Molecular cloning and characterization of the promoter of <Emphasis Type="Italic">SmGGPPs</Emphasis> and its expression pattern in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. It regulates the formation of diterpenoid, such as tanshinones. We cloned a gene for GGPP synthase SmGGPPs involved in diterpenoid biosynthesis from Salvia miltiorrhiza. At 2,767 bp long, this gene comprises an intron and two exons that encode a polypeptide of 364 amino acid residues. Then the 5′ flanking sequence of SmGGPPs was characterized by bioinformatics method. Deletion analysis of the promoter of SmGGPPs using tobacco plant displayed that the promoter was induced by heat and cold. To further search these cis-elements involved in induction regulation in the 5′ flanking sequence of SmGGPPs, many putative cis-elements were predicted with the PlantCARE and PLACE databases. A group of putative cis-acting elements are involved in induction regulation, including G-Box, WRKY, MYC and ATCT motifs. Real-time PCR analysis revealed that SmGGPPs is mainly expressed in the leaves and can also be induced by various factors, such as NaCl, wounding, high temperature, darkness, pathogen, methyl jasmonate, abscisic acid, salicylic acid, and gibberellins. This study provides useful information for further study of SmGGPPs and its regulator effect on the biosynthetic process of tanshinones so that researchers can improve the tanshinone contents in S. miltiorrhiza.     

Dissection of genetic overlap of drought and low-temperature tolerance QTLs at the germination stage using backcross introgression lines in soybean

Northeast of China is the main soybean production area, drought and low-temperature tolerance are both main factors involved in reducing soybean yield and limiting planting regions, the most effective way to solve this problem is to breed cultivars with drought and low-temperature tolerance. A set of the BC₂F₃ lines was constructed with Hongfeng 11 as recurrent parent and Harosoy as donor parent, and screened in drought and low-temperature condition at the germination stage. Related QTLs were obtained by Chi-test and ANOVA analysis with genotypic and phenotypic data. Eighteen QTLs of drought tolerance and 23 QTLs of low-temperature tolerance were detected. Among them, 12 QTLs were correlated with both drought and low-temperature tolerance, which showed a partial genetic overlap between drought and low-temperature tolerance at the germination stage in soybean. Among the 12 genetic overlap QTLs, Satt253, Satt513, Satt693, Satt240, Satt323, and Satt255 were detected by at least one method for both drought and low-temperature tolerance. Satt557, Satt452, Sat_331, Satt338, Satt271, and Satt588 were detected by only one analysis method. The QTLs detected above were significant loci for drought or low-temperature tolerance in soybean. This will play an important role in MAS for development of both drought and low-temperature tolerance variety.     

Synergistic inhibitory effects by the combination of gefitinib and genistein on NSCLC with acquired drug-resistance in vitro and in vivo

In clinical practice, most patients with non small cell lung cancer (NSCLC) who respond to tyrosine kinase inhibitors eventually progress because of an acquired resistance mutation, T790M, in epidermal growth factor receptor (EGFR). Thus, it is important to identify a new drug to reduce resistance. The aim of this study was to test whether genistein combined with gefitinib is effective against NSCLC in a cell line carrying T790M, and to clarify the underlying mechanisms. The human lung cancer cell line H1975 was used as an in vitro and in vivo model. Cells were treated with gefitinib, genistein, or a combination at a range of concentrations. Cell proliferation was calculated to assess the anticancer effects of the compounds in vitro. Flow cytometry and Western blotting were employed to determine the inhibitory effects on proliferation and the induction of apoptosis. The in vivo effects of the compounds were examined using a xenografted nude mouse model for validation. Gefitinib together with genistein enhanced both growth inhibition and apoptosis; however, the greatest synergistic effect was observed at low concentrations. p-EGFR, p-Akt, and p-mTOR expressions in vitro were reduced more by the combined use of the drugs, whereas caspase-3 and PARP activities were increased. Significantly more tumor growth inhibition was detected following combination treatment in the in vivo model. These findings suggest that genistein enhanced the antitumor effects of gefitinib in a NSCLC cell line carrying the T790M mutation. This synergistic activity may be due to increased inhibition of the downstream molecular and pro-apoptotic effects of EGFR.     

The cytotoxicity and anticancer mechanisms of alterporriol L, a marine bianthraquinone, against MCF-7 human breast cancer cells

Alterporriol L, a new bianthraquinone derivative, was isolated from a marine fungus Alternaria sp. ZJ9-6B. The cytotoxic activity and anticancer mechanisms of alterporriol L towards breast cancer cells lines were detected using MTT assay, immunofluorescence, and flow cytometry. Simultaneously, the changes in morphological properties of cells were detected before and after treatment with alterporriol L by atomic force microscope (AFM) at a nanometer scale. MTT assay showed that alterporriol L could effectively inhibit the growth and proliferation, and there was a dose-dependent manner of cell death. Moreover, the alterporriol L could induce cancer cell apoptosis or necrosis. Furthermore, the reactive oxygen species, mitochondrial membrane potential, and cytosolic free calcium level were changed after treatment with alterporriol L, suggesting that alterporriol L played vital roles in breast cancer cells through destroying the mitochondrial. And all these alterations are in accord with changes of morphology detected by AFM, which suggested that the AFM is a useful tool to detect the morphological changes of the cancer cells.     

Improvement of bacterial cellulose production by manipulating the metabolic pathways in which ethanol and sodium citrate involved

Nowadays, bacterial cellulose has played more and more important role as new biological material for food industry and medical and industrial products based on its unique properties. However, it is still a difficult task to improve the production of bacterial cellulose, especially a large number of byproducts are produced in the metabolic biosynthesis processes. To improve bacterial cellulose production, ethanol and sodium citrate are added into the medium during the fermentation, and the activities of key enzymes and concentration of extracellular metabolites are measured to assess the changes of the metabolic flux of the hexose monophosphate pathway (HMP), the Embden–Meyerhof–Parnas pathway (EMP), and the tricarboxylic acid cycle (TCA). Our results indicate that ethanol functions as energy source for ATP generation at the early stage of the fermentation in the HMP pathway and the supplementation of ethanol significantly reduces glycerol generation (a major byproduct). While in the EMP pathway, sodium citrate plays a key role, and its supplementation results in the byproducts (mainly acetic acid and pyruvic acid) entering the gluconeogenesis pathway for cellulose synthesis. Furthermore, by adding ethanol and sodium citrate, the main byproduct citric acid in the TCA cycle is also reduced significantly. It is concluded that bacterial cellulose production can be improved by increasing energy metabolism and reducing the formation of metabolic byproducts through the metabolic regulations of the bypasses.     

Insulin-producing cells from human adipose tissue-derived mesenchymal stem cells detected by atomic force microscope

We successfully differentiated human adipose tissue-derived mesenchymal stem cells (haMSCs) into insulin-producing cells (IPCs) in vitro and did not use any insulin which might be absorbed by cells during in vitro culture. Expression of insulin gene was massively increased by 28,000-fold at day 12 compared with haMSCs (P < 0.05). IPCs could secrete insulin after glucose was stimulated. The higher the concentration of glucose, the more production of insulin was noted. We reported AFM images of IPCs for the first time. AFM images showed that the sizes of cells were similar to each other, and all IPC surface had a porous structure in the cytoplasm area. In sugar-free group, the size of holes was similar (diameter, 1,086.98 ± 156.70 nm; depth, 185.22 ± 52.14 nm). In higher sugar-stimulated group, there were more holes with bigger diameter and smaller depth. (diameter, 3,183.65 ± 2,229.18 nm; depth 109.42 ± 56.26 nm, P < 0.05). We found that the hole diameter and depth could change with the concentration of glucose in media. Concurrently, laser scanning confocal microscopy images indicated that cortical actin network beneath plasma membrane in IPCs was dense and continuous. After glucose stimulation, we found the actin web depolymerized and became discontinuous in IPCs. We speculated that diameter augmentation of holes located in the cytoplasm area in IPCs was one manifestation of excytosis increase.     

组蛋白变异体H2A.Z的研究进展

H2A.Z是组蛋白H2A的变异体之一,是高度保守的组蛋白变异体,参与保护常染色体,防止形成异染色质;并且与转录调节、抗沉默、沉默和基因组稳定性有关。组蛋白变异体H2A.Z可能与染色体形成独立的结构域,从而调节染色质结构功能。但是,H2A.Z对染色体结构功能的作用机制还不是很清楚。组蛋白变异体H2A.Z和它的表观遗传修饰对染色体动态结构和功能起重要的作用。该文将对组蛋白变异体H2A.Z进行综述。