RESUMO
Although previous bacterial typing methods have been informative about potential relatedness of isolates collected during outbreaks, next-generation sequencing has emerged as a powerful tool to not only look at similarity between isolates, but also put differences into biological context. In this study, we have investigated the whole genome sequence of five Pseudomonas aeruginosa isolates collected during a persistent six-year outbreak at Nottingham University Hospitals National Health Service (NHS) Trust City Campus, United Kingdom. Sequencing, using both Roche 454 and Illumina, reveals that most of these isolates are closely related. Some regions of difference are noted between this cluster of isolates and previously published genome sequences. These include regions containing prophages and prophage remnants such as the serotype-converting bacteriophage D3 and the cytotoxin-converting phage phi CTX. Additionally, single nucleotide polymorphisms (SNPs) between the genomic sequence data reveal key single base differences that have accumulated during the course of this outbreak, giving insight into the evolution of the outbreak strain. Differentiating SNPs were found within a wide variety of genes, including lasR, nrdG, tadZ, and algB. These have been generated at a rate estimated to be one SNP every four to five months. In conclusion, we demonstrate that the single base resolution of whole genome sequencing is a powerful tool in analysis of outbreak isolates that can not only show strain similarity, but also evolution over time and potential adaptation through gene sequence changes.
Assuntos
Surtos de Doenças , Genoma Bacteriano/genética , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Monitoramento Epidemiológico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Hospitais , Humanos , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Polimorfismo de Nucleotídeo Único , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Fatores de Tempo , Reino Unido/epidemiologiaRESUMO
We determined the genome sequence of the type strain of Helicobacter canadensis, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places H. canadensis close to Wolinella succinogenes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Helicobacter/genética , Análise de Sequência de DNA , Animais , Infecções por Helicobacter/microbiologia , Humanos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência , Wolinella/genéticaRESUMO
One of the primary objectives of the Oncology Pathology Working Group (OPWG), a joint initiative of the Veterinary Cancer Society and the American College of Veterinary Pathologists, is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for oncologic pathology. Consensus is established through review of relevant peer-reviewed literature relative to a subgroup's particular focus. In this document, the authors provide descriptions of the literature reviewed, the review process, and a summary of the information gathered on immunocytochemistry. The intent of this publication is to help educate practitioners and pathologists on the process of immunocytochemistry and to provide a guide for the use of this technique in veterinary medicine. This document represents the opinions of the working group and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists or the Veterinary Cancer Society.
Assuntos
Imuno-Histoquímica/veterinária , Neoplasias/veterinária , Patologia Veterinária/métodos , Animais , Anticorpos/imunologia , Imuno-Histoquímica/métodos , Imuno-Histoquímica/tendências , Neoplasias/imunologia , Neoplasias/patologia , Patologia Veterinária/tendências , Guias de Prática Clínica como AssuntoRESUMO
The multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for understanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.
Assuntos
Proteínas de Ligação ao GTP , Família Multigênica , Receptores de Superfície Celular , Alinhamento de Sequência , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência MolecularRESUMO
Inactivation or loss of tumor-suppressor genes is believed to lead to the development or progression of malignancies. To determine whether a tumor-suppressor gene is located on chromosome 8, DNA was extracted from tumor and normal tissue of colorectal, gastric, and pancreatic specimens, and allele loss was investigated by Southern hybridization techniques with the chromosome 8 probe D8S7. Twenty-five percent of pancreatic carcinomas, 50% of gastric carcinomas, and 50% of colorectal carcinomas were found to have lost an allele on chromosome 8. These findings suggest the presence of a tumor-suppressor gene on chromosome 8, which is involved in colorectal carcinoma, gastric carcinoma, and pancreatic carcinoma. Definition of the frequency with which this tumor-suppressor gene is involved in gastrointestinal malignancies will await the study of many patients who are classified as informative and the use of multiple probes for chromosome 8.
Assuntos
Adenocarcinoma/genética , Cromossomos Humanos Par 8/fisiologia , Neoplasias Gastrointestinais/genética , Genes Supressores de Tumor/fisiologia , Neoplasias Pancreáticas/genética , Alelos , Southern Blotting , Deleção Cromossômica , Sondas de DNA , HumanosRESUMO
We investigated the distribution of the two dopamine D2 receptor mRNA splice variants in the rat using a sensitive and quantitative solution hybridization/nuclease protection assay. In all brain and endocrine regions studied, both splice variants were detected and the mRNA of the longer form (D2L) was more abundant than that of the shorter form (D2S). The lowest percentages of D2S were found in the pituitary and adrenal glands.
Assuntos
Splicing de RNA , RNA Mensageiro/metabolismo , Receptores Dopaminérgicos/genética , Glândulas Suprarrenais/metabolismo , Animais , Autorradiografia , Encéfalo/metabolismo , DNA/metabolismo , Masculino , Hibridização de Ácido Nucleico , Hipófise/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos , Receptores Dopaminérgicos/biossíntese , Receptores de Dopamina D2Assuntos
Drosophila melanogaster/metabolismo , Biossíntese de Proteínas , Trifosfato de Adenosina/farmacologia , Aminoácidos/farmacologia , Animais , Cátions Monovalentes/farmacologia , Sistema Livre de Células , Ácidos Cicloexanocarboxílicos/farmacologia , Ditiotreitol/farmacologia , Drosophila melanogaster/citologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/embriologia , Eletroforese Descontínua , Fluoretos/farmacologia , Guanosina Trifosfato/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Leucina/metabolismo , Magnésio/farmacologia , Fosfoenolpiruvato/farmacologia , Potássio/farmacologia , Frações Subcelulares/metabolismo , TrítioAssuntos
Plantas/metabolismo , RNA de Transferência/biossíntese , Aminoacil-tRNA Sintetases/metabolismo , Animais , Benzoatos , Radioisótopos de Carbono , Cloroplastos/metabolismo , Cromatografia DEAE-Celulose , Cromatografia por Troca Iônica , Grão Comestível/metabolismo , Escherichia coli/enzimologia , Euglena/enzimologia , Fenilalanina , Desenvolvimento Vegetal , Plantas/enzimologia , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Fatores de Tempo , TrítioAssuntos
Alquilantes/farmacologia , Azirinas/farmacologia , Aberrações Cromossômicas/efeitos dos fármacos , Mutação/efeitos dos fármacos , Ácidos Sulfônicos/farmacologia , Animais , Drosophila , Genes Letais/efeitos dos fármacos , Genes Recessivos/efeitos dos fármacos , Masculino , Cromossomos Sexuais/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosAssuntos
Obtenção de Tecidos e Órgãos/normas , Morte Encefálica , Sistema Cardiovascular , Olho , Hospitais , Humanos , Rim , Pulmão , Ohio , Preservação de Órgãos , Relações Profissional-FamíliaRESUMO
BACKGROUND: Tissue factor (TF) is expressed widely at the subluminal surface of blood vessels and serves as the primary cellular initiator of the extrinsic pathway of blood coagulation. Lack of TF in mice resulted in lethality in utero, but human TF (huTF) expressed at low levels from a human minigene rescued null mice from prenatal death. Although these low-TF expressing transgenic mice developed to term, they had a significantly shorter life span and exhibited hemorrhage and fibrosis in the heart. METHODS: Human TF knock-in (TFKI) mice were generated by replacing the first two exons of the mouse (murine) TF (muTF) gene with the huTF complete coding sequence, thus placing it under the control of the endogenous muTF promoter. RESULTS: Expression of huTF in the TFKI mice was similar to muTF in wild-type (wt) mice. The TFKI mice showed no microscopic evidence of spontaneous hemorrhage in the heart, nor cardiac fibrosis at up to 18 months of age. Immunohistochemistry showed that huTF was expressed in cells surrounding blood vessels in TFKI mice. Coagulation activity of brain homogenates from TFKI mice was comparable with that from wt brain. Cardiac hemorrhage similar to that of the low-TF transgenic mice occurred in the TFKI mice when huTF was blocked by a neutralizing anti-huTF monoclonal antibody. CONCLUSION: We generated a transgenic mouse line that expresses huTF under the control of the endogenous muTF promoter at physiological levels. Our results suggest that huTF can fully reconstitute the murine coagulation system and mediate normal hemostasis.
Assuntos
Hemostasia/fisiologia , Tromboplastina/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/toxicidade , Encéfalo/metabolismo , Cardiomiopatias/induzido quimicamente , Epitopos/imunologia , Feminino , Regulação da Expressão Gênica , Genes Sintéticos , Hemorragia/induzido quimicamente , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/toxicidade , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/metabolismo , Especificidade de Órgãos , Pericitos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Especificidade da Espécie , Tromboplastina/antagonistas & inibidores , Tromboplastina/biossíntese , Tromboplastina/genética , Tromboplastina/imunologiaRESUMO
A cDNA for the rat dopamine D3 receptor containing a 113 bp deletion has been isolated. The segment deleted, encompassing the first extracellular loop and third transmembrane domain, alters the reading frame, introducing 19 amino acids not found in the full length receptor followed by a premature stop codon. This novel mRNA encodes a 109 amino acid protein containing two putative transmembrane domains. A similar variant cDNA for the human D3 receptor has also been identified.
Assuntos
Splicing de RNA , RNA Mensageiro/genética , Receptores de Dopamina D2 , Receptores Dopaminérgicos/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Deleção Cromossômica , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Endogâmicos , Receptores de Dopamina D3 , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in Neisseria gonorrhoeae and Neisseria meningitidis. The three additional ORFs, internal to the dcw cluster, are not homologous to dcw-related genes present in other bacterial species. Analysis of the N. meningitidis strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial dcw gene cluster. Reverse transcription-PCR analysis of RNA extracted from N. gonorrhoeae strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (dca, for division cluster competence associated), located between murE and murF, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that dca encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains dca is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of N. meningitidis, suggesting that dca is phase variable in this species. A deletion and insertional mutation was made in the dca gene of N. gonorrhoeae strain FA19 and N. meningitidis strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the dca-encoded gene product is an essential competence factor for gonococci.
Assuntos
Proteínas de Bactérias , Proteínas de Membrana/genética , Família Multigênica , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética , Transformação Bacteriana , Sequência de Aminoácidos , Transferência Genética Horizontal , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Mononucleosomes prepared from Drosophila melanogaster nuclei contain the four core histones H2A, H2B, H3, and H4 plus an additional histone-like, acid-soluble, chromosomal protein. It is probably the protein designated D2 by Alfageme et al. [Alfageme, C.R., Zweidler, A., Mahowald, A. & Cohen, L.H. (1974) J. Biol. Chem. 249, 3729-3736]. D2 elutes with histone H2A from a Bio-Gel P-100 column, but can be distinguished electrophoretically from H2A and from the other standard Drosophila core histones. The amino acid composition of D2 resembles the compositions of H2A and H2B. However, peptide mapping reveals that D2 is not a simple sequence variant of either H2A or H2B. D2 is present in nuclei from embryos and adult heads, and thus is not restricted to a narrowly defined developmental period. It is present in D. melanogaster and D. virilis, and thus appears to be conserved during the evolution of Drosophila. D2 is present in D. melanogaster chromatin with an approximate frequency of one molecule per five nucleosomes, and must therefore be associated with a subset of nucleosomes. The function of this protein is not known. Its presence in nucleosomes, evolutionary conservation, and comparatively large abundance all suggest that it is an important nucleosomal element. It will be interesting to learn whether this histone-like protein is encoded in a subset of the Drosophila histone gene cluster or is encoded separately.
Assuntos
Drosophila melanogaster/ultraestrutura , Histonas/metabolismo , Nucleossomos/ultraestrutura , Aminoácidos/análise , Animais , Drosophila melanogaster/embriologia , Cabeça , Fragmentos de Peptídeos/análiseRESUMO
It is concluded that chromatin fragments derived from irradiated chicken spermatozoa are not viable vectors for gene transfer. In three experiments conducted at sequential intervals over a period of 1 1/2 years, no marker traits were found in 1,065 G0 progeny from irradiated spermatozoa of Minnesota Dominant Marker males inseminated into recessive Rhode Island Red and White Leghorn females. The inability to secure transformants is ascribed to the following factors: a maximum of five and probably fewer potential vector fragments for each G0 progeny because of irradiation effect on spermatozoan ability to enter the germinal disc; uncertainty of DNA integrity from highly irradiated chromatin; no known mechanism for release of chromatin fragments from irradiated spermatozoa supernumerary pronuclei; and the uncertainty of selective integration into the zygotic nucleus.
RESUMO
The purpose of this study was to determine the prevalence of p53 overexpression in feline oral squamous cell carcinomas (SCC) and to determine, if any, the association between p53 overexpression and lifestyle factors and environmental exposures, including exposure to environmental tobacco smoke (ETS). Questionnaires concerning exposure to ETS and other environmental factors were sent to owners of cats presenting to the Harrington Oncology Program with a diagnosis of oral SCC between 1991 and 2000. Additionally, 23 formalin-fixed biopsy samples from these cats, with information regarding ETS, were evaluated immunohistochemically for p53 expression using the CM-1 clone and the avidin-biotin-horseradish peroxidase method. Of the 23 samples evaluated, 15 (65%) showed positive nuclear staining for the CM-1 clone. Tumor biopsy samples from cats exposed to any ETS were 4.5 times more likely to overexpress p53 than were tumors from unexposed cats (P = 0.19). Among cats with any ETS exposure, those with 5 years or longer of exposure were 7.0 times more likely to overexpress p53 (P = 0.38). Longhaired cats (P = 0.18) and female cats (P = 0.35) were also more likely to show p53 expression in their tumors. These results provide additional support for a relationship between oral SCC development and exposure to household ETS and may implicate p53 as a potential site for carcinogen-related mutation in this tumor.
Assuntos
Carcinoma de Células Escamosas/veterinária , Doenças do Gato/metabolismo , Expressão Gênica , Genes p53/fisiologia , Neoplasias Bucais/veterinária , Poluição por Fumaça de Tabaco , Animais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Doenças do Gato/etiologia , Doenças do Gato/patologia , Gatos , Imuno-Histoquímica , Neoplasias Bucais/etiologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Inquéritos e QuestionáriosRESUMO
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.