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PubMed2001

Epidemiology of the genetic elements responsible for acquired glycopeptide resistance in enterococci.

Five genotypes of acquired glycopeptide resistance have been documented in enterococci, with vanA and vanB being the most globally widespread and prevalent. Resistance results from the production of peptidoglycan precursors with reduced binding affinity for glycopeptides and is encoded by complex clusters of van genes. The prototype VanA element is Tn1546, a 10.8-kb transposon that carries the vanRSHAXYZ genes. Diverse VanA elements exist, but all share the vanRSHAX cluster and are believed to be derived from a Tn1546 progenitor. The sequences of these genes are remarkably conserved, with only a few point mutations identified. VanA elements do however vary by the presence of deletions and insertion sequences (IS) in nonessential genes (orf1, orf2, vanY, and vanZ) and intergenic regions. IS transposition probably plays a key role in VanA element evolution. By contrast, vanB gene clusters show greater sequence divergence. Three vanB alleles have been reported, of which vanB2 appears to be the most widespread, generally as part of Tn5382 and related elements. To date, only four Enterococcus faecium strains with VanD resistance have been reported, and each contained a distinct vanD allele. The VanE and VanG types have each been identified in single strains of Enterococcus faecalis. The existence of distinct genotypes, together with the allelic nature of vanB and vanD, suggests that van clusters have transferred to enterococci on multiple occasions from undefined donor species, with subsequent horizontal dissemination, particularly of VanA and VanB elements, among enterococci. Characterization of glycopeptide resistance elements yields information on their evolution and broadens our insights into the epidemiology of resistant enterococci.

Anti-Bacterial Agents↗
PubMed1997

Intraperitoneal injection of genetically modified, human mesothelial cells for systemic gene therapy.

An ideal cell type for ex vivo gene therapy should be easy to biopsy, propagate, and genetically engineer in culture, should be transplantable using simple procedures, and should express therapeutic proteins at useful levels. The mesothelial cell appears to satisfy these criteria. Several thousand proliferative mesothelial cells were present in typical specimens of nonpathologic human peritoneal fluid obtained by needle aspiration. These divided rapidly in a specialized medium to yield pure cultures of approximately 10(7) cells within 2 weeks. The replicative lifespan of mesothelial cells cultured from adults was approximately 42-52 population doublings, permitting expansion and cryopreservation of a lifetime supply of autologous cells from one fluid sample. Cells transduced with a human growth hormone (hGH) adenoviral vector secreted 100-300 microg of hGH/10(6) cells per day for at least 6 weeks in culture when maintained at quiescence. Intraperitoneal injection of transduced cells into athymic mice resulted in rapid systemic delivery of hGH, with peak plasma levels of 0.1-1 microg/ml declining over 3 weeks to <1 ng/ml. Mice receiving a second injection of engineered cells displayed the same plasma hGH levels and duration as naive mice. Cells labeled with a beta-galactosidase vector were identifiable by in situ enzymatic staining as clusters attached to peritoneal surfaces at multiple sites for at least 19 days after injection. Cells serially passaged through about three-quarters of their lifespan before transduction and injection were as effective at hGH delivery as earlier-passage cells. These results indicate the clinical potential for ex vivo gene therapy using mesothelial cells.

Adenoviridae↗
PubMed1987

Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic messenger RNA encoding the structural proteins E1, E2 and C.

The complete nucleotide sequence of the 24S subgenomic mRNA of wild-type M33 strain rubella virus has been determined. This RNA is 3,383 nucleotides in length excluding the 3'-terminal poly(A) tract. After the three multiple in-phase termination codons clustered in the 5' terminus of this RNA, there are 81 nucleotides of nontranslated nucleic acid followed by a reading frame of 2,978 nucleotides that encodes the 110 kD precursor of the structural proteins. The 3'-untranslated region is 263 nucleotides. The 110 kD polyprotein is processed to produce nucleocapsid C, the glycoproteins E2 and E1 in that order. Sites of post-translational cleavage to produce E2 and E1 were located using available N-terminal amino acid sequences. RNAs synthesized by transcription in vitro are effective messengers in the rabbit reticulocyte cell-free translation system. Post-translational processing of the structural proteins was observed in the cell-free system supplemented with microsomes from dog pancreas.

Amino Acid Sequence↗
PubMed2006

Minimal metastatic disease in sentinel lymph nodes in breast carcinoma: some modest proposals to refine criteria for "isolated tumor cells".

Axillary lymph node status is one of the most important prognostic factors in breast carcinoma. The weight of cumulative evidence suggests that the development of the sentinel lymph node (SLN) biopsy procedure has not only allowed for accurate lymph node-staging but has also helped avoid the morbidity of a full axillary dissection in those patients who are unlikely to have metastatic tumor in that location. The detection of metastases in SLNs is facilitated by the, now relatively routine, enhanced histopathologic examination via step-sectioning and immunohistochemistry. In clinical terms, the finding of a metastatic deposit that measures between 0.2 and 2 mm, that is, "micrometastasis" in a SLN is largely noncontroversial; however, the presence of smaller metastatic foci detected either by routine hematoxylin and eosin stain or by cytokeratin immunostain [<0.2 mm, ie, so-called "isolated tumor cells (ITCs)"] has remained problematic since the advent of the SLN biopsy. In this communication, attention is drawn to the broad morphologic range of metastatic disease in SLN that may be placed in the category of so-called ITC. To facilitate the reproducible classification of the various strata of minimal metastasis in sentinel lymph nodes, we recommend the following: (1) the term "isolated tumor cell" (note singular form) be restricted to cases that show the presence of only a single tumor cell. (2) In situations where there are multiple isolated single cells and/or cell cluster(s) present and each cluster measures<0.2 mm, the term "submicroscopic metastasis" be adopted and an actual count of tumor cells present may be given. (3) Restrict the use of the term micrometastasis to cases wherein the largest metastatic focus is larger than 0.2 mm but smaller than 2.0 mm.

Axilla↗
PubMed1999

HIV-1 subtyping in Salvador, Bahia, Brazil: a city with African sociodemographic characteristics.

To investigate the prevalence of the HIV-1 subtypes in different populations from Salvador, Bahia, Brazil, blood samples from 72 HIV-1-seropositive injecting drug users (IDUs) and 62 individuals infected sexually were analyzed using the heteroduplex mobility assay (HMA). In the IDU group, 89.5% were classified as subtype B, 3% as subtype F, and 7.5% showed a B/F HMA profile. In the sexual transmission (ST) group, 95% were identified as B subtype, 3.4% showed a B/F profile, and 1.6% a B/C/E HMA profile. All Brazilian samples that showed multiple reactivities in the HMA analysis clustered on sequencing with B North American/ European HIV-1 isolates in the phylogenetic analysis, whereas the F subtypes clustered with F Brazilian HIV-I isolates. Serologic reactivities of IDU's sera were examined using a panel of synthetic V3 loop peptides representative of the different HIV-1 subtypes. No difference in serologic reactivity between F and B subtype plasma could be observed. Predominance of HIV-I subtype B was identified in both study groups, whereas subtype F was detected only among IDUs in a frequency lower than described for other Brazilian regions.

Adolescent↗
PubMed2004

Predictors of disruptive behavior, developmental delays, anxiety, and affective symptomatology among institutionally reared romanian children.

OBJECTIVE: To examine the associations between duration of institutionalization, age at abandonment, and mental and physical health outcomes of young institutionalized children in Romania and to examine patterns of associations between placement history, physical growth variables, and psychiatric symptoms. METHOD: Institutionalized children (ages 2-6 years) in a Romanian orphanage were studied through caregiver reports. Orphanage staff completed questionnaires on the children's psychiatric symptoms and physical growth measurements and placement histories on the children. RESULTS: Children showed high rates of developmental delays, anxiety/affective symptoms, and physical growth delays. Patterns of associations between history and growth variables (duration of institutionalization, age at abandonment, and physical stature) and psychiatric symptom clusters were examined using three sequential multiple regressions. Longer duration of institutionalization and shorter physical stature were significantly associated with anxiety/affective symptoms (F3,47=6.49, p <.01). Physical stature was significantly associated with developmental delays (F3,47=5.603, p <.01) and disruptive behaviors (F3,47=2.832, p <.05), with smaller stature being associated with greater developmental delays and fewer disruptive behavior problems. CONCLUSIONS: Institutionalized children demonstrate high rates of psychiatric symptoms. Duration of institutionalization, physical stature, and age at abandonment differentially relate to psychiatric symptoms. Findings are interpreted in light of implications for intervention and social policy.

Affect↗
PubMed2000

Tissue gene expression analysis using arrayed normalized cDNA libraries.

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference.

Algorithms↗
PubMed2003

Dynamical mean-field theory of spiking neuron ensembles: response to a single spike with independent noises.

A semianalytical dynamical mean-field theory has been developed for a study of dynamics of an ensemble of N-unit FitzHugh-Nagumo neurons subject to white noises. Assuming weak noises and the Gaussian distribution of state variables, we have driven equations of motions for moments of local and global variables. Dynamical mean-field approximation (DMA) has replaced original, 2N-dimensional stochastic differential equations (DEs) by eight-dimensional deterministic DEs, whereas the conventional moment method yields N(2N+3)-dimensional deterministic DEs for local variables. We have discussed the dependence of the spike firing precision and the synchronization on the noise intensity, synaptic coupling, and the size of the neuron ensemble. The spike timing precision is shown to be improved by increasing the size of the neuron ensemble, even when there are no couplings among neurons. When the coupling is introduced, neurons in ensembles respond to an input spike with a partial synchronization. Results calculated by our DMA theory are in good agreement with those obtained by direct simulations. DMA theory is extended to a large cluster which can be divided into multiple subclusters according to their functions. A model calculation has demonstrated that when the noise intensity is moderate, the spike propagation with a fairly precise timing is possible among noisy subclusters with feed-forward couplings, as in the synfire chain. We have compared DMA theory with the conventional moment method, showing that the former may be alternatively derived from the latter by a reduction in the number of moments with the mean-field approximation.

Animals↗
PubMed2002

Structural analysis of the maize rp1 complex reveals numerous sites and unexpected mechanisms of local rearrangement.

Rp1 is a complex disease resistance locus in maize that is exceptional in both allelic variability and meiotic instability. Genomic sequence analysis of three maize BACs from the Rp1 region of the B73 inbred line revealed 4 Rp1 homologs and 18 other gene-homologous sequences, of which at least 16 are truncated. Thirteen of the truncated genes are found in three clusters, suggesting that they arose from multiple illegitimate break repairs at the same sites or from complex repairs of each of these sites with multiple unlinked DNA templates. A 43-kb region that contains an Rp1 homolog, six truncated genes, and three Opie retrotransposons was found to be duplicated in this region. This duplication is relatively recent, occurring after the insertion of the three Opie elements. The breakpoints of the duplication are outside of any genes or identified repeat sequence, suggesting a duplication mechanism that did not involve unequal recombination. A physical map and partial sequencing of the Rp1 complex indicate the presence of 15 Rp1 homologs in regions of approximately 250 and 300 kb in the B73 inbred line. Comparison of fully sequenced Rp1-homologous sequences in the region demonstrates a history of unequal recombination and diversifying selection within the Leu-rich repeat 2 region, resulting in chimeric gene structures.

Chromosome Mapping↗
PubMed1993

On naevi and melanomas in dysplastic naevus syndrome patients.

Cutaneous melanoma may occur as isolated, so-called 'sporadic' cases or in association with multiple atypical naevi and in familial clusters, in which case it is referred to as the familial dysplastic naevus syndrome (DNS). In this retrospective study (a) the number and body distribution of naevocytic naevi and (b) the body distribution of malignant melanoma (MM) in individuals with familial DNS were compared in order to study their association. In 45 patients with familial DNS aged 20-39 years naevus counts on trunk and lower extremities were compared with melanoma data and distributions from a second group of 43 patients from the same DNS families aged 12-66 years. Men had significantly more naevi of a size > or = 2 mm or > or = 5 mm on the back than women (P = 0.02). Women showed a tendency towards a greater number of naevi on the lower extremities than men, but in women no significant difference in naevi between the lower extremities and the back was found. The total number of naevi on the trunk and lower extremities in familial DNS patients was higher than that in the general population. In conclusion, it was found that predilection sites for melanoma in familial DNS patients of both sexes correspond with the distribution of naevi; in males naevi and melanoma counts and percentage distributions were higher on the back, in females both the back and the lower extremities were affected. These findings strongly suggest an association between naevus distribution and melanoma occurrence and site in familial DNS, analogous to earlier reports on sporadic melanoma.

Adolescent↗
PubMed1991

Individual germinal centres of myasthenia gravis human thymuses contain polyclonal activated B cells that express all the Vh and Vk families.

Using in situ hybridization, we analysed the immunoglobulin repertoire expressed by the B cells present in myasthenia gravis thymuses from four patients. B cells, mostly in activated state, were clustered in germinal centres, in which multiple isotypes were identified. A majority of cells expressed IgG as compared with IgM, with a roughly similar contribution of kappa and lambda chains. Hybridization with the six VH and the 4 VK human family probes was observed in serial sections, providing additional evidence that individual germinal centres were polyclonal. The thymic B cell repertoire closely reflected the VH and the VK family usage of normal peripheral blood lymphocytes with the preferential utilization of VH3, VK1 and VK3.

Adult↗
PubMed2004

A group of related cDNAs encoding secreted proteins from Hessian fly [Mayetiola destructor (Say)] salivary glands.

A group of cDNAs has been isolated and characterized from Hessian fly [Mayetiola destructor (Say)] salivary glands. Members in this group appear to encode proteins with secretion signal peptides at the N-terminals. The mature putative proteins are small, basic proteins with calculated molecular weights that ranged from 8.5 to 10 kDa, and isoelectric points from 9.92 to 10.90. Sequence analysis indicated a strong selection for mutations that generate amino acid changes within the coding region. Northern blot analysis revealed that these genes are expressed only in the first instar larvae, a critical stage that determines if the interaction between a specific Hessian fly biotype and a specific wheat cultivar is compatible. Genomic analysis demonstrated that multiple copies of similar genes are clustered within a short region on chromosome 2A. This is the same arm in which two avirulence genes have been mapped.

Amino Acid Sequence↗
PubMed2004

A regulation cascade controls expression of Porphyromonas gingivalis fimbriae via the FimR response regulator.

Little is known about how Porphyromonas gingivalis, a Gram-negative oral anaerobe, senses environmental changes, and how such information is transmitted to the cell. The production of P. gingivalis surface fimbriae is regulated by FimS-FimR, a two component signal transduction system. Expression of fimA, encoding the fimbrilin protein subunit of fimbriae, is positively regulated by the FimR response regulator. In this study we investigated the molecular mechanisms of FimR regulation of fimA expression. Comparative transcription profiling of fimR wild-type and mutant strains shows that FimR controls the expression of several genes including five clustered around the fimA locus. Chromatin immunoprecipitation assays and electrophoretic mobility shift assays identify and confirm that FimR binds to the promoter region of the first gene in the fimA cluster. Gene expression analyses of mutant strains reveal a transcriptional cascade involving multiple steps, with FimR activating expression of the first gene of the cluster that encodes a key regulatory protein.

Animals↗
PubMed1993

Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein domains and relationship to other chitinases.

Streptomyces olivaceoviridis efficiently degrades chitin. Shotgun cloning of partially Sau3A-cleaved DNA using the multicopy vector pIJ702 and Streptomyces lividans 66 as host resulted in the identification of the plasmid pCHI O1 which harbours an insert of 4.6 kb. In the presence of chitin as sole carbon source, transformants of S. lividans 66 carrying pCHI O1 or its derivatives with smaller inserts overproduced an exochitinase which was purified to homogeneity. The chitin-inducible enzyme with an isoelectric point of 4.0 shows optimal activity at pH 7.3 and 55 degrees C, has an apparent molecular mass of 47 kDa and is competitively inhibited by the pseudosugar allosamidin. The enzyme was identified as an exochitinase since it generates exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high-molecular mass chitin. Sequence analysis of a reading frame of 1794 base pairs and comparison of the deduced amino-acid sequence allowed the identification of the putative catalytic domain, one region with significant similarity to the type-III module of fibronectin and one domain of unknown function. Multiple sequence alignment and hydrophobic-cluster analysis of 25 chitinolytic enzymes from bacteria, fungi and plants allowed the identification of their characteristic domains. The exochitinase from S. olivaceoviridis shares highest similarity with the chitinase D from Bacillus circulans.

Acetylglucosamine↗
PubMed2006

Biogeography of the marine actinomycete Salinispora.

Marine actinomycetes belonging to the genus Salinispora were cultured from marine sediments collected at six geographically distinct locations. Detailed phylogenetic analyses of both 16S rRNA and gyrB gene sequences reveal that this genus is comprised of three distinct but closely related clades corresponding to the species Salinispora tropica, Salinispora arenicola and a third species for which the name 'Salinispora pacifica' is proposed. Salinispora arenicola was cultured from all locations sampled and provides clear evidence for the cosmopolitan distribution of an individual bacterial species. The co-occurrence of S. arenicola with S. tropica and S. pacifica suggests that ecological differentiation as opposed to geographical isolation is driving speciation within the genus. All Salinispora strains cultured to date share greater than 99% 16S rRNA gene sequence identity and thus comprise what has been described as a microdiverse ribotype cluster. The description of this cluster as a new genus, containing multiple species, provides clear evidence that fine-scale 16S rDNA sequence analysis can be used to delineate among closely related species and that more conservative operational taxonomic unit values may significantly underestimate global species diversity.

Biodiversity↗
PubMed1998

Epidemiological investigation of vaginal Saccharomyces cerevisiae isolates by a genotypic method.

Saccharomyces cerevisiae is a ubiquitous, ascomycetous yeast, and vaginitis caused by this organism has been reported only very rarely. The aim of the present investigation was to assess the epidemiological relatedness of a group of vaginal and commercial S. cerevisiae isolates by a previously reported genetic typing method, which divided the isolates into two broad groups with numerous subtypes. Nineteen S. cerevisiae isolates obtained from patients suffering from vaginitis and four isolates from commercial products in the same city were analyzed. The cellular DNA from each isolate was digested with the restriction endonuclease EcoRI, and restriction fragment length polymorphisms were generated by horizontal gel electrophoresis. The results showed that although vaginal isolates did not cluster in any particular genetic subtype, multiple patients were infected with indistinguishable strains (there were nine distinct strains among 23 isolates). For two of three patients, all three with two episodes of S. cerevisiae vaginitis, different strains were isolated during the recurrence of this disease. Three other patients with indistinguishable isolates were epidemiologically related in that two were practitioners in the same clinic and the third was a patient at this clinic. We also found that one commercial strain was indistinguishable from the strain isolated from three different women at the time that they were suffering from vaginitis. The findings of the present study suggest that some S. cerevisiae strains may possess properties permitting persistence in the human host. Furthermore, person-to-person contact and the proliferation of the use of S. cerevisiae as a health-food product, in home baking, and in home brewing may be a contributing factor in human colonization and infection with this organism.

Adult↗
PubMed1994

Phylogenetic associations of human and simian T-cell leukemia/lymphotropic virus type I strains: evidence for interspecies transmission.

Homologous env sequences from 17 human T-leukemia/lymphotropic virus type I (HTLV-I) strains from throughout the world and from 25 simian T-leukemia/lymphotropic virus type I (STLV-I) strains from 12 simian species in Asia and Africa were analyzed in a phylogenetic context as an approach to resolving the natural history of these related retroviruses. STLV-I exhibited greater overall sequence variation between strains (1 to 18% compared with 0 to 9% for HTLV-I), supporting the simian origin of the modern viruses in all species. Three HTLV-I phylogenetic clusters or clades (cosmopolitan, Zaire, and Melanesia) were resolved with phenetic, parsimony, and likelihood analytical procedures. Seven phylogenetic clusters of STLV-I were resolved with the most primitive (deeply rooted) divergence involving several STLV-I strains from Asian primate species. Combined analysis of HTLV-I and STLV-I revealed that neither STLV-I clusters nor HTLV-I clusters recapitulated host species specificity; rather, multiple clades from the same species were closer to clades from other species than to each other. We interpret these evolutionary associations as support for the occurrence of multiple discrete interspecies transmissions of ancestral viruses between primate species (including human) that led to recognizable phylogenetic clades that persist in modern species. Geographic concordance of divergent host species that harbor closely related viruses reinforces that physical feasibility for hypothesized interspecies virus transmission in the past and in the present.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of the duck hepatitis B virus capsid protein associated with conformational changes in the C terminus.

The capsid protein of duck hepatitis B virus (DHBV) is phosphorylated at multiple sites during viral infection. A cluster of sites is located near the C terminus of the 262-amino-acid protein. We have used site-directed mutagenesis to show that three serines and one threonine serve as phosphate acceptor amino acids in the C terminus. An additional six potential phosphate acceptor sites in this region were apparently not utilized. Each serine or threonine that served as a phosphate acceptor was adjacent to a downstream proline, while all six serines that were not acceptors for phosphate residues lacked adjacent downstream prolines. Mutation of the downstream proline to glycine at each site had the same effect as mutating the serine itself, suggesting an SP or TP motif as an essential feature for capsid protein phosphorylation. Phosphorylation at these four sites resulted in complex shifts in electrophoretic mobility in sodium dodecyl sulfate gels of the capsid protein or of a C-terminal peptide containing the phosphorylated sites, suggesting that specific conformations of the C terminus are associated with different combinations of phosphorylated serines. We speculate that distinct functions of the C terminus may be associated with different phosphorylated domains on the intact capsid.

Amino Acid Sequence↗