Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.     

28-homobrassinolide improves growth and photosynthesis in <Emphasis Type="Italic">Cucumis sativus</Emphasis> L. through an enhanced antioxidant system in the presence of chilling stress

The ameliorative role of 28-homobrassinolide under chilling stress in various growth, photosynthesis, enzymes and biochemical parameters of cucumber (Cucumis sativus L.) were investigated. Cucumber seedlings were sprayed with 0 (control), 10⁻⁸, or 10⁻⁶ M of 28-homobrassinolide at the 30-day stage. 48 h after treatment plants were exposed for 18 h to chilling temperature (10/8°C, 5/3°C). The most evident effect of chilling stress was the marked reduction in plant growth, chlorophyll (Chl) content, and net photosynthetic rate, efficiency of photosystem II and activities of nitrate reductase and carbonic anhydrase. Moreover, the activities of antioxidant enzymes; catalase (E.C. 1.11.1.6), peroxidase (E.C.1.11.1.7), superoxide dismutase (E.C. 1.15.1.1) along with the proline content in leaves of the cucumber seedlings increased in proportion to chilling temperature. The stressed seedlings of cucumber pretreated with 28-homobrassinolide maintained a higher value of antioxidant enzymes and proline content over the control suggesting the protective mechanism against the ill-effect caused by chilling stress might be operative through an improved antioxidant system. Furthermore, the protective role of 28-homobrassinolide was reflected in improved growth, water relations, photosynthesis and maximum quantum yield of photosystem II both in the presence and absence of chilling stress.     

Protein and DNA destabilization by osmolytes: the other side of the coin

Osmolytes are naturally occurring small molecules accumulated intracellularly to protect organisms from various denaturing stresses. Similar to the two faces of a coin, several of these osmolytes are stabilizing and destabilizing proteins depending on the concentrations and/or solvent conditions. For example, the well known stabilizing osmolyte, trehalose destabilizes some proteins at high concentration and/or high pH. In spite of the fact that destabilizing aspects of osmolytes can modulate many cellular processes including regulation of protein homeostasis (proteostasis), protein-protein interaction, and protein-DNA interaction, researchers have mostly focused on the stabilizing aspects of osmolytes. Thus, it is important to look into both aspects of osmolytes to determine their precise role under physiological conditions. In this article, we have discussed both stabilizing and destabilizing/denaturant aspects of osmolytes to uncover both sides of the coin.     

Hydroxyl-substituted sulfonylureas as potent inhibitors of specific [3H]glyburide binding to rat brain synaptosomes

We are seeking to discover potent CNS-active sulfonylureas with structural features that allow for the formation of several types of prodrugs. We report herein the syntheses of compounds comprising an initial series of hydroxyl-substituted analogues of the potent ATP-sensitive potassium channel blockers glyburide (glibenclamide) and gliquidone. Somewhat unexpectedly, several of the compounds were found to be comparably potent to glyburide as inhibitors of specific [(3)H]glyburide binding in rat brain preparations.     

Soluble T cell immunoglobulin and mucin domain (TIM)-1 and -4 generated by A Disintegrin And Metalloprotease (ADAM)-10 and -17 bind to phosphatidylserine

T cell immunoglobulin and mucin domain 1 and 4 (TIM-1 and -4) proteins serve as phosphatidylserine receptors to engulf apoptotic cells. Here we show that human TIM-1 and TIM-4 proteins are targets of A Disintegrin And Metalloprotease (ADAM)-mediated ectodomain shedding resulting in soluble forms of TIM-1 and TIM-4. We identified ADAM10 and ADAM17 as major sheddases of TIM-1 and TIM-4 as shown by protease-specific inhibitors, the ADAM10 prodomain, siRNA and ADAM10/ADAM17 deficient murine embryonic fibroblasts (MEFs). TIM-1 and TIM-4 lacking the intracellular domain were efficiently cleaved after ionomycin- and PMA-treatment, indicating that the intracellular domain was not necessary for ectodomain shedding. Soluble TIM-1 and -4 were able to bind to phosphatidylserine, suggesting that soluble TIM-1 and -4 might act as negative regulators of cellular TIM-1 and -4. In summary, we describe TIM-1 and TIM-4 as novel targets for ADAM10- and ADAM17-mediated ectodomain shedding.     

Calcium malate overproduction by Penicillium viticola 152 using the medium containing corn steep liquor

In this study, after screening of eight fungal strains for their ability to produce calcium malate, it was found that Penicillium viticola 152 isolated from marine algae among them could produce the highest titer of calcium malate. At the same time, it was found that corn steep liquor (CSL) could stimulate calcium malate production and 0.5 % (v/v) CSL was the most suitable for calcium malate production. Under the optimal conditions, a titer of calcium malate in the supernatant was 132 g/l at flask level. During a 10-l fermentation, a titer of 168 g/l, a yield of 1.28 g/g of glucose, and a productivity of 1.75 g/l/h were reached within 96 h of the fermentation, and 93.4 % of the sugar was used for calcium malate production and cell growth, demonstrating that the titer, yield, and productivity of calcium malate by this fungal strain were very high and the fermentation period was very short. After analysis of the partially purified product with high-performance liquid chromatography, it was found that the main product was calcium malate. The results demonstrated that P. viticola 152 obtained in this study was the most suitable for developing a novel one-step fermentation process for calcium malate production from glucose on a large scale.     

Association of mutation and hypermethylation of p21 gene with susceptibility to breast cancer: a study from north India

p21 gene located at chromosome 6p21.2 is a possible tumour suppressor gene involved in the pathogenesis of breast cancer. Both genetic and epigenetic alterations in p21 have been implicated in breast carcinoma. In the present study, our main aim was to study the impact of these two kinds of alterations of p21 gene in Indian female breast cancer patients. A total of 150 female breast cancer patients of north India were screened by PCR-SSCP followed by direct sequencing and methylation specific PCR. Mutational screening of p21 gene revealed significant amount of mutations [32.66 % (49/150)] in exon 2, whereas p21 promoter was found hypermethylated in 42 of 150 (28 %) breast cancer patients in our population. The intriguing feature of the study was the G>T transition (GAG>TAG) at codon 107 and the A>C transition (AGC>CGC) at codon 146 possibly rendering p21 completely ineffective in its anti- proliferative activity. Our results suggest a significant association between the mutational and hypermethylation profile of p21 gene. Therefore, we show for the first time that the significant association of p21 mutation and hypermethylation leads to the complete inactivation of p21 gene in Indian female breast cancer patients. Complete silencing of the p21 gene seems to be the result not only of genetic alterations but also of epigenetic modification.     

Expression line approach to recombinant human epidermal growth factor into the yeast,Pichia pastoris from Huh-7 cell line

Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.