Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions

Background  

Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM) of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation.     

Effects of elevated CO2 concentration, supplemental irrigation and nitrogenous fertilizer application on rain-fed spring wheat yield

It is expected that the CO₂ concentration of the Earth’s atmosphere will reach 600–1000 ppm by the end of the 21st century. Therefore, in this study, we evaluated the effects of elevated CO₂ concentrations on the development of rain-fed spring wheat in an attempt to identify a practical pathway to increase crop production. To accomplish this, a field experiment was conducted at Guyuan Experimental Station in a semiarid region of China during 2005–2007. During this experiment, the CO₂ concentration was increased to 40.0 ppm and supplemental irrigation and nitrogenous fertilizer (N fertilizer) were applied. The experimental results showed that the elevated CO₂ concentration significantly improved the thousand-grain weight and the grain number per spike. Furthermore, supplemental irrigation and N fertilizer application during the elongation and booting stage of rain-fed spring wheat in conjunction with an elevated CO₂ concentration improved the water use efficiency (WUE), nitrogen use efficiency (NUE), thousand-grain weight, and the yield by 14.6%, 39.6%, 9.3%, and 14.7%, respectively, when compared to groups subjected to the same treatment but not grown under elevated CO₂ concentrations. Furthermore, the spring wheat yield was improved by 81.8% in response to an elevated CO₂ concentration, 60 mm of supplemental irrigation and applied N fertilizer (37.5 g m^?2 NH₄NO₃). However, the presence of an elevated CO₂ concentration without supplemental irrigation and N fertilizer only resulted in an increase in the wheat yield of 7.8%. Consequently, the combination of elevated CO₂ concentration, supplemental irrigation and N fertilizer application played an important role in the improvement of WUE, NUE, thousand-grain weight, and grain yield of rain-fed spring wheat in this region.     

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

A clinically-related animal model of Parkinson''s disease (PD) may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as dopamine (DA) neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. To overcome these limitations, we developed a rat model by stereotaxically (ST) infusing small doses of the mitochondrial complex-I inhibitor, rotenone, into two brain sites: the right ventral tegmental area and the substantia nigra. Four weeks after ST rotenone administration, tyrosine hydroxylase (TH) immunoreactivity in the infusion side decreased by 43.7%, in contrast to a 75.8% decrease observed in rats treated systemically with rotenone (SYS). The rotenone infusion also reduced the DA content, the glutathione and superoxide dismutase activities, and induced alpha-synuclein expression, when compared to the contralateral side. This ST model displays neither peripheral toxicity or mortality and has a high success rate. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD research.     

Unifying Fluorescence Microscopy and Mass Spectrometry for Studying Protein Complexes in Cells

We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. After this step we tested the constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosis in Saccharomyces cerevisiae, with a variety of fluorescent and affinity probes. Among the modular tags produced we found several combinations that were optimal for determining subcellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and dephosphorylation. Our results indicate that this approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.Fluorescent proteins have become invaluable probes for studying molecular processes in living cells with light microscopy techniques (1–3). Proteins, organelles, and entire cells can be selectively visualized using a variety of fluorescent proteins fused to the proteins of interest (1–6). Combined with genetics and molecular biology techniques fluorescence microscopy provides an efficient tool for observing molecular phenotypes useful for dissecting the pathways of cell cycle progression and cell response to internal and external signals (7). However, understanding the mechanism controlling the properties of proteins in cells can be a challenging task, frequently requiring a comprehensive characterization of the proteins at the molecular level.The proteins tagged with green fluorescent protein (GFP)¹ can be also purified using GFP antibodies. Cheeseman and Desai (8) and Cristea et al. (9) have enriched GFP-tagged proteins and protein complexes for further detailed analysis by MS. The MS-based methods for protein analysis are fast, sensitive, and able to identify both proteins in complex protein mixtures and residues bearing post-translational modifications (10, 11). Thus, the addition of affinity purification and mass spectrometry steps enabled the researchers to study protein interactions and the post-translational modifications in the context of the protein subcellular localization. Juxtaposition of the protein localization, composition of the protein complexes, and post-translational modifications frequently yield a unique perspective of the cellular processes and the molecular mechanisms of their regulation (12, 13).Using fluorescent proteins also as affinity probes can be problematic in several instances. First of all, the good quality antibodies against the rapidly increasing number of fluorescent proteins (3, 6) are not yet readily available. Furthermore raising antibodies specifically recognizing fluorescent proteins originating from the same organism but fluorescing a different color can be difficult or even impossible because such proteins frequently differ by mutations of only a few amino acids (1–6). Thus, we seek an alternative approach to the design of tags suitable for subcellular localization and purification of proteins and protein complexes that is 1) independent of the availability of antibody to a specific form of a fluorescent protein, 2) suitable for multiplexing, i.e. simultaneous observation of subcellular localization of several proteins and affinity purification of the proteins and stably associated protein complexes, and 3) flexible and easy to modify to incorporate better versions of fluorescent proteins and affinity tags after they are discovered.One possible solution that satisfies the stated requirements is to use a modular tag containing a version of a fluorescent protein fused to an affinity epitope. In this case we can decouple requirements for both modules and optimize the performance of each one independently for fluorescence microscopy and affinity purification experiments. To our knowledge, this possibility was first realized by Thorn and co-worker (14) who have fused 3HA (three repeats of YPYDVPDYA epitope from hemagglutinin protein) and 13MYC (13 repeats of EQKLISEEDL epitope, corresponding to a stretch of the C-terminal amino acids of the human c-MYC protein) tags to several variants of fluorescent proteins. The authors have argued that the fusion of the fluorescent proteins to the affinity epitopes may enable fluorescence and immunochemical analysis but did not test this idea. Cheeseman and Desai (8) fused the S-peptide and hexahistidine epitopes to the GFP protein to enable additional tandem purification steps. Su and co-workers (15) also fused a hexahistidine tag (His₆) to GFP to purify recombinantly produced proteins. Although hexahistidine tag performs well for isolation of overexpressed recombinant proteins, it works poorly for affinity purification of low abundance, endogenously expressed proteins (16). A double affinity tag containing a single MYC epitope and hexahistidine was also used to purify recombinantly produced fluorescent proteins (6).Here we describe the design and implementation of the modular fluorescent and affinity tags. These tags contain a variety of fluorescent proteins, which can be used exclusively for obtaining subcellular visualization, and several small epitope tags that can be utilized to perform two-step affinity purification. To test the performance of the constructs produced, we tagged two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, with a variety of modular tags.Eisosomes are large heterodimeric protein complexes recently discovered in Saccharomyces cerevisiae (17). There are ∼50–100 eisosomes in each mature yeast cell distributed uniformly in a characteristic dotted pattern at the cell surface periphery. Each eisosome contains ∼2000–5000 copies of Pil1 and Lsp1. It was shown that eisosomes serve as portals of endocytosis in yeast. The function of eisosomes is regulated by reversible phosphorylation (18, 19).Among the constructs tested, we found several combinations of fluorescent protein and affinity tags that were optimal for determining subcellular localization and purification of the proteins and protein complexes. We applied these tags to further investigate eisosomes and found several new protein components of the complexes and obtained new insights into molecular mechanisms regulating eisosome integrity by reversible phosphorylation and dephosphorylation. Our results indicate that an approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.     

CCL2 Accelerates Microglia-Mediated Aβ Oligomer Formation and Progression of Neurocognitive Dysfunction

Background

The linkages between neuroinflammation and Alzheimer''s disease (AD) pathogenesis are well established. What is not, however, is how specific immune pathways and proteins affect the disease. To this end, we previously demonstrated that transgenic over-expression of CCL2 enhanced microgliosis and induced diffuse amyloid plaque deposition in Tg2576 mice. This rodent model of AD expresses a Swedish β-amyloid (Aβ) precursor protein mutant.

Methodology/Principal Findings

We now report that CCL2 transgene expression accelerates deficits in spatial and working memory and hippocampal synaptic transmission in β-amyloid precursor protein (APP) mice as early as 2–3 months of age. This is followed by increased numbers of microglia that are seen surrounding Aβ oligomers. CCL2 does not suppress Aβ degradation. Rather, CCL2 and tumor necrosis factor-α directly facilitated Aβ uptake, intracellular Aβ oligomerization, and protein secretion.

Conclusions/Significance

We posit that CCL2 facilitates Aβ oligomer formation in microglia and propose that such events accelerate memory dysfunction by affecting Aβ seeding in the brain.     

The influence of hydrological regimes on sex ratios and spatial segregation of the sexes in two dioecious riparian shrub species in northern Sweden

River management practices have altered the hydrological regimes of many rivers and also altered the availability of regeneration niches for riparian species. We investigated the impact of changed hydrological regimes on the sex ratios and the Spatial Segregation of the Sexes (SSS) in the dioecious species Salix myrsinifolia Salisb.–phylicifolia L. and S. lapponum L. by studying the free-flowing Vindel River and the regulated Ume River in northern Sweden. We surveyed sex ratios of these species in 12 river reaches on the Vindel River and in 17 reaches on the Ume River. In addition, we surveyed the sex and location above mean river stage of 1,002 individuals across both river systems to investigate the SSS of both species. Cuttings were collected from male and female individuals of S. myrsinifolia–phylicifolia from both rivers and subjected to four different water table regimes in a greenhouse experiment to investigate growth response between the sexes. We found an M/F sex ratio in both river systems similar to the regional norm of 0.62 for S. myrsinifolia–phylicifolia and of 0.42 for S. lapponum. We found no evidence of SSS in either the free-flowing Vindel River or the regulated Ume River. In the greenhouse experiment, hydrological regime had a significant effect on shoot and root dry weight and on root length. Significantly higher shoot dry weights were found in females than in males and significantly different shoot and root dry weights were found between cuttings taken from the two rivers. We concluded that changed hydrological regimes are likely to alter dimensions of the regeneration niche and therefore to influence sex ratios and SSS at an early successional stage, making it difficult to find clear spatial patterns once these species reach maturity and can be sexed.     

Testing the higher-taxon approach: a case study of aquatic marcophytes in China’s northwest arid zone and its implications for conservation

Assessments of biodiversity are time-consuming and require a high level of expert knowledge. A reduced set of taxonomic ranks other than species has been proved to be useful for rapid and cost-effective assessment of biodiversity. However, few studies have examined how well this method performs for aquatic plant group that of enormous ecological importance. We studied the aquatic plant flora in the arid zone of China and examined whether the distribution of species α- and β-diversity could be predicted well from genus-, and family-levels. Analyses of 3 years field data showed that significant and positive relations exist between α-diversity of species and α-diversity of genera and family in both entire arid zone and five sub-zones. In contrast, β-diversity at species level is difficult to predict from β-diversity indexes at higher taxonomic level. The results indicate that higher-taxon α-diversity, especially at the generic level in our research, can be useful surrogates of species α-diversity for aquatic plants conservation.     

The discovery of a potent and selective lethal factor inhibitor for adjunct therapy of anthrax infection

A potent and selective anthrax LF inhibitor 40, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, was identified through SAR study of a high throughput screen lead. It has an IC50 of 54 nM in the enzyme assay and an IC50 of 210 nM in the macrophage cytotoxicity assay. Compound 40 is also effective in vivo in several animal model studies.     

The structure of a filamentous bacteriophage

Many thin helical polymers, including bacterial pili and filamentous bacteriophage, have been seen as refractory to high-resolution studies by electron microscopy. Studies of the quaternary structure of such filaments have depended upon techniques such as modeling or X-ray fiber diffraction, given that direct visualization of the subunit organization has not been possible. We report the first image reconstruction of a filamentous virus, bacteriophage fd, by cryoelectron microscopy. Although these thin ( approximately 70 A in diameter) rather featureless filaments scatter weakly, we have been able to achieve a nominal resolution of approximately 8 A using an iterative helical reconstruction procedure. We show that two different conformations of the virus exist, and that in both states the subunits are packed differently than in conflicting models previously proposed on the basis of X-ray fiber diffraction or solid-state NMR studies. A significant fraction of the population of wild-type fd is either disordered or in multiple conformational states, while in the presence of the Y21M mutation, this heterogeneity is greatly reduced, consistent with previous observations. These results show that new computational approaches to helical reconstruction can greatly extend the ability to visualize heterogeneous protein polymers at a reasonably high resolution.