Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs.

Identifying molecular features that distinguish fluvastatin-sensitive breast tumor cells.

Statins, routinely used to treat hypercholesterolemia, selectively induce apoptosis in some tumor cells by inhibiting the mevalonate pathway. Recent clinical studies suggest that a subset of breast tumors is particularly susceptible to lipophilic statins, such as fluvastatin. To quickly advance statins as effective anticancer agents for breast cancer treatment, it is critical to identify the molecular features defining this sensitive subset. We have therefore characterized fluvastatin sensitivity by MTT assay in a panel of 19 breast cell lines that reflect the molecular diversity of breast cancer, and have evaluated the association of sensitivity with several clinicopathological and molecular features. A wide range of fluvastatin sensitivity was observed across breast tumor cell lines, with fluvastatin triggering cell death in a subset of sensitive cell lines. Fluvastatin sensitivity was associated with an estrogen receptor alpha (ERα)-negative, basal-like tumor subtype, features that can be scored with routine and/or strong preclinical diagnostics. To ascertain additional candidate sensitivity-associated molecular features, we mined publicly available gene expression datasets, identifying genes encoding regulators of mevalonate production, non-sterol lipid homeostasis, and global cellular metabolism, including the oncogene MYC. Further exploration of this data allowed us to generate a 10-gene mRNA abundance signature predictive of fluvastatin sensitivity, which showed preliminary validation in an independent set of breast tumor cell lines. Here, we have therefore identified several candidate predictors of sensitivity to fluvastatin treatment in breast cancer, which warrant further preclinical and clinical evaluation.

Hypocholesterolemic effect of rice protein is due to regulating hepatic cholesterol metabolism in adult rats.

Aging is one of major risk factors for developing hypercholesterolemia. To elucidate the cholesterol-lowering mechanism exerted by rice protein (RP), the effects on hepatic cholesterol outputs and cholesterol metabolism related enzymes were investigated in adult rats, which were fed by casein (CAS) and RP without cholesterol in diets. After 2 weeks of feeding, the significant cholesterol-lowering effect was observed in adult rats fed by RP compared to CAS. The hepatic total- and VLDL-cholesterol secretions into circulation were significantly depressed in RP group, whereas biliary outputs of bile acids and cholesterol were effectively stimulated by RP-feeding, causing an increase in fecal sterol excretion compared to CAS. As a result, the apparent cholesterol absorption was significantly inhibited by RP. RP-feeding significantly increased the activity and gene expression of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase-2 activity and gene expression were significantly decreased by RP as compared with CAS. Neither activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP did differ from CAS in the liver. The present study demonstrates that rice protein can prevent hypercholesterolemia through modifying hepatic cholesterol metabolism under cholesterol-free dietary condition. The findings suggest that hypocholesterolemic action induced by rice protein is attributed in part to the inhibition of cholesterol absorption during the adult period.

Molecular characterization of three 3-hydroxy-3-methylglutaryl-CoA reductase genes including pathogen-induced Hmg2 from pepper (Capsicum annuum).

Sesquiterpene phytoalexins, a class of plant defense metabolites, are synthesized from the cytosolic acetate/mevalonate pathway in isoprenoids biosynthetic system of plants. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the synthesis of mevalonate, which is the specific precursor of this pathway, as a multi gene family. Three kinds of cDNA clones encoding HMGR were isolated from Korean red pepper (Capsicum annuum L. cv. NocKwang) and the HMGR2 gene (Hmg2) was especially obtained from a cDNA library constructed with Phytophthora capsici-infected pepper root RNAs. The Hmg2 encoding a 604-amino-acid peptide had typical features as an elicitor-induced isoform among HMGRs on its gene structure and had a predicted amino acid sequence homology. In addition, the expression of Hmg2 was rapidly induced within 1 h in response to a fungal pathogen and continuously increased up to 48 h. Together with sesquiterpene cyclase gene that was strongly induced 24 h after pathogen-infection, the Hmg2 and farnesyl pyrophosphate synthase gene were coordinately and sequentially regulated for the biosynthesis of defense-related sesquiterpene phytoalexins in pepper.