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Biomedical subjects

G Bradley

Publications and source records attributed to G Bradley.

At least 19 recordsLinked to original sources
PubMed1996

Ostrich (Struthio camelus) carboxypeptidase B: purification, kinetic properties and characterization of the pancreatic enzyme.

Carboxypeptidase B has been isolated from numerous mammalian and invertebrate species. In contrast, very little is known about carboxypeptidases of avian origin. To provide information for a comparative study, we have undertaken an investigation of the kinetic and physical properties of ostrich carboxypeptidase B. Carboxypeptidase B from the pancreas of the ostrich was purified by water extraction of acetone powder and aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. The effects of pH and temperature on CPB activity were examined. K(i)-values for numerous inhibitors (PCI, ABSA, hipp-D-lys, epsilon-aminocaproic acid, D-arg and 3-phenylproprionic acid) and kinetic parameters (K(m), k(cat) and k(cat)/K(m)) for several substrates (hipp-arg, hipp-lys, FAAA, FAAL and hipp-AA) were determined. N-terminal sequencing and amino acid analysis were also performed. Purified ostrich carboxypeptidase B was assessed to be homogeneous by SDS-PAGE with a M(r) value of approx. 35,000. For ostrich carboxypeptidase B the K(m) values for the different substrates were of the same order as those reported for other species, whereas the k(cat) values were 8- to 21-fold lower than the reported values. FAAA and hipp-AA were the preferred substrates. PCI was the most effective inhibitor, with a K(i) in the nM region, and no inhibition was shown with 3-phenylpropionic acid. The N-terminal sequence showed a high degree of homology when aligned with CPB from other species. Amino acid analysis showed significantly lower levels of Asx and Cyh and higher levels of Trp and Leu when compared with other species. Ostrich carboxypeptidase B would appear to show many physical, chemical and kinetic properties similar to those of other known carboxypeptidases.

Amino Acid Sequence↗
PubMed1995

Overexpression of the class II P-glycoprotein gene in primary rat hepatocyte culture: evidence for increased mRNA stability.

The overexpression of P-glycoprotein (Pgp) appears to be responsible for multidrug resistance in some human cancers. The molecular basis of this overexpression is not understood. We have used primary monolayer cultures of adult rat hepatocytes as a model system to study the regulation of Pgp gene expression (Lee et al., J. Cell. Physiol., 157: 392-402, 1993). We observed a dramatic and specific overexpression of class II Pgp as a function of the time in culture. This isoform of Pgp, which is expressed at a very low level in normal liver, has also been shown to be predominantly overexpressed in several models of rat liver carcinogenesis. In the present study, we have used nuclear run-on assays and mRNA decay studies to investigate the mechanism for the overexpression of class II Pgp in cultured hepatocytes. We conclude that an increased mRNA stability is the major factor involved in the increased expression of class II Pgp. Studies using various drugs indicate that the integrity of the cytoskeleton is important for the maintenance of high expression of class II Pgp. Disruption of the cytoskeleton in cultured hepatocytes with cytochalasin D did not affect the transcriptional activity of the class II Pgp gene but rapidly destabilized its mRNA. This raises the possibility that an association between class II Pgp mRNA and cytoskeletal elements may underlie the mechanism that regulates class II Pgp mRNA stability. These findings have important implications for our understanding of the overexpression of class II Pgp during liver carcinogenesis.

Actins↗
PubMed1994

P-glycoprotein, multidrug resistance and tumor progression.

P-glycoprotein (Pgp) is a plasma membrane protein that was first characterised in multidrug resistant cell lines. The occurrence of Pgp in clinical tumors has been widely studied. Recent investigations have begun to focus on the relationship between Pgp detection in tumors and treatment outcome. In several types of tumors, detection of Pgp correlates with poor response to chemotherapy and shorter survival. P-glycoprotein over-expression often occurs upon relapse from chemotherapy but may also occur at the time of diagnosis. Studies of experimental rat liver carcinogenesis have shown that Pgp expression increases in late stages of carcinogenesis, suggesting that Pgp may be involved in tumor progression. While some of the Pgp isoforms are known to transport hydrophobic chemotherapeutic drugs out of tumor cells, the biologic effects of Pgp overexpression in tumor cells are not fully understood, because the spectrum of substrates for Pgp-mediated transport has not been determined. In the rat liver carcinoma model, strong expression of Pgp is associated with a highly vascular stroma, suggesting that Pgp in tumor cells may affect the connective tissue stroma. The regulation of Pgp appears to be complex, and little is known about how it is up-regulated during carcinogenesis. Further studies of the role of Pgp in malignancy may contribute to our understanding of molecular mechanisms which underlie tumor progression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Ostrich (Struthio camelus) carboxypeptidase A: purification, kinetic properties and characterization of the pancreatic enzyme.

1. Carboxypeptidase A beta and carboxypeptidase A tau-type from the pancreas of the ostrich were purified by water extraction of acetone powder, aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. 2. The final preparations were homogeneous when subjected to SDS-PAGE and PAGE. The M(r) values obtained from SDS-PAGE for CPA beta and CPA tau-type were 34,600 and 34,400, respectively. 3. The effects of inhibitors (1,10 phenanthroline and indole-3-acetic acid), pH and temperature on CPA activity were examined. Ki-values for CPI, PPA, D-phe, D-trp and aminobenzylsuccinic acid were determined. 4. Km, kcat and kcat/Km values were determined for hipp-phe, cbz-gly-phe, cbz-(gly)2-phe, cbz-gly-leu, cbz-(gly)2-leu and cbz-(gly)2-val. 5. N-terminal sequencing and amino acid analysis were performed for CPA beta and CPA tau-type.

Amino Acid Sequence↗
PubMed1994

A sensitive multilayer immunoalkaline phosphatase method for detection of P-glycoprotein in leukemic and tumor cells in the bone marrow.

BACKGROUND: Relatively low levels of the multidrug resistance P-glycoprotein have correlated with poor prognosis in rhabdomyosarcoma, neuroblastoma, acute myelogenous leukemia, lymphoma, myeloma and breast carcinoma. A sensitive, nonradioactive method, less costly and time-consuming than the present molecular biologic techniques, is desirable for direct measurement of P-glycoprotein in tumor cells versus normal cells. EXPERIMENTAL DESIGN: We have devised an immunoalkaline phosphatase method using four antibody layers to amplify the primary signal considerably and refined staining conditions to optimize the 'signal-to-noise' ratio. Immunoalkaline phosphatase is preferred to immunoperoxidase for testing leukemic and tumor cells in bone marrow, because it avoids myeloperoxidase staining in myeloblasts and myeloid progenitors that interferes with P-glycoprotein interpretation. RESULTS: Multilayer immunoalkaline phosphatase detected low levels of P-glycoprotein overexpression, that could not be identified by conventional immunoperoxidase or immunoblot, in a low-resistance (8-fold) cell line with a barely detectable transcript. Our technique also detected increased P-glycoprotein in malignant cells in bone marrow of relapsed acute lymphoblastic leukemia (10/11), acute myelogenous leukemia (2/2), lymphoma (1/1), neuroblastoma (7/7), and rhabdomyosarcoma (2/2). Increased P-glycoprotein was not identified at diagnosis in 6 patients with acute lymphoblastic leukemia, and two with stage IV and three with stage IVS neuroblastoma that remained relapse-free in the long-term, but was detected in 4 patients with stage IV neuroblastoma and three with rhabdomyosarcoma who ultimately relapsed. CONCLUSIONS: Our new technique is more sensitive than conventional immunoperoxidase and immunoblot for assaying P-glycoprotein in low-resistance cell lines. It may be potentially applicable for detecting low levels of P-glycoprotein overexpression in leukemic and tumor cells in bone marrow. Early identification of low levels of multidrug resistance may be clinically relevant by allowing poor-prognostic patients to receive alternative therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Differential expression of P-glycoprotein genes in primary rat hepatocyte culture.

The multidrug resistance (MDR)-associated protein, P-glycoprotein (Pgp), is expressed on the bile canalicular surface of hepatocytes, where it is thought to function in the detoxification of xenobiotics or in the transport of specific metabolites. Several studies have shown that Pgp expression in rat liver can be perturbed in vivo and in vitro; however, it is not known which of the 3 Pgp genes (class I, II, or III) are involved. In rodents, the class I and II Pgp genes have been shown to mediate MDR while the class III gene apparently does not. In this report, we have used gene-specific probes generated from the 3'-untranslated regions of the three rat Pgp genes (Deuchars et al.: Biochim. Biophys. Acta, 1130:157-165, 1992) to investigate Pgp gene expression in primary rat hepatocytes. We observed that the class II Pgp mRNA, the least abundant in the intact liver, is dramatically increased in culture over a 48 h period, while the class I Pgp showed only a modest increase in mRNA level. In contrast, the class III Pgp mRNA, which is the most abundant in the intact liver, exhibited a gradual decline. In rat liver hepatocytes, different culture conditions, as well as drugs such as cytochalasin D and colchicine, appear to affect the level of the class II Pgp gene expression. Moreover, under all these conditions, there is a strong correlation between the level of the class II Pgp and cytoskeletal (actin and tubulin) mRNAs. Thus, there may be a common mechanism regulating the expression of cytoskeletal protein genes and the class II Pgp gene. These findings have implications for our understanding of the regulation of Pgp gene expression in normal and malignant tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

P-glycoprotein expression during tumor progression in the rat liver.

P-Glycoprotein (Pgp) has been shown to mediate multidrug resistance in tumor cell lines. Overexpression of Pgp has been detected in clinical cancer samples of many histological types. The basis and biological significance of such increases in Pgp expression are not well understood. In this study, the expression of Pgp during stepwise progression to rat liver cancer was examined to investigate the possible role of Pgp in carcinogenesis. An immunohistochemical technique was used to detect Pgp at the single-cell level, in a large number of liver nodules, hepatocellular carcinoma, and in distant metastases of the carcinomas. The results showed that distinct changes in Pgp expression occurred during stepwise liver carcinogenesis and that these changes were closely associated with the microscopic anatomy of the lesions. In contrast to gamma-glutamyl transpeptidase and glutathione S-transferase-7.7, whose expression appeared to correlate with the early steps of liver carcinogenesis, Pgp expression was higher in the large hyperplastic nodules and in hepatocellular carcinomas than in the early microscopic lesions. A particularly striking finding was the consistent expression of Pgp in the lung metastases. These findings suggested that Pgp was associated with a more progressed malignant phenotype in liver carcinogenesis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Novel antagonists of the 5-HT3 receptor. Synthesis and structure-activity relationships of (2-alkoxybenzoyl)ureas.

A series of benzoylureas derived from bicycle amines were prepared and evaluated for 5-HT3 antagonist activity on the rat isolated vagus nerve. From among these compounds, those analogues which were ortho substituted by an alkoxy group on the benzoyl function were shown to be potent 5-HT3 antagonists with similar or greater potency than the standard agent ondansetron. NMR and X-ray crystallography studies showed these o-alkoxy compounds to exist as a planar, hydrogen-bonded, tricyclic ring system. In molecular modeling studies on endo-N-[[(8-methyl-8-azabicyclo[3.2.1]octan-3-yl-amino] carbonyl]-2-(cyclopropylmethoxy)benzamide (30) the central hydrogen-bonded ring was able to mimic an aromatic ring present in previously reported 5-HT3 antagonists.

Animals↗
PubMed1992

Partial characterisation of human and porcine adipose acidic protease activity.

1. An aspartic protease was isolated from human and porcine white adipose tissue and from isolated human adipocytes. The three preparations appeared to represent the same enzyme. 2. Electrofocusing of all three preparations revealed two bands corresponding to a pI of 3.6 and 4.4. respectively. On PAGE a single band in the same position was obtained in all three cases. 3. Both the porcine and human fractions were optimally active at pH 3.4, using acid denatured haemoglobin as substrate, and both activities were strongly inhibited by pepstatin and iodoacetate. 4. The Km values for haemoglobin for the porcine and human proteases were 0.16 and 0.14 mM respectively, whereas Vmax values of 30 and 33 units.nmol-1, respectively, were obtained.

Adipose Tissue↗
PubMed1992

Isolation and characterization of cDNAs from BamHI-H gene family RNAs associated with the tumorigenicity of Marek's disease virus.

It has been reported that loss of the tumorigenic potential of attenuated Marek's disease virus (MDV) is strongly associated with amplification of the 132-bp repeat sequences found within the BamHI-D and BamHI-H fragments contained within the long terminal repeat and the long internal repeat, respectively. The expansion of this region results in loss of transcripts that are 3.8, 3.0, and 1.8 kbp long that are produced by tumorigenic strains of MDV. This evidence suggests that production of one or more of these three RNAs is strongly associated with the tumorigenic potential of the virus. In this study, we have cloned and sequenced 1.69-, 1.5-, 1.9-, and 2.2-kbp cDNAs from the BamHI-H gene family RNAs associated with tumorigenicity. The 1.69- and 2.2-kbp cDNAs are derived from nonspliced transcripts, whereas the 1.5- and 1.9-kbp cDNAs are from single spliced mRNAs spanning the BamHI-H and BamHI-I2 fragments of MDV DNA. Sequence analysis has shown two potential open reading frames in each of the cDNAs. The putative 63-amino-acid protein encoded by the first open reading frame in the 1.69-kbp cDNA and a putative 75-amino-acid protein encoded by the first open reading frame in the 1.5-kbp cDNA showed limited homology with the mouse T-cell lymphoma oncogene and the fes/fps family of kinase-related transforming proteins.

Amino Acid Sequence↗
PubMed1991

Inhibition of human immunodeficiency virus forward and reverse transcription by PC6, a natural product from cones of pine trees.

We have previously shown that PC6, a natural product extracted from cones of Pinus parviflora Sieb et Zucc, can inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in CD4+ T cells and in monocyte/macrophage cell lines. Here, we show by immunoprecipitation of HIV-1 proteins with a specific pooled serum that PC6 inhibited the expression of all HIV-1 proteins in CEM cells. PC6 did not affect the posttranslational processing of the HIV-1 proteins. Northern, Southern, and kinetics analyses revealed that PC6 inhibited HIV-1 reverse and forward transcription in CEM cells. Transient transfection of CEM cells with HIV-1 long terminal repeat (LTR) linked CAT DNA also showed that PC6 inhibited LTR-driven gene expression at the transcriptional level.

Antiviral Agents↗
PubMed1991

Inverted repeat regions of Marek's disease virus DNA possess a structure similar to that of the a sequence of herpes simplex virus DNA and contain host cell telomere sequences.

The genomic structure of Marek's disease virus (MDV) is similar to those of the alphaherpesviruses herpes simplex virus (HSV) types 1 and 2. Sequence analysis of the junction region between the long component (L) and the short component (S) revealed the existence of an a-like sequence, similar in structure to the a sequence of HSV-1. Further study revealed that the MDV genome contains five copies of the a-like sequence within the long terminal repeat region as well as in the short terminal repeat region. The junction between the L and S components was found to contain 10 copies of the a-like sequence. Within the a-like sequence, a structure homologous to the DR2 of HSV was found to contain 17 copies of the telomeric sequence, GGGGTTA. There appears to be little to no sequence homology between the HSV a sequence and the MDV a-like sequence; however, the strong physical homology to its counterpart in HSV-1 suggests that the MDV a-like sequence may have the same functional homology (the domain for cleavage/packaging of the DNA into the viral capsids and for genomic inversion) as well.

Animals↗
PubMed1990

Sex-dependent and independent expression of the P-glycoprotein isoforms in Chinese hamster.

P-glycoprotein (Pgp) is a small family of membrane proteins which belongs to a superfamily of energy-dependent membrane transport proteins identified in phylogenetically distant species, from bacteria to man. Among mammalian species, some of the Pgp isoforms can mediate multidrug resistance by acting as an energy-dependent drug efflux pump. However, the physiologic functions of the Pgp isoforms have not been defined. In this study we examined the expression of the three hamster Pgp isoforms in normal hamster tissues, by using isoform-specific monoclonal antibodies in a competitive immunohistochemical assay. We showed that each Pgp isoform is predominantly expressed in a small, distinct group of differentiated cells, where it is likely to function in specific secretory pathways. The expression of the Pgp isoforms appears to be tightly regulated and, at least in some cells, under complex hormonal control. Furthermore, there is a striking sex difference in Pgp content of the adrenal cortex. These findings are important for the ultimate understanding of the normal physiologic roles of the Pgp gene family members.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Interaction of Epstein-Barr viral (EBV) origin of replication (oriP) with EBNA-1 and cellular anti-EBNA-1 proteins.

We have previously shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates expression of the latent genome of the Epstein-Barr virus (EBV) in Burkitt lymphoma cells induces the synthesis of two cellular anti-EBNA-1 competitor proteins, anti-EBNA-1.1 and anti-EBNA-1.2. Both anti-EBNA-1 proteins can uncouple the specific binding of the EBNA-1 to the region required for EBV plasmid maintenance (oriP). Here, we show by DNase I footprinting that the binding sites on oriP for the EBNA-1 and the anti-EBNA-1 proteins were indistinguishable. The proteins bound to the 30-bp tandem repeats of the oriP. Glycerol-gradient centrifugation and gel retardation assay revealed that a 60-kDa protein formed the anti-EBNA-1.1-DNA complex and a 40-kDa protein formed the anti-EBNA-1.2-DNA complex.

Antigens, Viral↗