RESUMO
Sea turtles are vulnerable to climate change since their reproductive output is influenced by incubating temperatures, with warmer temperatures causing lower hatching success and increased feminization of embryos. Their ability to cope with projected increases in ambient temperatures will depend on their capacity to adapt to shifts in climatic regimes. Here, we assessed the extent to which phenological shifts could mitigate impacts from increases in ambient temperatures (from 1.5 to 3°C in air temperatures and from 1.4 to 2.3°C in sea surface temperatures by 2100 at our sites) on four species of sea turtles, under a "middle of the road" scenario (SSP2-4.5). Sand temperatures at sea turtle nesting sites are projected to increase from 0.58 to 4.17°C by 2100 and expected shifts in nesting of 26-43 days earlier will not be sufficient to maintain current incubation temperatures at 7 (29%) of our sites, hatching success rates at 10 (42%) of our sites, with current trends in hatchling sex ratio being able to be maintained at half of the sites. We also calculated the phenological shifts that would be required (both backward for an earlier shift in nesting and forward for a later shift) to keep up with present-day incubation temperatures, hatching success rates, and sex ratios. The required shifts backward in nesting for incubation temperatures ranged from -20 to -191 days, whereas the required shifts forward ranged from +54 to +180 days. However, for half of the sites, no matter the shift the median incubation temperature will always be warmer than the 75th percentile of current ranges. Given that phenological shifts will not be able to ameliorate predicted changes in temperature, hatching success and sex ratio at most sites, turtles may need to use other adaptive responses and/or there is the need to enhance sea turtle resilience to climate warming.
Assuntos
Tartarugas , Animais , Tartarugas/fisiologia , Temperatura , Mudança Climática , Reprodução , Razão de MasculinidadeRESUMO
BACKGROUND: In this work, we assessed the efficacy and safety of brentuximab vedotin (BV) plus ESHAP (BRESHAP) as second-line therapy for Relapsed/Refractory Hodgkin lymphoma (RRHL) to improve the results before autologous stem-cell transplantation (ASCT). PATIENTS AND METHODS: This was a multicenter, open-label, phase I-II trial of patients with RRHL after first-line chemotherapy. Treatment had three 21-day cycles of etoposide, solumedrol, high-dose AraC, and cisplatin. BV was administered at three dose levels (0.9, 1.2, and 1.8 mg/kg) intravenous on day â1 to 3 + 3 cohorts of patients. Final BV dose was 1.8 mg/kg. Responding patients proceeded to ASCT, followed by three BV courses (1.8 mg/kg, every 21 days). Main end points for evaluation were maximum tolerable dose and overall and complete response (CR) before ASCT. RESULTS: A total of 66 patients were recruited (median age 36 years; range 18-66): 40 were primary refractory, 16 early relapse and 10 late relapse. There were 39 severe adverse events were reported in 22 patients, most frequently fever (n = 25, 35% neutropenic), including 3 deaths. Grade 3-4 hematological toxicity presented in 28 cases: neutropenia (n = 21), thrombocytopenia (n = 14), and anemia (n = 7). Grade ≥3-4 extrahematological adverse events (≥5%) were non-neutropenic fever (n = 13) and hypomagnesaemia (n = 3). Sixty-four patients underwent stem-cell mobilization; all collected >2×10e6/kg CD34+ cells (median 5.75; range 2.12-33.4). Overall response before transplant was 91% (CI 84% to 98%), including 70% (CRs 95% CI 59% to 81%). 60 patients were transplanted with no failure engraftments. Post-transplant response was CR in 49 patients (82% CI 73% to 91%) and partial responses in six (10% CI 5% to 15%). After a mean follow-up of 27 months, the 30-month time to treatment to failure was 74% (95% CI 68% to 80%), progression-free survival 71% (95% CI 65% to 77%), and overall survival 91% (CI 84% to 98%). CONCLUSION: BRESHAP looks a safe and effective pre-transplant induction regimen, does not jeopardize transplant and allows long-term remissions and survival.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Brentuximab Vedotin/administração & dosagem , Neutropenia Febril Induzida por Quimioterapia/epidemiologia , Doença de Hodgkin/terapia , Recidiva Local de Neoplasia/terapia , Terapia de Salvação/métodos , Administração Intravenosa , Adolescente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Brentuximab Vedotin/efeitos adversos , Neutropenia Febril Induzida por Quimioterapia/etiologia , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Citarabina/administração & dosagem , Citarabina/efeitos adversos , Relação Dose-Resposta a Droga , Esquema de Medicação , Etoposídeo/administração & dosagem , Etoposídeo/efeitos adversos , Feminino , Seguimentos , Transplante de Células-Tronco Hematopoéticas , Doença de Hodgkin/mortalidade , Doença de Hodgkin/patologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante/efeitos adversos , Terapia Neoadjuvante/métodos , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Intervalo Livre de Progressão , Terapia de Salvação/efeitos adversos , Transplante Autólogo , Adulto JovemRESUMO
Autologous hematopoietic cell transplantation (AHCT) is the standard of care for young patients with relapsed/refractory (R/R) Hodgkin's lymphoma (HL). However, there is limited experience of its efficacy and feasibility in older patients. The characteristics and outcomes of 121 patients aged ≥50 years (42 of them are ≥60 years old) with R/R HL who underwent AHCT were reviewed. After a median follow-up of 3.1 years, overall survival (OS) and progression-free survival (PFS) at 5 years were 64 and 55 %, respectively, with no differences between 50-59-year-old and ≥60-year-old patients. Hematological and extra-hematological toxicities after AHCT were comparable between the two groups of age. In univariate analysis, poorer OS and PFS were associated with disease status other than complete remission, hematopoietic cell transplantation comorbidity index (HCT-CI) scores >1, and Charlson Comorbidity Index (CCI) scores >1. HCT-CI scores >1 were also associated with a higher risk of grade 3-4 extrahematologic toxicity. In multivariate analysis, HCT-CI and CCI remained significantly associated with OS and PFS after adjustment for disease status. Our data show that AHCT can be performed in selected patients with R/R HL ≥50 years with acceptable outcome and toxicity. Comorbidities appear to impact AHCT outcome more than age.
Assuntos
Transplante de Células-Tronco Hematopoéticas/tendências , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/terapia , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/terapia , Fatores Etários , Idoso , Comorbidade , Intervalo Livre de Doença , Feminino , Transplante de Células-Tronco Hematopoéticas/mortalidade , Doença de Hodgkin/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Valor Preditivo dos Testes , Estudos Retrospectivos , Transplante Autólogo/mortalidade , Transplante Autólogo/tendências , Resultado do TratamentoAssuntos
Ácidos Nucleicos Livres , Neoplasias , Metilação de DNA , Detecção Precoce de Câncer , HumanosRESUMO
Along the Pacific coast of the Baja California Peninsula (Mexico), abalone represents one of the most lucrative fisheries. As wild populations are currently depleted, abalone farm production aims to balance the decreasing populations with the increasing demand. The Mexican abalone aquaculture is almost entirely based on red abalone (Haliotis rufescens). However, the increasing frequency of extreme temperature events is hampering this activity. The use interspecific hybrids can potentially improve abalone culture, as species have differences in their thermal tolerance. Therefore, the hybrid progeny between H. rufescens (â) and pink abalone H. corrugata (â), a temperate and a warmer water abalone species, respectively, will naturally support higher temperature. To test this hypothesis, growth rate, mortality and metabolic rate of both pure (RR) and hybrid abalone (RP) were assessed under the H. rufescens' optimum (18 °C) and thermally stressed (22 °C) conditions. To unveil the molecular pathways involved in the heat response, transcriptional profiling of both crosses was also investigated. At high temperature, we observed constrained growth and survival in RR while RP showed a significant increase in both rates, supporting the improved performance of the hybrid compared. These results match with the transcriptional profiling of hybrids showing higher expression of genes involved in growth and calcification, whereas in the pure red progeny, the transcriptional profile was mainly associated with the regulation of necroptosis process. Our results may contribute to propose new management plans to increase farm abalone production in Baja California.
Assuntos
Gastrópodes/crescimento & desenvolvimento , Gastrópodes/genética , Hibridização Genética , Animais , Aquicultura , Metabolismo Basal/genética , Calcificação Fisiológica/genética , Feminino , Gastrópodes/metabolismo , Expressão Gênica , Temperatura Alta , Masculino , TranscriptomaRESUMO
Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.
Assuntos
Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas/metabolismo , Transporte Biológico/genética , Citosol/metabolismo , Citosol/fisiologia , Retículo Endoplasmático/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Microssomos/metabolismo , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Ligação Proteica/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Proteínas/genética , Canais de Translocação SEC , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Deleção de Sequência/genética , Especificidade por Substrato/genéticaRESUMO
The aim of this study was to evaluate the effect of chronic consumption of the GABAB agonist baclofen on temperature perception in humans. We investigated temperature perception thresholds to detect warm and cold stimuli in a group of 21 patients with spinal cord injury, who were chronically consuming oral baclofen at different daily doses to treat spasticity. Temperature perception thresholds were assessed above the level of the lesion, using a psychophysical approach based on the ability of the subjects to perceive precisely quantified sensory stimuli (quantitative sensory testing, QST). The data were compared with a control group of healthy subjects, not receiving baclofen. We found that chronic baclofen consumption increased temperature perception thresholds for both cold and warm stimuli in a dose-dependent manner. Temperature perception thresholds did not depend on the level of the lesion nor on the duration of baclofen treatment, suggesting that our finding represent normal GABAB-mediated modulation in spared nervous structures. We conclude that GABAB therefore plays a role in temperature perception in humans.
Assuntos
Baclofeno/farmacologia , Agonistas GABAérgicos/farmacologia , Limiar Sensorial/efeitos dos fármacos , Sensação Térmica/efeitos dos fármacos , Adolescente , Adulto , Análise de Variância , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estimulação Física , Psicofísica/métodos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Recently in Mexico the number of cosmetic surgeries has increased. These procedures are often carried out by unqualified people using obsolete and contraindicated products such as injectable oil, which cause uncorrectable disfigurement or more serious complications, even death, after reaching the systemic circulation. We report the case of a fat embolism syndrome (FES) caused by injections of vitamin E (tocopherol) in order to increase the volume of the buttocks. This case of a FES caused by injections of vitamin E was confirmed by gas chromatography coupled to mass spectrometry.
Assuntos
Nádegas , Embolia Gordurosa/induzido quimicamente , Vitamina E/intoxicação , Adulto , Cosméticos , Evolução Fatal , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Injeções , México , Síndrome , Vitaminas/intoxicação , Adulto JovemRESUMO
In bacteria, horizontal gene transfer (HGT) is widely recognized as the mechanism responsible for the widespread distribution of antibiotic resistance genes, gene clusters encoding biodegradative pathways and pathogenicity determinants. We propose that HGT is also responsible for speciation and sub-speciation in bacteria, and that HGT mechanisms exist in eukaryotes.
Assuntos
Bactérias/genética , Recombinação Genética , Bactérias/metabolismo , Bactérias/patogenicidade , Biodegradação Ambiental , Resistência Microbiana a Medicamentos , Evolução Molecular , VirulênciaRESUMO
alpha-Haemolysin is a protein toxin (107 kDa) secreted by some pathogenic strains of E. coli. It binds to mammalian cell membranes, disrupting cellular activities and lysing cells. This paper describes the mechanism of alpha-haemolysin-induced membrane leakage, from experiments in which extrusion large unilamellar vesicles, loaded with fluorescent solutes, are treated with purified toxin. The results show that the toxin does not require of any membrane receptor to exert its activity, that vesicles become leaky following an 'all-or-none' mechanism, and that leakage occurs through a non-osmotic detergent-like bilayer disruption induced by the protein. Small pores formed by monomeric alpha-haemolysin, as described by other authors, do not appear to be related to the process of membrane disruption. Instead, the experimental data would be in agreement with the idea of oligomeric assemblies being required to produce release of solutes from a single vesicle.
Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Proteínas de Escherichia coli , Proteínas Hemolisinas/farmacologia , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Detergentes/farmacologia , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Bicamadas Lipídicas/química , Lipídeos de Membrana/análise , Receptores de Superfície Celular/fisiologiaRESUMO
The region of the IncW plasmid R388 involved in conjugal DNA metabolism and mobilization (MOBw) has been analyzed by Tn5tac1 insertion mutagenesis, genetic complementation and DNA sequencing. Three genes (trwA, trwB and trwC) were mapped within MOBw. They are transcribed from the same strand and away from oriT. The predicted products of trwA, trwB and trwC are proteins of 121, 507 and 966 amino acids, respectively. The three proteins were visualized in a minicell expression system, showing apparent molecular masses of 13.5, 55 and 105 kDa, respectively. The deduced amino acid sequence of TrwA shows significant similarity to TraJ of the IncP plasmids RP4 and R751, to NikA of the IncI plasmid R64 and to MobB of plasmid pTF-FC2. The amino acid sequence of TrwB predicts an integral membrane protein which contains an NTP-binding motif. It shows 28% to 29% identity with TraD of plasmids F and R100, 23% identity with TraG of plasmids RP4 and R751 and 20% identity with VirD4 of the Ti plasmids of Agrobacterium tumefaciens. The amino acid sequence of TrwC shows the characteristic motifs of the Rep family of DNA helicases. It shows 33% identity with the sequence of helicase I (TraI) of plasmid F. The similarity is highest in the N-terminal segments of the proteins, which show conservation of characteristic amino acid motifs of a family of DNA-relaxases, including VirD2 of the Ti plasmid. The conserved features of these three proteins among the different transfer systems suggest that a very widespread conjugal DNA mobilization mechanism is shared by the transfer apparatuses of IncF, IncI, IncP, IncW and Ti plasmids.
Assuntos
Conjugação Genética/genética , Escherichia coli/genética , Genes Bacterianos/genética , Plasmídeos/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Mapeamento Cromossômico , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos/metabolismoRESUMO
TrwC is required for conjugal DNA transfer of the broad host range plasmid R388. The purified protein shows in vitro DNA helicase activity. Here we report that it also has in vitro oriT-endonuclease activity. TrwC specifically nicks oriT-containing supercoiled plasmid DNA in the presence of Mg2+, and the nicked DNA can be visualized after treatment with SDS. Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence. Both the 5' and the 3' ends of the nick were mapped. The 5' end was not accesible to phosphorylation by T4 polynucleotyde kinase, suggesting a covalent association with TrwC. Analysis of a collection of deletions in oriT indicated that the nucleotide sequences immediately surrounding the nic site are important, but not the only essential feature, for the nicking reaction. Comparison of the R388 nic site with previously published nic DNA sequences suggests that IncF, IncN and IncW plasmids form a family of related nic sites. During the course of this work we have also demonstrated a terminal transferase activity of Sequenase Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems.
Assuntos
Conjugação Genética/genética , DNA Nucleotidiltransferases/metabolismo , Desoxirribonuclease I/metabolismo , Integrases , Fatores R/genética , Sequência de Bases , DNA Super-Helicoidal/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Dados de Sequência Molecular , Fatores R/metabolismo , Recombinases , Alinhamento de Sequência , Deleção de Sequência/fisiologiaRESUMO
Plasmid R6K contains two functional origins or transfer (oriT), in contrast to previously characterized conjugative plasmids. The oriTs are formed by 98 bp palindromic sequences invertedly orientated with respect to each other and located in the immediate vicinity of the alpha and beta origins of replication. The gene for R6K oriT-nickase, taxC, was identified by transposon mutagenesis and sequenced, revealing that TaxC belongs to the VirD2 nickase family. The protein was overproduced and purified. It catalysed a cleaving-joining reaction on single-stranded DNA containing its target sequence. Identification of the nic sites suggested that the R6K oriTs belong to the RP4/VirD2 oriT family. Cleavage was highly specific and did not occur with oligonucleotides cleaved by related nickases like TraI of RP4 or VirD2 of the Ti plasmid. nic cleavage of in vivo preassembled relaxation complexes was induced by incubation of plasmid cleared lysates with ethidium bromide. Nicked molecules obtained in this way were treated with snake venom phosphodiesterase to produce double strand cleavages at the nic sites. 35% of the molecules were cleaved simultaneously at both nic sites, both in the case of R6K and of R6Kdrd1, a derepressed mutant whose frequency of transfer is 1000-fold higher. This figure represents the minimum percentage of individual R6K molecules containing two pre-assembled relaxation complexes.
Assuntos
Conjugação Genética/genética , Proteínas de Ligação a DNA/genética , Plasmídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos/metabolismo , Diester Fosfórico Hidrolases , Plasmídeos/metabolismo , Origem de Replicação , Análise de Sequência de DNARESUMO
TrwA protein was purified from an overproducing Escherichia coli strain and characterized as a 53 kDa tetrameric DNA-binding protein. Gel shift assays showed that TrwA bound specifically to the oriT sequence of plasmid R388. DNAse I footprinting analysis defined two DNA regions within oriT (sites A and B) that were protected by TrwA. At low TrwA concentrations only region A was protected (K(D) = 4 x 10(-8) M) while region B required higher TrwA concentrations (K(D) = 4 x 10(-7) M). As a result of its binding to oriT, TrwA was found to perform two biochemical activities related to its role in R388 conjugation. First, TrwA binding to oriT resulted in transcriptional repression of the trwABC operon as indicated by its effect on the beta-galactosidase activity of transcriptional fusions in trwB and trwC, and by direct measurement of the trwA mRNA levels by hybridization. This result was further confirmed by the fact that TrwA overexpression resulted in lowered conjugation frequencies. Second, TrwA enhanced the relaxation activity of TrwC in vitro. This effect was correlated to a 10(5)-fold increase in the frequency of conjugation in vivo and was shown to be independent of the regulation of transcription. Thus, TrwA shows functional similarities to protein TraY of F-like plasmids, that could be correlated to a structural similarity in their DNA-binding motifs.
Assuntos
Proteínas de Bactérias/genética , Conjugação Genética , Proteínas de Escherichia coli , Escherichia coli/genética , Integrases , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Recombinases , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genéticaRESUMO
TrwC is a bifunctional enzyme that displays two biochemical activities essential for plasmid R388 conjugation: oriT-specific DNA strand-transferase and DNA helicase activities. We overproduced and purified different segments of the protein allowing us to map the relaxase and DNA helicase activities to separate regions of the protein. A peptide comprising the N-terminal 275 amino acid residues of the protein was able to catalyze DNA cleavage and strand-transfer reactions when using oligonucleotides encompassing the nic site, although a longer fragment of TrwC (348 amino acid residues) was required to produce the nick on a supercoiled double-stranded DNA substrate. The segment of the protein between amino acid residues 192 and 966 contained the ATPase and DNA helicase activities, while a peptide consisting of amino acid residues 346 to 966 lost both activities. The dimerization region lay in the 495 C-terminal amino acid residues. Two peptides containing the DNA strand-transferase and DNA helicase activities, respectively, could functionally substitute for TrwC in R388 conjugation although at a 10,000-fold lower efficiency. Thus, integrity of the covalent structure of the protein was required for efficient DNA transfer. It can be assumed that the covalent linkage increases the efficiency of conjugation by increasing the effective concentration of one component (presumably the DNA helicase) at its site of action.
Assuntos
DNA Helicases/química , DNA Helicases/genética , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Integrases , Plasmídeos/genética , Sequência de Bases , Conjugação Genética , DNA Helicases/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Teste de Complementação Genética , Estrutura Molecular , Oligodesoxirribonucleotídeos/genética , Recombinases , Origem de Replicação , Especificidade por SubstratoRESUMO
Protein TrwC is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. Site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxation domain of the protein. Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein TrwC on oligonucleotides containing the nic site. Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide-protein covalent complexes that constitute presumed intermediates of these reactions. This hypothesis was confirmed by the observation of Y18 and Y26-specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives. Finally mutation Y18F, but not mutation Y26F, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT). These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation. The model suggests a control mechanism that can be effective at each conjugative replication cycle.
Assuntos
Conjugação Genética/genética , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/metabolismo , Escherichia coli/enzimologia , Integrases , Plasmídeos/genética , Tirosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sítios de Ligação , Catálise , Adutos de DNA/química , Adutos de DNA/genética , Adutos de DNA/metabolismo , DNA Nucleotidiltransferases/genética , DNA Super-Helicoidal/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Oligodesoxirribonucleotídeos/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Recombinases , Sequências Reguladoras de Ácido Nucleico/genética , Alinhamento de Sequência , Fatores de Tempo , Tirosina/genéticaRESUMO
OBJECTIVE: Repetitive transcranial magnetic stimulation (rTMS) has become a useful tool for investigating and even modulating human brain function. RTMS of the human motor cortex can produce changes in excitability that outlast the period of stimulation. To investigate the persistent effect of high-frequency rTMS of sensorimotor cortex (SM1) on somatosensory function. METHODS: We evaluated the thermal thresholds (cold and warm sensation) in 14 normal subjects before and after a short train of 5Hz rTMS over the SM1 or occipital cortex (OC). RESULTS: Threshold for cold perception was increased immediately after rTMS of the left SM1 and no effects at all were noticed after OC stimulation. There was a slight, not significant, increase of warm threshold immediately after the rTMS of the left SM1 and no effects at all were noticed after OC stimulation. CONCLUSIONS: High frequency rTMS over primary sensorimotor cortex seems to modulate sensory function related to thermal (cold) perception. SIGNIFICANCE: The method may be useful for both the study of normal human physiology of temperature perception and for rTMS based manipulation of brain plasticity in patients with sensory disturbances.
Assuntos
Estimulação Elétrica/efeitos adversos , Hipestesia/etiologia , Magnetismo , Córtex Somatossensorial/fisiopatologia , Temperatura , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Percepção/fisiologia , Limiar Sensorial/fisiologiaRESUMO
The immune system in marine invertebrates is mediated through cellular and humoral components, which act together to address the action of potential pathogenic microorganisms. In bivalve mollusks biomolecules implicated in oxidative stress and recognition of pathogens have been involved in the innate immune response. To better understand the molecular basis of the immune response of surf clam Mesodesma donacium, qPCR approaches were used to identify genes related to its immune response against Vibrio anguillarum infection. Genes related to oxidative stress response and recognition of pathogens like superoxide dismutase (MdSOD), catalase (MdCAT), ferritin (MdFER) and filamin (MdFLMN) were identified from 454-pyrosequencing cDNA library of M. donacium and were evaluated in mantle, adductor muscle and gills. The results for transcripts expression indicated that MdSOD, MdFLMN and MdFER were primarily expressed in the muscle, while MdCAT was more expressed in gills. Challenge experiments with the pathogen V. anguillarum had showed that levels of transcript expression for MdSOD, MdCAT, MdFER, and MdFLMN were positively regulated by pathogen, following a time-dependent expression pattern with significant statistical differences between control and challenge group responses (p<0.05). These results suggest that superoxide dismutase, catalase, ferritin and filamin, could be contributing to the innate immune response of M. donacium against the pathogen V. anguillarum.
Assuntos
Bivalves/genética , Bivalves/imunologia , Imunidade Inata/genética , Vibrio/imunologia , Animais , Sequência de Bases , Bivalves/microbiologia , Catalase/genética , Catalase/metabolismo , DNA Complementar/genética , Ferritinas/genética , Ferritinas/metabolismo , Filaminas/genética , Filaminas/metabolismo , Dados de Sequência Molecular , Músculos/metabolismo , Estresse Oxidativo/genética , Análise de Sequência de DNA , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
A new series of vectors, pSU2716, pSU2717, pSU2718, and pSU2719, has been constructed. The plasmids contain (i) the P15A replicon, (ii) the chloramphenicol acetyl transferase (CAT)-coding gene from Tn9, and (iii) the HaeII fragment which carries the multiple cloning site and the lacZ alpha reporter gene of pUC8, pUC9, pUC18 and pUC19, respectively. These vectors allow rapid and simple transfer of inserts from pUC plasmids, have an intermediate copy number (which allows regulated expression from the lac promoter), and are compatible with ColE1-derived vectors (and, therefore, can be used in studies requiring the joint expression of two genes, for example, in genetic complementation analysis). Furthermore, the accumulation of CAT instead of beta-lactamase, allows an easy visualization in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of proteins of 28-35 kDa, which can otherwise be obscured by the beta-lactamase.