RESUMO
For millennia, numerous cultures and civilizations have relied on traditional remedies derived from plants to treat a wide range of conditions and ailments. Here, we systematically analyzed ethnobotanical patterns across taxonomically related plants, demonstrating that congeneric medicinal plants are more likely to be used for treating similar indications. Next, we reconstructed the phytochemical space covered by medicinal plants to reveal that (i) taxonomically related medicinal plants cover a similar phytochemical space, and (ii) chemical similarity correlates with similar therapeutic usage. Lastly, we present several case scenarios illustrating how mining this information can be used for drug discovery applications, including: (i) investigating taxonomic hotspots around particular indications, (ii) exploring shared patterns of congeneric plants located in different geographic areas, but which have been used to treat the same indications, and (iii) showing the concordance between ethnobotanical patterns among non-taxonomically related plants and the presence of shared bioactive phytochemicals.
RESUMO
Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.9 milligrams per deciliter lower LDL-C (P = 4.1 × 1019) and 29 milligrams per deciliter lower plasma fibrinogen (P = 1.3 × 105). B4GALT1 genebased analysis in 544,955 subjects showed an association with decreased coronary artery disease (odds ratio = 0.64, P = 0.006). The mutant protein had 50% lower galactosyltransferase activity compared with the wild-type protein. N-linked glycan profiling of human serum found serine 352 allele to be associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. B4galt1 353Ser knock-in mice showed decreases in LDL-C and fibrinogen. Our findings suggest that targeted modulation of protein galactosylation may represent a therapeutic approach to decreasing cardiovascular disease.
Assuntos
LDL-Colesterol/sangue , Fibrinogênio/análise , Galactosiltransferases/genética , Mutação de Sentido Incorreto , Animais , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/prevenção & controle , Feminino , Galactose/metabolismo , Galactosiltransferases/metabolismo , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Glicoproteínas/sangue , Glicosilação , Humanos , Fígado/enzimologia , Masculino , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/sangue , Sequenciamento Completo do GenomaRESUMO
A radiological dispersive device (RDD) spreads radioactive material, complicates the treatment of physical injuries, raises cancer risk, and induces disproportionate fear. Simulating such an event enables more effective and efficient utilization of the triage and treatment resources of staff, facilities, and space. Fast simulation can give detail on events in progress or future events. The resources for triage and treatment of contaminated trauma victims can differ for pure exposure individuals, while discouraging the "worried well" from presenting in the crisis phase by media announcement would relieve pressure on hospital facilities. The proposed methodology integrates capabilities from different platforms in a convergent way composed of three phases: (a) scenario simulation, (b) data generation, and (c) risk assessment for triage focused on follow-up epidemiological assessment. Simulations typically indicate that most of the affected population does not require immediate medical assistance. Medical triage for the few severely injured and the radiological triage to diminish the contamination with radioactivity will always be the priority. For this study, however, higher priorities should be given to individuals from radiological "warm" and "hot" zones as required by risk criteria. The proposed methodology could thus help to (a) filter and reduce the number of individuals to be attended, (b) optimize the prioritization of medical care,
Assuntos
Radioisótopos de Césio/toxicidade , Planejamento em Desastres , Saúde Pública , Liberação Nociva de Radioativos , Medição de Risco , Triagem , Adulto , Feminino , Humanos , Masculino , Densidade Demográfica , Terrorismo , Adulto JovemRESUMO
BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but â¼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Farmacorresistência Viral/genética , HIV-1/metabolismo , Humanos , Succinatos/farmacologia , Triterpenos/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
A radiological dispersal device (RDD) is a simple weapon capable of causing human harm, environmental contamination, disruption, area denial, and economic cost. It can affect small, large, or long areas depending on atmospheric stability. The risk of developing a radio-induced cancer depends on exposure, and an effective response depends upon available timely guidance. This article proposes and demonstrates a convergence of three different capabilities to assess risk and support rapid safe resource efficient response. The three capabilities that are integrated are Hotspot for dispersion, RERF for epidemiological risk, and RESRAD-RDD for response guidance. The combined methodology supports decisions on risk reduction and resource allocation through work schedules, the designation and composition of response teams, and siting for operations. In the illustrative RDD scenario, the contamination area for sheltering, evacuation, and long-term public concern was greatest for calm atmospheric conditions, whilst close-quarter responders faced highest dose rates for neutral atmospheric conditions. Generally, the risks to women responders were found to be significantly greater than for men, and the risks to 20-year-old responders were three times that of their 60-year-old counterparts for similar exposure.
Assuntos
Liberação Nociva de Radioativos , Medição de Risco/métodos , Fatores Etários , Planejamento em Desastres , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Neoplasias Induzidas por Radiação/diagnóstico , Neoplasias Induzidas por Radiação/epidemiologia , Exposição Ocupacional , Fatores Sexuais , Tempo (Meteorologia) , Adulto JovemRESUMO
OBJECTIVES: In an 8 day monotherapy study of subjects infected with HIV-1 (subtype B) (NCT01009814), BMS-626529 (an attachment inhibitor that binds to HIV-1 envelope glycoprotein gp120), administered as the prodrug BMS-663068, produced substantial declines in plasma HIV-1 RNA. However, large variability in susceptibility to BMS-626529 was noted and virus with low susceptibility was less likely to be suppressed by BMS-663068 administration. The current analysis sought to investigate the genotypic correlates of susceptibility to BMS-626529. METHODS: In vitro selection experiments, evaluation of clinical samples of subtype B from the monotherapy study and evaluation of intrinsically resistant subtype AE viruses were conducted. Reverse genetics was used to identify key substitutions in envelope clones responsible for reduced susceptibility. RESULTS: An M426L or S375M change were the major substitutions associated with reductions in susceptibility to BMS-626529 in baseline samples of subtype B viruses from the monotherapy study, with M434I and M475I contributing to a lesser extent. Class resistance in subtype AE viruses was mapped to 375H and 475I substitutions, found in the vast majority of these viruses. Analysis of multiple envelope clones from infected subjects showed higher intrasubject variability in susceptibility to BMS-626529 compared with other classes of entry inhibitors. CONCLUSIONS: These data define key genotypic substitutions in HIV-1 gp120 that could confer phenotypic resistance to BMS-626529.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , Organofosfatos/farmacologia , Piperazinas/farmacologia , Pró-Fármacos/farmacologia , Triazóis/farmacologia , Substituição de Aminoácidos , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Organofosfatos/uso terapêutico , Piperazinas/uso terapêutico , Pró-Fármacos/uso terapêutico , Genética Reversa , Análise de Sequência de DNA , Triazóis/uso terapêuticoRESUMO
BACKGROUND: BMS-663068 is the phosphonooxymethyl prodrug of BMS-626529, a small-molecule attachment inhibitor that targets the HIV-1 envelope glycoprotein gp120 preventing it from binding to CD4 T cells. In vitro investigations have demonstrated considerable variation in susceptibility of different HIV-1 isolates to BMS-626529. BMS-663068 monotherapy in HIV-1-infected subjects produced a mean maximum change from baseline of -1.64 log10 copies per milliliter, but the response was variable. METHODS: In this analysis, baseline and day 8 samples were analyzed for susceptibility to BMS-626529 and the presence of known HIV-1 attachment inhibitor resistance mutations. In addition, predictors of virological response (maximal HIV-1 RNA decline ≥1 log10 copies per milliliter) and resistance selection were investigated. RESULTS: The only factor associated with reduced virological response was low baseline susceptibility to BMS-626529. There was no apparent relationship between virological response and baseline treatment experience, coreceptor tropism, plasma HIV-1 RNA level, or CD4 T-cell count. Examination of all positions with known BMS-626529 resistance mutations based on in vitro selection studies showed that gp120 M426L was the primary substitution most clearly associated with nonresponse to BMS-663068. There was minimal change in susceptibility to BMS-626529 over the course of the study and no clear evidence of emergence of a known HIV-1 attachment inhibitor resistance mutation in the majority of subjects as measured by standard population-based phenotypic and genotypic approaches. CONCLUSIONS: Nonresponse to BMS-663068 was associated with low baseline susceptibility to BMS-626529 and the presence of M426L. In this short-term trial, there was minimal evidence of selection for BMS-626529 high-level resistance over 8 days of monotherapy with BMS-663068 by population-based approaches.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/efeitos dos fármacos , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Organofosfatos/uso terapêutico , Piperazinas/uso terapêutico , Pró-Fármacos/farmacologia , Triazóis/uso terapêutico , Carga Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Suscetibilidade a Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Feminino , Inibidores da Fusão de HIV/administração & dosagem , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Masculino , Mutação de Sentido Incorreto , Pró-Fármacos/uso terapêutico , RNA Viral/sangue , Resultado do TratamentoRESUMO
Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.
Assuntos
Antivirais/farmacologia , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Cristalografia por Raios X , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Hidrodinâmica , Camundongos , Modelos Moleculares , Nucleoproteínas/ultraestrutura , Orthomyxoviridae/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Bibliotecas de Moléculas Pequenas/uso terapêutico , SoluçõesRESUMO
Common bone and soft tissue tumors in the foot and ankle are described in this article, and x-ray and magnetic resonance imaging characteristics are given. Ultrasound can be used for limited indications only, noting that ultrasound features are nonspecific. Of the bone and soft tissue tumors, approximately 7% occur in the foot and ankle. Soft tissue tumors are more common than bone tumors. Tumors of the foot and ankle are generally benign or nonneoplastic. Patients with suspected malignant lesions should be referred to a specialized bone tumor unit before biopsy.
Assuntos
Tornozelo/patologia , Neoplasias Ósseas/diagnóstico , Pé/patologia , Neoplasias de Tecidos Moles/patologia , Tornozelo/diagnóstico por imagem , Neoplasias Ósseas/diagnóstico por imagem , Diagnóstico por Imagem , Pé/diagnóstico por imagem , Humanos , RadiografiaRESUMO
BACKGROUND: The purpose of this study was to reconsider current recommended treatment guidelines for vasculogenic claudication by examining the contemporary results of surgical intervention. STUDY DESIGN: We performed a retrospective review of 1,000 consecutive limbs in 669 patients treated for medically refractory vasculogenic claudication and prospectively followed. Outcomes measured included procedural complication rates, reconstruction patency, limb salvage, maintenance of ambulatory status, maintenance of independent living status, survival, symptom resolution, and symptom recurrence. RESULTS: Of the 1,000 limbs treated, endovascular therapy was used in 64.3% and open surgery in 35.7% of patients; aortoiliac occlusive disease was treated in 70.1% and infrainguinal disease in 29.9% of patients. The overall 30-day periprocedural complication rate was 7.5%, with no notable difference in complication rates when comparing types of treatment or levels of disease. Overall reconstruction primary patency rates were 87.7% and 70.8%; secondary patencies were 97.8% and 93.9%; limb salvage, 100% and 98.8%; and survivals, 95.4% and 76.9%, at 1 and 5 years, respectively. More than 96% of patients maintained independence and ambulatory ability at 5 years. Overall symptom resolution occurred in 78.8%, and symptom recurrence occurred in 18.1% of limbs treated, with slightly higher resolution and recurrence noted in patients treated with endovascular therapy. CONCLUSIONS: Contemporary treatment of vasculogenic claudication is safe, effective, and predominantly endovascular. These data support a more liberal use of revascularization for patients with claudication and suggest that current nonoperative treatment guidelines may be based more on surgical dogma than on achievable outcomes.
Assuntos
Claudicação Intermitente/cirurgia , Extremidade Inferior/irrigação sanguínea , Adulto , Idoso , Idoso de 80 Anos ou mais , Angioplastia com Balão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Medição de Risco , Stents , Resultado do Tratamento , Procedimentos Cirúrgicos VascularesRESUMO
BACKGROUND: Superior mesenteric vein injuries are rare and incur high mortality. Given their low incidence, little data exist delineating indications for when to institute primary repair versus ligation. The purposes of this study are to review our institutional experience, to determine the additive effect on mortality of associated vascular injuries, to correlate mortality with the American Association for the Surgery of Trauma-Organ Injury Scale (AAST-OIS) for abdominal vascular injury and to examine and define the indications and outcomes for primary repair versus ligation. MATERIAL: Retrospective 156 months study (January 1992 through December 2004) in a large Level I urban trauma center of all patients admitted with superior mesenteric vein injuries. Patients were stratified, according to surgical technique employed to deal with their injuries, into those undergoing primary repair versus ligation to determine outcomes and define the surgical indications of these methods. The main outcome measure was overall survival. Cases of survival were stratified according to surgical method: primary repair versus ligation. RESULTS: There were 51 patients with a mean Injury Severity Score of 25 +/- 12. Mechanism of injury was penetrating for 38 (76%), blunt for 13 (24%), and patients undergoing emergency department thoracotomy for 4 (8%). Surgical management was ligation for 30 (59%), primary repair for 16 (31%), and 5 (10%) patients were exsanguinated before repair. The overall survival rate was 24/50 (47%). The survival rate excluding patients undergoing emergency department thoracotomy was 51%. The survival rate excluding patients that sustained greater than 3 to 4 associated vessels injured was 65%. The survival rates of patients with superior mesenteric vein and superior mesenteric artery was 55% and superior mesenteric vein and portal vein (PV) was 40%. The survival rate of patients with isolated superior mesenteric vein injuries was 55%. Mortality stratified to AAST-OIS grade III, 44%; grade IV, 42%; and grade V, 42%. Survival rates stratified to method of management consisted of primary repair (60%) versus ligation (40%). CONCLUSIONS: SMV injuries are highly lethal. Multiple associated vessel injuries increase mortality. Mortality correlates well with the American Association for the Surgery of Trauma-Organ Injury Scale for abdominal vascular injuries. Patients undergoing primary repair have higher survival rates (63%) and lesser numbers of associated vascular and nonvascular injuries; whereas those undergoing ligation have a smaller survival rate (40%) and higher number of associated vascular and nonvascular injuries. Ligation appears to be safe and should be selected for hemodynamically unstable patients with a large number of associated injuries.
Assuntos
Veias Mesentéricas/lesões , Veias Mesentéricas/cirurgia , Abdome/irrigação sanguínea , Traumatismos Abdominais/etiologia , Traumatismos Abdominais/mortalidade , Adulto , Vasos Sanguíneos/lesões , Feminino , Humanos , Escala de Gravidade do Ferimento , Ligadura , Masculino , Taxa de Sobrevida , Traumatismos Torácicos/etiologia , Traumatismos Torácicos/mortalidade , Procedimentos Cirúrgicos Vasculares , Ferimentos e Lesões/mortalidadeRESUMO
BLAST is a widely used genetic sequence comparison program developed at the National Center for Biotechnology Information (NCBI). In this unit, three Basic Protocols and one Support Protocol are provided for general-purpose BLAST searches on the NCBI and ENSEMBL Web-accessible BLAST servers. Key parameters affecting how the search algorithm works are reviewed, with advice on modifying search parameters for specific situations. Many other public and private Web sites offer BLAST interfaces which may differ from those described in this unit, but the general principles will be similar. The Support Protocol describes how to obtain sequences in various formats from NCBI for use in BLAST searches. It is emphasized that no algorithm can be a substitute for biological understanding; performing a BLAST search takes only a few minutes but understanding the implications of the results takes much longer.
Assuntos
Biologia Computacional/métodos , Alinhamento de Sequência/métodos , Software , Algoritmos , Interface Usuário-ComputadorRESUMO
BACKGROUND: The authors compared the microtensile bond strength of teeth restored with four adhesives at the gingival and pulpal cavity walls of Class II resin-based composite restorations. METHODS: Five pairs of extracted third molars received two Class II preparations/restorations in each tooth. The authors randomly assigned each preparation to one of four adhesive groups: Adper Scotchbond Multipurpose Dental Adhesive (SBMP) (3M ESPE, St. Paul, Minn.), Clearfil SE Bond (CFSE) (Kuraray America, New York City), Prime & Bond NT (PBNT) (Dentsply Caulk, Milford, Del.) and PQ1 (Ultradent, South Jordan, Utah). They restored the teeth and obtained microtensile specimens from each cavity wall. Specimens were tested on a testing machine until they failed. RESULTS: The mean (+/- standard deviation) bond strengths (in megapascals) were as follows: SBMP (pulpal), 36.4 (17.2); SBMP (gingival), 29.7 (15.3); CFSE (pulpal), 50.8 (13.6); CFSE (gingival), 50.2 (14.0); PBNT (pulpal), 38.3 (19.2); PBNT (gingival), 38.9 (17.7); PQ1 (pulpal), 58.7 (8.7); and PQ1 (gingival), 54.5 (18.5). A two-way analysis of variance found an adhesive effect (P < .001) but no location effect (P >.05). CONCLUSIONS: PQ1 and CFSE performed the best. The results showed no significant difference in microtensile bond strength at the gingival wall versus the pulpal wall. CLINICAL IMPLICATIONS: Under in vitro conditions, a total-etch ethanol-based adhesive (PQ1) failed cohesively more often than did the other adhesives tested.
Assuntos
Adesivos/química , Colagem Dentária , Restauração Dentária Permanente/classificação , Adesivos Dentinários/química , Dentina/ultraestrutura , Condicionamento Ácido do Dente , Resinas Compostas/química , Preparo da Cavidade Dentária/classificação , Polpa Dentária , Etanol/química , Gengiva , Humanos , Teste de Materiais , Ácidos Polimetacrílicos/química , Cimentos de Resina/química , Solventes/química , Estresse Mecânico , Resistência à TraçãoRESUMO
The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.
Assuntos
Antifúngicos/farmacologia , Candida albicans/genética , Sequência Conservada , Desenho de Fármacos , Genes Fúngicos , Regiões Promotoras Genéticas/genética , Sequência de Bases , Candida albicans/efeitos dos fármacos , Biologia Computacional/métodos , Testes de Sensibilidade Microbiana/métodos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.
Assuntos
Proteínas de Bactérias/química , Metiltransferases/química , Streptococcus pneumoniae/enzimologia , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
The Framesearch algorithm includes the possibility of a frameshift error in its alignment algorithm, and therefore can find alignments that span different reading frames. Protocols in this unit describe the use of Framesearch to search a protein sequence database for sequences that are similar to a query nucleotide sequence, and to search a nucleotide sequence database for sequences that are similar to a query protein sequence. Three alternate protocols describe ways to improve the speed of Framesearch and thus make it practical for routine use. Framesearch is especially appropriate for low-quality single-read nucleotide sequence data, such as ESTs (expressed sequence tags) or early drafts of genomic sequences; it does not offer any significant advantage over less CPU-intensive algorithms for relatively high-quality nucleotide sequences without many single-nucleotide insertion or deletion errors.