Biochemical changes during germination and seedling growth in Cuscuta campestris

Changes in the contents of starch, protein, DNA, RNA, total phosphorus, acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis.
Starch content in Cuscuta seeds showed a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination and then decreased. Total phosphorus in the seeds remained almost unchanged during the period of study. Both trichloroacetic acid soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed.     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.     

Inhibition of hydrogen sulfide production by gene silencing attenuates inflammatory activity of LPS-activated RAW264.7 cells

Hydrogen sulfide is an inflammatory mediator and is produced by the activity of the enzyme cystathionine γ-lyase (CSE) in macrophages. Previously, pharmacological inhibition of CSE has been reported to have conflicting results, and this may be due to the lack of specificity of the pharmacological agents. Therefore, this study used a very specific approach of small interfering RNA (siRNA) to inhibit the production of the CSE in an in vitro setting. We found that the activation of macrophages by lipopolysaccharide (LPS) resulted in higher levels of CSE mRNA and protein as well as the increased production of proinflammatory cytokines and nitric oxide (NO). We successfully used siRNA to specifically reduce the levels of CSE mRNA and protein in activated macrophages. Furthermore, the levels of proinflammatory cytokines in LPS-activated macrophages were significantly lower in siRNA-transfected cells compared to those in untransfected controls. However, the production levels of NO by the transfected cells were higher, suggesting that CSE activity has an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages, and its activity has an inhibitory effect on NO production by these cells.     

A Simulation Optimization Approach to Epidemic Forecasting

Reliable forecasts of influenza can aid in the control of both seasonal and pandemic outbreaks. We introduce a simulation optimization (SIMOP) approach for forecasting the influenza epidemic curve. This study represents the final step of a project aimed at using a combination of simulation, classification, statistical and optimization techniques to forecast the epidemic curve and infer underlying model parameters during an influenza outbreak. The SIMOP procedure combines an individual-based model and the Nelder-Mead simplex optimization method. The method is used to forecast epidemics simulated over synthetic social networks representing Montgomery County in Virginia, Miami, Seattle and surrounding metropolitan regions. The results are presented for the first four weeks. Depending on the synthetic network, the peak time could be predicted within a 95% CI as early as seven weeks before the actual peak. The peak infected and total infected were also accurately forecasted for Montgomery County in Virginia within the forecasting period. Forecasting of the epidemic curve for both seasonal and pandemic influenza outbreaks is a complex problem, however this is a preliminary step and the results suggest that more can be achieved in this area.     

Hydrogen sulfide acts as a mediator of inflammation in acute pancreatitis: in vitro studies using isolated mouse pancreatic acinar cells

Hydrogen sulphide (H(2)S) is synthesized from L-cysteine via the action of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS). We have earlier shown that H(2)S acts as a mediator of inflammation. However the mechanism remains unclear. In this study, we investigated the presence of H(2)S and the expression of H(2)S synthesizing enzymes, CSE and CBS, in isolated mouse pancreatic acini. Pancreatic acinar cells from mice were incubated with or without caerulein (10(-7) M for 30 and 60 min). Caerulein increased the levels of H(2)S and CSE mRNA expression while CBS mRNA expression was decreased. In addition, cells pre-treated with DL-propargylglycine (PAG, 3 mM), a CSE inhibitor, reduced the formation of H(2)S in caerulein treated cells, suggesting that CSE may be the main enzyme involved in H(2)S formation in mouse acinar cells. Furthermore, substance P (SP) concentration in the acini and expression of SP gene (preprotachykinin-A, PPT-A) and neurokinin-1 receptor (NK-1R), the primary receptor for SP, are increased in secretagogue caerulein-treated acinar cells. Inhibition of endogenous production of H(2)S by PAG significantly suppressed SP concentration, PPT-A expression and NK1-R expression in the acini. To determine whether H(2)S itself provoked inflammation in acinar cells, the cells were treated with H(2)S donor drug, sodium hydrosulphide (NaHS), (10, 50 and 100 muM), that resulted in a significant increase in SP concentration and expression of PPT-A and NK1-R in acinar cells. These results suggest that the pro-inflammatory effect of H(2)S may be mediated by SP-NK-1R related pathway in mouse pancreatic acinar cells.     

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini.