Molecular cloning, purification, and characterization of the Caenorhabditis elegans Phosphodiesterase 3 (CEPDE3) gene

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of the second messenger molecules cAMP and cGMP by controlling their rates of degradation. In humans, there are 11 different PDE genes, and each gene produces several different isoforms and splice variants. The PDEs are a medically important class of proteins that regulate signaling across a range of tissues, and inhibition of these enzymes is clinically important in a wide array of human diseases. The human PDE3 gene produces two distinct, but related isoforms PDE3A and PDE3B. PDE3A is further subdivided into three isoforms with masses averaging at ~125 kDa. PDE3 play an important role in insulin signaling pathways, cardiovascular tissues, platelets, adipocytes, and oocyte maturation. In particular, PDE3A isoforms are prominently expressed in myocardium and vascular smooth muscle cells, hence, the inhibition of PDE3 activity is particularly useful in treatment of cardiac disease. The focus of this project is the PDE3 gene present in C. elegans. Although PDE3 activity has been widely studied from cell and tissue extracts from mammals, there is little data on purified protein for detailed biochemical analysis and characterization of the PDE3 gene and, based on the literature, it suggests that these are difficult proteins to express and purify. There is also very little structural data on the PDE family of enzymes. What exists is mostly on partial proteins, and none at all on the PDE3s. The aims of this project were to clone and overexpress affinity-tagged recombinant proteins of isoforms of the C. elegans PDE3 gene to analyze their catalytic properties and attempt crystallization of the isoforms. The CEPDE3 gene is a homolog of the mammalian PDE3 family encoding two different CEPDE3 isoforms; CEPDE3-LF (long form) codes for a 63.5 kDa protein, and CEPDE3-SF (short form) codes for a 54.2 kDa protein. Sequence homology alignments of CEPDE3 with human PDE sequences indicate the mammalian and the nematode gene have ~ 47% homology. The advantages of this approach are that the C. elegans isoforms are smaller in mass, making them potentially easier to purify. Preliminary experimental data has suggested that the nematode CEPDE3 gene exhibits catalytic properties and inhibitor sensitivities that are characteristic of the mammalian PDE3 gene family. A crystal structure would enable the determination of structural elements of the PDE3 catalytic pocket which could contribute to the design of more effective small molecule modulators of PDE3 catalytic activity. Initially, the CEPDE3-SF isoform was expressed as a His-tag protein from an existing clone. However, due to low protein expression, solubility, and enzyme activity, the use of a GST-tag was attempted since GST-tags are reported to improve solubility in some studies. Initial cloning using traditional PCR and restriction/ligation cloning was attempted to clone both isoforms into the plasmid pGEX-6P1 with little success. To overcome the problem, two alternative approaches to traditional cloning were attempted. The long form was amplified by PCR and was cloned by recombination using the TOPO vector system, prior to subcloning into the expression vector pGEX-6P1. The short form was made synthetically and shipped as a codon optimized insert in a vector and also subcloned into the expression vector pGEX-6P1. Both isoforms were expressed as a GST-tag protein in E. coli and, despite the change of affinity tag, the solubility was still poor using standard expression conditions. The solubility of these proteins was a significant barrier to purification, and this was improved by the optimization of the growth conditions, IPTG concentration, and cell lysis. Optimal solubility was achieved by a novel expression protocol which included an incubation of cells at 4°C prior to expression. Both isoforms were then purified by optimizing conditions using the batch method of purification and using glutathione sepharose 4B. Cleavage of the GST-tag was achieved using PreScission Protease. The purified CEPDE3-LF and CEPDE3-SF after enzymatic cleavage from GST-tag correspond to the predicted size of 63.5 kDa and 54.2 kDa, respectively. Some initial biochemistry was performed on the proteins. CEPDE3-LF and SF lysate and purified protein were assayed for PDE3 enzyme activity by measuring breakdown of cAMP to adenosine. CEPDE3-SF purified protein showed an increase in enzyme activity (24 pmoles/min/mg) compared to crude lysate (5.6 pmoles/min/mg), which is a ~5-fold increase. CEPDE3-LF showed an increase from 2.8 pmoles/min/mg in lysate to 37.5 pmoles/min/mg in purified protein; a ~ 7-fold increase in PDE3 enzyme activity. The CEPDE3-LF and SF isofoms expressed in Sf21 insect cells were assayed with a range of doses of the PDE3 specific inhibitor cilostamide to determine the capacity of enzyme inhibition. A 0.01 μM cilostamide treatments showed a 10-20% inhibition of activity and 5 μM of cilostamide showed ~95% inhibition. Hence both isoforms were inhibited by PDE3 specific inhibitor. The CEPDE3 isoforms expressed in insect cells were also assayed for cAMP and cGMP hydrolysis. The CEPDE3-LF actively hydrolyzed cAMP substrate with a Vmax of 70.4 pmoles/min/mg, Km of 0.11 μM and Kcat of 0.26 seconds-1. In comparison, the CEPDE3-SF assay did not reach saturation under the same experimental conditions. Therefore, CEPDE3-SF was less efficient in cAMP hydrolysis. Meanwhile, CEPDE3-SF actively hydrolyzed cGMP substrate with a Vmax of 232 pmoles/min/mg, Km of 0.37 μM and Kcat of 0.84 seconds-1. However, CEPDE3-LF assay did not reach saturation under the same experimental conditions. While both CEPDE3 isoforms have dual specificity for cAMP and cGMP, in this study, the long isoform hydrolyzes cAMP more efficiently short isoform more efficiently hydrolyze

Genetic Studies in Advanced Sugarcane Mid-Late Clones through Yield and Quality Traits

Sugarcane varietal development is frequently aimed at increasing yield and sucrose quality. Effective crop genetic evolution requires knowledge of the numerous traits that contribute to the present diversity by genetic analysis. Keeping in view, this experiment was conducted using a randomized block design with three replications and the trial consisted of nine mid-late sugarcane genotypes. Data on cane yield and quality traits were used to estimate the genetic variability parameters, heritability, and genetic advance (GA). Analysis of variance revealed highly significant and significant differences for all studied traits. Evaluated characters exhibited different levels of variability, heritability, and genetic advance among the studied genotypes. Low to high phenotypic coefficient of variation (PCV) and genotypic coefficient of variation (GCV) were recorded. The moderate GCV and PCV values were found particularly for Sugar Yield at harvest (18.09% and 21.64%) and Cane Yield at harvest (16.62% and 20.14%) respectively, whereas the lowest GCV and PCV (1.43% and 2.26% respectively) manifested for Purity at the 12 months stage. The highest broad sense heritability value manifested for Pol in juice at 12 months stage (%) (86.47%) followed by CCS at 12 months stage (%) (85.43%), while the lowest heritability (35.00%) revealed only for Germination % at 30 DAP. In the present study, high heritability and genetic advance as a percentage of the mean (>50) was recorded for Millable canes at harvest (000/ha) and single cane weight at harvest (kg) indicating a predominance of additive gene action for these characters. Therefore the result of this study suggests the existence of variability for cane yield and quality traits in these sugarcane genotypes, which should be exploited in future breeding

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used