Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis

Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type (WT) and Syt VII knockout (KO) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.     

Marker assisted breeding for transformability in maize

Corn lines with improved culturability and transformability were produced using Marker Assisted Breeding (MAB) to introgress specific regions from the highly transformable hybrid, Hi-II, into the elite line, FBLL that responds very poorly in culture. FBLL is a female inbred parental stiff-stalk line that has been used to produce a series of some of DEKALB’s historically best selling hybrids. Five unlinked regions important for culturability and transformability were identified by segregation distortion analysis and introgressed into FBLL to produce the highly transformable FBLL-MAB lines. Agrobacterium mediated transformation was used to screen the FBLL-MAB lines and select the most efficient lines for transformation using immature embryo explants. Two highly efficient transformation systems were developed using kanamycin and glyphosate as selective agents. To evaluate agronomics, two testcross hybrids were produced for each of the three lead FBLL-MAB lines. A 25-location, 3-replication yield trial was used to evaluate grain yield, yield stability, and agronomic characteristics of the hybrids. Yields were found to be 2–5% lower and more stable (across a diverse set of environments) among hybrids produced with the FBLL-MAB lines as compared to the same hybrids produced with FBLL.     

Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

Plasma membrane calcium pumps in smooth muscle: from fictional molecules to novel inhibitors

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.     

sIR: siRNA Information Resource,a web-based tool for siRNA sequence design and analysis and an open access siRNA database

Background  

RNA interference has revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In this report we describe a web-based computational tool, siRNA Information Resource (sIR), which consists of a new open source database that contains validation information about published siRNA sequences and also provides a user-friendly interface to design and analyze siRNA sequences against a chosen target sequence.     

Induction and submerged cultivation of Valeriana jatamansi adventitious root cultures for production of valerenic acids and its derivatives

Plant Cell, Tissue and Organ Culture (PCTOC) - In vitro adventitious roots were induced from leaves of Valeriana jatamansi to assess their potential as a sustainable alternative to extract...     

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP?+?1.4 µM GA₃ enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.

     

The red seaweed Kappaphycus alvarezii antiporter gene (KaNa+/H+) confers abiotic stress tolerance in transgenic tobacco

Molecular Biology Reports - Plant establishment, growth, development and productivity are adversely affected by abiotic stresses that are dominant characteristics of environmentally...