Enhanced spore production ofBacillus thuringiensis by fed-batch culture

Summary Fed-batch culture was carried out to increase cell mass followed by batch culture for spore production ofbacillus thuringiensis. High cell mass obtained by increasing the feeding glucose concentration in constant fed-batch culture which supported fast cell growth resulted in good sporulation during subsequent batch culture, and the maximum cell mass of 72.6 g/L and spore concentration of 1.25×10¹⁰ spores/mL could be obtained.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Candidate Markers That Associate with Chemotherapy Resistance in Breast Cancer through the Study on Taxotere-Induced Damage to Tumor Microenvironment and Gene Expression Profiling of Carcinoma-Associated Fibroblasts (CAFs)

Recently, emerging evidence has suggested that carcinoma-associated fibroblasts (CAFs) could contribute to chemotherapy resistances in breast cancer treatment. The aim of this study is to compare the gene expression profiling of CAFs before and after chemotherapy and pick up candidate genes that might associate with chemotherapy resistance and could be used as predictors of treatment response. CAFs were cultured from surgically resected primary breast cancers and identified with immunohistochemistry (IHC) and Flow cytometry (FCM). MDA-MB-231 cells were cultured as the breast cancer cell line. Cell adhesion assay, invasion assay, and proliferation assay (MTT) were performed to compare the function of MDA-MB-231 cells co-cultured with CAFs and MDA-MB-231 cells without co-culture, after chemotherapy. Totally 6 pairs of CAFs were prepared for microarray analysis. Each pair of CAFs were obtained from the same patient and classified into two groups. One group was treated with Taxotere (regarded as after chemotherapy) while the other group was not processed with Taxotere (regarded as before chemotherapy). According to our study, the primary-cultured CAFs exhibited characteristic phenotype. After chemotherapy, MDA-MB-231 cells co-cultured with CAFs displayed increasing adhesion, invasiveness and proliferation abilities, compared with MDA-MB-231 cells without CAFs. Moreover, 35 differentially expressed genes (absolute fold change >2) were identified between CAFs after chemotherapy and before chemotherapy, including 17 up-regulated genes and 18 down-regulated genes. CXCL2, MMP1, IL8, RARRES1, FGF1, and CXCR7 were picked up as the candidate markers, of which the differential expression in CAFs before and after chemotherapy was confirmed. The results indicate the changes of gene expression in CAFs induced by Taxotere treatment and propose the candidate markers that possibly associate with chemotherapy resistance in breast cancer.     

Plasmodium vivax: molecular cloning, expression and characterization of glutathione S-transferase

Malaria parasite glutathione S-transferases (GSTs) are postulated to be essential for parasite survival by protecting the parasite against oxidative stress and buffering the detoxification of heme-binding compounds; therefore, GSTs are considered potential targets for drug development. In this study, we identified a Plasmodium vivax gene encoding GST (PvGST) and characterized the biochemical properties of the recombinant enzyme. The PvGST contained 618 bp that encoded 205 amino acids and shared a significant degree of sequence identity with GSTs from other Plasmodium species. The recombinant homodimeric enzyme had an approximate molecular mass of 50kDa and exhibited GSH-conjugating and GSH-peroxidase activities towards various model substrates. The optimal pH for recombinant PvGST (rPvGST) activity was pH 8.0, and the enzyme was moderately unstable at 37 degrees C. The K(m) values of rPvGST with respect to GSH and CDNB were 0.17+/-0.09 and 2.1+/-0.4mM, respectively. The significant sequence homology and similar biochemical properties of PvGST and Plasmodium falciparum GST (PfGST) indicate that they may have similar molecular structures. This information may be useful for the design of specific inhibitors for plasmodial GSTs as potential antimalarial drugs.     

果蝇原生殖细胞特化的分子机制

原生殖细胞在许多有性生殖动物的胚胎发育早期就已特化出来,并进一步分化为生殖细胞以产生新的子代。动物原生殖细胞的特化主要有生殖质决定和诱导两种模式,果蝇原生殖细胞的特化模式属于前者。研究表明,果蝇原生殖细胞特化过程中生殖质组装的关键基因是osk,其调控下游基因转录产物的定位和翻译,如vas和tud。此外,基因转录沉默是原生殖细胞特化过程的一个重要特征,其与生殖质中的成分如基因nos、gcl、pgc的表达产物密切相关。现对果蝇原生殖细胞特化分子机制进行综述。     

Effect of ionic liquids on enzymatic synthesis of caffeic acid phenethyl ester

Although caffeic acid phenethyl ester (CAPE), an active flavonoid, plays an important role in the antioxidant activity of honeybee propolis, the isolation of CAPE from honeybee propolis is time-consuming due to wide variety of impurities present. Therefore, biochemical method to synthesize CAPE was investigated in this study. Since ionic liquids (ILs) possess some unique characteristics as appreciated alternatives to conventional solvents for certain biotransformation, the effect of ILs as reaction media for enzymatic synthesis of CAPE was assessed. Several factors including substrate molar ratio, and reaction temperature affecting the conversion yield of lipase-catalyzed CAPE synthesis were also investigated. Reaction yields were significantly higher in hydrophobic ILs than in hydrophilic ILs (almost zero). Among nine hydrophobic ILs tested, the highest conversion of synthetic reaction was obtained in 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]). A reaction temperature of 70 °C was found to give high conversion. In addition, optimal substrate molar ratio between phenethyl alcohol and caffeic acid (CA) was decreased significantly from 92:1 to 30:1 when ILs were used instead of isooctane.     

Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species

Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.