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  • Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). 14576842

    Observations of functional adenosine triphosphate (ATP)-dependent drug efflux in certain multidrug-resistant cancer cell lines without overexpression of P-glycoprotein or multidrug resistance protein (MRP) family members suggested the existence of another ATP-binding cassette (ABC) transporter capable of causing cancer drug resistance. In one such cell line (MCF-7/AdrVp), the overexpression of a novel member of the G subfamily of ABC transporters was found. The new transporter was termed the breast cancer resistance protein (BCRP), because of its identification in MCF-7 human breast carcinoma cells. BCRP is a 655 amino-acid polypeptide, formally designated as ABCG2. Like all members of the ABC G (white) subfamily, BCRP is a half transporter. Transfection and enforced overexpression of BCRP in drug-sensitive MCF-7 or MDA-MB-231 cells recapitulates the drug-resistance phenotype of MCF-7/AdrVp cells, consistent with current evidence suggesting that functional BCRP is a homodimer. BCRP maps to chromosome 4q22, downstream from a TATA-less promoter. The spectrum of anticancer drugs effluxed by BCRP includes mitoxantrone, camptothecin-derived and indolocarbazole topoisomerase I inhibitors, methotrexate, flavopiridol, and quinazoline ErbB1 inhibitors. Transport of anthracyclines is variable and appears to depend on the presence of a BCRP mutation at codon 482. Potent and specific inhibitors of BCRP are now being developed, opening the door to clinical applications of BCRP inhibition. Owing to tissue localization in the placenta, bile canaliculi, colon, small bowel, and brain microvessel endothelium, BCRP may play a role in protecting the organism from potentially harmful xenobiotics. BCRP expression has also been demonstrated in pluripotential "side population" stem cells, responsible for the characteristic ability of these cells to exclude Hoechst 33342 dye, and possibly for the maintenance of the stem cell phenotype. Studies are emerging on the role of BCRP expression in drug resistance in clinical cancers. More prospective studies are needed, preferably combining BCRP protein or mRNA quantification with functional assays, in order to determine the contribution of BCRP to drug resistance in human cancers.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4155
    Product Catalog Name:
    Anti-BCRP1 Antibody, clone 5D3

  • StyA1 and StyA2B from Rhodococcus opacus 1CP: a multifunctional styrene monooxygenase system. 20675468

    Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH2 provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH2, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH2-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view.
    Document Type:
    Reference
    Product Catalog Number:
    MAB318
    Product Catalog Name:
    Anti-Tyrosine Hydroxylase Antibody, clone LNC1

  • A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells ... 23722522

    DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4381
    Product Catalog Name:
    Anti-TRA-1-81 Antibody, clone TRA-1-81

  • Microfabricated intracortical extracellular matrix-microelectrodes for improving neural interfaces. 31057918

    Intracortical neural microelectrodes, which can directly interface with local neural microcircuits with high spatial and temporal resolution, are critical for neuroscience research, emerging clinical applications, and brain computer interfaces (BCI). However, clinical applications of these devices remain limited mostly by their inability to mitigate inflammatory reactions and support dense neuronal survival at their interfaces. Herein we report the development of microelectrodes primarily composed of extracellular matrix (ECM) proteins, which act as a bio-compatible and an electrochemical interface between the microelectrodes and physiological solution. These ECM-microelectrodes are batch fabricated using a novel combination of micro-transfer-molding and excimer laser micromachining to exhibit final dimensions comparable to those of commercial silicon-based microelectrodes. These are further integrated with a removable insertion stent which aids in intracortical implantation. Results from electrochemical models and in vivo recordings from the rat's cortex indicate that ECM encapsulations have no significant effect on the electrochemical impedance characteristics of ECM-microelectrodes at neurologically relevant frequencies. ECM-microelectrodes are found to support a dense layer of neuronal somata and neurites on the electrode surface with high neuronal viability and exhibited markedly diminished neuroinflammation and glial scarring in early chronic experiments in rats.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Tissue-type plasminogen activator: characteristics, applications and production technology. 14537155

    Plasminogen activators have immense clinical significance as thrombolytic agents for management of stroke and myocardial infarction. Tissue-type plasminogen activator (tPA) is generally preferred as being effective and safer than either urokinase or streptokinase type activators. Large-scale production of tPA became possible through groundbreaking developments in cell lines and bioprocess technology. Nevertheless, at thousands of dollars per treatment, tPA remains expensive. Enhancing cellular productivity and downstream product recovery through new approaches continue to be major challenges as discussed in this review. Recent clinical experience suggests the need for yet better fibrinolytic agents and attempts are underway to modify the tPA molecule to second generation products. Emerging trends in this field are outlined.
    Document Type:
    Reference
    Product Catalog Number:
    05-883
    Product Catalog Name:
    Anti-tPA (Tissue Plasminogen Activator) Antibody, clone GMA-043

  • Effective enhancement of fluorescence detection efficiency in protein microarray assays: application of a highly fluorinated organosilane as the blocking agent on the bac ... 19705849

    Protein microarrays are emerging as an important enabling technology for the simultaneous investigation of complicated interactions among thousands of proteins. The solution-based blocking protocols commonly used in protein microarray assays often cause cross-contamination among probes and diminution of protein binding efficiency because of the spreading of blocking solution and the obstruction formed by the blocking molecules. In this paper, an alternative blocking process for protein microarray assays is proposed to obtain better performance by employing a vapor-phase deposition method to form self-assembled surface coatings using a highly fluorinated organosilane as the blocking agent on the background surfaces. Compared to conventional solution-based blocking processes, our experimental results showed that this vapor-phase process could shorten the blocking time from hours to less than 10 min, enhance the binding efficiency by up to 6 times, reduce the background noise by up to 16 times, and improve the S/N ratio by up to 64 times. This facile blocking process is compatible with current microarray assays using silica-based substrates and can be performed on many types of silane-modified surfaces.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Chromosome 7 and 19 trisomy in cultured human neural progenitor cells. 19898616

    Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations.While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC(+7)) and trisomy 19 (hNPC(+19)), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC(+7) and hNPC(+19) cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (beta(III)-tubulin) in hNPC(+7) and hNPC(+19), using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50-60 population doublings and never showed neoplastic changes. Although hNPC(+7) and hNPC(+19) survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line.We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Hematopoietic stem cell development requires transient Wnt/β-catenin activity. 22802352

    Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that β-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly, Wnt/β-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of β-catenin from the embryonic endothelium stage (using VE-cadherin-Cre recombinase), but not from embryonic hematopoietic cells (using Vav1-Cre), precludes progression of mutant cells toward the hematopoietic lineage; however, these mutant cells still contribute to the adult endothelium. Together, those findings indicate that Wnt/β-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos.
    Document Type:
    Reference
    Product Catalog Number:
    05-665
    Product Catalog Name:
    Anti-Active-β-Catenin (Anti-ABC) Antibody, clone 8E7

  • Recovery of live virus after storage at ambient temperature using ViveST™. 23046621

    A major impediment to performing virological field studies in developing nations is the lack of ultra-low freezers as well as the expense and difficulty of shipping frozen samples. A commercially available product, ViveST™, was developed to preserve nucleic acids at ambient temperature for use in specimen storage and transportation. However, its applications as a viral storage, transport and recovery device have not been evaluated.To examine the ability of ViveST to preserve live virus following storage at ambient temperature.A panel of six viruses was stored at ambient temperature (~22°C) in ViveST with fetal bovine serum (FBS), or ViveST with minimal essential media (MEM) and compared with virus stored in universal transport media (M4RT), MEM, and FBS alone. Stored viruses included: human adenovirus (14p), dengue virus 2 (16608), echovirus 3 (Morrisey), human rhinovirus 15 (1734), Coxsackie virus B5 (Faulkner), and herpes simplex virus 1 (HF). After 7 days storage at ambient temperature, virus recovery was measured via titration using viral plaque assays or focus-forming unit assays.Viral titer studies indicate that ViveST with either FBS or M4RT preserved/recovered 5 different viruses for 1 week at ambient temperature. MEM preserved 4 viruses while FBS and ViveST with MEM preserved 3 viruses each. Statistical analyses indicate that M4RT and ViveST with FBS preserved significantly more virus than the other treatments.These data suggest that ViveST with either FBS or M4RT may be useful in field specimen collection scenarios where ultra-cold storage is not available.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Tetrahydroberberine blocks ATP-sensitive potassium channels in dopamine neurons acutely-dissociated from rat substantia nigra pars compacta. 20804776

    Tetrahydroberberine (THB) exhibits neuroprotective effects but its targets and underlying mechanisms are largely unknown. Emerging evidence indicates that ATP-sensitive potassium (K(ATP)) channels in the substantia nigra pars compacta (SNc) promote Parkinson disease (PD) pathogenesis, thus blocking K(ATP) channels may protect neurons against neuronal degeneration. In the present study, we tested a hypothesis that THB blocks K(ATP) channels in dopaminergic (DA) neurons acutely dissociated from rat SNc. Using perforated patch-clamp recording in current-clamp mode, the functional K(ATP) channels can be opened by persistent perfusion of rotenone, an inhibitor of complex I of the mitochondrial respiratory chain. Bath-application of THB reversibly blocks opened K(ATP) channels in a concentration-dependent manner, which is comparable to a classical K(ATP) channel blocker, Tol. Compared to THB analogs, l-stepholidine (l-SPD) or l-tetrahydropalmatine (l-THP), THB exhibits more profound blockade in K(ATP) channels. In addition, exposure of THB alone to the recorded neuron increases action potential firing, and THB also restores rotenone-induced membrane hyperpolarization in the presence of dopamine D2 receptor antagonist (sulpiride), suggesting that THB exhibits an excitatory effect on SNc DA neurons through the block of K(ATP) channels. Collectively, the blockade of neuronal K(ATP) channels by THB in SNc DA neurons is a novel pharmacological mechanism of THB, which may contribute to its neuroprotective effects in PD.
    Document Type:
    Reference
    Product Catalog Number:
    AB152
    Product Catalog Name:
    Anti-Tyrosine Hydroxylase Antibody