Product Change Notification: Ultracel® 100K Regenerated Cellulose Membrane Disc Products
- Document Type:
- User Guide
nitrocellulose membrane disc
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Product Change Notification MF-Millipore Mixed Cellulose Ester (MCE) 0.45 and 0.8 μm Black Membrane Disc Products
- Document Type:
- User Guide
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Naturally occurring and bioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL: the abnormal HDL phenotypes can be corrected by treatmen ...
- In the present study we have used adenovirus-mediated gene transfer of apoA-I (apolipoprotein A-I) mutants in apoA-I-/- mice to investigate how structural mutations in apoA-I affect the biogenesis and the plasma levels of HDL (high-density lipoprotein). The natural mutants apoA-I(R151C)Paris, apoA-I(R160L)Oslo and the bioengineered mutant apoA-I(R149A) were secreted efficiently from cells in culture. Their capacity to activate LCAT (lecithin:cholesterol acyltransferase) in vitro was greatly reduced, and their ability to promote ABCA1 (ATP-binding cassette transporter A1)-mediated cholesterol efflux was similar to that of WT (wild-type) apoA-I. Gene transfer of the three mutants in apoA-I-/- mice generated aberrant HDL phenotypes. The total plasma cholesterol of mice expressing the apoA-I(R160L)Oslo, apoA-I(R149A) and apoA-I(R151C)Paris mutants was reduced by 78, 59 and 61% and the apoA-I levels were reduced by 68, 64 and 55% respectively, as compared with mice expressing the WT apoA-I. The CE (cholesteryl ester)/TC (total cholesterol) ratio of HDL was decreased and the apoA-I was distributed in the HDL3 region. apoA-I(R160L)Oslo and apoA-I(R149A) promoted the formation of prebeta1 and alpha4-HDL subpopulations and gave a mixture of discoidal and spherical particles. apoA-I(R151C)Paris generated subpopulations of different sizes that migrate between prebeta and alpha-HDL and formed mostly spherical and a few discoidal particles. Simultaneous treatment of mice with adenovirus expressing any of the three mutants and human LCAT normalized plasma apoA-I, HDL cholesterol levels and the CE/TC ratio. It also led to the formation of spherical HDL particles consisting mostly of alpha-HDL subpopulations of larger size. The correction of the aberrant HDL phenotypes by treatment with LCAT suggests a potential therapeutic intervention for HDL abnormalities that result from specific mutations in apoA-I.
- Document Type:
- Reference
- Product Catalog Number:
- AB740
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Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model.
- We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.
- Document Type:
- Reference
- Product Catalog Number:
- MAB4155F
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Controlling gene loss of function in newts with emphasis on lens regeneration.
- Here we describe a protocol for gene loss of function during regeneration in newts, specifically applied to lens regeneration. Knockdown with the use of morpholinos can be achieved both in vitro and in vivo, depending on the experimental design. These methods achieve desirable levels of gene knockdown, and thus can be compared with methods developed for use in other animals, such as zebrafish. The technology has been applied to study molecular mechanisms during the process of lens regeneration by knocking down genes at specific stages and examining their effects on other genes and lens differentiation. The protocol can take a few days or up to 20 d to complete, depending on the duration of the experiment.
- Document Type:
- Reference
- Product Catalog Number:
- S7110
- Product Catalog Name:
- ApopTag® Fluorescein In Situ Apoptosis Detection Kit - (ApopTag® Fluorescein In Situ Apoptosis Detection Kit)
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Sp1 and Sp3 recruit histone deacetylase to repress transcription of human telomerase reverse transcriptase (hTERT) promoter in normal human somatic cells.
- Activation of telomerase is crucial for cells to gain immortality. In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In most normal human somatic cells, hTERT is not expressed, and its suppression acts as an important gatekeeper against tumorigenesis. Here we describe the systematic analyses of hTERT promoter to understand the transcriptional repression mechanism of the hTERT gene in normal human somatic cells. Through the serial deletion analysis of hTERT promoter in normal human fibroblasts, we identified a critical repressive element on the hTERT promoter. The repressive element formed DNA-protein complexes with Sp1 and Sp3 in nuclear extracts. Using formaldehyde cross-linked chromatin immunoprecipitation analysis, we found that Sp1 and Sp3 were associated with the endogenously repressed hTERT promoter in human fibroblasts. Furthermore, Sp1 and Sp3 interacted with histone deacetylase (HDAC) in these cells. Overexpression of dominant-negative mutants of Sp1 and Sp3, which contained mainly the HDAC2-binding domain, relieved the HDAC-mediated repression of the hTERT promoter. Taken together, these results suggest that Sp1 and Sp3 associate with the hTERT promoter, recruiting HDAC for the localized deacetylation of nucleosomal histones and transcriptional silencing of the hTERT gene in normal human somatic cells.
- Document Type:
- Reference
- Product Catalog Number:
- 07-107
- Product Catalog Name:
- Anti-Sp3 Antibody - (Anti-Sp3 Antibody)
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Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines.
- NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.
- Document Type:
- Reference
- Product Catalog Number:
- MAB363
- Product Catalog Name:
- Anti-NMDAR1 Antibody, clone 54.1 - (Anti-NMDAR1 Antibody, clone 54.1)
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T-cell lymphomas mask slower developing B-lymphoid and myeloid tumours in transgenic mice with broad haemopoietic expression of MYC.
- Deregulation of MYC expression occurs in many haematological malignancies. Previous studies modelling MYC-induced lymphomagenesis in the mouse used transgenic vectors that directed MYC overexpression in a lineage-specific manner. Here, we describe a transgenic mouse strain in which constitutive MYC expression is driven broadly in haemopoiesis by a vector containing regulatory elements of the Vav gene. Healthy young VavP-MYC17 mice had multiple haemopoietic abnormalities, most notably increased size and numbers of B-lymphoid cells, monocytes and megakaryocytes. The mice rapidly developed tumours and, surprisingly, these were exclusively T-cell lymphomas, mostly of mature CD4(+) CD8(-) T cells, a tumour type that is seldom seen in mouse models. To examine tumour development in the absence of the susceptible T cells, we bred VavP-MYC17 mice lacking the Rag1 recombinase. They survived longer and succumbed to tumours of several different haemopoietic cell types: pre-T cells, pro-B cells, macrophages and unusual progenitor cells. Thus, although T-lineage cells have the shortest latent period to transformation, the VavP-MYC17 transgene drives malignant transformation of multiple cell types and VavP-MYC17 mice provide a new model for tumours of multiple haemopoietic lineages.
- Document Type:
- Reference
- Product Catalog Number:
- 06-340
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The interaction of estrogen receptor alpha and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes.
- Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor alpha in cardiomyocytes, we investigate whether the membrane estrogen receptor alpha associates with caveolin-3 and whether this association is linked to the 17beta-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor alpha co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor alpha was not seen following treatment with the cholesterol-depleting agent methyl-beta-cyclodextrin (5mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor alpha. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor alpha, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17beta-estradiol (200 nM). The effect of 17beta-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor alpha docking at caveolae, which provide a unique compartment for conveying 17beta-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.
- Document Type:
- Reference
- Product Catalog Number:
- AP132F
- Product Catalog Name:
- Goat Anti-Rabbit IgG Antibody, FITC conjugate - (Goat Anti-Rabbit IgG Antibody, FITC conjugate)
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Differential response of cancer cells to HDAC inhibitors trichostatin A and depsipeptide.
- Over the last decade, several drugs that inhibit class I and/or class II histone deacetylases (HDACs) have been identified, including trichostatin A, the cyclic depsipeptide FR901228 and the antibiotic apicidin. These compounds have had immediate application in cancer research because of their ability to reactivate aberrantly silenced tumour suppressor genes and/or block tumour cell growth. Although a number of HDAC inhibitors are being evaluated in preclinical cancer models and in clinical trials, little is known about the differences in their specific mechanism of action and about the unique determinants of cancer cell sensitivity to each of these inhibitors.Using a combination of cell viability assays, HDAC enzyme activity measurements, western blots for histone modifications, microarray gene expression analysis and qRT-PCR, we have characterised differences in trichostatin A vs depsipeptide-induced phenotypes in lung cancer, breast cancer and skin cancer cells and in normal cells and have then expanded these studies to other HDAC inhibitors.Cell viability profiles across panels of lung cancer, breast cancer and melanoma cell lines showed distinct sensitivities to the pan-inhibitor TSA compared with the class 1 selective inhibitor depsipeptide. In several instances, the cell lines most sensitive to one inhibitor were most resistant to the other inhibitor, demonstrating these drugs act on at least some non-overlapping cellular targets. These differences were not explained by the HDAC selectivity of these inhibitors alone since apicidin, which is a class 1 selective compound similar to depsipeptide, also showed a unique drug sensitivity profile of its own. TSA had greater specificity for cancer vs normal cells compared with other HDAC inhibitors. In addition, at concentrations that blocked cancer cell viability, TSA effectively inhibited purified recombinant HDACs 1, 2 and 5 and moderately inhibited HDAC8, while depsipeptide did not inhibit the activity of purified HDACs in vitro but did in cellular extracts, suggesting a potentially indirect action of this drug. Although both depsipeptide and TSA increased levels of histone acetylation in cancer cells, only depsipeptide decreased global levels of transcriptionally repressive histone methylation marks. Analysis of gene expression profiles of an isogenic cell line pair that showed discrepant sensitivity to depsipeptide, suggested that resistance to this inhibitor may be mediated by increased expression of multidrug resistance genes triggered by exposure to chemotherapy as was confirmed by verapamil studies.Although generally thought to have similar activities, the HDAC modulators trichostatin A and depsipeptide demonstrated distinct phenotypes in the inhibition of cancer cell viability and of HDAC activity, in their selectivity for cancer vs normal cells, and in their effects on histone modifications. These differences in mode of action may bear on the future therapeutic and research application of these inhibitors.
- Document Type:
- Reference
- Product Catalog Number:
- Multiple
- Product Catalog Name:
- Multiple