Comparison of the effects of trypsin and chymotrypsin on thylakoid membrane function

The thylakoid membranes are the functional units upon which the photosynthetic energy-tansducing reactions occur. There is a strict relationship between the structure of these membranes and their functional capabilities; this is because the components of the photosynthetic electron-transport chain are bound to proteins in the membrane in a specific manner. Functional changes can be induced by using protediytic enzymes to alter the structure of the proteins. Two enzymes, trypsin (which digests bonds involving arginine and lysine) and chymotrypsin (which digest bonds involving tryptophan, tryosine and phenylalanine) have been used to investigate the functions of the thylakoid membrane. The rationale was that because these enzymes have different specificities for the bonds which they attack they may induce different functional changes in the membrane. The effects of these enzymes are compared. Measurements of light-induced oxygen evolution rates in the presence of exogenous electron acceptors showed that both trypsin and chymotrypsin altered the electron transport reactions in a number of ways. At higher concentrations both trypsin and chymotrypsin treatment of thylakoids caused the rate of electron transport to be dependent on the type of electron acceptor present and rendered electron transport insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea which inhibits photosystem II. These effects of trypsin have previously been shown to be due to partial digestion of a 32KDa protein on the acceptor side of photosystem II. Thus chymotrypsin may also affect this polypeptide. At lower concentrations trypsin, and to a lesser extent chymotrypsin, stimulated the rate of electron transport. This suggested that both of these enzymes tend to uncouple the thylakoid membranes. Trypsin and chymotrypsin altered the flash-induced field indicating absorption change measured at 520 nm (the electrochromic bandshift) in two ways : 1) the half-time of decay was decreased suggesting increased trans-membrane ion flux and 2) the extent was reduced. The decrease in the half-time of the decay of the electrochromic bandshift was correlated with uncoupling, by trypsin and to a lesser extent by chymotrypsin, of the thylakoid membrane. This proposal was supported by phosphorylation measurements which showed that both trypsin and chymotrypsin can, at least partially, inhibit ATP synthesis. But after brief trypsin incubation in the light a stimulation in the rate of phosphorylation and oxygen evolution (water to methyl viologen) was evident. It is possible that trypsin affects the e sub-unit, believed to be an inhibitor of ATPase function, however, no evidence to support this proposal was seen in polyacrylamide gel electrophoresis analysis of the ATPase. (Abstract shortened by ProQuest.)

Arabidopsis CP12 mutants have reduced levels of phosphoribulokinase and impaired function of the Calvin–Benson cycle

CP12 is a small, redox-sensitive protein, the most detailed understanding of which is the thioredoxin-mediated regulation of the Calvin–Benson cycle, where it facilitates the formation of a complex between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in response to changes in light intensity. In most organisms, CP12 proteins are encoded by small multigene families, where the importance of each individual CP12 gene in vivo has not yet been reported. We used Arabidopsis thaliana T-DNA mutants and RNAi transgenic lines with reduced levels of CP12 transcript to determine the relative importance of each of the CP12 genes. We found that single cp12-1, cp12-2, and cp12-3 mutants do not develop a severe photosynthetic or growth phenotype. In contrast, reductions of both CP12-1 and CP12-2 transcripts lead to reductions in photosynthetic capacity and to slower growth and reduced seed yield. No clear phenotype for CP12-3 was evident. Additionally, the levels of PRK protein are reduced in the cp12-1, cp12-1/2, and multiple mutants. Our results suggest that there is functional redundancy between CP12-1 and CP12-2 in Arabidopsis where these proteins have a role in determining the level of PRK in mature leaves and hence photosynthetic capacity

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.Biotechnological and Biological Sciences Research Council (BBSRC). Grant Numbers: BB/K005952/1, BB/L02182X/1 Synthetic Biology Research Centre ‘OpenPlant’ award. Grant Number: BB/L014130/1 Spanish MINECO. Grant Number: BIO2013‐42193‐R Engineering Nitrogen Symbiosis for Africa (ENSA) The Bill & Melinda Gates Foundation US Department of Energy, Office of Biological and Environmental. Grant Number: DE‐AC02‐05CH1123 COST Action. Grant Number: FA100

Standards for plant synthetic biology: a common syntax for exchange of DNA parts

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering

Optimization of Metformin in the GRADE Cohort: Effect on Glycemia and Body Weight

ObjectiveWe evaluated the effect of optimizing metformin dosing on glycemia and body weight in type 2 diabetes.Research design and methodsThis was a prespecified analysis of 6,823 participants in the Glycemia Reduction Approaches in Diabetes: A Comparative Effectiveness Study (GRADE) taking metformin as the sole glucose-lowering drug who completed a 4- to 14-week (mean ± SD 7.9 ± 2.4) run-in in which metformin was adjusted to 2,000 mg/day or a maximally tolerated lower dose. Participants had type 2 diabetes for <10 years and an HbA1c ≥6.8% (51 mmol/mol) while taking ≥500 mg of metformin/day. Participants also received diet and exercise counseling. The primary outcome was the change in HbA1c during run-in.ResultsAdjusted for duration of run-in, the mean ± SD change in HbA1c was -0.65 ± 0.02% (-7.1 ± 0.2 mmol/mol) when the dose was increased by ≥1,000 mg/day, -0.48 ± 0.02% (-5.2 ± 0.2 mmol/mol) when the dose was unchanged, and -0.23 ± 0.07% (-2.5 ± 0.8 mmol/mol) when the dose was decreased (n = 2,169, 3,548, and 192, respectively). Higher HbA1c at entry predicted greater reduction in HbA1c (P < 0.001) in univariate and multivariate analyses. Weight loss adjusted for duration of run-in averaged 0.91 ± 0.05 kg in participants who increased metformin by ≥1,000 mg/day (n = 1,894).ConclusionsOptimizing metformin to 2,000 mg/day or a maximally tolerated lower dose combined with emphasis on medication adherence and lifestyle can improve glycemia in type 2 diabetes and HbA1c values ≥6.8% (51 mmol/mol). These findings may help guide efforts to optimize metformin therapy among persons with type 2 diabetes and suboptimal glycemic control