RESUMO
Real time measurement of melt rheology has been investigated as a Process Analytical Technology (PAT) to monitor hot melt extrusion of an Active Pharmaceutical Ingredient (API) in a polymer matrix. A developmental API was melt mixed with a commercial copolymer using a heated twin screw extruder at different API loadings and set temperatures. The extruder was equipped with an instrumented rheological slit die which incorporated three pressure transducers flush mounted to the die surface. Pressure drop measurements within the die at a range of extrusion throughputs were used to calculate rheological parameters, such as shear viscosity and exit pressure, related to shear and elastic melt flow properties, respectively. Results showed that the melt exhibited shear thinning behavior whereby viscosity decreased with increasing flow rate. Increase in drug loading and set extrusion temperature resulted in a reduction in melt viscosity. Shear viscosity and exit pressure measurements were found to be sensitive to API loading. These findings suggest that this technique could be used as a simple tool to measure material attributes in-line, to build better overall process understanding for hot melt extrusion.
Assuntos
Química Farmacêutica/métodos , Composição de Medicamentos/instrumentação , Composição de Medicamentos/métodos , Reologia/métodos , Varredura Diferencial de Calorimetria , Elasticidade , Polímeros , Pressão , Temperatura , Termogravimetria , ViscosidadeRESUMO
The next generation of gene-based crop models offers the potential of predicting crop vegetative and reproductive development based on genotype and weather data as inputs. Here, we illustrate an approach for developing a dynamic modular gene-based model to simulate changes in main stem node numbers, time to first anthesis, and final node number on the main stem of common bean (Phaseolus vulgaris L.). In the modules, these crop characteristics are functions of relevant genes (quantitative trait loci (QTL)), the environment (E), and QTL × E interactions. The model was based on data from 187 recombinant inbred (RI) genotypes and the two parents grown at five sites (Citra, FL; Palmira, Colombia; Popayan, Colombia; Isabela Puerto Rico; and Prosper, North Dakota). The model consists of three dynamic QTL effect models for node addition rate (NAR, No. d- 1), daily rate of progress from emergence toward flowering (RF), and daily maximum main stem node number (MSNODmax), that were integrated to simulate main stem node number vs. time, and date of first flower using daily time steps. Model evaluation with genotypes not used in model development showed reliable predictions across all sites for time to first anthesis (R2 = 0.75) and main stem node numbers during the linear phase of node addition (R2 = 0.93), while prediction of the final main stem node number was less reliable (R2 = 0.27). The use of mixed-effects models to analyze multi-environment data from a wide range of genotypes holds considerable promise for assisting development of dynamic QTL effect models capable of simulating vegetative and reproductive development.
RESUMO
Bordetella bronchiseptica PagP (PagPBB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection. B. parapertussis contains a putative pagP homolog (encoding B. parapertussis PagP [PagPBPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-type B. parapertussis lipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagPBPa is required for the modification of lipid A with palmitate. While PagPBB transfers a single palmitate to the lipid A C-3' position, PagPBPa transfers palmitates to the lipid A C-2 and C-3' positions. The addition of two palmitate acyl chains is unique to B. parapertussis. Mutation of pagPBPa resulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.
Assuntos
Aciltransferases/metabolismo , Bordetella parapertussis/enzimologia , Lipídeo A/metabolismo , Palmitatos/metabolismo , Aciltransferases/genética , Bordetella parapertussis/química , Bordetella parapertussis/genética , Análise Mutacional de DNA , Lipídeo A/química , Espectrometria de MassasRESUMO
Dialkyl lecithin dispersions in water exhibit two phase transitions upon cooling from the lamellar phase (L(α)). At the main transition (T(M)) the L(α) phase changes to a ripple (gel) phase (P(ß')) which then transforms to a second gel phase (L(ß')) at the "pretransition" (T(P)). We have made accurate density measurements through the various phases for two lecithins having unequal chains: 1-myristoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (MSPC) and 1-stearoyl-2-myristoyl-sn-glycero-3-phosphatidylcholine (SMPC). The measurements were carried out over five heat/cool cycles from 5 to 55 °C, followed by cooling back to 5 °C. The samples were then held at 50 °C for 24 hours, followed by a further three cool/heat cycles. For SMPC we observe an increase in density of the gel phases over the first 5 cycles, followed by much smaller changes after incubation at 50 °C. The lamellar phase also shows an increase in density, albeit much smaller. This parallels the behaviour of 1,2-di-palmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-di-myristoyl-sn-glycero-3-phosphatidylcholine (DMPC) reported earlier (Jones et al., Liquid Crystals 32, 1465 (2005)). For MSPC we observe a decrease in density within the gel phases while T(P) almost disappears after the first cycle. The lamellar phase shows little evidence of any change with each cycle. Within the lamellar phases there is a marked reduction in density on approaching T(M), which is attributed to the formation of transitory gel phase domains. Additional measurements by DSC and X-ray diffraction show that the changes in densities are not accompanied by large changes in transition enthalpies or phase structures. NMR data indicate that the pretransitional event within the L(α) phase is accompanied by ordering of the alkyl chains. The results indicate that the exact nature of the lipid alkyl chains could play a key role in the formation of gel phase patches within membrane bilayers. Their detailed chemical structures merit more attention than by simply assuming a uniform "bending energy" to describe the behaviour.
Assuntos
Géis/química , Fosfatidilcolinas/química , Varredura Diferencial de Calorimetria , Espectroscopia de Ressonância Magnética , Transição de Fase , Difração de Raios XRESUMO
PagL and LpxO are enzymes that modify lipid A. PagL is a 3-O deacylase that removes the primary acyl chain from the 3 position, and LpxO is an oxygenase that 2-hydroxylates specific acyl chains in the lipid A. pagL and lpxO homologues have been identified in the genome of Bordetella bronchiseptica, but in the current structure for B. bronchiseptica lipid A the 3 position is acylated and 2-OH acylation is not reported. We have investigated the role of B. bronchiseptica pagL and lpxO in lipid A biosynthesis. We report a different structure for wild-type (WT) B. bronchiseptica lipid A, including the presence of 2-OH-myristate, the presence of which is dependent on lpxO. We also demonstrate that the 3 position is not acylated in the major WT lipid A structures but that mutation of pagL results in the presence of 3-OH-decanoic acid at this position, suggesting that lipid A containing this acylation is synthesized but that PagL removes most of it from the mature lipid A. These data refine the structure of B. bronchiseptica lipid A and demonstrate that pagL and lpxO are involved in its biosynthesis.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/enzimologia , Bordetella bronchiseptica/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Lipídeo A/biossíntese , Lipídeo A/química , Oxigenases/metabolismo , Proteínas de Bactérias/genética , Bordetella bronchiseptica/genética , Hidrolases de Éster Carboxílico/genética , Ácidos Láuricos/análise , Miristatos/análise , Oxigenases/genéticaRESUMO
Clinical governance is the structured approach to maintaining and improving the quality of patient care and is a vital part of global surgery. BFIRST and BSSH closely collaborate with local doctors on a number of overseas projects, seeking to strengthen and develop local knowledge and skills, aiming for an independent local practice in reconstructive and upper limb surgery. Thoughts on essential requirements, improvements and pitfalls in the ethical approach to global collaboratives are presented.
Assuntos
Missões Médicas/organização & administração , Procedimentos de Cirurgia Plástica/normas , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Assistência ao Convalescente , Fortalecimento Institucional/organização & administração , Saúde Global , Humanos , Consentimento Livre e Esclarecido , Garantia da Qualidade dos Cuidados de Saúde/métodosRESUMO
The solution and liquid crystalline phases formed by dissolution of the dye Edicol Sunset Yellow (ESY) in water have been examined using optical microscopy, multinuclear NMR (1H, 2H, 13C, 23Na), and X-ray diffraction. From the solution 1H and 13C spectra (particularly 13C), it is clear that the tautomeric form present in all these phases is the hydrazone, NH, structure, not the usually given azo, OH, form. Two chromonic mesophases occur: a nematic (N) phase at approximately 30-40 wt % and a hexagonal (M) phase at approximately 40-45 wt %. X-ray diffraction data show that the aggregates in the mesophases are single molecule stacks, with a typical spacing of approximately 3.5 angstroms, as expected for these systems. The NMR quadrupole splittings (2H2O, 23Na) are similar to those observed for surfactant lyotropic mesophases, suggesting that there are no water molecules or counter ions that are tightly bound to the ESY aggregates. An unusual feature of the X-ray diffraction pattern of the mesophases is the occurrence of diffuse off-axis reflections at approximately 6.8 angstroms. It is proposed that these arise from a head-to-tail packing of the molecules within the stacks.
RESUMO
Soil carbon (C) sequestration has been proposed as a transitional win-win strategy to help replenish organic-matter content in depleted agricultural soils and counter increases in atmospheric greenhouse gases. Data assimilation and remote sensing can reduce uncertainty in sequestered C mass estimates, but simple soil organic carbon (SOC) models are required to make operational predictions of tradeable amounts over large, heterogenous areas. Our study compared the performance of RothC26.3 and a reduced compartmental model on an 11-year fertilizer trial in subhumid West Africa. Root mean square error (RMSE) differences of 0.05 Mg C/ha between models on total SOC predictions suggest that for contractual purposes, SOC dynamics can be simulated by a two-pool structure with labile and stable components. Faster (seasonal) and slower (semicentennial and beyond) rates can be approximated by constants as instantaneous and infinite decay. In these systems, simulations indicate that cereal residue incorporation holds most potential for mitigation of transient C loss associated with recent land conversion to agriculture.
Assuntos
Biomassa , Carbono/metabolismo , Ecossistema , Modelos Biológicos , Solo/análise , Monitoramento AmbientalRESUMO
Mosquitoes were collected in the Amazon Basin, near Iquitos, Peru, and used in experimental studies to evaluate their susceptibility to strains of eastern equine encephalitis virus (EEEV) that were isolated from mosquitoes captured within 20 km of Iquitos. When fed on hamsters or chickens with a viremia of 4105 plaque-forming units (PFU) of EEEV/ml, Culex pedroi Sirivanakarn and Belkin, Aedesfulvus (Wiedemann), Psorophora albigenu (Peryassu), and Psorophoraferox (Von Humboldt) were susceptible to infection, whereas none of the Aedes serratus (Theobald), Culex vomerifer Komp, Culex gnomatos Sallum, Huchings, and Ferreira, Culex portesi Senevet and Abonnenc, or Culex coronator Dyar and Knab became infected, even though they fed on the same viremic blood sources. When these mosquito species fed on animals with viremias of approximately 10(8) PFU/ml, Cx. pedroi, Ae.II (Brazil-Peru) and a lineage III (Argentina-Panama) isolate of EEEV. This study, combined with the repeated isolation of strains of EEEV from Cx. pedroi captured in the Amazon Basin region of Peru, suggests that Cx. pedroi may be the primary enzootic vector of EEEV in this region.
Assuntos
Culicidae/virologia , Vírus da Encefalite Equina do Leste/patogenicidade , Aedes/crescimento & desenvolvimento , Aedes/virologia , Animais , Galinhas , Cricetinae , Culex/crescimento & desenvolvimento , Culex/virologia , Culicidae/crescimento & desenvolvimento , Encefalomielite Equina do Leste/prevenção & controle , Encefalomielite Equina do Leste/transmissão , Encefalomielite Equina do Leste/veterinária , Feminino , Predisposição Genética para Doença , PeruRESUMO
DETAILED STUDIES OF THE HIGH DENSITY LIPOPROTEINS FROM THREE PATIENTS WITH ABETALIPOPROTEINEMIA HAVE REVEALED THE FOLLOWING PRINCIPAL ABNORMALITIES: 1) High density lipoprotein 3 (HDL3) is reduced in both absolute and relative concentration, although HDL2 is present in normal amounts. 2) The phospholipid distribution of both HDL fractions is abnormal, with low concentrations of lecithin and an increased percentage (though normal absolute quantity) of sphingomyelin. 3) In both HDL fractions, lecithin contains less linoleate and more oleate than normal. The cholesteryl esters are also low in linoleic acid, and the sphingomyelin is high in nervonic acid. Dietary intake influences the linoleic acid concentration within 2 weeks, and perhaps sooner, but the elevated sphingomyelin nervonic acid is little affected by up to 6 months of corn oil supplementation. Qualitatively similar changes in fatty acid composition, but not phospholipid distribution, are also found in other malabsorption states. The available evidence suggests that the abnormally low levels of HDL3 and the deranged phospholipid distribution are more specific for abetalipoproteinemia than the fatty acid abnormalities. However, the absence of these abnormalities in obligate heterozygous subjects makes their relationship to the primary defect of abetalipoproteinemia difficult to assess.
Assuntos
Abetalipoproteinemia/sangue , Proteínas Sanguíneas/análise , Colesterol/análise , Ácidos Graxos/sangue , Glicerídeos/sangue , Lisofosfatidilcolinas/sangue , Fosfatidilcolinas/sangue , Fosfatidiletanolaminas/sangue , Esfingomielinas/sangue , Adulto , Criança , Cromatografia , Cromatografia em Camada Fina , Feminino , Humanos , Masculino , Triglicerídeos/sangueRESUMO
The nature of the gastrointestinal absorptive defect for triglyceride in three subjects with abetalipoproteinemia has been investigated by studying peroral biopsies of the gastrointestinal mucosa. The following conclusions were reached.1) In confirmation of other studies, the abnormal vacuoles within the duodenal absorptive cells of these individuals were lipophilic.2) On chemical analysis there was significantly more mucosal lipid than found in normal fasting specimens, and almost the entire increase was due to triglyceride.3) This excess mucosal lipid was reduced by a low fat diet, but even after 34 days on such a diet there was still an excess of lipophilic material near the villus tip and increased quantities of total lipid and triglyceride when compared with material from normal subjects similarly treated.4) Although there are demonstrable qualitative changes in mucosal and plasma lipids after an acute fat load, they are not quantitatively as great as in normal individuals. Fat balance studies and the qualitative changes in plasma and tissue lipids that do occur after more extended periods on different types of dietary fat do indicate that a considerable percentage of the dietary fat is assimilated. The route by which it is absorbed remains to be clarified.
Assuntos
Abetalipoproteinemia , Glicerídeos/farmacologia , Absorção Intestinal , Mucosa Intestinal/citologia , Biópsia , Cromatografia em Camada Fina , Citoplasma/análise , Gorduras na Dieta/metabolismo , Ácidos Graxos/análise , Humanos , Jejuno/citologia , Lipídeos/análise , Fosfolipídeos/análise , Triglicerídeos/farmacologiaRESUMO
For the development of community-based vector control programs for dengue prevention, one of the key components is to formulate an adequate classification scheme for the different containers in which immature Aedes mosquitoes develop. Such a standardized scheme would permit more efficient targeting of efforts and resources in the most productive way possible. Based on field data from Kamphaeng Phet, Thailand, we developed a classification method that consists of the shape (S), use (U), and material (M) of the container (SUM-method). We determined that by targeting the four container classes that held the most Ae. aegypti pupae, adult mosquito production could theoretically be reduced by 70%. The classification method may be equally suitable for similar studies elsewhere in the world. Main advantages of the classification scheme are that categorization of containers does not need to be done a priori, that there is no "miscellaneous" class, and that different immature control strategies can be easily and prospectively tested with a local database. We expect that the classification strategy will 1) facilitate comparison of results among different ecological and geographic settings and 2) simplify communication among vector control personnel and affected communities.
Assuntos
Aedes/fisiologia , Utensílios Domésticos , Controle de Mosquitos , Água/parasitologia , Animais , Dinâmica Populacional , TailândiaRESUMO
Given that tools for dengue emergency control are limited, continuous evaluation of the effectiveness of insecticide applications in the field is of utmost importance. Such studies will provide a sound basis for defining spraying schemes for public health authorities in dengue-affected countries. In this article, we address the following research questions: How do different space spraying strategies affect Aedes aegypti (L.) (Diptera: Culicidae) populations in both space and time? More specifically, how well are these mosquitoes killed, and how quickly do their populations recover and from where? Field trials were carried out with ultralow volume sprayers in Kamphaeng Phet province, Thailand, with a pyrethrin mixture that was applied 1) indoors only, 2) indoors plus outdoors, 3) indoors with a doubled spraying time, and 4) indoors with doubled spraying time plus outdoors. We found that within 7 d, Ae. aegypti populations recovered to approximately 50% of their original numbers. Spraying the outdoor area and doubling the time sprayed per room only had a significant impact on mosquito numbers 1 d after spraying. Two and 7 d after spraying, these effects were no longer detected. By investigating the spatial arrangement of Ae. aegypti numbers, we found that during the first 2 d after spraying immigration from untreated areas extended approximately 15 m into the sprayed area, whereas after 7 d this effect extended up to 50 m. Results are discussed in relation to ongoing dengue control efforts in Thailand.
Assuntos
Aedes/fisiologia , Insetos Vetores/fisiologia , Inseticidas , Piretrinas , Animais , Feminino , Modelos Logísticos , Controle de Mosquitos/normas , Densidade Demográfica , Fatores de TempoRESUMO
There has been increasing interest in functional heterogeneity along the septotemporal, dorsal-ventral (D-V) axis of the hippocampus. Although anatomical connectivity and lesion studies point to discrete roles for these sub-regions, the contribution of differential gene expression across this axis has not been systematically studied. Here we present findings from an Affymetrix microarray screen aimed at identifying genes in the CA1 region of the adult murine hippocampus that show significant differential expression along the D-V axis. Our results indicate that the vast majority of monitored genes (>90%) had tissue expression levels that differed by less than 20% between regions, while less than 0.1% of genes had expression levels that varied more than three-fold by sub-region. Only 23 probes showed a CA1 dorsoventral signal intensity ratio greater than three: 18 enriched dorsally and five enriched ventrally. Probes with the greatest difference in expression levels represent a range of genes with known functions in patterning and signaling, as well as genes without known function. Selective screening with digoxigenin-labeled in situ hybridization confirms the existence of CA1 sub-regionalized expression, with some genes exhibiting a graded expression pattern across the D-V axis, and others restricted to a discrete region. Our findings demonstrate that there are gene expression differences across the D-V axis of the adult murine hippocampus within traditionally recognized cytoarchitecturally defined boundaries. Combined with the previously recognized differences in connectivity and results from lesion studies, our data further confirm the existence of functional heterogeneity along the D-V axis.
Assuntos
Perfilação da Expressão Gênica , Hipocampo/anatomia & histologia , Hipocampo/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Hibridização In Situ , Masculino , CamundongosRESUMO
OBJECTIVES: We sought to assess the variability of results obtained with thallium scintigraphy as a method for tracking the extent of myocardial ischemia in medically refractory patients with angina who are not suitable for coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty. BACKGROUND: New therapies are being evaluated for patients with "no option" angina in whom medical therapy has failed. Nuclear techniques, like thallium scintigraphy, are used in multicenter trials to evaluate whether such therapies improve myocardial perfusion. However, the variability of test results is unknown in this patient group in a multicenter study. METHODS: The Angina Treatments: Lasers And Normal Therapies In Comparison (ATLANTIC) study was a randomized trial of transmyocardial laser revascularization (n = 182). Patients underwent dipyridamole thallium stress tests at baseline and 3, 6 and 12 months after enrollment. The control group (n = 90) was treated with constant medical therapy during the study and is a relevant group to investigate test variability. Test variability over time was quantified by the mean absolute change in the percentage of reversible perfusion defects between baseline and follow-up. RESULTS: Baseline percent myocardium with ischemia averaged 17.0 +/- 13.7% and did not change during follow-up. However, variations in the percent myocardium with reversible perfusion defects over time amounted to an average of 6 to 8 percentage points, or 43% to 55% of the baseline value. Only approximately 13% of this variability was attributable to variability in image reconstruction and analysis. CONCLUSIONS: As demonstrated in the ATLANTIC study, percent myocardial ischemia in control subjects receiving constant medical therapy varied in individual patients by an average of approximately 50%. This may limit the utility of thallium scintigraphy to detect improved myocardial perfusion over time in response to therapy.
Assuntos
Doença das Coronárias/diagnóstico por imagem , Coração/diagnóstico por imagem , Radioisótopos de Tálio , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Valor Preditivo dos Testes , Cintilografia , Ensaios Clínicos Controlados Aleatórios como Assunto , Medição de RiscoRESUMO
Evidence for a transcription attenuation site downstream from the leu promoter was obtained by transcription experiments in vitro. Most transcription initiated in vitro from leuP is terminated prematurely, resulting in the synthesis of a 160 nucleotide leader RNA. We define here the point at which transcription is initiated in vitro and in vivo and demonstrate that the site of premature termination is between the promoter and the first structural gene (leuA). Additional nucleotide sequences are presented that extend the known sequence 200 base-pairs upstream and 300 base-pairs downstream from leuP. The location of the promoter-proximal end of cistron leuA was deduced by comparing nucleotide sequence data with the sequence of the ten amino acids at the N-terminus of alpha-isopropylmalate synthase. To facilitate the isolation of quantities of material for sequencing experiments, the enzyme was isolated from a plasmid-containing strain, CV605, grown under conditions of leucine limitation. Under such conditions, about 20% of the total soluble protein of strain CV605 is alpha-isopropylmalate synthase and another 20% is beta-isopropylmalate dehydrogenase (leuB product).
Assuntos
Escherichia coli/genética , Leucina/genética , Óperon , Iniciação Traducional da Cadeia Peptídica , Salmonella typhimurium/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos , DNA Bacteriano , Desoxirribonucleotídeos/análise , Eletroforese em Gel de Poliacrilamida , Plasmídeos , RNA Bacteriano , Ribonucleotídeos/análise , Transcrição GênicaRESUMO
Seven different revertants of bacteriophage phi X174am16 (AB5276G leads to T) have been isolated and the nature of the reversions determined by sequencing their DNA. The revertants each differ from am16 by just a single base substitution. These may be distinguished with varying degrees of ease by characteristic temperature sensitivities of growth. This has facilitated the determination of the frequency at which DNA polymerase III catalyses different types of substitution mutations in copying phi X174 DNA in vitro and in vivo. During the replicative form (RF) leads to single-stranded (SS) stage of replication in vitro, four different revertants may be readily produced according to well-defined rate laws on biasing the concentrations of dNTPs. Transversion mutations are found to be formed predominantly by purine x purine mismatching, whilst transitions are formed predominantly by G x T mismatching. The substitutions via G x T and G x A mismatches are estimated to occur at similar frequencies in vivo. The two most common revertants isolated in vivo, however, are not those readily produced during the RF leads to SS stage in vitro but are those produced on purine x purine mismatching in the SS leads to RF stage. The accuracy of the DNA polymerase in vitro appears to be similar to that in this stage in vivo. However, the overall accuracy of the RF leads to SS replication in vivo is more accurate than predicted from the measurements of the accuracy in vitro.
Assuntos
Bacteriófago phi X 174/genética , Replicação do DNA , DNA Viral/biossíntese , Replicação Viral , Aminoácidos/análise , Bacteriófago phi X 174/classificação , Bacteriófago phi X 174/metabolismo , Composição de Bases , Sequência de Bases , DNA de Cadeia Simples/genética , Desoxirribonucleotídeos/genética , Mutação , TemperaturaRESUMO
The effects of deoxynucleoside monophosphates on the 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme have been correlated with their effects on the fidelity of DNA replication. In particular, dGMP inhibits the proofreading activity of the enzyme and decreases the fidelity in those cases where a "following nucleotide effect" is also noted. This is strong evidence for proofreading. However, the absence of the effects of proofreading inhibitors or following nucleotides need not be evidence against the occurrence of proofreading: a theoretical analysis shows that these effects may not be observed even though there is active proofreading. This is suggested to be the case with the phage T4 enzyme system. The proofreading activity of Pol III appears to be directed primarily towards removing purine x pyrimidine-mediated rather than purine x purine-mediated misincorporations. recA protein inhibits the proofreading activity of Pol III on synthetic templates containing mismatched 3' termini. This is paralleled by a decrease in the fidelity of DNA replication in vitro. The inhibition is increased in the presence of dGMP or dAMP but there is no further increase in the infidelity of replication. The presence of both dNMPs and recA protein does not enable Pol III to copy past pyrimidine photodimers.
Assuntos
DNA Polimerase III/genética , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Proteínas de Bactérias/farmacologia , Bacteriófago phi X 174/genética , Composição de Bases , DNA Polimerase III/antagonistas & inibidores , Desoxirribonucleotídeos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonuclease V , Exodesoxirribonucleases/antagonistas & inibidores , Cinética , Recombinases Rec A , Replicação Viral/efeitos dos fármacosRESUMO
As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.
Assuntos
Arbovírus/isolamento & purificação , Culicidae/virologia , Meio Ambiente , Animais , Arbovírus/classificação , Arbovírus/genética , Chlorocebus aethiops , Técnica Direta de Fluorescência para Anticorpo , Peru , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estações do Ano , Especificidade da Espécie , Células VeroRESUMO
This study was conducted to investigate the effect of starch in the preservation solution during hypothermic machine perfusion (HMP) on endothelial cell and hepatocyte functions in an isolated perfused rat liver model. Livers isolated from male Sprague-Dawley rats were perfused with the University of Wisconsin (UW) solution (HMP + starch group); modified UW solution (starch omitted) (HMP - starch group) at 0.4 mL/min per g liver; or simply stored in the UW solution (SCS group) at 4 degrees C for 24 hours. Following preservation, livers from HMP + starch, HMP - starch, SCS, and control group (without preservation) were perfused with Krebs-Henseleit Buffer solution at 37 degrees C for 30 minutes. Samples were taken every 10 minutes during 30-minute warm perfusion to assess hepatocyte and endothelial cell function and damage. After 24 hours of hypothermic preservation and 30 minutes rewarming, livers in the HMP + starch group displayed significantly lower lactate dehydrogenase levels and higher bile production. Endothelial cell function was also improved as indicated by hyaluronic acid uptake and shorter transient time for albumin observed in a multiple indicator dilution study. Liver wet and dry ratio and histological findings confirmed reduced edema formation in the tissue of the HMP + starch group livers compared with that of the HMP - starch and SCS group livers. These results suggest that HMP with the UW solution containing starch improve endothelial cell function and induce less hepatocellular damage following 24-hour preservation compared to SCS and HMP with the starch-free UW solution. These results also suggest that oncotic support may be an important component in preserving hepatic microcirculation in HMP.