Enhancing the production of poly-γ-glutamate in Bacillus subtilis ZJS18 by the heat- and osmotic shock and its mechanism.

Poly-γ-glutamate (γ-PGA) is a natural macromolecule peptide, and is widely used in the food, medicine, and pharmaceutical industries. In this study, heat- and osmotic shock were used to improve the production of γ-PGA in Bacillus subtilis ZJS18, and its molecular mechanism was explored. The results indicated that the heat- and osmotic shock significantly promoted the production of γ-PGA owing to the stress response of B. subtilis cells to adverse environment. The highest concentrations of γ-PGA reached 14.53 and 15.98 g/l under heat- and osmotic shock, respectively. The activities of five enzymes related to the metabolism of the endogenous glutamate were determined and analyzed. It was found that the activities of glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glutamate dehydrogenase and glutamate synthase were significantly altered during heat- and osmotic shock, while the activity of α-ketoglutarate dehydrogenase only showed a little alteration. This study provides a basis for the industrial production and use of γ-PGA, and for understanding its biosynthetic mechanism in B. subtilis ZJS18.

Early warning of MIB episode based on gene abundance and expression in drinking water reservoirs.

Cellular 2-methylisoborneol (MIB) yield of cyanobacteria varies under different conditions according to culture studies and field investigations, the causal mechanism remains unclear and results in ineffective MIB prediction. Through an intensive field survey during an MIB episode produced by Pseudanabaena cinerea in QCS reservoir, we demonstrated that MIB synthesis (mic) gene abundance (DNA) and expression (RNA) might be useful as parameters for early warning of MIB production. It was found that the abundance of mic DNA and RNA peaked ahead of MIB concentrations by 10 and 7 days, respectively. In addition, the RNA abundance (R2 = 0.45, p < 0.01) showed a slightly higher correlation with MIB compared to DNA abundance (R2 = 0.37, p < 0.01), suggesting that the conditions for the growth of Pseudanabaena cinerea might be slightly different from those for mic gene expression, which was verified by a culture experiment. The highest cell growth was obtained under 36 µmol photons m-2 s-1, while the highest cellular MIB yield and mic gene expression level were obtained under 85 µmol photons m-2 s-1. Our results clearly supported that light intensity was the virtual regulator governing the mic gene expression within the controlled culture experiment and the actual MIB episode in the reservoir. Besides these results, we developed an early warning model using mic gene abundance as an indicator of MIB episodes, which was verified in two other reservoirs. Our findings highlight the effect of light intensity on mic gene expression and MIB synthesis and provide an early warning tool targeting MIB episode prediction, which therefore should be of importance for source water authorities.

Down-regulation of Kir6.2 affects calcium influx and insulin secretion in HIT-T15 cells.

In pancreatic beta cells, ATP-sensitive potassium (K(ATP)) channels are metabolic sensors that couple cell metabolism to electrical activity, and therefore K(ATP) channels regulate insulin secretion. We assume that down-regulating the expression of Kir6.2 subunits of K(ATP) channels may change calcium influx induced by glucose and insulin secretion regulated by K(ATP) channels. In our study, we employ Kir6.2-shRNA plasmid to downregulate Kir6.2 expression in HIT-T15 cells. Then, we research the effect of downregulation of Kir6.2 on K(ATP) current, cytoplasmic free Ca2+ concentration and insulin secretion. All results illustrate that downregulation of Kir6.2 subunits of K(ATP) channels in HIT-T15 cells affects K(ATP) current and insulin secretion, and fails to promote calcium influx. The results demonstrate the function of Kir6.2 subunits in electrophysiology characteristic, insulin secretion and calcium influx, and RNA interference provides a feasible alternative to study the function of Kir6.2 subunits in K(ATP) channels in different kinds of diabetes.

Inhibition of Stat3 expression induces apoptosis and suppresses proliferation in human leukemia HL-60 cells.

Acute myeloid leukemia (AML) is the most common myeloid leukemia. It is highly malignant, thus, most patients with AML will relapse and die after traditional treatment. Stat3, a member of the signal transducer and activator of transcription (Stat) family, is involved in the development and progression of many tumors. The purpose of the study was to investigate whether the down-regulation of Stat3 expression by RNA interference is effective against human leukemia HL-60 cells. The results indicated that constitutively expressed Stat3 is present in human leukemia HL-60 cells, and the down-regulation of Stat3 expression caused significant induction of apoptosis as well as inhibition of proliferation in HL-60 cells. These data further demonstrated that Stat3 plays a critical role in human leukemia HL-60 cell apoptosis and proliferation. Thus, targeting Stat3 may be a useful adjunctive treatment strategy in AML.

Down-regulation of Stat3 decreases invasion activity and induces apoptosis of human glioma cells.

Gliomas are the most common type of primary brain tumors. Despite the improvement in current treatments for gliomas, including surgical resection, radiation, and chemotherapy, there has been very little progress in curing this kind of disease. Stat3 is a member of signal transducer and activator of transcription family. It plays an important role in regulating cell survival, invasion, and apoptosis. This study investigated the influence of low-level expression of Stat3 on invasion and apoptosis in U251 cells. Our data showed that Stat3 is constitutively expressed in human gliomas cell line U251. The invasion activity in U251 cells was weakened and the apoptosis in U251 cells was induced after down-regulation of Stat3. In addition, down-regulation of Stat3 can suppress the expression of MMP-2, Bcl-xL and survivin but not 67LR. These results further indicate that Stat3 plays a key role in the invasion and apoptosis of human glioma cell line U251.