Competitive metal-binding stoichiometry between calcium and strontium by cell wall proteins of Neurospora crassa.

Cell wall proteins from Neurospora crassa were isolated and evaluated to demonstrate their metal ability to bind Ca2+ /Sr2+ by loading the solubilized protein fraction on to immobilized metal affinity chromatography (IMAC) column pre-equilibrated with Ca2+ /Sr2+ . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis IMAC eluent, revealed ∼18 proteins with a similarity in the proteome pattern of Ca2+ /Sr2+ fractions. Diethyl aminoethyl chromatography showed five proteins in common in binding to Ca2+ and Sr2+ , were subjected to N-terminal sequencing. The sequence analysis was studied for the determination of metal-binding site prediction by CHED software indicating that all five were found to have a high affinity toward Ca2+ . From these five, two were randomly selected and denoted as CWP-A (possess five Ca binding sites of six metal-binding sites) and CWP-B (possess six binding sites of eight metal-binding sites). They were selected for further characterization studies to determine their Ca2+ bound Sr2+ binding properties. Surprisingly, these proteins were able to bind Sr2+ ions (29 µmol) with equal affinity as to Ca2+ ions (42 µmol) by means of direct binding, and/or by displacing calcium as observed in metal-dependent proteolytic protection, fluorescence-based metal exchange assays, and molecular simulation studies. From the results, we demonstrate for the first time, that there is a stoichiometry between Ca2+ (an essential macro elemental metal ion) and Sr2+ ions (a nonessential element for which no reported metabolic activity is reported) for the metal-binding sites on cell wall proteins. This stoichiometry could be due to similar atomic dimensions and metal-protein structure stabilizing properties of Sr2+ compared to Ca2+ .

Zinc dyshomeostasis in azoxymethane-induced colonic precancerous and cancerous lesions in Fischer rats.

Zinc is an essential micronutrient involved in various biological processes. It is also argued that tumors need zinc for maintenance and proliferation and tumor cell apoptosis. Zinc homeostasis is regulated by the gastrointestinal tract and involves interplay of host, dietary, environmental and social factors such as alcohol consumption. The DNA alkylation agent azoxymethane (AOM), which is primarily activated in the liver, induces a high incidence of initiation and promotion steps of precancerous lesions in the colon of rats. The altered expression of hepatic zinc transporters by AOM may lead to zinc dyshomeostasis in liver. Decreased serum zinc concentration, despite increased liver zinc also indicates altered liver zinc mobilization and failure to regulate zinc homeostasis. During the transformation from normal colonic mucosa to colonic epithelial hyperplasia and aberrant crypt formation, a reduction in zinc concentration is observed. It will be interesting to study further if the same trend continues throughout tumor progression towards adenocarcinomas. Lowered local zinc concentrations in the colon epithelium may not just reflect a bystander effect, but may induce cell proliferation and compromise DNA integrity due to impairment of zinc-containing proteins. In congruence with the tissue zinc concentrations, metallothionein levels were found to be less induced in AOM -administered colon compared to normal healthy colon. Lowered tissue zinc levels in small and large intestine were also associated with increased expression of mRNA and protein ZnT1. In this regard, the mode of zinc responsiveness to ZnT1 mimics that of metallothionein, albeit at a lower level for ZnT1.

Overexpression of the ascorbate peroxidase through enhancer-trapped pSB111 bar vector for alleviating drought stress in rice.

Rice (Oryza sativa L.) plant growth and productivity is adversely affected by various stress factors. Overexpression of drought tolerance-related genes is one of the best approaches for developing drought-resistant transgenics. Agrobacterium tumefaciens has been widely used in generating transgenic plants through plasmid vector to obtain desired characteristics and to know the specific expression profiles of genes in the plant. The enhancer trap method was developed to know the specific expression of genes at different stages of growth by entrapping the genes of an organism. In the present study, we designed a vector molecule with a feature of promoting the expression of a specific gene more than four times than its normal expression and it is useful for efficient transformation to higher plants by utilizing the trans configuration of vir genes of the plasmid A. tumefaciens, to transfer right and left sequence bordered of transferred DNA (T-DNA) into the nuclear genome of plants. We developed a binary vector consisting of 1.8-kb green fluorescent protein (GFP) cassette as a reporter gene and 1.4-kb tetramer of CaMv35S enhancer (4XEn) were cloned at HindIII site of pSB11 bar intermediate vector to tag and know the genes and their expression profiles, then mobilized into A. tumefaciens to produce a super-binary vector pSB111-bar-4XEn-GFP. The resultant construct was confirmed by polymerase chain reaction and restriction digestion methods. Finally, we discuss the role of overexpressed ascorbate peroxidase in drought stress.