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PubMed1995

High specificity of Trypanosoma cruzi epimastigote ribonucleoprotein as antigen in serodiagnosis of Chagas' disease.

We assessed the performance of an enzyme-linked immunosorbent assay (ELISA) with the Trypanosoma cruzi epimastigote ribosomal fraction (Tulahuen and Y strains) in order to improve the diagnostic specificity of the test. A total of 100 serum samples from patients with chronic Chagas' disease from Brazil and Argentina were studied. Sera from 116 patients, without Chagas' disease, including 10 with active mucocutaneous leishmaniasis and 20 with visceral leishmaniasis, were used as controls. Immunoglobulin G (IgG) antibodies against the ribosomal fraction (ribonucleoproteins [RNPs]) in the ELISA were found in 97% of samples from patients with Chagas' disease. A total of 99% of the sera from patients without the disease were negative, including sera from patients with mucocutaneous and visceral leishmaniases. The distribution of IgG isotypes in randomly chosen serum samples was determined by ELISA; IgG1 and IgG3 were predominant (100% exhibited IgG1 and 85% exhibited IgG3, and 50% also presented the IgG2 isotype. The distribution of the IgG subclasses was confirmed by the Western blot (immunoblot) technique. When total IgG was assayed by Western blot assay, no correlation was found between the pattern of serum reactivity and the clinical features of the patients with Chagas' disease. Therefore, no typical profile of polypeptide recognition could be associated with any clinical form of Chagas' disease (cardiomyopathy or megaviscera). Our results showed that sera from patients with Chagas' disease react with ribosomal antigens and display a typical profile of IgG isotypes (IgG1 plus IgG3). The RNP ELISA seems to have improved specificity compared with those of routine techniques such as the indirect immunofluorescence assay and hemagglutination because it better discriminates between patients with Chagas' disease and patients without the disease. Since sera from patients with leishmaniasis failed to show cross-reactivity with this antigen, the ELISA seems useful for detecting Chagas' disease as well as confirming the nature of sera, when it is doubtful whether the patients has Chagas' disease, by the isotype distribution of IgG.

Animals↗
PubMed2005

Assessment of the efficacy of telithromycin simulating human exposures against S. pneumoniae with ribosomal mutations in a murine pneumonia model.

Telithromycin (TEL) is a ketolide antimicrobial agent with in vitro activity against Streptococcus pneumoniae (SPN), including macrolide resistant strains. The purpose of this study was to assess the efficacy of TEL against clinical SPN isolates with various genotypic mutations including the newly recognized ribosomal mutations. Pneumonia was induced in either immunocompetent and immunosuppressed mice. Six isolates were included in the study and all were resistant to azithromycin (AZI) by MIC testing. Three oral regimens of TEL were chosen to simulate the human pharmacokinetic (PK) exposures observed in young healthy, healthy elderly (> or =65 years), and infected subjects. An additional group was given AZI in human simulated doses. Bacterial density in lung was determined after each treatment. Telithromycin administered simulating infected patients showed greater efficacy (i.e., change in logCFU) than the azithromycin treated group for all isolates except P1660008. The immune system was responsible for increased efficacy (ranging from 45-146%) for all but one of the telithromycin treatment regimens. Unlike other isolates studied in this in vivo model, P1660008 displayed a highly variable response to therapy, such that the reductions in CFU were not consistent with the microbiological and PK profiles of either compound. For all other isolates, the activity of AZI was comparable with untreated controls. Human simulated exposures of TEL displayed 0.5-3.4 log kill in vivo despite the ribosomal mutations studied. These data support the in vivo efficacy of TEL against a variety of genotypic resistance profiles observed in pneumococci, however, additional studies are required to fully characterize the killing profile of the compound against these recently determined ribosomal mutations.

Administration, Oral↗
PubMed2002

Expression profile of active genes in the human pituitary gland.

To characterize transcripts abundantly expressed in the human pituitary gland in general as well as to isolate novel transcripts expressed specifically in this gland, we generated an expression profile of the active genes transcribed in it. A total of 1015 randomly collected 3prime prime or minute expressed sequence tags (ESTs) (gene signatures, GSs) were grouped into 527GS species. The results showed the relative expression levels of genes in the pituitary gland. The genes comprising more than 1% of total mRNA were prolactin, growth hormone and chromogranin B genes. When known genes were categorized, the genes for pituitary hormones were the most actively transcribed, followed by the genes for ribosomal proteins, nuclear proteins and secretory granule proteins. Through comparison of this gene expression profile with the BodyMap database containing profiles generated from 63 other human tissues, we obtained 11 genes which appeared to be specifically expressed in the pituitary gland. In addition to the eight known genes, we identified three novel pituitary-specific transcripts which encode putative proteins: pituitary gland specific factor 1a (PGSF1a), PGSF1b and PGSF2. This expression profile method is a novel approach to the isolation of pituitary-specific genes that may have important functions.

Amino Acid Sequence↗
PubMed1993

Biotype and molecular fingerprints of metronidazole-resistant strains of Helicobacter pylori from antral gastric mucosa.

Biotypes, ribosomal RNA gene restriction patterns (ribopatterns), whole-cell protein patterns and plasmid profiles of paired Helicobacter pylori isolates from 17 patients were examined. Each pair comprised a pre- and a post-treatment isolate; nine of the 17 post-treatment isolates were obtained after treatment with tripotassium dicitrate bismuthate (De-Nol) and metronidazole. All strains of H. pylori had identical biotypes, but exhibited diversity between pairs in their molecular fingerprints. Each of the 17 strain pairs had unique ribopatterns; the pre- and post-treatment isolates in most pairs (16 of 17) were similar or identical, irrespective of metronidazole susceptibility. DNA subtype variants were detected in three patient sets. Although nine post-treatment isolates had acquired resistance to metronidazole, most (six of nine) resembled the pre-treatment isolates in their ribopattern, protein and plasmid profiles. No significant correlation was observed between metronidazole resistance and plasmid content in these H. pylori isolates. Emergence of post-treatment metronidazole-resistant isolates of H. pylori was associated only rarely with colonisation by a novel strain or acquisition of a plasmid and, in most patients, probably resulted from spontaneous emergence of resistance in the original infecting strain.

Bacterial Proteins↗
PubMed1997

Outbreak of septicaemia in neonates caused by Acinetobacter junii investigated by amplified ribosomal DNA restriction analysis (ARDRA) and four typing methods.

Septicaemia caused by Acinetobacter occurred in six infants in the neonatal unit. A total of 18 acinetobacters were isolated from blood cultures, cultures of intravascular catheters, and surveillance cultures. Twelve isolates from the six affected infants were identified as Acinetobacter junii by the use of a novel method, amplified ribosomal DNA restriction analysis (ARDRA). Typing of the organisms using the biochemical profiles of the API 20NE system, antibiogram typing, cell envelope protein electrophoresis, and PCR fingerprinting with two primer sets, ERIC1/ERIC2 and ERIC2/ 1026, showed that these 12 isolates were indistinguishable, whereas the remaining six isolates were different. The six infants recovered after therapy with ciprofloxacin alone in five cases and with a combination of ciprofloxacin and gentamicin in one case. This study showed that A. junii is capable of causing a serious, though non-fatal infection in neonates. The combined use of genotypic and phenotypic methods allowed the rapid separation of epidemic from non-epidemic isolates. It is concluded that for a better understanding of the role of the various Acinetobacter genomic species in human pathology, identification of acinetobacters according to the recent taxonomy is imperative.

Acinetobacter↗
PubMed1999

Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status.

The most primitive hematopoietic stem cells capable of longterm reconstitution of the entire hematopoietic system following transplantation are characterized by their ability to exclude both Rhodamine 123 and Hoechst 33342 dyes (Rh/Ho(dull)), and are an appropriate target population for the determination of stem cell ultrastructure. We have used a fluorescence-activated cell sorter to enrich to near purity these rare, highly quiescent cells. Analysis of the in vitro growth characteristics of Rh/Ho(dull) cells demonstrated an obligatory requirement for multiple growth factors, with 62% of the sorted population having the capacity to form colonies in the presence of CSF-1 + IL-1alpha + IL-3 + SCF. The Rh/Ho(dull) cells were small, with profiles having a mean diameter of 4.6 microm. Ultrastructural examination showed numerous ribosomes and several mitochondria in the thin rim of cytoplasm surrounding the nucleus, with other cytoplasmic organelles revealed in serial sections. The cells were generally homogeneous in appearance apart from the nucleus, which had an irregular shape with a single deep indentation. The heterochromatin around the margin was distinctly more pronounced in about 50% of nuclei. The findings provide a basis for studying the structural changes that occur with progressive differentiation of early hematopoietic cells.

Animals↗
PubMed1978

Sedimentation characteristics of the scrapie agent from murine spleen and brain.

Sedimentation profiles of the scrapie agent in extracts of murine spleen and brain were determined by analytical differential centrifugation. Infectivity profiles of the agent from the two tissues were similar. Sedimentation of the agent was not substantially altered by detergent treatment with sodium deoxycholate. In the presence of detergent, centrifugation at an omega2t value of 3.0 x 1010 rad2/s in a fixed-angle rotor sedimented 90% of the agent. Comparative studies with radioisotopically labeled Simian virus 40 showed that centrifugation at an omega2t value of 1.6 x 10(10) rad2/s removed 90% of the virions. The sedimentation profile of the scrapie agent was similar to that observed for cellular ribosomal RNA. Heating infectious extracts of spleen to 80 degrees C for 30 min resulted in the destruction of 95% of the RNA while sedimentation of the scrapie agent was unchanged. These studies establish a limited range of particle sizes for the scrapie agent.

Animals↗
PubMed2004

Clustering binary fingerprint vectors with missing values for DNA array data analysis.

Oligonucleotide fingerprinting is a powerful DNA array-based method to characterize cDNA and ribosomal RNA gene (rDNA) libraries and has many applications including gene expression profiling and DNA clone classification. We are especially interested in the latter application. A key step in the method is the cluster analysis of fingerprint data obtained from DNA array hybridization experiments. Most of the existing approaches to clustering use (normalized) real intensity values and thus do not treat positive and negative hybridization signals equally (positive signals are much more emphasized). In this paper, we consider a discrete approach. Fingerprint data are first normalized and binarized using control DNA clones. Because there may exist unresolved (or missing) values in this binarization process, we formulate the clustering of (binary) oligonucleotide fingerprints as a combinatorial optimization problem that attempts to identify clusters and resolve the missing values in the fingerprints simultaneously. We study the computational complexity of this clustering problem and a natural parameterized version and present an efficient greedy algorithm based on MINIMUM CLIQUE PARTITION on graphs. The algorithm takes advantage of some unique properties of the graphs considered here, which allow us to efficiently find the maximum cliques as well as some special maximal cliques. Our preliminary experimental results on simulated and real data demonstrate that the algorithm runs faster and performs better than some popular hierarchical and graph-based clustering methods. The results on real data from DNA clone classification also suggest that this discrete approach is more accurate than clustering methods based on real intensity values in terms of separating clones that have different characteristics with respect to the given oligonucleotide probes.

Algorithms↗
PubMed2003

Comprehensive aligned sequence construction for automated design of effective probes (CASCADE-P) using 16S rDNA.

MOTIVATION: Prokaryotic organisms have been identified utilizing the sequence variation of the 16S rRNA gene. Variations steer the design of DNA probes for the detection of taxonomic groups or specific organisms. The long-term goal of our project is to create probe arrays capable of identifying 16S rDNA sequences in unknown samples. This necessitated the authentication, categorization and alignment of the >75 000 publicly available '16S' sequences. Preferably, the entire process should be computationally administrated so the aligned collection could periodically absorb 16S rDNA sequences from the public records. A complete multiple sequence alignment would provide a foundation for computational probe selection and facilitates microbial taxonomy and phylogeny. RESULTS: Here we report the alignment and similarity clustering of 62 662 16S rDNA sequences and an approach for designing effective probes for each cluster. A novel alignment compression algorithm, NAST (Nearest Alignment Space Termination), was designed to produce the uniform multiple sequence alignment referred to as the prokMSA. From the prokMSA, 9020 Operational Taxonomic Units (OTUs) were found based on transitive sequence similarities. An automated approach to probe design was straightforward using the prokMSA clustered into OTUs. As a test case, multiple probes were computationally picked for each of the 27 OTUs that were identified within the Staphylococcus Group. The probes were incorporated into a customized microarray and were able to correctly categorize Staphylococcus aureus and Bacillus anthracis into their correct OTUs. Although a successful probe picking strategy is outlined, the main focus of creating the prokMSA was to provide a comprehensive, categorized, updateable 16S rDNA collection useful as a foundation for any probe selection algorithm.

Algorithms↗
PubMed2003

Kinetic Outlier Detection (KOD) in real-time PCR.

Real-time PCR is becoming the method of choice for precise quantification of minute amounts of nucleic acids. For proper comparison of samples, almost all quantification methods assume similar PCR efficiencies in the exponential phase of the reaction. However, inhibition of PCR is common when working with biological samples and may invalidate the assumed similarity of PCR efficiencies. Here we present a statistical method, Kinetic Outlier Detection (KOD), to detect samples with dissimilar efficiencies. KOD is based on a comparison of PCR efficiency, estimated from the amplification curve of a test sample, with the mean PCR efficiency of samples in a training set. KOD is demonstrated and validated on samples with the same initial number of template molecules, where PCR is inhibited to various degrees by elevated concentrations of dNTP; and in detection of cDNA samples with an aberrant ratio of two genes. Translating the dissimilarity in efficiency to quantity, KOD identifies outliers that differ by 1.3-1.9-fold in their quantity from normal samples with a P-value of 0.05. This precision is higher than the minimal 2-fold difference in number of DNA molecules that real-time PCR usually aims to detect. Thus, KOD may be a useful tool for outlier detection in real-time PCR.

Algorithms↗
PubMed2004

Bacterial diversity in marine hydrocarbon seep sediments.

Marine seeps introduce significant amounts of hydrocarbons into oceans and create unusual habitats for microfauna and -flora. In the vicinity of chronic seeps, microbes likely exert control on carbon quality entering the marine food chain and, in turn, hydrocarbons could influence microbial community composition and diversity. To determine the effects of seep oil on marine sediment bacterial communities, we collected sediment piston cores within an active marine hydrocarbon seep zone in the Coal Oil Point Seep Field, at a depth of 22 m in the Santa Barbara Channel, California. Cores were taken adjacent to an active seep vent in a hydrocarbon volcano, on the edge of the volcano, and at the periphery of the area of active seepage. Bacterial community profiles were determined by terminal restriction fragment length polymorphisms (TRFLPs) of 16S ribosomal genes that were polymerase chain reaction (PCR)-amplified with eubacterial primers. Sediment carbon content and C/N ratio increased with oil content. Terminal restriction fragment length polymorphisms suggested that bacterial communities varied both with depth into sediments and with oil concentration. Whereas the apparent abundance of several peaks correlated positively with hydrocarbon content, overall bacterial diversity and richness decreased with increasing sediment hydrocarbon content. Sequence analysis of a clone library generated from sediments collected at the periphery of the seep suggested that oil-sensitive species belong to the gamma Proteobacteria and Holophaga groups. These sequences were closely related to sequences previously recovered from uncontaminated marine sediments. Our results suggest that seep hydrocarbons exert a strong selective pressure on bacterial communities in marine sediments. This selective pressure could, in turn, control the effects of oil on other biota in the vicinity of marine hydrocarbon seeps.

Bacteria↗
PubMed1973

Ribonucleic acid and protein synthesis in guinea pig liver during Q fever.

Protein and ribonucleic acid (RNA) syntheses increase in the liver during Q fever, coincident with progressive increments in cortisol levels. Cell-free protein-synthesizing systems indicate that the pH 5 enzyme fraction was chiefly responsible for enhanced activity. Although the polysome profiles from normal and infected livers were similar, the number of liver ribosomes doubled during infection. There was a concomitant increase in orotate incorporation in the 28s, 18s, and 4s RNA species of infected liver.

Amino Acids↗
PubMed2003

Linear and circular plasmid content in Borrelia burgdorferi clinical isolates.

The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host.

Bacteremia↗
PubMed2003

Clustering binary fingerprint vectors with missing values for DNA array data analysis.

Oligonucleotide fingerprinting is a powerful DNA array based method to characterize cDNA and ribosomal RNA gene (rDNA) libraries and has many applications including gene expression profiling and DNA clone classification. We are especially interested in the latter application. A key step in the method is the cluster analysis of fingerprint data obtained from DNA array hybridization experiments. Most of the existing approaches to clustering use (normalized) real intensity values and thus do not treat positive and negative hybridization signals equally (positive signals are much more emphasized). In this paper, we consider a discrete approach. Fingerprint data are first normalized and binarized using control DNA clones. Because there may exist unresolved (or missing) values in this binarization process, we formulate the clustering of (binary) oligonucleotide fingerprints as a combinatorial optimization problem that attempts to identify clusters and resolve the missing values in the fingerprints simultaneously. We study the computational complexity of this clustering problem and a natural parameterized version, and present an efficient greedy algorithm based on MINIMUM CLIQUE PARTITION on graphs. The algorithm takes advantage of some unique properties of the graphs considered here, which allow us to efficiently find the maximum cliques as well as some special maximal cliques. Our experimental results on simulated and real data demonstrate that the algorithm runs faster and performs better than some popular hierarchical and graph-based clustering methods. The results on real data from DNA clone classification also suggest that this discrete approach is more accurate than clustering methods based on real intensity values, in terms of separating clones that have different characteristics with respect to the given oligonucleotide probes.

Algorithms↗
PubMed1988

Interaction between macrophage and parasite cells in lobomycosis. The thickened cell wall of Paracoccidioides loboi exhibits apertures to the extracellular milieu.

Lesioned, cutaneous, tissue fragments from five indians of the Caiabi tribe with lobomycosis, living in the Xingu National Park (Central Brazil), are analyzed by light and electron microscopy. Clusters of macrophages filled with parasite and/or cell wall debris, separated by collagen fiber bundles, characterize the morphological pattern seen in thick and thin sections. Paracoccidioides loboi within the phagocytic cells are multinucleate organisms whose cytoplasm contains mitochondria with few cristae, ribosomes and vacuoles; a large, dense, globular body and peculiarly curved mitochondrial profiles are described. From the outer portion of the double layered parasite cell wall, radial projections commonly emerge, rendering the structure conspicuously thicker and more irregularly surfaced than that seen in many other phagocytized yeast cell species. The cell wall layers from fungi possessing distinct subcellular organization show a weak or no reaction for acid phosphatase. Most of the foamy cells commonly seen by light microscopy are macrophages filled with fungal cell wall remnants which exhibit marked acid phosphatase activity. Occasionally, microchannels extending from the outer layer of the parasite cell wall to the macrophage surface and exocytic-like openings, possibly derived from the fusion of the macrophage membrane covering the parasite cell wall and the macrophage plasmalemma can be seen. Through such routes, material of texture and density similar to that of the outermost cell wall layer appears to be deposited extracellularly.

Adult↗
PubMed1994

Elongation factor-1 alpha mRNA is selectively translated following mitogenic stimulation.

Stimulation of quiescent Swiss 3T3 cells to proliferate leads to a selective 6-fold increase in the rate of translation of protein synthesis elongation factor eEF-1 alpha. Northern blot and solution hybridization protection studies show that levels of eEF-1 alpha mRNA remain constant following serum stimulation, demonstrating that eEF-1 alpha transcripts are not degraded following mitogenic stimulation and that the increase in eEF-1 alpha synthesis is accounted for by pre-existing mRNA. Localization of these transcripts in resting cells shows that they are largely distributed equally between stored mRNA protein particles and monosomes/disomes. Following serum addition, eEF-1 alpha transcripts present in mRNA protein particles redistribute to large polysomes rather than to monosomes and disomes as would be expected. The same is true for those transcripts present in monosomes and disomes. Salt-shift and translational runoff studies indicate that eEF-1 alpha transcripts sedimenting with monosomes and disomes in quiescent cells are associated with actively translating ribosomes. The results suggest that a specific transcript can move within polysome profile as a function of the affinity of translational apparatus for that transcript.

3T3 Cells↗
PubMed2002

Resistance to macrolides in clinical isolates of Streptococcus pyogenes due to ribosomal mutations.

OBJECTIVE: Two clinical strains of Streptococcus pyogenes, 237 and 544, one isolated in Slovakia and the other in Croatia, that were resistant to azithromycin (MIC 8 and 2 mg/L, respectively) but susceptible to erythromycin (MIC 0.5 and 0.12 mg/L, respectively) did not contain any gene known to confer macrolide resistance by ribosomal modification (erm gene) or efflux [mef(A) and msr(A) genes]. The aim of the study was to determine the mechanisms of macrolide resistance in both strains. METHODS: Portions of genes encoding ribosomal proteins L22 and L4, and 23S rRNA (domains II and V) in the two macrolide-resistant strains and in control strains susceptible to macrolides, were analysed by PCR and single-strand conformational polymorphism, to screen for mutations. The DNA sequences of amplicons from resistant strains that differed from those of susceptible strains, in terms of their electrophoretic migration profiles, were determined. RESULTS: S. pyogenes 237 displayed a KG insertion after position 69 in ribosomal protein L4. S. pyogenes 544 contained a C2611U mutation in domain V of 23S rRNA. CONCLUSION: Mutations at a similar position in ribosomal protein L4 and 23S rRNA have been reported previously in macrolide-resistant pneumococci. This report shows that similar mutations can be found in macrolide-resistant S. pyogenes.

Adult↗