Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.     

Targeting monocarboxylate transporter by α-cyano-4-hydroxycinnamate modulates apoptosis and cisplatin resistance of Colo205 cells: implication of altered cell survival regulation

The present investigation was undertaken to study the effect of in vitro exposure of Colo205, colonadenocarcinoma cells, to monocarboxylate transporter inhibitor α-cyano-4-hydroxycinnamate (αCHC) on cell survival and evolution of resistance to chemotherapeutic drug cisplatin. αCHC-treated Colo205 cells showed inhibition of survival accompanied by an augmented induction of apoptosis. Changes in cell survival properties were associated with alterations in lactate efflux, pH homeostasis, expression of glucose transporters, glucose uptake, HIF-1α, generation of nitric oxide, expression pattern of some key cell survival regulatory molecules: Bcl2, Bax, active caspase-3 and p53. Pretreatment of Colo205 cells with αCHC also altered their susceptibility to the cytotoxicity of cisplatin accompanied by altered expression of multidrug resistance regulating MDR1 and MRP1 genes. This study for the first time deciphers some of the key molecular events underlying modulation of cell survival of cancer cells of colorectal origin by αCHC and its contribution to chemosensitization against cisplatin. Thus these findings will be of immense help in further research for optimizing the use of αCHC for improving the chemotherapeutic efficacy of anticancer drugs like cisplatin.     

Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation

Degradation of streptokinase (SK) has been frequently observed during large-scale protein production. An enhanced susceptibility of SK to degradation has been correlated with its existence in a partially unfolded state. The influence of the carbohydrate moiety on the stability and functional characteristics of SK has been examined by obtaining the glycoform of SK following its secretion through the methylotrophic yeast Pichia pastoris. Secretion of the protein product was achieved by replacing the native secretion signal codons of SK with those from α-factor leader peptide and expressing the fusion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P. pastoris after its integration into the host chromosome. Western blot and zymographic analysis of proteins secreted from the recombinant P. pastoris indicated that SK was glycosylated by the host cells, which resulted in the appearance of a SK species migrating slowly, corresponding to a 55-kDa protein product as compared to the 47-kDa native SK. The glycosylated SK retained a plasminogen activation capability identical to that of its unglycosylated counterpart. Glycoform SK exhibited an enhanced stability profile at 25 °C and 37 °C and improved resistance towards protease treatment compared to unglycosylated SK secreted through P. pastoris after tunicamycin treatment or that secreted from the recombinant Escherichia coli. The results presented thus illustrate that N-linked glycosylation of SK results in 30–40% enhancement of the protein stability and resistance towards degradation but does not interfere with its fibrinolytic function. Received: 1 March 1999 / Received last revision: 5 October 1999 / Accepted: 10 October 1999     

NO and ROS implications in the organization of root system architecture

Over the past decades the role of nitric oxide (NO) and reactive oxygen species (ROS) in signaling and cellular responses to stress has witnessed an exponential trend line. Despite advances in the subject, our knowledge of the role of NO and ROS as regulators of stress and plant growth and their implication in signaling pathways is still partial. The crosstalk between NO and ROS during root formation offers new domains to be explored, as it regulates several plant functions. Previous findings indicate that plants utilize these signaling molecules for regulating physiological responses and development. Depending upon cellular concentration, NO either can stimulate or impede root system architecture (RSA) by modulating enzymes through post-translational modifications. Similarly, the ROS signaling molecule network, in association with other hormonal signaling pathways, control the RSA. The spatial regulation of ROS controls cell growth and ROS determine primary root and act in concert with NO to promote lateral root primordia. NO and ROS are two central messenger molecules which act differentially to upregulate or downregulate the expression of genes pertaining to auxin synthesis and to the configuration of root architecture. The investigation concerning the contribution of donors and inhibitors of NO and ROS can further aid in deciphering their role in root development. With this background, this review provides comprehensive details about the effect and function of NO and ROS in the development of RSA.     

Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.     

Local repeat sequence organization of an intergenic spacer in the chloroplast genome ofChlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: A complex mode of “copychoice replication”?

Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA ofChlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt⁺) in a cross between a pair of polymorphic interfertile strains ofChlamydomonas (C. reinhardtii andC. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, “P2” (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3′ end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, “P2” seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely “sequence-scrambled” product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a "unique" new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a “complex path” of copy-choice replication.