Status and plans of NASA's Materials Science and Manufacturing in Space (MS/MS) program

A description is given of a research and development program on the space shuttle mission designed to prepare the way for possible commercial manufacturing operations on permanently orbiting space stations

Neurotransmitter release regulated by a MALS–liprin-α presynaptic complex

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli–CASK–Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-α as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS–CASK–liprin-α complex recruits components of the synaptic release machinery to adhesive proteins of the active zone

P110 and P140 Cytadherence-Related Proteins Are Negative Effectors of Terminal Organelle Duplication in Mycoplasma genitalium

BACKGROUND:The terminal organelle is a complex structure involved in many aspects of the biology of mycoplasmas such as cell adherence, motility or cell division. Mycoplasma genitalium cells display a single terminal organelle and duplicate this structure prior to cytokinesis in a coordinated manner with the cell division process. Despite the significance of the terminal organelle in mycoplasma virulence, little is known about the mechanisms governing its duplication. METHODOLOGY/PRINCIPAL FINDINGS:In this study we describe the isolation of a mutant, named T192, with a transposon insertion close to the 3' end of the mg192 gene encoding for P110 adhesin. This mutant shows a truncated P110, low levels of P140 and P110 adhesins, a large number of non-motile cells and a high frequency of new terminal organelle formation. Further analyses revealed that the high rates of new terminal organelle formation in T192 cells are a direct consequence of the reduced levels of P110 and P140 rather than to the expression of a truncated P110. Consistently, the phenotype of the T192 mutant was successfully complemented by the reintroduction of the mg192 WT allele which restored the levels of P110 and P140 to those of the WT strain. Quantification of DAPI-stained DNA also showed that the increase in the number of terminal organelles in T192 cells is not accompanied by a higher DNA content, indicating that terminal organelle duplication does not trigger DNA replication in mycoplasmas. CONCLUSIONS/SIGNIFICANCE:Our results demonstrate the existence of a mechanism regulating terminal organelle duplication in M. genitalium and strongly suggest the implication of P110 and P140 adhesins in this mechanism

Analysis of sludge from Hanford K East Basin canisters

Sludge samples from the canisters in the Hanford K East Basin fuel storage pool have been retrieved and analyzed. Both chemical and physical properties have been determined. The results are to be used to determine the disposition of the bulk of the sludge and to assess the impact of residual sludge on dry storage of the associated intact metallic uranium fuel elements. This report is a summary and review of the data provided by various laboratories. Although raw chemistry data were originally reported on various bases (compositions for as-settled, centrifuged, or dry sludge) this report places all of the data on a common comparable basis. Data were evaluated for internal consistency and consistency with respect to the governing sample analysis plan. Conclusions applicable to sludge disposition and spent fuel storage are drawn where possible

Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding

Analysis of Inducible Nitric Oxide Synthase Gene Polymorphisms in Vitiligo in Han Chinese People

Background: Vitiligo is a chronic depigmented skin disorder with regional melanocytes depletion. The pathogenesis was not completely clarified. Recently, more and more evidence suggested that polymorphisms of some genes are associated with vitiligo risk. Here, we want to examine the association between the inducible nitric oxide synthase (iNOS) gene polymorphisms and the risk of vitiligo in Chinese populations. Methods and Principal Findings: In a hospital-based case-control study of 749 patients with vitiligo and 763 age- and sexmatched healthy controls, three polymorphisms of iNOS gene were genotyped by using the PCR-restriction fragment length polymorphism (PCR-RFLP) and mutagenically separated PCR (MS-PCR) methods, respectively. We found the iNOS-954 polymorphism was associated with a significantly higher risk of vitiligo (adjusted OR = 1.36, 95 % CI = 1.02–1.81). Furthermore, this association is more pronounced in vulgaris vitiligo, active vitiligo and vitiligo without other autoimmune diseases in the stratification study. Analysis of haplotypes showed increased risk for the C-1173C-954CEx16+14 (OR = 1.44, 95% CI = 1.01–1.74). In addition, the serum iNOS activity is significantly associated with iNOS-954 combined genotype (GC+CC) and is much higher in vitiligo patients than in the controls (P,0.01). Logistic regression analysis of iNOS activity showed increased risk between higher activity and iNOS-954 GRC variant genotype carriers (Ptrend,0.001). Conclusions and Significance: INOS gene polymorphisms may play an important role in the genetic susceptibility to th

Science To Support DOE Site Cleanup: The Pacific Northwest National Laboratory Environmental Management Science Program Awards

Pacific Northwest National Laboratory (PNNL) was awarded ten Environmental Management Science Program (EMSP) research grants in fiscal year 1996, six in fiscal year 1997, nine in fiscal year 1998, seven in fiscal year 1999, and five in fiscal year 2000. All of the fiscal year 1996 award projects have published final reports. The 1997 and 1998 award projects have been completed or are nearing completion. Final reports for these awards will be published, so their annual updates will not be included in this document. This section summarizes how each of the 1999 and 2000 grants address significant U.S. Department of Energy (DOE) cleanup issues, including those at the Hanford Site. The technical progress made to date in each of these research projects is addressed in more detail in the individual progress reports contained in this document. The 1999 and 2000 EMSP awards at PNNL are focused primarily in two areas: Tank Waste Remediation, and Soil and Groundwater Cleanup

Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved