA mRNA in the absence of CspA production, which in turn traps all the cellular ribosomes in a non-adaptive form incapable of forming initiation complexes with other cellular mRNAs. LACE may provide a novel approach to the development of a new antibiotic.

Bacterial Proteins↗
PubMed1968

Changes in the ribonucleic acid metabolism of aging mouse tissues with particular reference to the prostate gland.

1. The synthesis of RNA in purified nuclei of tissues from aged mice is substantially diminished. 2. As judged by the recovery of RNA in 105000g supernatants of tissue homogenates, the synthesis of ribosomal RNA may be particularly affected. 3. The decrease in RNA synthesis may be due to changes in the composition of the nuclear-associated protein with age, which result in changes in the priming ability of tissue chromatin. 4. Aging is accompanied by marked changes in the sedimentation profiles of ribonucleoprotein particles. These can be attributed to an age-associated depletion in messenger RNA or to an increased susceptibility to enzymic disaggregation.

Aging↗