Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The herpes simplex virus thymidine kinase (HSV-tk) gene conferring ganciclovir (GCV)-specific sensitivity to transduced cells might control Graft-versus-Leukemia (GvL)/Graft-versus-Host Disease (GvHD). Human T lymphocytes were engineered with an LSN-tk retroviral vector encoding tk and neomycin resistance (NeoR) genes. A total of 80 x 10(6) tk(+) lymphocytes were injected intraperitoneally in NOD-SCID mice. Engraftment was evaluated by human CD45(+)/CD3(+) cytofluorimetric analysis and NeoR-based polymerase chain reaction (PCR) on peripheral blood, bone marrow, liver, thymus, and spleen on day +5. After 14 days, GCV (10 mg/kg daily) cytofluorimetric analysis and PCR were repeated (day +19). Immunohistological studies with anti-CD3 monoclonal antibody followed by alkaline phosphatase and monoclonal anti-alkaline phosphatase staining were performed on spleen and liver at the same time points. Human CD45(+)/CD3(+) cells were engrafted in all tissues on day +5 according to cytofluorimetry, immunohistology, and PCR. Lymphocytes "homed" to the white pulp T-cell area and to the red pulp; liver localization is prevalently at the periportal area. After GCV (day +19), cytofluorimetry and immunohistology showed very few CD3(+) cells. PCR identified the transgene in 22% tissue samples (positive only in thymus and spleen). GvHD did not occur in any animal. These data demonstrate elevated doses of human-transduced CD3(+) cells engraft in NOD/SCID mice; after GCV, very few CD3(+) cells can be detected and those that escape treatment can be found in the thymus and in the spleen on day +19. Lack of full response to GCV may account for cases of GvHD in patients receiving tk-transduced T lymphocytes.
...
PMID:Homing and survival of thymidine kinase-transduced human T cells in NOD/SCID mice. 1218 25

A cell line modified genetically (Vero-ICP10-SEAP) that responds to infection by herpes simplex virus (HSV) was established. The cell line was constructed by stable transfection of Vero cell with a plasmid encoding the secreted alkaline phosphatase (SEAP) driven by the promoter of the HSV-2 ICP10 gene. Following infection with HSV, the stable line secretes a high level of the SEAP in the supernatants as measured by a chemiluminescence-based assay. The detection system is sensitive to an HSV titer as low as a single plaque-forming unit (PFU), with a linear range up to the equivalent of 2.5 x 10(4) PFU inoculum after infection for 24 h. There was no detectable enhancement in SEAP activities following inoculations with several viruses other than HSV. The Vero-ICP10-SEAP cell line was also utilized to develop an assay for determination of antiviral susceptibility given that the induced SEAP activity appeared to reflect the numbers of plaque. Evaluations of the stable line with representative acyclovir (ACV)-sensitive and-resistant HSV isolates demonstrated that their drug susceptibilities were determined accurately. In summary, this novel SEAP reporter system is a sensitive means for rapid diagnosis, quantitation, and drug susceptibility testing for HSV, with potential to the development of an automated assay.
...
PMID:A cell line that secretes inducibly a reporter protein for monitoring herpes simplex virus infection and drug susceptibility. 1237 69

In previous studies, we developed mouse genetic models and discovered genetic components of quantitative trait loci on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and fracture healing. However, these regions contain dozens of genes in several overlapping bacterial artificial chromosomes (BACs) and are difficult to clone by physical cloning strategies. A feasible and efficient approach of identifying candidate genes is to transfer the genomic loci in BAC clones into mammalian cells for functional studies. In this study, we retrofitted a BAC construct into herpes simplex virus-1 amplicon and packaged it into an infectious BAC (iBAC) to test gene function in a cell-based system, using a 128-kb clone containing the complete bone morphogenetic protein-2 (BMP-2) gene. We transduced MC3T3-E1 cells with the iBAC bearing BMP-2 gene and examined transgene expression and function. Our results have demonstrated that an iBAC can efficiently deliver a BMP-2 genomic locus into preosteoblast cells and express functional BMP-2 protein, inducing a phenotype of cell differentiation, as indicated by an increase in alkaline phosphatase activity. Therefore, this experimental system provides a rapid, efficient cell-based model of high-throughput phenotypic screening to identify the BAC clones from physically mapped regions that are important for osteoblast differentiation. It also illustrates the potential of iBAC technology in functional testing of single nucleotide polymorphisms located in the distal promoter or/and intron regions responsible for low bone density.
...
PMID:HSV-1 amplicon-mediated transfer of 128-kb BMP-2 genomic locus stimulates osteoblast differentiation in vitro. 1518 51

Virus culture has played significant roles in basic and clinical virology, with a number of advantages that cannot be attainable by modern molecular techniques. However, virus culture is generally a slower process, as it inevitably takes the period of a full replication cycle of a given virus. A genetically modified cell culture with a virus-inducible marker is described here, using a frequently isolated DNA virus (herpes simplex virus) as a model. The assay system relies on expression of the reporter gene driven by a specific viral promoter that is triggered early in the course of viral infection. The reporter gene employed was green fluorescent protein (GFP) or secreted alkaline phosphatase (SEAP), whose assays offer real-time detection or quantification, respectively. This cell-based assay is simple, rapid, sensitive, specific, and quantitative and serves as a phenotypic method for determination of antiviral susceptibilities.
...
PMID:Reporter cell lines for the detection of herpes simplex viruses. 1550 2

Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Vectors based on herpes simplex virus type-1 (HSV-1) have been demonstrated to mediate efficient gene transfer into hepatocytes both in vitro and in vivo. Large transgene capacity and extrachromosomal persistence make HSV-1/EBV hybrid amplicon vectors an attractive candidate for hepatic gene replacement therapy. To assess liver-directed gene transfer, we constructed (i) a conventional HSV-1 amplicon vector encoding a secreted reporter protein (secreted alkaline phosphatase, SEAP) under the control of the HSV-1 immediate-early 4/5 promoter; (ii) a HSV-1 amplicon encoding SEAP under the control of the artificial CAG promoter (the chicken beta-actin promoter and cytomegalovirus (CMV) immediate-early enhancer); and (iii) a HSV-1/EBV hybrid amplicon, also encoding SEAP under the control of the CAG promoter. While all three vector constructs yielded high SEAP concentrations in vitro and in vivo, use of HSV-1/EBV hybrid amplicon vectors significantly prolonged the duration of gene expression. Using conventional amplicon vectors in cultured hepatocytes, SEAP was detected for two weeks, whereas SEAP was detected for at least six weeks when HSV-1/EBV amplicons were used. Intraparenchymal injection into the liver of SICD mice yielded high (up to 77 ng of SEAP per milliliter serum) and sustained (greater than three weeks) expression of SEAP. Serum transaminases (ALT/AST) were measured at different time points to monitor for hepatocellular damage. While initially elevated four times above baseline, the transaminase levels returned to normal after three to seven days. These results demonstrate the usefulness of HSV-1-based amplicons and SEAP for the evaluation of gene replacement strategies in the liver.
...
PMID:Gene transfer into hepatocytes mediated by herpes simplex virus-Epstein-Barr virus hybrid amplicons. 1558

Nutrient and oxygen availability are key metabolic parameters for biopharmaceutical manufacturing. In order to enable mammalian cells to manifest their intracellular nutrient and oxygen levels we engineered a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a robust reporter gene expression readout. Capitalizing on the Streptomyces coelicolor redox control system, consisting of REX modulating ROP-containing promoters in an NADH-dependent manner, we designed a mammalian dual sensor transcription control system by fusing REX to the generic VP16 transactivation domain of Herpes simplex, which reconstitutes an artificial transactivator (REDOX) able to bind and activate chimeric promoters assembled by placing a ROP operator module 5' of a minimal eukaryotic promoter (P(ROP)). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent P(ROP)-driven expression of secreted (human-secreted alkaline phosphatase; SEAP) or intracellular (Renilla reniformis luciferase; rLUC) reporter genes was high as a consequence of increased REDOX-P(ROP) affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX-P(ROP) interaction mediated low-level transgene expression in Chinese hamster ovary (CHO-K1) cells. Other molecules (for example, 2,4-dinitrophenol, cyanide or hydrogen peroxide) which are known to imbalance the intracellular NADH/NAD+ poise could also be detected using the REDOX-P(ROP) sensor circuitry. REDOX's sensor capacity (nutrient and oxygen levels) operated seamlessly in transgenic CHO-K1 cell derivatives adapted for growth in serum-free suspension cultures and enabled precise monitoring of the population's metabolic state. As the first genetic metabolic sensor designed for mammalian cells, REDOX may foster advances in process development and biopharmaceutical manufacturing.
...
PMID:A genetic redox sensor for mammalian cells. 1647 37

We demonstrated a novel application of transient coulostatic pulse technique for the detection of label free DNA hybridization on nm-sized gold interdigitated ultramicroelectrode arrays (Au-IDA) made in silicon technology. The array consists of eight different positions with an Au-IDA pair at each position arranged on the Si-based Biochip. Immobilization of capture probes onto the Au-IDA was accomplished by self-assembling of thiol-modified oligonucleotides. Target hybridization was indicated by a change in the magnitude of the time dependant potential relaxation curve in presence of electroactive Fe(CN)(6)(3-) in the phosphate buffer solution. While complementary DNA hybridization showed 50% increase in the relaxation potential, the non-complementary DNA showed a negligible change. A constant behaviour was noted for all positions. The dsDNA specific intercalating molecule, methylene blue, was found to be enhancing the discrimination effect. The changes in the relaxation potential curves were further corroborated following the ELISA like experiments using ExtraAvidine alkaline phosphatase labelling and redox recycling of para-aminophenol phosphate at IDAs. The coulostatic pulse technique was shown to be useful for identifying DNA sequences from brain tumour gene CK20, human herpes simplex virus, cytomegalovirus, Epstein-Barr virus and M13 phage. Compared to the hybridization of short chain ONTs (27 mers), the hybridization of long chain M13 phage DNA showed three times higher increase in the relaxation curves. The method is fast enough to monitor hybridization interactions in milli or microsecond time scales and is well suitable for miniaturization and integration compared to the common impedance techniques for developing capacitative DNA sensors.
...
PMID:DNA hybridization detection on electrical microarrays using coulostatic pulse technique. 1657 97

For optimal compatibility with biopharmaceutical manufacturing and gene therapy, heterologous transgene control systems must be responsive to side-effect-free physiologic inducer molecules. The arginine-inducible interaction of the ArgR repressor and the ArgR-specific ARG box, which synchronize arginine import and synthesis in the intracellular human pathogen Chlamydia pneumoniae, was engineered for arginine-regulated transgene (ART) expression in mammalian cells. A synthetic arginine-responsive transactivator (ARG), consisting of ArgR fused to the Herpes simplex VP16 transactivation domain, reversibly adjusted transgene transcription of chimeric ARG box-containing mammalian minimal promoters (P(ART)) in an arginine-inducible manner. Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology. ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types. Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses. Using a physiologic inducer, such as the amino acid l-arginine, to control heterologous transgenes in a seamless manner which is devoid of noticeable metabolic interference will foster novel opportunities for precise expression dosing in future gene therapy scenarios as well as the manufacturing of difficult-to-produce protein pharmaceuticals.
...
PMID:An engineered L-arginine sensor of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice. 1794 34

Kaposi's sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.
...
PMID:The HIV protease inhibitor nelfinavir inhibits Kaposi's sarcoma-associated herpesvirus replication in vitro. 2140 41

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities.
...
PMID:A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus. 2154 82


<< Previous 1 2 3 4 5 6 Next >>

---