Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

The CipA scaffoldin protein plays a key role in the Clostridium thermocellum cellulosome. Previous studies have revealed that mutants deficient in binding or solubilizing cellulose also exhibit reduced expression of CipA. To confirm that CipA is, in fact, necessary for rapid solubilization of crystalline cellulose, the gene was deleted from the chromosome using targeted gene deletion technologies. The CipA deletion mutant exhibited a 100-fold reduction in cellulose solubilization rate, although it was eventually able to solubilize 80% of the 5 g/liter cellulose initially present. The deletion mutant was complemented by a copy of cipA expressed from a replicating plasmid. In this strain, Avicelase activity was restored, although the rate was 2-fold lower than that in the wild type and the duration of the lag phase was increased. The cipA coding sequence is located at the beginning of a gene cluster containing several other genes thought to be responsible for the structural organization of the cellulosome, including olpB, orf2p, and olpA. Tandem mass spectrometry revealed a 10-fold reduction in the expression of olpB, which may explain the lower growth rate. This deletion experiment adds further evidence that CipA plays a key role in cellulose solubilization by C. thermocellum, and it raises interesting questions about the differential roles of the anchor scaffoldin proteins OlpB, Orf2p, and SdbA

Cytochrome P450associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.Fil: Balabanidou, Vasileia. Foundation for Research and Technology-Hellas; Grecia. Universidad de Creta; GreciaFil: Kampouraki, Anastasia. Universidad de Creta; GreciaFil: Mac Lean, Marina. University of Nevada; Estados UnidosFil: Blomquist, Gary J.. University of Nevada; Estados UnidosFil: Tittiger, Claus. University of Nevada; Estados UnidosFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Mijailovsky, Sergio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Chalepakis, George. Universidad de Creta; GreciaFil: Anthousi, Amalia. Universidad de Creta; GreciaFil: Lynd, Amy. Liverpool School of Tropical Medicine; Reino UnidoFil: Antoine, Sanou. Liverpool School of Tropical Medicine; Reino UnidoFil: Hemingway, Janet. Liverpool School of Tropical Medicine; Reino UnidoFil: Ranson, Hilary. Liverpool School of Tropical Medicine; Reino UnidoFil: Lycett, Gareth J.. Liverpool School of Tropical Medicine; Reino UnidoFil: Vontas, John. Foundation for Research and Technology-Hellas; Grecia. Agricultural University of Athens; Greci

Efeito da fertilidade do solo no crescimento, nodulação e bioquímica dos nódulos de Cratylia floribunda Benth

The effect of fertility treatments on regrowth vigor, nodulation, nitrogenase, nodule enzymes and carbohydrates of Cratylia floribunda was evaluated when cultivated on a Dark-Red Latosol (Typic Eutrustox). P augmented regrowth of aerial parts, root dry weight, and number and weight of the nodules, as well as their levels of protein and glucose. Increased nitrogenase activity was observed in pots fertilized with P (µmoles C2H4/pot/hr), but non-significant effects were found when results were expressed as µmoles C2H4/g fresh nodule/hr, and a negative effect was noted if nitrogenase was calculated as enzyme specific activity (µmoles C2H4/mg protein/hr). The same effect was observed on glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and the transaminases glutamate-oxaloacetate and glutamate-pyruvate. Mg had a beneficial effect on herbage yield, but depressed the nodule enzymatic activity. S and K increased herbage production but showed little effect on nodule enzymatic activity, and Ca augmented glutamate synthase activity, levels of α - Ketoglutarate, pyridoxyl phosphates, soluble protein and starch, but depressed herbage production on the first two clippings, root growth and levels of glucose and sucrose in the nodules.Avaliou-se o efeito de tratamentos de fertilidade sobre o vigor de rebrota, nodulação, atividade da nitrogenase e algumas enzimas e carboidratos dos nódulos de Cratylia floribunda Benth, quando cultivada em Latossolo Vermelho-Escuro (Typic Eutrustox). O P aumentou a rebrota da parte aérea, o peso seco das raízes, e o número e peso dos nódulos e seus teores de proteína e glicose. A atividade da nitrogenase aumentou nos vasos fertilizados com P (µmoles C2H4/vaso/h), mas este efeito não existiu quando os resultados foram expressos como µmoles C2H4/g de nódulo fresco/h, e foi negativo quando calculado como atividade enzimática específica (µmoles C2H4/mg de proteína/min). Este efeito também foi observado na desidrogenase do glutamato, na sintetase da glutamina, na sintase do glutamato, e nas transaminases glutamato-oxaloacetato e glutamato-piruvato. O Mg beneficiou a produção de forragem, mas deprimiu as atividades enzimáticas, O S e o K aumentaram a produção de forragem em alguns cortes, mas pouco afetaram as características fisiológicas dos nódulos. O Ca aumentou a atividade da sintase do glutamato, os níveis de α cetoglutarato, fosfatos de piridoxal, proteína solúvel e do amido, mas diminuiu a produção de forragem nos dois primeiros cortes, o crescimento radicular e os níveis de glicose e sucrose

Fixation and Release of Potassium in Several Eastern Oklahoma Soils

The Oklahoma Agricultural Experiment Station periodically issues revisions to its publications. The most current edition is made available. For access to an earlier edition, if available for this title, please contact the Oklahoma State University Library Archives by email at [email protected] or by phone at 405-744-6311

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA, Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at ∼90% theoretical yield and titer of 70 g/L. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ∼50%, whereas multiple gene deletions of all annotated alcohol dehydrogenases except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked ADH activity (acetaldehyde-reducing) by 93%

Achieving provider engagement: providers' perceptions of implementing and delivering integrated care

The literature on integrated care is limited with respect to practical learning and experience. Although some attention has been paid to organizational processes and structures, not enough is paid to people, relationships, and the importance of these in bringing about integration. Little is known, for example, about provider engagement in the organizational change process, how to obtain and maintain it, and how it is demonstrated in the delivery of integrated care. Based on qualitative data from the evaluation of a large-scale integrated care initiative in London, United Kingdom, we explored the role of provider engagement in effective integration of services. Using thematic analysis, we identified an evolving engagement narrative with three distinct phases: enthusiasm, antipathy, and ambivalence, and argue that health care managers need to be aware of the impact of professional engagement to succeed in advancing the integrated care agenda

Future Impact of Various Interventions on the Burden of COPD in Canada: A Dynamic Population Model

Background: Chronic obstructive pulmonary disease (COPD) is a growing economic burden worldwide. Smoking cessation is thought to be the single most effective way of reducing the economic burden of COPD. The impact of other strategies such as interventions that predict risk of disease, reduce progression of disease, or reduce exacerbations has not been systematically studied. Objectives: We estimated the economic and clinical burden of COPD over the next 25 years in Canada and the impact of three potential interventions (screening test for predisposition to COPD, new drugs to avoid progression into more severe disease stages, and predictive test for exacerbations) on COPD burden. Methods Using a dynamic simulation model, we projected the total burden of COPD (cost, morbidity, and mortality) from 2011 to 2035 using the population of Canada as a case study. The model stratified population based on sex, age, smoking status, respiratory symptoms, and their COPD stage. The cost and quality adjusted life years (QALYs) associated with each intervention were estimated. Results: The model indicates that annual societal cost of COPD is $4.52 billion (B) Canadian dollars in 2011 and will reach$3.61B ($7.33B undiscounted) per year in 2035. Over the next 25 years, COPD will be responsible for approximately$101.4B in societal costs ($147.5B undiscounted) and 12.9 million QALYs lost (19.0 million undiscounted). Our results suggested that the best strategy to reduce the financial burden of COPD is by reducing exacerbations. Smoking cessation, while it is the cornerstone of COPD prevention, has only a modest effect in attenuating the financial burden of COPD over the next 25 years in Western countries such as Canada. Conclusion: Our data suggest that any intervention that can reduce the number of exacerbations has a substantial impact on morbidity and costs of COPD and should be considered in conjunction with the ongoing efforts to reduce smoking rates