Mouse cytoplasmic dynein intermediate chains: identification of new isoforms, alternative splicing and tissue distribution of transcripts

BACKGROUND: Intracellular transport of cargoes including organelles, vesicles, signalling molecules, protein complexes, and RNAs, is essential for normal function of eukaryotic cells. The cytoplasmic dynein complex is an important motor that moves cargos along microtubule tracks within the cell. In mammals this multiprotein complex includes dynein intermediate chains 1 and 2 which are encoded by two genes, Dync1i1 and Dync1i2. These proteins are involved in dynein cargo binding and dynein complexes with different intermediate chains bind to specific cargoes, although the mechanisms to achieve this are not known. The DYNC1I1 and DYNC1I2 proteins are translated from different splice isoforms, and specific forms of each protein are essential for the function of different dynein complexes in neurons. METHODOLOGY/PRINCIPAL FINDINGS: Here we have undertaken a systematic survey of the dynein intermediate chain splice isoforms in mouse, basing our study on mRNA expression patterns in a range of tissues, and on bioinformatics analysis of mouse, rat and human genomic and cDNA sequences. We found a complex pattern of alternative splicing of both dynein intermediate chain genes, with maximum complexity in the embryonic and adult nervous system. We have found novel transcripts, including some with orthologues in human and rat, and a new promoter and alternative non-coding exon 1 for Dync1i2. CONCLUSIONS/SIGNIFICANCE: These data, including the cloned isoforms will be essential for understanding the role of intermediate chains in the cytoplasmic dynein complex, particularly their role in cargo binding within individual tissues including different brain regions

TDP-43 is a culprit in human neurodegeneration, and not just an innocent bystander

In 2006 the protein TDP-43 was identified as the major ubiquitinated component deposited in the inclusion bodies found in two human neurodegenerative diseases, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenesis of both disorders is unclear, although they are related by having some overlap of symptoms and now by the shared histopathology of TDP-43 deposition. Now, in 2008, several papers have been published in quick succession describing mutations in the TDP-43 gene, showing they can be a primary cause of amyotrophic lateral sclerosis. There are many precedents in neurodegenerative disease in which rare single-gene mutations have given great insight into understanding disease processes, which is why the TDP-43 mutations are potentially very important

Description of Transport Tunnel in Haloalkane Dehalogenase Variant LinB D147C+L177C from Sphingobium japonicum

The activity of enzymes with active sites buried inside their protein core highly depends on the efficient transport of substrates and products between the active site and the bulk solvent. The engineering of access tunnels in order to increase or decrease catalytic activity and specificity in a rational way is a challenging task. Here, we describe a combined experimental and computational approach to characterize the structural basis of altered activity in the haloalkane dehalogenase LinB D147C+L177C variant. While the overall protein fold is similar to the wild type enzyme and the other LinB variants, the access tunnels have been altered by introduced cysteines that were expected to form a disulfide bond. Surprisingly, the mutations have allowed several conformations of the amino acid chain in their vicinity, interfering with the structural analysis of the mutant by X-ray crystallography. The duration required for the growing of protein crystals changed from days to 1.5 years by introducing the substitutions. The haloalkane dehalogenase LinB D147C+L177C variant crystal structure was solved to 1.15 angstrom resolution, characterized and deposited to Protein Data Bank under PDB ID 6s06

Precision mass measurements on neutron-rich rare-earth isotopes at JYFLTRAP - reduced neutron pairing and implications for the rr-process calculations

The rare-earth peak in the $r$-process abundance pattern depends sensitively on both the astrophysical conditions and subtle changes in nuclear structure in the region. This work takes an important step elucidating the nuclear structure and reducing the uncertainties in $r$-process calculations via precise atomic mass measurements at the JYFLTRAP double Penning trap. $^{158}$Nd, $^{160}$Pm, $^{162}$Sm, and $^{164-166}$Gd have been measured for the first time and the precisions for $^{156}$Nd, $^{158}$Pm, $^{162,163}$Eu, $^{163}$Gd, and $^{164}$Tb have been improved considerably. Nuclear structure has been probed via two-neutron separation energies $S_{2n}$ and neutron pairing energy metrics $D_n$. The data do not support the existence of a subshell closure at $N=100$. Neutron pairing has been found to be weaker than predicted by theoretical mass models. The impact on the calculated $r$-process abundances has been studied. Substantial changes resulting in a smoother abundance distribution and a better agreement with the solar $r$-process abundances are observed.Comment: 8 pages, 4 figures, accepted for publication in Physical Review Letter

Compressed gas domestic aerosol valve design using high viscous product

Most of the current universal consumer aerosol products using high viscous product such as cooking oil, antiperspirants, hair removal cream are primarily used LPG (Liquefied Petroleum Gas) propellant which is unfriendly environmental. The advantages of the new innovative technology described in this paper are: i. No butane or other liquefied hydrocarbon gas is used as a propellant and it replaced with Compressed air, nitrogen or other safe gas propellant. ii. Customer acceptable spray quality and consistency during can lifetime iii. Conventional cans and filling technology There is only a feasible energy source which is inert gas (i.e. compressed air) to replace VOCs (Volatile Organic Compounds) and greenhouse gases, which must be avoided, to improve atomisation by generating gas bubbles and turbulence inside the atomiser insert and the actuator. This research concentrates on using “bubbly flow” in the valve stem, with injection of compressed gas into the passing flow, thus also generating turbulence. The new valve designed in this investigation using inert gases has advantageous over conventional valve with butane propellant using high viscous product (> 400 Cp) because, when the valving arrangement is fully open, there are negligible energy losses as fluid passes through the valve from the interior of the container to the actuator insert. The use of valving arrangement thus permits all pressure drops to be controlled, resulting in improved control of atomising efficiency and flow rate, whereas in conventional valves a significant pressure drops occurs through the valve which has a complex effect on the corresponding spray

Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system

Standardization of cytokine flow cytometry assays

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4(+)cytokine(+ )cells and CD8(+)cytokine(+ )cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays

Global profiling of co- and post-translationally N-myristoylated proteomes in human cells

Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells

Understanding biological responses to degraded hydromorphology and multiple stresses. Deliverable 3.2 of REFORM (REstoring rivers FOR effective catchment Management), a Collaborative project (large-scale integrating project) funded by the European Commission within the 7th Framework Programme under Grant Agreement 282656

The aim of this deliverable is to conceptually model and empirically test the response of biota to the effects of both hydromorphological pressures acting in concert with one another or with other types of pressures. Best use is made of existing large national monitoring datasets (Denmark, UK, Finland, France, Germany, Austria & WISER datasets), case studies and modeling to provide evidence of multiple stressors interacting to alter river biota (Biological Quality Elements: BQE)

Cytotaxonomic studies on some Vicia L. species growing in the eastern Mediterranean and southern Aegean regions II.

In this study, some natural Vicia L. species growing naturally in eastern Mediterranean and southern Aegean Regions have been investigated morphological and cytotaxonomic point of view. Morphology, chromosome number and morphometries of the 9 taxa belonging to the Vicia L. genus including Vicia peregrina, V. lutea var. hirta, V. anatolica, V. hybrida, V. grandiflora var. grandiflora, V. grandiflora var. dissecta, V. cuspidata, V. sativa subsp. sativa, V. sativa subsp. nigra var. segatalis have been studied by using karyological and numerical taxonomic techniques. Data obtained chromosome measurements were analysed by using cluster analysis. The chromosome number of studied Vicia species have been found as 2n = 10, 12 and 14. The member of Vicia section, related taxa were compared with respect to their chromosomal and morphological characteristics. Some karyological relationships among the studied Vicia taxa were discussed with help of taxonomic history and evolutionary data