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At least 217 records · Page 12Linked to original sources
PubMed2003

Pharmacogenomic analysis of mechanisms mediating ethanol regulation of dopamine beta-hydroxylase.

We previously showed that ethanol regulates dopamine beta-hydroxylase (DBH) mRNA and protein levels in human neuroblastoma cells (Thibault, C., Lai, C., Wilke, N., Duong, B., Olive, M. F., Rahman, S., Dong, H., Hodge, C. W., Lockhart, D. J., and Miles, M. F. (2000) Mol. Pharmacol. 58, 1593-1600). DBH catalyzes norepinephrine synthesis, and several studies have suggested a role for norepinephrine in ethanol-mediated behaviors. Here, we performed a detailed analysis of mechanism(s) underlying ethanol regulation of DBH expression in SH-SY5Y cells. Transient transfection analysis showed that ethanol (25-200 mM) caused concentration- and time-dependent increases in DBH gene transcription. Progressive deletions identified ethanol-responsive sequences in the -262 to -142 bp region of the DBH gene promoter. Mutagenesis of cAMP-response element (CRE) sequences in this region abolished ethanol responsiveness while maintaining responsiveness to phorbol esters. Coexpression of dominant-negative CRE-binding protein greatly reduced ethanol induction of DBH. Inhibitors of protein kinase A, casein kinase II, and MAPK reduced ethanol induction of DBH promoter activity. Pharmacogenomic studies with microarrays showed that protein kinase A, MEK, and casein kinase II inhibitors blocked induction of DBH and a large subset of ethanol-responsive genes. These genes had diverse functional groupings, including multiple members of the MAPK and phosphatidylinositol signaling cascades. Real-time PCR analysis validated select microarray results. Taken together, these results suggest that ethanol regulation of DBH requires a functional CRE and its binding protein and may require interaction of multiple kinase pathways. This mechanism may also mediate ethanol responsiveness of a complex subset of genes in neural cells. These studies may have implications for behavioral responses to ethanol or mechanisms underlying ethanol-related neurological disease.

Casein Kinase II↗
PubMed1994

Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.

Animals↗
PubMed1996

B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new "speed congenic" stock of NOD.Ig mu null mice.

The T lymphocytes mediating autoimmune destruction of pancreatic beta cells in the nonobese diabetic (NOD) mouse model of insulin-dependent diabetes mellitus (IDDM) may be generated due to functional defects in hematopoietically derived antigen-presenting cells (APC). However, it has not been clear which particular subpopulations of APC (B lymphocytes, macrophages, and dendritic cells) contribute to the development and activation of diabetogenic T cells in NOD mice. In the current study we utilized a functionally inactivated immunoglobulin (Ig) mu allele (Ig mu null) to generate a "speed congenic" stock of B lymphocyte-deficient NOD mice that are fixed for linkage markers delineating previously identified diabetes susceptibility (Idd) genes. These B lymphocyte NOD.Ig mu null mice had normal numbers of T cells but were free of overt IDDM and insulitis resistant, while the frequency of disease in the B lymphocyte intact segregants was equivalent to that of standard NOD mice in our colony. Thus, B lymphocytes play a heretofore unrecognized role that is essential for the initial development and/or activation of beta cell autoreactive T cells in NOD mice.

Age Factors↗
PubMed1993

[Experimental analysis of cytotoxicity mediated by activated macrophages against glioma cells in mice].

Macrophage (M phi) has a very important role in host natural defence and tumor cell killing. Activated M phi with tumor cytotoxicity can be induced by various lymphokines, such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-4, granulocyte macrophage-colony stimulating factor (GM-CSF), and macrophage-colony stimulating factor (M-CSF), but also by various bacterial products, such as lipopolysaccharide (LPS), Bacillus Calmette Guérin (BCG), corynebacterium parvum (C.p.), and muramyl-dipeptide (MDP). Although the brain is considered as an "immunologically privileged site", it has been demonstrated that in malignant brain tumors infiltration of lymphocytes or M phi can be seen in tissue samples. This may suggest that immune surveillance exists in the brain. But the biological significance of infiltrative M phi is unclear. The purpose of this study is to confirm the significant generation of activated M phi with cytotoxicity to glial tumor cells and to elucidate the cytotoxic mechanism associated with cellular interaction between effector M phi and target cells. This is the first report on experimental analysis of cytotoxicity mediated by activated M phi against glioma cells in mice. The cytotoxicity mediated by murine peritoneal activated M phi was examined against 3 kinds of murine glioma cell lines; VM-Glioma (spontaneously occurring astrocytoma of the VM mouse origin, H-2b), RSV-M (Schmitt-Ruppin Rous sarcoma virus-induced malignant glioma of the C3H/He mouse origin, H-2k), and 203-Glioma (methylcholanthrene-induced ependymoblastoma of the C57BL/6 mouse origin, H-2b). Activated M phi were obtained from peritoneal exudate cells of 4 strains of mice, C57BL/6 (H-2b), C3H/He (H-2k), DBA/2 (H-2d), and BALB/c (H-2d), following intraperitoneal injection of (1) LPS 200 micrograms, (2) BCG 200 micrograms, (3) C.p. 200 micrograms, (4) MDP 350 micrograms, and (5) IFN-gamma 10(3) units, 7 days prior to 20 hr 51Cr release-cytotoxicity assay. Of the various combination of mouse strains and activating agents tested, that of activated M phi of the C3H/He mouse with induction by LPS had the most tumoricidal effect against the glioma cells, which was not MHC restricted. Although LPS-activated M phi underwent marked loss of cytotoxicity within 24 hr following initiation of in vitro culture, this 20 hr pretreatment with IFN-gamma or TNF-alpha inhibited this reduction in tumoricidal effects in a dose dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157.

The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.

2,6-Dichloroindophenol↗
PubMed2004

Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer.

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays and array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.

Cell Line, Tumor↗
PubMed1991

Topical thrombin-induced IgE-mediated anaphylaxis: RAST analysis and skin test studies.

Bovine topical thrombin (BTT) is a heterologous plasma thrombin concentrate that has been frequently used for the hemostasis since the 1940s. Recently, three patients in Japan went into shock after the topical application of BTT at lesion sites, and two of these patients had received BTT repeatedly. The clinical symptoms and the increased anti-BTT percent RAST counts suggest that these reactions were shock mediated by anti-BTT IgE antibodies. The RAST-inhibition analysis suggested that the antigenic substance(s) were bovine-specific moiety(ies) mainly involved in the contaminant rather than bovine thrombin itself. The skin tests were studied to predict such allergic reactions. The intracutaneous test provoked nonspecific reactions even at the low concentrations of BTT. The prospective study on the predictive value of the prick test with 1000 U/ml (1 mg/ml) of BTT in 192 patients suggested that it is useful to detect highly sensitive patients. In addition, the increased levels of anti-BTT IgE antibodies in patients 1 month after the single administration of BTT suggested the immunogenicity of the topical application of BTT.

Administration, Topical↗
PubMed1998

Semi-automated analysis of NMDA-mediated toxicity in digitised colour images from rat hippocampus.

The evaluation of neuronal cell survival after, for example, mechanical, hypoxic or drug-mediated injury requires the analysis of a high number of histological specimens. Since this is a time-consuming occupation, we have developed a semi-automated analysis routine for the determination of the distribution of live and dead cells. After digitalization of the histological preparations, 8-bit colour bitmaps were assessed using a compiled image-analysis programme of the software package Khoros. In the current study a detailed example of the application of this image-processing approach is described for the investigation of the cell survival after intraventricular application of N-methyl-D-aspartate (NMDA). The samples were prepared as fuchsin acid/toluidine blue stained hippocampal thin slices. The calculated areas of the live and dead cells were highly correlated with manual counts of live and dead cells in the 100 samples examined in this study. Twenty-four hours following NMDA-treatment animals (n = 5) were found to have significantly fewer live and more dead hippocampal cells than the saline-treated animals (n = 5), using either automated or manual examination techniques. The automated technique also revealed that NMDA treatment resulted in a reduction in the density of live cell distribution.

Animals↗
PubMed1994

BvgAS-mediated signal transduction: analysis of phase-locked regulatory mutants of Bordetella bronchiseptica in a rabbit model.

Members of the Bordetella genus alternate between two distinct phenotypic phases in response to changes in their environment. This switch, termed phenotypic modulation, is mediated by the BvgAS sensory transduction system. We developed an animal model based on the interaction of Bordetella bronchiseptica with one of its natural hosts, the rabbit. To investigate the importance of BvgAS signal transduction, we constructed constitutive (RB53) and Bvg- (RB54) phase-locked derivatives of a wild-type strain, RB50. RB50 and RB53, but not RB54, established respiratory infections in B. bronchiseptica-free rabbits with an intranasal 50% infective dose of less than 200 organisms, and the course of the infection closely resembled that observed with naturally infected rabbits. Bacteria were recovered from the nasal cavity, larynx, trachea, and lungs in similar numbers from RB50- and RB53-infected rabbits, yet no pathology was detected by histological examination of lung and tracheal sections. The antibody responses in rabbits inoculated with RB50 or RB53 were quantitatively and qualitatively indistinguishable; high titers of antibodies were generated primarily against Bvg(+)-phase-specific antigens. No response against flagella, a Bvg- phase factor, was detected. Assessment of bacteria associated with alveolar macrophages indicated that only a small percentage of bacteria, if any, appear to be residing within lung macrophages. We also tested the ability of these strains to survive in a nutrient poor environment, conditions which may be encountered within certain niches in the host or in an environmental reservoir. The Bvg- phase was advantageous for growth under these conditions. Our results indicate the Bvg+ phase is sufficient for establishment of respiratory tract infection in the rabbit and the normal BvgAS-mediated response to environmental signals is not required during initial colonization. The Bvg- phase may play a role at later stages of infection, including persistence, transmission, or survival in the environment.

Animals↗
PubMed1994

Tetracyanoquinodimethane-mediated flow injection analysis electrochemical sensor for NADH coupled with dehydrogenase enzymes.

A flow injection analysis (FIA) sensor for the oxidation of NADH is reported. The system utilizes a graphite paste electrode modified with the electroactive material tetracyanoquinodimethane (TCNQ). TCNQ acts as an efficient mediator for the oxidation of NADH to biologically active NAD+. Alcohol dehydrogenase/lactate dehydrogenase and NAD+ were coimmobilized in TCNQ-modified graphite paste using polyethylenimine to develop a FIA sensor for ethanol/lactate. The system responded rapidly with wide linearity. Response curves for ethanol/lactate and NADH are reported.

Alcohol Dehydrogenase↗
PubMed1997

Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis.

Reabsorption of Pi in the proximal tubule of the kidney is an important determinant of Pi homoeostasis. At least three types (types I-III) of high-affinity Na+-dependent Pi co-transporters have been identified in mammalian kidneys. The relative roles of these three types of Na+/Pi co-transporters in Pi transport in mouse kidney cortex have now been investigated by RNase H-mediated hybrid depletion. Whereas isolated brush-border membrane vesicles showed the presence of two kinetically distinct Na+/Pi co-transport systems (high Km-low Vmax and low Km-high Vmax), Xenopus oocytes, microinjected with polyadenylated [poly(A)+] RNA from mouse kidney cortex, showed only the high-affinity Pi uptake system. Kidney poly(A)+ RNA was incubated in vitro with antisense oligonucleotides corresponding to Npt-1 (type I), NaPi -7 (type II) or Glvr-1 (type III) Na+/Pi co-transporter mRNAs, and then with RNase H. Injection of such treated RNA preparations into Xenopus oocytes revealed that an NaPi-7 antisense oligonucleotide that resulted in complete degradation of NaPi-7 mRNA (as revealed by Northern blot analysis), also induced complete inhibition of Pi uptake. Degradation of Npt-1 or Glvr-1 mRNAs induced by corresponding antisense oligonucleotides had no effect on Pi transport, which was subsequently measured in oocytes. These results indicate that the type II Na+/Pi co-transporter NaPi-7 mediated most Na+-dependent Pi transport in mouse kidney cortex.

Animals↗
PubMed2003

Laser-mediated microdissection facilitates analysis of area-specific gene expression in rheumatoid synovium.

OBJECTIVE: Current approaches to analyzing gene expression in rheumatoid arthritis (RA) synovium are based on RNA isolated either from cultured synovial cells or from synovial biopsy specimens. This strategy does not, in general, allow distinction of specific gene expression between cells originating from different synovial areas, due to potential mixture of expression profiles. Therefore, we established the combination of laser-mediated microdissection (LMM) and differential display to analyze profiles of gene expression in histologically defined areas of rheumatoid synovium. The present study was undertaken to establish parameters for this technique and assess its usefulness for gene expression analysis. METHODS: Cryosections derived from RA synovial tissues were used to obtain cell samples from synovial lining versus sublining, using a microbeam laser microscope. RNA was isolated and analyzed by nested RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) for differential display fingerprinting. Differentially expressed bands were cut out, and PCR products were eluted, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry analysis. RESULTS: Microdissected sections of RA synovial tissue containing approximately 600 cells yielded enough RNA to produce a reproducible RNA fingerprint pattern. Several genes could be identified as being expressed differentially between the synovial lining and the sublining, and their expression could be confirmed at the messenger RNA and protein levels. CONCLUSION: The combination of LMM and RAP-PCR presents a valuable tool to obtain novel insights into the area-dependent differential regulation of gene expression in RA synovium. Both known and previously unknown genes were revealed with this technique. This study is the first to demonstrate the potential of this analytic strategy in the investigation of a nonmalignant, multifactorial, inflammatory disease.

Arthritis, Rheumatoid↗
PubMed2005

Analysis of inflammatory mediators in temporomandibular joint synovial fluid lavage samples of symptomatic patients and asymptomatic controls.

PURPOSE: It was our purpose to study IgA, IgG, and beta-glucuronidase levels in temporomandibular joint fluid lavage samples in order to find a correlation between biochemical markers and joint pathology. MATERIALS AND METHODS: Our patient population included 20 patients (18 female and 2 male) with severe pain and limitation of mandibular movement that failed to improve with at least 3 months of a full course of nonsurgical therapy. After institutional review board approval, 13 control subjects (6 female and 7 male), with no history of temporomandibular joint pain/dysfunction, were obtained. Arthroscopic examination of the patient and control groups involved classification of joints for osteoarthritis and synovitis using a visual grading system. All synovial fluid samples were analyzed for beta-glucuronidase, IgA, and IgG using fluorometric assay and enzyme-linked immunosorbent assay. RESULTS: beta-Glucuronidase was significantly elevated (P<.05, t test) in the patient group compared with the control group (4.13+/-8.30 versus 0.9+/-0.83 [mean+/-SD]). The difference in the IgG level was also statistically significant (31,638+/-70,714 versus 4,407+/-1,324) (P<.05, t test). IgA level showed a similar trend between the patient and control groups (6,315+/-19,037 versus 425+/-192) (P=.10, t test). CONCLUSIONS: Quantitative synovial fluid analysis of beta-glucuronidase, IgA, and IgG demonstrates elevated levels of inflammatory mediators in diseased joints compared with asymptomatic nondiseased joints. We hypothesize that high levels of IgA, IgG, and beta-glucuronidase in the TMJ synovial fluids are due to infiltration from the sera and chronic inflammatory cells residing in the synovium. High levels of immunoglobulins in a closed joint space can elicit strong inflammatory reaction and cause destruction to the joint tissues via complement activation and immune complex deposition. Complement activation increases blood vessel permeability to enable recruitment of neutrophilic leukocytes, which then liberate various lysosomal enzymes and damage the articular cartilage.

Adult↗
PubMed1998

Cationic lipid-mediated gene transfer: analysis of cellular uptake and nuclear import of plasmid DNA.

Cationic lipids are widely used for gene transfer in vitro and show promise as vectors for in vivo gene therapy applications. However, there is limited understanding of the cellular mechanisms involved in nonviral gene transfer. We investigated two major steps that could be limiting barriers to cationic lipid-mediated gene transfer in vitro. We used a fluorescent plasmid to study the cellular uptake and the intracellular fate of lipoplexes during in vitro transfection of fibroblast cells and found that 100% of the cells take up lipoplexes. The intracellular staining observed with lipoplexes was clearly different from that obtained with endocytosed fluorescent dextran. This suggests that cells readily take up lipoplexes by a mechanism that could be different from endocytosis in our conditions. However, the escape of DNA from intracellular vesicles could be a major limiting barrier to gene transfer. Direct injection of plasmid DNA into the nucleus and cytoplasm of cells indicated that DNA traffic from the cytoplasm to the nucleus might be also an important limiting step.

Animals↗
PubMed1999

The structural analysis of adhesions mediated by Ep-CAM.

The epithelial cell adhesion molecule Ep-CAM is capable of mediating Ca2+-independent homotypic cell-cell adhesion when introduced into cells lacking their own means of cell-cell interactions. We used (confocal) immunofluorescent and (immuno-) electron microscopy to investigate the structural organization of Ep-CAM-mediated adhesions and their relation to other types of intercellular adhesions. Ep-CAM-transfected cell lines, cells of epithelial origin, and epithelial tissues were analyzed. In transfected L cells Ep-CAM brings the opposing intercellular membranes into a close proximity (approximately 10-14 nm) at sporadic contacts; however, no structures resembling junctional complexes were observed. In L cells cotransfected with Ep-CAM and E-cadherin, both molecules localize at the sites of cell-cell contact, forming independent adhesion sites with no Ep-CAM detectable within the structurally distinguishable cadherin-mediated adherens junctions. In well-differentiated carcinoma cell lines Ep-CAM colocalized with E-cadherin practically along the whole lateral domain; however, no colocalization was observed between Ep-CAM and the components of the tight junction complex (occludin and ZO-1), desmosomes (desmoplakins I/II), or cell-substrate adhesions (beta1 integrins). This was confirmed by analysis of polarized epithelium of normal colon where Ep-CAM was present at the lateral membrane including the adherens junction areas, but was fully excluded from the apical cell membrane (microvilli), tight junctions, and desmosomes. We conclude that (1) Ep-CAM does not form junctional complexes in L cells, (2) in epithelial cells, cell surface Ep-CAM is present at the lateral cell membrane, but is excluded from tight junctions and desmosomes, and (3) in epithelial cells, Ep-CAM is present within adhesions mediated by the classic cadherins (especially E-cadherin) with both types of molecules remaining as independent clusters. The colocalization with cadherins might be important for the modulating effect of Ep-CAM on cadherin-mediated adhesions.

Animals↗
PubMed2001

A new technique for the analysis of endogenous mediators released following thermal injury.

Few techniques today enable us to measure the complex processes taking place inside a burn wound in vivo. The present in vivo technique was based on a standardised burn model in rat skin. A partial- or full-thickness burn was induced and resulted in a gelatinous oedema located between the skin and the underlying rectus muscle. The oedema has distinct borders to the surrounding connective tissue and is separated and removed easily for further analysis. Myeloperoxidase (MPO) activity used as indicator of neutrofil infiltration was increased significantly in the burn oedema versus non-burned skin. Leukocyte metabolic activity was high as shown by significantly higher free radical formation (ESR) in the oedema than in surrounding burned and non-burned tissue. Leukocyte viability measured by Trypan blue stain was 70% in the oedema of full-thickness burns. In order to decide whether processes taking place in the oedema communicate freely with systemic circulation, we conducted a number of experiments. Results show in burned animals in vivo that intravenous administration of indomethacin induced a strong inhibition of PGE(2) in the burn oedema as compared with saline but, as expected, had no significant effect on LTB(4) synthesis. In conclusion, the present technique allows us to analyse the processes taking place inside the burn wound in vivo and to evaluate the effects of various agents on these processes.

Albumins↗
PubMed2002

Analysis of pathways mediating preserved vision after striate cortex lesions.

This study investigated the neural substrates of preserved visual functioning in a patient with homonymous hemianopsia and Riddoch syndrome after a posterior cerebral artery stroke affecting the primary visual cortex (area V1). The limited visual abilities of this patient included above-chance verbal reports of movement and color change as well as discrimination of movement direction in the hemianopic field. Functional magnetic resonance imaging showed that motion and color-change stimuli presented to the hemianopic field produced activation in several extrastriate areas of the lesioned hemisphere that were defined using retinotopic mapping. Magnetoencephalographic recordings indicated that evoked activity occurred earlier in the higher-tier visual areas V4/V8 and V5 than in the lower-tier areas V2/V3 adjacent to the lesion. In addition, the functional magnetic resonance imaging analysis showed an increased functional connectivity between areas V4/V8 and V5 of the lesioned hemisphere in comparison with the same areas in the intact hemisphere during the presentation of color changes. These results suggest that visual perception after the V1 lesion in Riddoch syndrome is mediated by subcortical pathways that bypass V1 and project first to higher-tier visual areas V5 and V4/V8 and subsequently to lower-tier areas V2/V3.

Adult↗