Biomedical subjects

Q Li

Publications and source records attributed to Q Li.

At least 613 records · Page 34Linked to original sources

PubMed1997

Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cellular cholesterol efflux in macrophages.

Extracellular apolipoprotein A-I removed cholesterol and phospholipid from cholesterol-loaded mouse peritoneal macrophage and thereby generated a prebeta high-density lipoprotein (HDL) particle having a weight ratio of cholesterol to phosphatidylcholine of approximately 1:1. Treatment of the cells with phorbol myristate slightly increased cholesterol efflux by this mechanism without influencing the nonspecific cholesterol efflux to the lipid microemulsion. When the cells were treated by protein kinase C (PKC) inhibitors, H7 and staurosporine, apolipoprotein-mediated cellular cholesterol efflux was substantially reduced without a significant change in phosphatidylcholine efflux, resulting in generation of cholesterol-poor prebeta-HDL particles having a weight ratio of cholesterol to phosphatidylcholine as low as 1:10. In spite of this change, specific binding of apoA-I to the cellular surface was unaffected. Cellular cholesterol available for acylCoA:cholesterol acyltransferase (ACAT) was rapidly depleted by adding apoA-I to the medium, and the PKC inhibitor treatment reversed this effect. In contrast, nonspecific cellular cholesterol efflux to the lipid microemulsion did not influence the ACAT-available cellular cholesterol pool, and it was not influenced by the PKC inhibitors. Thus, we concluded that apolipoprotein-mediated cellular cholesterol efflux is linked to mobilization of cholesterol from an intracellular pool used by ACAT to a specific pool for apolipoprotein-mediated prebeta-HDL generation, in response to apolipoprotein-cell interaction and subsequent intracellular signaling. Binding of apolipoprotein to the cell surface is required for assembly of the prebeta-HDL particle with cellular phospholipid, and the intracellular cholesterol mobilization is needed for enrichment with cholesterol of the prebeta-HDL. These reactions are largely independent of diffusion-mediated nonspecific cell cholesterol efflux.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Difference in brown adipose tissue effector response and associated thermoresponsiveness of ventromedial hypothalamic (VMH) neurons of 21 degrees C vs. 4 degrees C acclimatized rats to scrotal thermal stimulation.

The present study was designed (1) to determine if scrotal thermal stimulation would activate brown adipose tissue (BAT) thermogenesis, indicated by increases in interscapular BAT temperature (TIBAT) of cold acclimatized (CA, kept at 4 degrees C for 4 weeks) and room temperature acclimatized rats (RA, kept at 21 degrees C for 4 weeks) and (2) to compare the thermoresponsiveness of VMH neurons of both groups to scrotal heating and cooling. VMH extracellular activity was recorded in male RA and CA Sprague-Dawley rats when scrotal temperatures (Tsc) were changed between 5-40 degrees C via localized thermode (3 mm2) along with measurements of TscS and TIBATS. The CA-group showed significant increases in TIBATS during scrotal cooling compared to respective TIBATS of the RA-group. The ratio of VMH warm responsive (WRN), cold responsive (CRN) and temperature non-responsive (TNRN) neurons in the CA-group changed compared to that in the RA-group as a greater percentage of CRNs occurred in the CA-group. Also, the thermoresponsiveness of individual VMH CRNs of the CA rats was significantly increased compared to VMH CRNs of the RA-group. The results indicated that localized scrotal cooling of CA-rats (not RA-rats) can activate BAT thermogenesis. Furthermore, VMH CRNs increased their thermoresponsiveness with chronic cold exposure which may be an important neuronal adaptive response for the subsequent enhanced BAT thermogenic effector response seen in that group.

Acclimatization↗

PubMed1997

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

Amino Acid Sequence↗

PubMed1997

Autoradiographic evidence for differential G-protein coupling of 5-HT1A receptors in rat brain: lack of effect of repeated injections of fluoxetine.

The present study examined the distribution of [3H]8-OH-DPAT-labeled 5-HT1A receptors and their degree of coupling to G proteins in the hypothalamus and several other brain regions. In addition, we also investigated the effects of repeated injections of fluoxetine on the density and G protein coupling of 5-HT1A receptors in hypothalamic nuclei and other brain regions using autoradiography. Male rats received daily injections of either fluoxetine (10 mg/kg, ip) for 3, 7, 14 and 22 days, or saline for 22 days. 5-HT1A receptors were labeled by 2 nM [3H]8-hydroxy-2-(dipropylamino)tetralin ([3H]8-OH-DPAT) in the absence or presence of guanylylimidodiphosphate (Gpp(NH)p, 10[-5] M) to determine the percentage of 5-HT1A receptors coupled to G proteins. 5-HT1A receptor densities ranged from 7 to 63 fmol/mg tissue equivalent among hypothalamic nuclei. Similarly, the degree of G protein coupling to 5-HT1A receptors varied markedly among hypothalamic nuclei (from 14% to 61%) and among other brain regions (from 17% to 85%). Fluoxetine did not alter the density or the degree of coupling of 5-HT1A receptors in any brain regions. These data indicate marked regional differences in the degree of G protein-coupled 5-HT1A receptors and suggest that fluoxetine-induced desensitization of hypothalamic 5-HT1A receptors is not mediated by changes in receptor density or G protein coupling.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1997

Molecular modeling and 3D-QSAR studies on the interaction mechanism of tripeptidyl thrombin inhibitors with human alpha-thrombin.

The mechanism of inhibition of peptidyl inhibitors with thrombin was studied using molecular modeling, molecular mechanics, and CoMFA statistical analysis. A new procedure for the elucidation of binding conformations, BCSPL, is described and was employed to obtain the binding conformers of a series of 18 tripeptidyl thrombin inhibitors. Energetic studies and QSAR analysis of the BCSPL-derived conformers indicated a modest correlation between the calculated binding energies of the title compounds and their inhibitory activities to human alpha-thrombin. CoMFA analysis of the BCSPL alignment resulted in a satisfactory model of the thrombin active site.

Antithrombins↗

PubMed1997

Communication between switch II and switch III of the transducin alpha subunit is essential for target activation.

Comparisons of the tertiary structures of the GDP-bound and guanosine 5'-O-(thiotriphosphate) (GTPgammaS)-bound forms of the alpha subunit of transducin (alphaT) indicate that there are three regions that undergo changes in conformation upon alphaT activation. Two of these regions, Switch I and Switch II, were originally identified in Ras, while Switch III appears to be unique to trimeric GTP-binding proteins (G proteins). We find that replacement of the Switch III region (aspartic acid 227 through asparagine 237) with a single alanine residue yields an alphaT subunit that fully binds and hydrolyzes GTP but no longer stimulates the activity of the cyclic GMP phosphodiesterase (PDE), the physiological target for transducin. We also show that changing glutamic acid 232 of alphaT to a leucine (E232L) had no effect on rhodopsin-stimulated GTP-GDP exchange nor on the GTP hydrolytic activity of alphaT. However, the GTPgammaS-bound form of the alphaTE232L mutant was unable to stimulate the activity of the cyclic GMP PDE. The lack of stimulation was not due to an inability of the alphaTE232L mutant to bind to the target. Taken together, these results indicate that glutamic acid 232 mediates a conformational coupling between Switch II and Switch III, which is essential for converting GTP-dependent G protein-target interactions into a stimulation of target/effector activity.

Animals↗

PubMed1997

Characterization of rabbit UDP-glucuronosyltransferase UGT1A7: tertiary amine glucuronidation is catalyzed by UGT1A7 and UGT1A4.

A rabbit liver UDP-glucuronosyltransferase cDNA that is related to human and rat UGT1A7 has been identified. The predicted amino acid sequence of the UGT1A7l displays 80% similarity to that encoded by human HP4 (UGT1A9), but 81% to that predicted for human UGT1A7 and 77% to the rat UGT1A7 (UGTA2). The exons encoding human UGT1A7 and rat UGTA2 are the seventh of the series of cassette exons that flank the 3' common exon series of the UGT1A locus. Southern blot analysis demonstrates that the exon sequence encoding UGT1A7l is part of a larger cluster of highly related genes. The UGT1A7l RNA is expressed in both neonatal and adult liver, and unlike rat UGT1A2 which is inducible with Ah receptor ligands such as polycyclic aromatic hydrocarbons, rabbit UGT1A7l is not regulated when animals are exposed to these inducers. Following expression of UGT1A7l in COS-1 cells, glucuronidation activity was identified for small phenolic molecules like 4-nitrophenol, bulky phenols as represented by 4-hydroxybiphenol and octylgallate, as well as 4-hydroxyestrone. In addition, UGT1A7l possesses catalytic activity toward tertiary amines like the tricyclic antidepressant imipramine. The pattern of UGT1A7l glucuronidation is similar to that observed for human UGT1A9, except tertiary amines are not subject to glucuronidation by human UGT1A9. Glucuronidation of tertiary amines is catalyzed principally by human UGT1A4 as well as rabbit UGT1A4. Although rabbit UGT1A7l catalyzes the formation of quarternary ammonium glucuronides, the Vmax is considerably less than that observed for rabbit UGT1A4. Overall, the characterization of rabbit UGT1A7l suggests that this protein represents the ortholog of the human UGT1A7, which to date has not been identified.

Amines↗

PubMed1997

Association between CpG island methylation and microsatellite instability in colorectal cancer.

De novo methylation of promoter region CpG islands has been increasingly associated with transcriptional inactivation of important genes in neoplasia. To study the potential mechanisms underlying aberrant methylation in cancer, we have determined the methylation patterns of selected genes in colorectal cancers with and without microsatellite instability (MI), which results from defects in one of several base mismatch repair genes. A total of 47 colorectal cancers were analyzed, of which 15 were MI+ (32%). We now report that both the frequency and the extent of de novo methylation are strikingly increased in MI+ cancers. Hypermethylation of the p16 gene was found in 60% of MI+ cancers, compared to only 22% in MI- cancers (P = 0.02). Similarly, hypermethylation of the thrombospondin-1 (TSP-1) gene, an angiogenesis inhibitor, was increased in MI+ cancers (27% versus 0%; P = 0.008). Extensive methylation of insulin-like growth factor II (IGF2) and hypermethylated in cancer-1 (HIC-1) genes was observed in 60 and 80% of MI+ cancers, respectively, as contrasted with 6 and 38% of MI- cancers (P = 0.0002 and 0.01, respectively). Furthermore, 60% of the MI+ cancers displayed the hypermethylation events at two or more loci in a concordant manner compared to only 9% of the MI- cancers (P < 0.001). These results demonstrate a strong link between promoter hypermethylation and genetic instability due to deficient DNA repair.

Carrier Proteins↗

PubMed1997

Measurement of frontal lobe volume on magnetic resonance imaging scans.

This article describes rules for measurement of the frontal lobe on thin SPGR (spoiled gradient recalled echo in steady state) MRI (magnetic resonance imaging) scans. Measurements were performed using a locally-developed software program that allows 3-dimensional reconstruction of images, 'painting' of landmarks on the surface of the brain, and reconstruction of 2-dimensional images in any plane with landmark 'paint' remaining on the surface of the brain. Excellent inter-rater reliability has been achieved for this method. The approach may be particularly useful for studies involving groups of patients whose brains are known to be dysmorphic and who may not, therefore, be appropriate for measurement methods that involve image warping or dependence on arbitrary landmarks for defining the posterior boundary of the frontal lobe.

Adult↗

PubMed1997

Molecular characterization of the promoter region of a neuroendocrine tumor marker, IA-1.

IA-1 is an intronless gene, which encodes a 510 amino acid protein with a zinc-finger DNA-binding motif that is expressed in tumors of neuroendocrine origin. The 5'-upstream region of the IA-1 gene was recently sequenced. In this paper, the regulatory elements and the promoter region of the 5'-upstream region were analyzed by use of a series of deletion mutants (ranging from +26 bp to -2090 bp upstream of the IA-1 gene), which were tested in a pituitary tumor cell line, AtT-20, and Hela cells by transient transfection assays. These experiments showed that a 506 base pair upstream sequence was sufficient for maximal expression of a reporter gene. Multiple known regulatory elements were found within this region including three E boxes and a clustered Sp-1 site. In addition, Southwestern blot analysis, using a radiolabeled promoter sequence (extending from -108 bp to -66 bp) and nuclear extracts from both neuroendocrine and non-neuroendocrine cell lines, revealed four promoter binding proteins designated PBP1, PBP2, PBP3 and PBP4 with molecular weights of 55 kD, 32 kD, 29 kD, and 27/28 kD, respectively. These studies suggest that several different regulatory elements in the 5'-upstream region of the IA-1 gene and at least four different nuclear proteins may be involved in the cell-specific expression of IA-1.

Binding Sites↗

PubMed1997

The 6H1 orphan receptor, claimed to be the p2y5 receptor, does not mediate nucleotide-promoted second messenger responses.

An orphan G protein-coupled receptor, termed 6H1, with approximately 30% sequence identity to P2Y receptors has been proposed to be a P2Y receptor (p2y5) based solely on a radioligand binding assay with [35S]dATP alphaS [Webb et al. (1997) Biochem. Biophys. Res. Commun. 219:105-110]. Previous work in our laboratory has shown that [35S]-dATP alphaS is not a general ligand for P2Y receptors, and thus inclusion of the p2y5 receptor in the family of P2Y receptors is questionable. To define unambiguously whether the p2y5 receptor is a P2Y receptor, we have cloned the turkey homologue of the chick p2y5 receptor. Sequence analysis indicated that the turkey receptor contains an additional 32 amino acids at its carboxy terminus compared to the published chick sequence. HA epitope-tagged turkey p2y5 receptors were stably expressed in 1321N1 human astrocytoma cells, and cells shown to express the HA-tagged p2y5 receptor by an intact cell-based ELISA were used to determine whether changes in second messenger levels occurred in response to a series of nucleotides. ATP, ADP, UTP, UDP, dATP alphaS, and A2P4 had no effect on either inositol phosphate or cyclic AMP concentrations in cells expressing the p2y5 receptor. Robust inositol phosphate and cyclic AMP responses occurred to other G protein-coupled receptors expressed in 1321N1 cells, which indicate that these cells contain all of the necessary signaling components to generate these second messenger responses. These data indicate that the 6H1/p2y5 receptor is not a member of the P2Y receptor family of signaling proteins.

Animals↗

PubMed1997

Suppression of tumor growth by the 3' untranslated region of mel-18 in 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18.

By introducing a cDNA library derived from rat embryonic fibroblast cells, we isolated several morphologically flat revertants of rat 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18 (HPV18). From one of the revertants, we recovered a 0.2-kb cDNA, N56, that suppresses the tumor growth of the transformed 3Y1 cells irrespective of the expression of the E6 and E7 genes. The nucleotide sequence of the cDNA was shown to be identical to that of the 3' untranslated region of a putative mammalian polycomb group gene, mel-18.

Animals↗

PubMed1997

Lack of nucleotide-promoted second messenger signaling responses in 1321N1 cells expressing the proposed P2Y receptor, p2y7.

A recently cloned G protein-coupled receptor (named the p2y7 receptor) with relatively low sequence identity to previously cloned P2Y receptors was proposed to be a member of this family of receptors on the basis of both a radioligand binding assay with [35S]dATP alphaS and an inositol phosphate response to ATP in COS-7 cells transiently transfected with receptor cDNA. Previous work in our laboratory has shown that [35S]dATP alphaS is not a general radioligand for the identification of P2Y receptors and that COS-7 cells express an endogenous P2Y receptor (P2Y2) that complicates the analysis of nucleotide-promoted inositol phosphate responses. Thus, data supporting inclusion of the p2y7 receptor in the P2Y family of receptors are equivocal. To determine unambiguously whether the p2y7 receptor is a P2Y receptor subtype, a p2y7 receptor bearing an epitope-tag at its NH2-terminus was expressed in 1321N1 cells and cell surface expression of the receptor was demonstrated by an intact cell-based ELISA. Cells shown to express epitope-tagged p2y7 receptors by ELISA were examined for their second messenger signaling properties in response to a range of nucleotides. ATP, UTP, ADP, UDP, and dATP alphaS had no effect on phospholipase C or adenylyl cyclase activities in cells expressing the p2y7 receptor. Experimental controls utilizing expression of other G protein-coupled receptors showed that 1321N1 cells displayed robust responses for each of these signaling pathways. These data, together with the low sequence identity of the p2y7 receptor to other P2Y receptors, indicate that the p2y7 is not a member of the P2Y family of signaling molecules.

Adenine Nucleotides↗

PubMed1997

Basal ganglia volume in adults with Down syndrome.

This study was designed to determine the effects of aging on the volume of the basal ganglia in individuals with Down syndrome (DS) and to examine the relationship between basal ganglia volumes, neuropsychological test performance, and dementia status in this population. Subjects were 32 adults with DS. Basal ganglia volumes from 22 of these subjects were compared with those of 22 cognitively-normal individuals, who were individually matched on age, sex, and race. Performance on neuropsychological tests was correlated with basal ganglia volumes for 32 individuals with DS, and basal ganglia volumes of five demented DS subjects were compared with those of 14 non-demented DS subjects. Results indicated larger putamen volumes in the DS subjects, despite significantly smaller total brain volumes. Volumes of caudate and globus pallidus did not differ between DS and control subjects. Although there were some significant correlations between basal ganglia volumes and age, neuropsychological test performance, and dementia status in the DS subjects, these associations appeared to be a reflection of neurodevelopmental or atrophic reductions in overall brain volume rather than a reflection of specific basal ganglia abnormality. Correlations between age and volumes of basal ganglia and total brain were not significantly greater in non-demented DS subjects than in control subjects. Results suggest that volume reductions of the basal ganglia are not a salient feature of aging or of the dementia associated with DS.

Adult↗

PubMed1997

Binary transgenic mouse model for studying the trans control of globin gene switching: evidence that GATA-1 is an in vivo repressor of human epsilon gene expression.

To test whether human GATA-1 (hGATA-1) is involved in the transcriptional control of globin gene switching, we produced transgenic mice overexpressing hGATA-1, crossed them with mice carrying a human beta-globin locus yeast artificial chromosome (beta YAC), and analyzed globin gene expression in their progeny. Mice carrying both the hGATA-1 and the beta YAC transgenes had normal levels of gamma- and beta-globin mRNA and no distortion in the rate or in the timing of gamma-to-beta switch, indicating that hGATA-1 is not involved in the developmental control of gamma- and beta-globin genes. In contrast, mice carrying the hGATA-1 and the beta YAC transgenes had 5- to 6-fold lower expression of the human epsilon globin gene compared with beta YAC mice lacking the hGATA-1 transgene. These results provide direct in vivo evidence that hGATA-1 is a specific repressor of human epsilon gene expression. Our findings also suggest that binary transgenic mouse systems based on overexpression of transcriptional factors can be used to investigate the trans control of human globin gene switching. Systems as the one we describe here should be useful in the study of any developmentally controlled human gene for which transgenic mice are available.

Animals↗

PubMed1997

The thermoresponsiveness of ventromedial hypothalamic (VMH) neurons following repeated scrotal thermal stimulation of rats maintained at normothermic or acutely hypothermic core temperatures.

Neurons within the central nervous system, including those within the ventromedial hypothalamic (VMH) nucleus, alter their neuronal activity in response to scrotal thermal stimulation. This study set out to establish if the thermoresponsiveness of VMH neurons becomes modified to repeated trials of scrotal thermal stimulation. VMH extracellular activity was recorded with a glass micropipette filled with 0.5 M sodium acetate in urethane-anaesthetized male Sprague-Dawley rats following 20 min scrotal heating (scrotal packs, 2 x 2 cm, filled with 40 degrees C hot water) and scrotal cooling (scrotal packs filled with ice). In study A, VMH neurons were tested to 2 trials of scrotal heating and cooling with colonic temperatures (T(c)s) servo-controlled at 37 degrees C during both trials. In study B, VMH neurons were tested for 3 trials of scrotal thermal stimulation, with T(c)s servo-controlled at 37, 35 and 33 degrees C during the 3 trials. In study A, 42 VMH neurons were isolated. Based on their thermal coefficients (TCs) to the 1st trial of scrotal heating and cooling, 12 VMH neurons were classified as warm-responsive neurons (WRNs), 10 as cold-responsive neurons (CRNs) and 20 were temperature-non-responsive neurons (TNRNs). Of the neurons recorded long enough to test their thermoresponsiveness to 2nd trial of scrotal thermal stimulation (9 out of 12 WRNs, 7 out of 10 CRNs and 18 out of 20 TNRNs), the mean TCs of each type of VMH neuron did not significantly change between the 2 trials. In study B, 65 VMH neurons were isolated and 11 out of 22 WRNs, 7 out of 13 CRNs and 15 out of 30 TNRNs had their thermoresponsiveness tested to 3 trials of scrotal heating and cooling, with T(c)s kept at 37, 35 and 33 degrees C, respectively, for these trials. The mean TCs for VMH WRNs, CRNs and TNRNs again did not change between the 3 trials of scrotal thermal stimulation. However, mean basal firing rates did increase significantly of all recorded VMH neurons between the 1st and 3rd trials of scrotal heating and cooling as T(c)s were acutely lowered from 37 to 33 degrees C. Results demonstrated that VMH thermoresponsive neurons retain their responsiveness to repeated trials of scrotal heating and cooling of animals maintained at normothermic (37 degrees C) or acutely hypothermic (35 and 33 degrees C) temperatures.

Anesthesia, General↗

PubMed1997

Expression and functional domains of rabbit liver UDP-glucuronosyltransferase 2B16 and 2B13.

Southern blot analysis has demonstrated that the 5' portion of the rabbit liver dexamethasone-inducible UDP-glucuronosyltransferase (UGT) 2B13 RNA is related in sequence to a family of UGT genes (Tukey, R. H., Pendurthi, U. R., Nguyen, N. T., Green, M. D., and Tephly, T. R. (1993) J. Biol. Chem. 268, 15260-15266). To identify these additional gene transcripts, rabbit liver cDNA libraries were screened with a 5' conserved 330-base pair UGT2B13 cDNA fragment, resulting in the isolation and characterization of several rabbit liver UGT cDNAs. One such clone, called pGT11, encodes a putative glycoprotein that is 78% similar to rabbit UGT2B13. The new UGT has been designated UGT2B16. The UGT2B16 gene is expressed as a single 4200-base RNA transcript that is regulated only in adult rabbits. The predicted NH2-terminal 25 amino acids of UGT2B16 are identical to that of rabbit liver UGT2B13, with the remainder of the protein being 77% similar to UGT2B13. Expressed UGT2B16 protein in COS-1 cells was active toward 4-hydroxybiphenyl, similar to that of UGT2B13. However, UGT2B16 efficiently conjugated 4-hydroxyestrone and 4-tert-butylphenol, substrates that are not efficiently catalyzed by UGT2B13. To further characterize the structural domains of UGT2B16 and UGT2B13, a series of chimeric cDNAs were constructed that contained portions of both UGT2B16 and UGT2B13. Chimeric 2B163002B13531, which contained the amino-terminal UGT2B16 amino acids 1-300 followed by amino acids 301-531 of UGT2B13, as well as chimeric 2B163582B13531 and 2B164342B13531 proteins, catalyzed the glucuronidation of 4-hydroxyestrone, indicating that the carboxyl terminus of UGT2B13 could substitute for those same regions on UGT2B16. However, the replacement of the carboxyl end of UGT2B13 with 2B16300-531 or 2B16434-531 dramatically impaired the catalytic function of the chimeric proteins. These results indicate that the carboxyl end of UGT2B13 plays an important role in the functional and possible conformational state of the protein.

Amino Acid Sequence↗

PubMed1997

Selective inhibition of cytosolic phospholipase A2 in activated human monocytes. Regulation of superoxide anion production and low density lipoprotein oxidation.

Our previous studies have shown that monocyte activation and release of O-2 are required for monocyte-mediated low density lipoprotein (LDL) lipid oxidation. We have also found that intracellular Ca2+ levels and protein kinase C activity are requisite participants in this potentially pathogenic process. In these studies, we further investigated the mechanisms involved in the oxidation of LDL lipids by activated human monocytes, particularly the potential contributions of the cytosolic phospholipase A2 (cPLA2) signaling pathway. The most well-studied cPLA2, has a molecular mass of 85 kDa and has been reported to be regulated by both Ca2+ and phosphorylation. We found that cPLA2 protein levels and cPLA2 enzymatic activity were induced upon activation of human monocytes by opsonized zymosan. Pharmacologic inhibition of cPLA2 activity by AACOCF3, which has been reported to be a specific inhibitor of cPLA2 as compared with sPLA2, caused a dose-dependent inhibition of cPLA2 enzymatic activity and LDL lipid oxidation by activated human monocytes, whereas sPLA2 activity was not affected. To corroborate these findings, we used specific antisense oligonucleotides to inhibit cPLA2. We observed that treatment with antisense oligonucleotides caused suppression of both cPLA2 protein expression and enzymatic activity as well as monocyte-mediated LDL lipid oxidation. Furthermore, antisense oligonucleotide treatment caused a substantial inhibition of O-2 production by activated human monocytes. In parallel experimental groups, cPLA2 sense oligonucleotides did not affect cPLA2 protein expression, cPLA2 enzymatic activity, O-2 production, or monocyte-mediated LDL lipid oxidation. These studies support the proposal that cPLA2 activity is required for activated monocytes to oxidize LDL lipids.

Cytosol↗