S-nitrosylation of N-ethylmaleimide sensitive factor mediates surface expression of AMPA receptors

Postsynaptic AMPA receptor (AMPAR) trafficking mediates some forms of synaptic plasticity that are modulated by NMDA receptor (NMDAR) activation and N-ethylmaleimide sensitive factor (NSF). We report that NSF is physiologically S-nitrosylated by endogenous, neuronally derived nitric oxide (NO). S-nitrosylation of NSF augments its binding to the AMPAR GluR2 subunit. Surface insertion of GluR2 in response to activation of synaptic NMDARs requires endogenous NO, acting selectively upon the binding of NSF to GluR2. Thus, AMPAR recycling elicited by NMDA neurotransmission is mediated by a cascade involving NMDA activation of neuronal NO synthase to form NO, leading to S-nitrosylation of NSF which is thereby activated, enabling it to bind to GluR2 and promote the receptor's surface expression.     

S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.     

Aberrant signal peptide cleavage of collagen X in Schmid metaphyseal chondrodysplasia. Implications for the molecular basis of the disease

Schmid metaphyseal chondrodysplasia results from mutations in the collagen X (COL10A1) gene. With the exception of two cases, the known mutations are clustered in the C-terminal nonhelical (NC1) domain of the collagen X. In vitro and cell culture studies have shown that the NC1 mutations result in impaired collagen X trimer assembly and secretion. In the two other cases, missense mutations that alter Gly(18) at the -1 position of the putative signal peptide cleavage site were identified (Ikegawa, S., Nakamura, K., Nagano, A., Haga, N., and Nakamura, Y. (1997) Hum. Mutat. 9, 131-135). To study their impact on collagen X biosynthesis using in vitro cell-free translation in the presence of microsomes, and cell transfection assays, these two mutations were created in COL10A1 by site-directed mutagenesis. The data suggest that translocation of the mutant pre-alpha1(X) chains into the microsomes is not affected, but cleavage of the signal peptide is inhibited, and the mutant chains remain anchored to the membrane of microsomes. Cell-free translation and transfection studies in cells showed that the mutant chains associate into trimers but cannot form a triple helix. The combined effect of both the lack of signal peptide cleavage and helical configuration is impaired secretion. Thus, despite the different nature of the NC1 and signal peptide mutations in collagen X, both result in impaired collagen X secretion, probably followed by intracellular retention and degradation of mutant chains, and causing the Schmid metaphyseal chondrodysplasia phenotype.     

Identification of four functionally important microRNA families with contrasting differential expression profiles between drought-tolerant and susceptible rice leaf at vegetative stage

Background

Developing drought-tolerant rice varieties with higher yield under water stressed conditions provides a viable solution to serious yield-reduction impact of drought. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success of rice molecular breeding programmes. microRNAs have received tremendous attention recently due to its importance in negative regulation. In plants, apart from regulating developmental and physiological processes, microRNAs have also been associated with different biotic and abiotic stresses. Hence here we chose to analyze the differential expression profiles of microRNAs in three drought treated rice varieties: Vandana (drought-tolerant), Aday Sel (drought-tolerant) and IR64 (drought-susceptible) in greenhouse conditions via high-throughput sequencing.

Results

Twenty-six novel microRNA candidates involved in the regulation of diverse biological processes were identified based on the detection of miRNA*. Out of their 110 predicted targets, we confirmed 16 targets from 5 novel microRNA candidates. In the differential expression analysis, mature microRNA members from 49 families of known Oryza sativa microRNA were differentially expressed in leaf and stem respectively with over 28 families having at least a similar mature microRNA member commonly found to be differentially expressed between both tissues. Via the sequence profiling data of leaf samples, we identified osa-miR397a/b, osa-miR398b, osa-miR408-5p and osa-miR528-5p as being down-regulated in two drought-tolerant rice varieties and up-regulated in the drought-susceptible variety. These microRNAs are known to be involved in regulating starch metabolism, antioxidant defence, respiration and photosynthesis. A wide range of biological processes were found to be regulated by the target genes of all the identified differentially expressed microRNAs between both tissues, namely root development (5.3–5.7 %), cell transport (13.2–18.4 %), response to stress (10.5–11.3 %), lignin catabolic process (3.8–5.3 %), metabolic processes (32.1–39.5 %), oxidation-reduction process (9.4–13.2 %) and DNA replication (5.7–7.9 %). The predicted target genes of osa-miR166e-3p, osa-miR166h-5p*, osa-miR169r-3p* and osa-miR397a/b were found to be annotated to several of the aforementioned biological processes.

Conclusions

The experimental design of this study, which features rice varieties with different drought tolerance and tissue specificity (leaf and stem), has provided new microRNA profiling information. The potentially regulatory importance of the microRNA genes mentioned above and their target genes would require further functional analyses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1851-3) contains supplementary material, which is available to authorized users.     

Coming together is a beginning: The making of an intervertebral disc

The intervertebral disc (IVD) is a complex fibrocartilaginous structure located between the vertebral bodies that allows for movement and acts as a shock absorber in our spine for daily activities. It is composed of three components: the nucleus pulposus (NP), annulus fibrosus, and cartilaginous endplate. The characteristics of these cells are different, as they produce specific extracellular matrix (ECM) for tissue function and the niche in supporting the differentiation status of the cells in the IVD. Furthermore, cell heterogeneities exist in each compartment. The cells and the supporting ECM change as we age, leading to degenerative outcomes that often lead to pathological symptoms such as back pain and sciatica. There are speculations as to the potential of cell therapy or the use of tissue engineering as treatments. However, the nature of the cells present in the IVD that support tissue function is not clear. This review looks at the origin of cells in the making of an IVD, from the earliest stages of embryogenesis in the formation of the notochord, and its role as a signaling center, guiding the formation of spine, and in its journey to become the NP at the center of the IVD. While our current understanding of the molecular signatures of IVD cells is still limited, the field is moving fast and the potential is enormous as we begin to understand the progenitor and differentiated cells present, their molecular signatures, and signals that we could harness in directing the appropriate in vitro and in vivo cellular responses in our quest to regain or maintain a healthy IVD as we age. Birth Defects Research (Part C) 102:83–100, 2014. © 2014 Wiley Periodicals, Inc.     

Profiling of recovery efficiencies for three standard protocols (FDA-BAM, ISO-11290, and modified USDA) on temperature-injured Listeria monocytogenes

There have been a number of studies conducted in order to compare the efficiencies of recovery rates, utilizing different protocols, for the isolation of L. monocytogenes. However, the severity of multiple cell injury has not been included in these studies. In the current study, L. monocytogenes ATCC 19112 was injured by exposure to extreme temperatures (60°C and -20°C) for a one-step injury, and for a two-step injury the cells were transferred directly from a heat treatment to frozen state to induce a severe cell injury (up to 100% injury). The injured cells were then subjected to the US Food and Drug Administration (FDA), the ISO-11290, and the modified United States Department of Agriculture (mUSDA) protocols, and plated on TSAyeast (0.6% yeast), PALCAM agar, and CHROMAgar Listeria for 24 h or 48 h. The evaluation of the total recovery of injured cells was also calculated based on the costs involved in the preparation of media for each protocol. Results indicate that the mUSDA method is best able to aid the recovery of heat-injured, freeze-injured, and heat-freeze-injured cells and was shown to be the most cost effective for heat-freeze-injured cells.     

Alpha-tomatine induces apoptosis and inhibits nuclear factor-kappa B activation on human prostatic adenocarcinoma PC-3 cells

Background

Alpha-tomatine (α-tomatine) is the major saponin in tomato (Lycopersicon esculentum). This study investigates the chemopreventive potential of α-tomatine on androgen-independent human prostatic adenocarcinoma PC-3 cells.

Methodology/Principal Findings

Treatment of highly aggressive human prostate cancer PC-3 cells with α-tomatine resulted in a concentration-dependent inhibition of cell growth with a half-maximal efficient concentration (EC₅₀) value of 1.67±0.3 µM. It is also less cytotoxic to normal human liver WRL-68 cells and normal human prostate RWPE-1 cells. Assessment of real-time growth kinetics by cell impedance-based Real-Time Cell Analyzer (RTCA) showed that α-tomatine exhibited its cytotoxic effects against PC-3 cells as early as an hour after treatment. The inhibitory effect of α-tomatine on PC-3 cancer cell growth was mainly due to induction of apoptosis as evidenced by positive Annexin V staining and decreased in mitochondrial membrane potential but increased in nuclear condensation, polarization of F-actin, cell membrane permeability and cytochrome c expressions. Results also showed that α-tomatine induced activation of caspase-3, -8 and -9, suggesting that both intrinsic and extrinsic apoptosis pathways are involved. Furthermore, nuclear factor-kappa B (NF-κB) nuclear translocation was inhibited, which in turn resulted in significant decreased in NF-κB/p50 and NF-κB/p65 in the nuclear fraction of the treated cells compared to the control untreated cells. These results provide further insights into the molecular mechanism of the anti-proliferative actions of α-tomatine.

Conclusion/Significance

α-tomatine induces apoptosis and inhibits NF-κB activation on prostate cancer cells. These results suggest that α-tomatine may be beneficial for protection against prostate cancer development and progression.     

A novel counter‐selection method for markerless genetic modification in Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is a photosynthetic organism capable of efficient harnessing of solar energy while capturing CO₂ from the environment. Methods to genetically alter its genomic DNA are essential for elucidating gene functions and are useful tools for metabolic engineering. In this study, a novel counter‐selection method for the genetic alteration of Synechocystis was developed. This method utilizes the nickel inducible expression of mazF, a general protein synthesis inhibitor, as a counter‐selection marker. Counter‐selection is particularly useful because the engineered strain is free of any markers which make further genetic modification independent of available antibiotic resistance genes. The usability of this method was further demonstrated by altering genes at several loci in two variants of Synechocystis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013