How should I select the appropriate Molecular Weight Cutoff (MWCO) for Amicon Ultra?…
- Select a Molecular Weight Cutoff that is 2x smaller that the Molecular Weight (MW) of the solute of interest.
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Phosphorylation does not prompt, nor prevent, the formation of alpha-synuclein toxic species in a rat model of Parkinson's disease.
- Phosphorylation is involved in numerous neurodegenerative diseases. In particular, alpha-synuclein is extensively phosphorylated in aggregates in patients suffering from synucleinopathies. However, the share of this modification in the events that lead to the conversion of alpha-synuclein to aggregated toxic species needed to be clarified. The rat model that we developed through rAAV2/6-mediated expression of alpha-synuclein demonstrates a correlation between neurodegeneration and formation of small filamentous alpha-synuclein aggregates. A mutation preventing phosphorylation (S129A) significantly increases alpha-synuclein toxicity and leads to enhanced formation of beta-sheet-rich, proteinase K-resistant aggregates, increased affinity for intracellular membranes, a disarrayed network of neurofilaments and enhanced alpha-synuclein nuclear localization. The expression of a mutation mimicking phosphorylation (S129D) does not lead to dopaminergic cell loss. Nevertheless, fewer but larger aggregates are formed, and signals of apoptosis are also activated in rats expressing the phosphorylation-mimicking form of alpha-synuclein. These observations strongly suggest that phosphorylation does not play an active role in the accumulation of cytotoxic pre-inclusion aggregates. Unexpectedly, the study also demonstrates that constitutive expression of phosphorylation-mimicking forms of alpha-synuclein does not protect from neurodegeneration. The role of phosphorylation at Serine 129 in the early phase of Parkinson's disease is examined, which brings new perspective to therapeutic approaches focusing on the modulation of kinases/phosphatases activity to control alpha-synuclein toxicity.
- 문서 타입:
- Reference
- 카탈로그 번호:
- AB5334P
- 제품명:
- Anti-Synuclein α Antibody
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Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system.
- After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2).We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents.RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.
- 문서 타입:
- Reference
- 카탈로그 번호:
- AB3080
- 제품명:
- Anti-Green Fluorescent Protein Antibody
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Presynaptic dopaminergic compartment determines the susceptibility to L-DOPA-induced dyskinesia in rats.
- Drug-induced dyskinesias in dopamine-denervated animals are known to depend on both pre- and postsynaptic changes of the nigrostriatal circuitry. In lesion models used thus far, changes occur in both of these compartments and, therefore, it has not been possible to dissect the individual contribution of each compartment in the pathophysiology of dyskinesias. Here we silenced the nigrostriatal dopamine neurotransmission without affecting the anatomical integrity of the presynaptic terminals using a short-hairpin RNA-mediated knockdown of tyrosine hydroxylase enzyme (shTH). This treatment resulted in significant reduction (by about 70%) in extracellular dopamine concentration in the striatum as measured by on-line microdialysis. Under these conditions, the animals remained nondyskinetic after chronic L-DOPA treatment, whereas partial intrastriatal 6-hydoxydopamine lesioned rats with comparable reduction in extracellular dopamine levels developed dyskinesias. On the other hand, apomorphine caused moderate to severe dyskinesias in both groups. Importantly, single-dose L-DOPA challenge in apomorphine-primed shTH animals failed to activate the already established abnormal postsynaptic responses. Taken together, these data provide direct evidence that the status of the presynaptic, DA releasing compartment is a critical determinant of both the induction and maintenance of L-DOPA-induced dyskinesias.
- 문서 타입:
- Reference
- 카탈로그 번호:
- MAB318
- 제품명:
- Anti-Tyrosine Hydroxylase Antibody, clone LNC1
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Cerebrospinal fluid acetylcholinesterase changes after treatment with donepezil in patients with Alzheimer's disease.
- We analyzed whether donepezil differently influences acetylcholinesterase (AChE) variants from cerebrospinal fluid (CSF) in patients with Alzheimer's disease (AD) after long-term treatment. Overall CSF-AChE activity in AD patients before treatment was not different from controls, but the ratio between the major tetrameric form, G(4), and the smaller G(1) and G(2) species was significantly lower. AChE levels at study outset were found to correlate positively with beta-amyloid (1-42) (Abeta42). When patients were re-examined after 12 months treatment with donepezil, there was a remarkable increase in both the G(4) and the lighter species of CSF AChE. As compared with placebo, donepezil caused decreases in the percentage of AChE that failed to bind to the lectin concanavalin A and the antibody AE1. These non-binding species comprised primarily a small subset of G(1) and G(2) forms. In treated patients, these light variants were the only subset of CSF AChE that correlated with CSF-Abeta42 levels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that a 77-kDa band, attributed in part to inactive AChE, was lower in AD patients than in controls. Unlike enzyme activity, the intensity of this band did not increase after donepezil treatment. The varying responses of different AChE species to ChE-I treatment suggest different modes of regulation, which may have therapeutic implications.
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- Reference
- 카탈로그 번호:
- MAB304
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Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies.
- The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar-Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair.
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- Reference
- 카탈로그 번호:
- ECM625
- 제품명:
- In Vitro Angiogenesis Assay Kit
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SIRT1 decreases Lox-1-mediated foam cell formation in atherogenesis.
- Aims Endothelial activation, macrophage infiltration, and foam cell formation are pivotal steps in atherogenesis. Our aim in this study was to analyse the role of SIRT1, a class III deacetylase with important metabolic functions, in plaque macrophages and atherogenesis. Methods and results Using partial SIRT1 deletion in atherosclerotic mice, we demonstrate that SIRT1 protects against atherosclerosis by reducing macrophage foam cell formation. Peritoneal macrophages from heterozygous SIRT1 mice accumulate more oxidized low-density lipoprotein (oxLDL), thereby promoting foam cell formation. Bone marrow-restricted SIRT1 deletion confirmed that SIRT1 function in macrophages is sufficient to decrease atherogenesis. Moreover, we show that SIRT1 reduces the uptake of oxLDL by diminishing the expression of lectin-like oxLDL receptor-1 (Lox-1) via suppression of the NF-kappaB signalling pathway. Conclusion Our findings demonstrate protective effects of SIRT1 in atherogenesis and suggest pharmacological SIRT1 activation as a novel anti-atherosclerotic strategy by reducing macrophage foam cell formation.
- 문서 타입:
- Reference
- 카탈로그 번호:
- 05-414
- 제품명:
- Anti-CrkL Antibody, clone 5-6
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The correlation between human adipose-derived stem cells differentiation and cell adhesion mechanism.
- In recent years, research in the areas of stem cells has dramatically increased, including studies of cellular adhesion to a substrate. We sought to determine the adhesive properties of human adipose-derived stem cells (hASCs) for extracellular matrix proteins. The adhesion of hASCs to collagens and laminin was completely inhibited by a monoclonal antibody, Mab 2253, which binds to the beta1 integrin subunit. These data indicate that hASC adhesion to collagens and laminin was exclusively mediated by an integrin. Cell adhesion on fibronectin (Fn) was inhibited by the heparin-binding peptide (HBP) in the presence of Mab 2253, but not by either Mab 2253 or HBP alone. These results indicate that both the beta1 subunit and the heparan sulfate proteoglycan participated in the cell adhesion to Fn. Microscopic views showed extensive spreading of hASCs cultured on Fn, whereas the cells maintained a round shape when cultured on a heparin-binding domain (HBD) substrate. hASCs differentiated into adipocytes, which stained positive for lipid vacuoles by Oil Red-O analysis, more readily on HBD substrate than on FN substrate. These results suggest that hASCs have an adhesion mechanism for the HBD of Fn and hASC morphology is controlled by the adhesion mechanism and strongly correlated with adipogenic differentiation.
- 문서 타입:
- Reference
- 카탈로그 번호:
- MAB2253
- 제품명:
- Anti-Integrin β1 Antibody, clone 6S6
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Identification of Contractile Vacuole Proteins in Trypanosoma cruzi.
- Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.
- 문서 타입:
- Reference
- 카탈로그 번호:
- AP180
- 제품명:
- Donkey Anti-Goat IgG Antibody, Species Adsorbed
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The C-terminal coiled-coil of the bacterial voltage-gated sodium channel NaChBac is not essential for tetramer formation, but stabilizes subunit-to-subunit interactions.
- The NaChBac is a prokaryotic homologue of the voltage-gated sodium channel found in the genome of the alkalophilic bacterium Bacillus halodurans C-125. Like a repeating cassette of mammalian sodium channel, the NaChBac possesses hydrophobic domains corresponding to six putative transmembrane segments and a pore loop, and exerts channel function by forming a tetramer although detailed mechanisms of subunit assembly remain unclear. We generated truncated mutants from NaChBac, and investigated their ability to form tetramers in relation to their channel functions. A mutant that deletes almost all of the C-terminal coiled-coil structure lost its voltage-dependent ion permeability, although it was properly translocated to the cell surface. The mutant protein was purified as a tetramer using a reduced concentration of detergent, but the association between the subunits was shown to be much weaker than the wild type. The chemical cross-linking, blue native PAGE, sedimentation velocity experiments, size exclusion chromatography, immunoprecipitation, and electron microscopy all supported its tetrameric assembly. We further purified the C-terminal cytoplasmic domain alone and confirmed its self-oligomerization. These data suggest that the C-terminal coiled-coil structure stabilizes subunit-to-subunit interactions of NaChBac, but is not critical for their tetramer formation.
- 문서 타입:
- Reference
- 카탈로그 번호:
- AB5510
- 제품명:
- Anti-Histidine Antibody