Biomedical subjects

L Wang

Publications and source records attributed to L Wang.

At least 1,189 records · Page 66Linked to original sources

PubMed1998

(+)-4-[2-[4-(8-Chloro-3,10-dibromo-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1,2-b]- pyridin-11(R)-yl)-1-piperidinyl]-2-oxo-ethyl]-1-piperidinecarboxamid e (SCH-66336): a very potent farnesyl protein transferase inhibitor as a novel antitumor agent.

We have previously shown that appropriate modification of the benzocycloheptapyridine tricyclic ring system can provide potent farnesyl protein transferase (FPT) inhibitors with good cellular activity. Our laboratories have also established that incorporation of either pyridinylacetyl N-oxide or 4-N-carboxamidopiperidinylacetyl moieties results in pharmacokinetically stable inhibitors that are orally efficacious in nude mice. We now demonstrate that further elaboration of the tricyclic ring system by introducing a bromine atom at the 7- or the 10-position of the 3-bromo-8-chlorotricyclic ring system provides compounds that have superior potency and selectivity in FPT inhibition. These compounds have good serum levels and half-lives when given orally to rodents and primates. In vitro and in vivo evaluation of a panel of these inhibitors has led to identification of 15 (SCH 66336) as a highly potent (IC50 = 1.9 nM) antitumor agent that is currently undergoing human clinical trials.

Alkyl and Aryl Transferases↗

PubMed1998

Identification and chromosomal localization of CTNNAL1, a novel protein homologous to alpha-catenin.

Sodium butyrate (NaB) was shown to induce growth inhibition and apoptosis in a human pancreatic cancer cell line (AsPC-1). To identify the genes that are differentially regulated by NaB, we generated subtracted EST libraries highly enriched for up- or down-regulated transcripts using the suppression subtraction hybridization technique. One of the ESTs identified in the down-regulated library showed significant homology to human alpha-catenin. A cDNA of 2.45 kb that codes for a putative protein of 734 aa was cloned. The cloned cDNA was used as a template in an in vitro transcription-translation reaction. This produced a protein product of about 82 kDa in size. The gene designated as CTNNAL1 (catenin (cadherin-associated protein), alpha-like 1) was found to be ubiquitously expressed in many tissues including pancreas, heart, and skeletal muscle. A human BAC clone containing the gene was isolated and used as a probe for fluorescence in situ hybridization (FISH). Both radiation hybrid and FISH analysis mapped the gene to chromosome band 9q31.2, a region where frequent abnormalities have been observed in bladder carcinoma, esophageal cancers, and several other tumors.

Amino Acid Sequence↗

PubMed1998

Abbreviated regimens of zidovudine prophylaxis and perinatal transmission of the human immunodeficiency virus.

BACKGROUND: The Pediatric AIDS Clinical Trials Group Protocol 076 reported a reduction in the rate of perinatal transmission of the human immunodeficiency virus (HIV) from 25.5 percent to 8.3 percent with a three-part regimen of zidovudine given ante partum, intra partum, and to the newborn. We examined the effects of abbreviated zidovudine regimens on perinatal HIV transmission using data from the HIV polymerase-chain-reaction (PCR) testing service of the New York State Department of Health. Pregnant women who received abbreviated regimens rather than the recommended regimens did so because of limited prenatal care or by choice. METHODS: The requisition form used by the PCR testing service included information on the demographic characteristics of the infants and the timing of any perinatal treatment with zidovudine. We also analyzed data on the timing of zidovudine prophylaxis collected by chart review in a subgroup of 454 infants as a means of validating the results in the entire cohort. RESULTS: From August 1, 1995, through January 31, 1997, specimens from 939 HIV-exposed infants who were 180 days of age or younger were submitted for PCR testing. The rates of perinatal HIV transmission varied depending on when zidovudine prophylaxis was begun. When treatment was begun in the prenatal period, the rate of HIV transmission was 6.1 percent (95 percent confidence interval, 4.1 to 8.9 percent); when begun intra partum, the rate was 10.0 percent (3.3 to 21.8 percent); when begun within the first 48 hours of life, the rate was 9.3 percent (4.1 to 17.5 percent); and when begun on day 3 of life or later, the rate was 18.4 percent (7.7 to 34.3 percent). In the absence of zidovudine prophylaxis, the rate of HIV transmission was 26.6 percent (21.1 to 32.7 percent). CONCLUSIONS: These results confirm the efficacy of zidovudine prophylaxis and suggest that there are reductions in the rates of perinatal transmission of HIV even with the use of abbreviated regimens that are begun intra partum or in the first 48 hours of life.

Anti-HIV Agents↗

PubMed1998

High-resolution analysis of cytosine methylation in the 5ĺong terminal repeat of retroviral vectors.

Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.

Animals↗

PubMed1998

Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

3T3 Cells↗

PubMed1998

[PCR in combination with silver as a method for the determination of apolipoprotein E alleles].

OBJECTIVE: To present a rapid, sensitive and safe procedure for the determination of human apolipoprotein E alleles. METHODS: Genomic DNA was extracted from 0.5ml whole blood by a non-phenol protocol. After PCR and restriction enzyme digestion,short DNA fragments were detected by a simplified silver staining method. This technique was used to assay apolipoprotein E alleles in 19 patients with sporadic Alzheimer's disease and 41 normal aged persons. RESULTS: The short DNA fragments were visualized clearly on polyacrylamide gel processed by silver staining. It took 24 hours from DNA extraction to the final result. The A4 allele frequency in patients with sporadic Alzheimer's disease was 0.29, much higher than that in control group (0.02, P<0.001). CONCLUSION: PCR in combination with silver staining can satisfy the need for high-resolution and high-efficiency in the determination of apolipoprotein E alleles and can be used as a routine procedure in clinical and epidemiological investigations.

Alleles↗

PubMed1998

[Studies on molecular teratogenic mechanism of methylmercury in early developing rat embryos].

The present study further revealed the teratogenic mechanism of methylmercury chloride (MMC) during rat neurulation by means of nonradioactive in situ hybridization (ISH), semiquantitative analysis of immunohistochemical staining and in vivo teratogenic test. The main aim was to teat the hypothesis that iNOS, HSP70, NT, TGF-beta and Bcl-2 genes contribute to MMC-induced day 9.5 embryonic damages. Results showed there were no obvious poisoning signs and death of pregnant female rats injected intraperitoncally with 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/kg MMC. While the doses increased, the total morphological scores decreased gradually, and the rates of embryo deformity and development delay increased step by step to 34% and 76% respectively. The levels of iNOS mRNA and protein and HSP70 mRNA increased, and NT mRNA with its protein became down, all these changes were concentration dependent. In addition, MMC could inhibit the level of TGF-beta mRNA, but no obvious influence on the levels of Bcl-2 mRNA and Bcl-2 protein. On the basis of parallel findings from embryos, genes and proteins, abnormal expression of genes in transcriptional level might be related to MMC-induced teratogenic insult.

Animals↗

PubMed1998

Novel configurations of the hammerhead ribozyme: increased activity and reduced magnesium ion requirement.

The hammerhead ribozyme is one of the smallest of known catalytic RNAs. Its structure and mechanism have been examined using a broad range of approaches. Recently, crystal structures of the hammerhead have been reported. Within the crystal the hammerhead exists in a Y-shaped configuration in which helices I and II form the adjacent upper arms, while helix III forms the lower leg of the Y. Based on these findings, hammerheads in which helix I and II are constrained to remain adjacent and roughly parallel are expected to be catalytically active. We have examined this possibility with two novel hammerhead configurations, circular and circular/hairpin. These circular and circular/hairpin hammerhead ribozymes possess activity comparable to, and in some cases even greater than, non-constrained hammerheads. Since these novel ribozymes are constrained into a closed, and active conformation, they offer advantages for structure/function studies. Additionally, these novel ribozymes will be adventitious for antisense mediated gene inhibition, since they possess increased activity and a reduced requirement for magnesium ion, and are expected to be more resistant to nuclease degradation.

Circular Dichroism↗

PubMed1998

Reconstitution of insulin signaling pathways in rat 3Y1 cells lacking insulin receptor and insulin receptor substrate-1. Evidence that activation of Akt is insufficient for insulin-stimulated glycogen synthesis or glucose uptake in rat 3Y1 cells.

Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1. To investigate the role of IR and IRS-1 in effects of insulin, we transfected IR and IRS-1 expression plasmids into cells and reconstituted the insulin signaling pathways. 3Y1 cells stably expressing the c-myc epitope-tagged glucose transporter type 4 (3Y1-GLUT4myc) exhibit no effects of insulin, at physiological concentrations. The 3Y1-GLUT4myc-IR cells expressing GLUT4myc and IR responded to phosphatidylinositol 3,4, 5-trisphosphate (PI-3,4,5-P3) accumulation, Akt activation, the stimulation of DNA synthesis, and membrane ruffling but not to glycogen synthesis, glucose uptake, or GLUT4myc translocation. The further expression of IRS-1 in 3Y1-GLUT4myc-IR cells led to stimulation of glycogen synthesis but not to glucose uptake or GLUT4myc translocation in response to insulin, although NaF or phorbol 12-myristate 13-acetate did trigger GLUT4myc translocation in the cells. These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for GLUT4 translocation.

Amino Acid Sequence↗

PubMed1998

NMR solution structure of type II human cellular retinoic acid binding protein: implications for ligand binding.

The structure of human apo-cellular retinoic acid binding protein II (apo-CRABPII) in solution at pH 7.3 has been determined by NMR spectroscopy. The sequential assignments of the 1H, 13C, and 15N resonances of apo-CRABPII were established by multinuclear, multidimensional NMR spectroscopy. The solution structure of apo-CRABPII was derived from 2382 experimental NMR restraints using a hybrid distance geometry-simulated annealing protocol. The root-mean-square deviation of the ensemble of 25 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 +/- 0.18 and 0.92 +/- 0.20 A for the backbone atoms and all heavy atoms, respectively, of all residues except Ala32-Pro39 and Thr57-Glu62, which are in disordered regions. The solution structure of apo-CRABPII is similar to the crystal structure of holo-CRABPII [Kleywegt, G. J., Bergfors, T., Senn, H., Le Motte, P., Gsell, B., Shudo, K., and Jones, T. A. (1994) Structure 2, 1241-1258] except the ligand entrance, which is sufficiently enlarged in the apoprotein to be readily accessible to retinoic acid. The enlargement of the ligand entrance of apo-CRABPII relative to that of holo-CRABPII is due mainly to a concerted conformational change in three structural elements, namely, the second helix, the betaC-betaD loop, and the betaE-betaF loop. Furthermore, the ligand-binding pocket of apo-CRABPII showed evidence of dynamic disorder; among the 21 residues that constitute this pocket, 16 residues had weak or no detectable cross-peaks in the two-dimensional 1H-15N HSQC spectrum recorded under conditions of minimal water saturation or dephasing. Apo-CRABPII is largely monomeric in solution, with no evidence for the dimeric structure shown in the crystal structure of apo-CRABPI which was suggested to be a prerequisite for ligand entry [Thompson, J. R., Bratt, J. M., and Banaszak, L. J. (1995) J. Mol. Biol. 252, 433-446]. Thus, the widening of the ligand entrance required for entry of retinoic acid appears to be a property of monomeric apo-CRABPII.

Crystallization↗

PubMed1998

NMR study suggests a major role for Arg111 in maintaining the structure and dynamical properties of type II human cellular retinoic acid binding protein.

The solution structure of a site-directed mutant of type-II human cellular retinoic acid binding protein (CRABPII) with Arg111 replaced by methionine (R111M) has been determined by NMR spectroscopy. The sequential assignments of the 1H and 15N resonances of apo-R111M were established by multinuclear multidimensional NMR. The solution structure was calculated from 2302 distance restraints and 77 phi dihedral restraints derived from the NMR data. The root-mean-square deviation of the ensemble of 28 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 +/- 0.26 and 0.98 +/- 0.23 A for the backbone atoms and all heavy atoms, respectively. The solution structure of apo-R111M is similar to that of wild-type apo-CRABPII. However, there are significant conformational differences between the two proteins, localized mainly to three segments (Leu19-Ala36, Glu73-Cys81, and Leu99-Pro105) clustered around the ligand entrance more than 17 A away from the point mutation. In apo-R111M, all the three segments move toward the center of the ligand entrance so that the opening of the ligand-binding pocket in apo-R111M is much smaller than that in wild-type apo-CRABPII. Furthermore, the ligand-binding pocket of apo-R111M, especially the ligand entrance, is much less flexible than that of apo-CRABPII. Surprisingly, apo-R111M is more similar to holo-CRABPII than to apo-CRABPII in both structure and dynamical properties. The conformational and dynamical changes caused by the mutation are similar to those induced by binding of RA, although the magnitudes of the changes caused by the mutation are smaller than those induced by binding of RA. The results suggest that Arg111 plays a critical role in determining the structure and dynamical properties of CRABPII.

Amino Acid Sequence↗

PubMed1998

Hippocampal morphometry in schizophrenia by high dimensional brain mapping.

Theories of the pathophysiology of schizophrenia have implicated the hippocampus, but controversy remains regarding hippocampal abnormalities in patients with schizophrenia. In vivo studies of hippocampal anatomy using high resolution magnetic resonance scanning and manual methods for volumetric measurement have yielded inconclusive results, perhaps because of the normal variability in hippocampal volume and the error involved in manual measurement techniques. To resolve this controversy, high dimensional transformations of a computerized brain template were used to compare hippocampal volumes and shape characteristics in 15 matched pairs of schizophrenia and control subjects. The transformations were derived from principles of general pattern matching and were constrained according to the physical properties of fluids. The analysis and comparison of hippocampal shapes based on these transformations were far superior to the comparison of hippocampal volumes or other global indices of hippocampal anatomy in showing a statistically significant difference between the two groups. In the schizophrenia subjects, hippocampal shape deformations were found to be localized to subregions of the structure that send projections to prefrontal cortex. The results of this study demonstrate that abnormalities of hippocampal anatomy occur in schizophrenia and support current hypotheses that schizophrenia involves a disturbance of hippocampal-prefrontal connections. These results also show that comparisons of neuroanatomical shapes can be more informative than volume comparisons for identifying individuals with neuropsychiatric diseases, such as schizophrenia.

Brain Mapping↗

PubMed1998

Differential in vivo recovery of sinusoidal endothelial cells, hepatocytes, and Kupffer cells after cold preservation and liver transplantation in rats.

BACKGROUND: The injury resulting from cold preservation/reperfusion primarily affects sinusoidal endothelial cells, while hepatocytes are thought to be less vulnerable; morphological changes and increased cytokine release suggest that Kupffer cells are activated. We evaluated the extent of functional damage to the different cell types in the liver after cold preservation and transplantation. Additionally, we analyzed in vivo the patterns of functional recovery of all three cell types over the first week after transplantation in Lewis rats. METHODS: We evaluated the in vivo uptake of hyaluronic acid, indocyanine green, and radio-labeled sulphur colloid to assess the function of sinusoidal endothelial cells, hepatocytes, and Kupffer cells, respectively. Measurements were performed immediately after transplantation using syngeneic grafts preserved in University of Wisconsin solution for different periods. Functional recovery was monitored in animals receiving grafts preserved for 24 hr over the first postoperative week. RESULTS: We found that hepatocyte were less affected compared with the profoundly damaged endothelial cells. The phagocytic ability of Kupffer cells was, however, also seriously compromised, which suggests a selective down-regulation. Functional recovery occurs in a differential manner during the first postoperative week starting with hepatocytes followed by sinusoidal endothelial cells. Phagocytic function further deteriorates after transplantation before showing improvement. CONCLUSIONS: In viable liver grafts, all cell types recover from preservation/reperfusion injury by the end of the first week after transplantation. The differential time courses of the recovery suggest that successful sinusoidal endothelial cell recovery may depend upon prior hepatocyte regeneration and may involve a paracrine interaction, via cytokines and growth factors.

Alanine Transaminase↗

PubMed1998

Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I.

Apoptosis is regulated by specific intracellular signaling pathways. The development of cardiomyopathy involves the apoptosis of cardiomyocytes; however, the details of their apoptotic signaling are not yet known. Insulin-like growth factor I (IGF I) is an important survival growth factor for myocardium and other tissues, but the effects of IGF I on apoptotic signaling remain largely unknown. To study apoptotic signaling pathways in cardiomyocytes and to understand IGF I actions on the apoptotic signaling of cardiac muscle cells, we have defined the effects of IGF I on Bcl-2, Bax, caspase 3, DNA fragmentation, and cell survival in primary cardiomyocytes. Compared with Bax levels, the levels of Bcl-2 were found to be quite low in these cells. Serum withdrawal and doxorubicin reduced cell viability, increased fragmentation of DNA, increased cellular contents of Bax, and activated caspase 3. IGF I enhanced cell viability, suppressed DNA fragmentation, attenuated Bax induction, and suppressed caspase 3 activation. The levels of Bcl-2-associated Bax were increased after serum withdrawal and incubation with doxorubicin and were reduced by IGF I. Thus, cardiomyocyte apoptosis induced by serum withdrawal and doxorubicin likely results, in part, from the induction of Bax and activation of caspase 3, but IGF I may inhibit cardiomyocyte apoptosis by attenuating Bax induction and caspase 3 activation. These findings provide new insight into the mechanisms of cardiomyocytes apoptosis and may help elucidate how IGF I modulates apoptotic signaling in cardiac muscle.

Animals↗

PubMed1998

Can one predict protein stability? An attempt to do so for residue 133 of T4 lysozyme using a combination of free energy derivatives, PROFEC, and free energy perturbation methods.

Free energy derivatives, pictorial representation of free energy changes (PROFEC) and free energy perturbation methods were employed to suggest the modifications that may improve the stability of a mutant T4 lysozyme with a S-2-amino-3-cyclopentylpropanoic acid residue (Cpe) at position 133. The free energy derivatives and PROFEC methods were used to locate promising sites where modifications may be introduced. The effects of several candidate modifications on the enzyme's stability were analyzed by the free energy perturbation method. We found that this scheme is able to effectively suggest modifications that may increase the enzyme's stability. The modifications investigated are the introduction of a methyl, a tert-butyl or a trifluoromethyl group at the Cepsilon2 position and a cyclopropyl group between the Cdelta2 and Cepsilon2 position on the cyclopentyl ring. The stereochemistry of the introduced groups (in the alpha or beta configurations) was studied. Our calculations predict that the introduction of a methyl group in the alpha configuration or a cyclopropyl group in the beta configuration will increase the stability of the enzyme; the introduction of the two groups in the other configurations and the other modifications will decrease the stability of the enzyme. The results indicate that packing interactions can strongly influence the stability of the enzyme.

Bacteriophage T4↗

PubMed1998

Functional reassembly of the anion transport domain of human red cell band 3 (AE1) from multiple and non-complementary fragments.

We constructed cDNA clones encoding N-terminal, C-terminal and internal polypeptide fragments of the human red cell anion exchanger (band 3; AE1). The internal fragments comprised between one and seven putative transmembrane spans with two or more spans deleted from both termini of the membrane domain of band 3. Sets of three, four or five complementary fragments, which together represented the complete amino acid sequence of the membrane domain, were co-expressed in Xenopus oocytes. Stilbene disulphonate-sensitive chloride uptake assays revealed that all six of the three-fragment combinations and two of the four-fragment combinations reassembled functionally in vivo. Unexpectedly, co-expression of a non-complementary pair of fragments comprising the first five and last seven putative transmembrane spans (i.e. entirely lacking spans six and seven) was also found to be sufficient to generate stilbene disulphonate-sensitive chloride uptake.

Animals↗

PubMed1998

The early stage of folding of villin headpiece subdomain observed in a 200-nanosecond fully solvated molecular dynamics simulation.

A new approach in implementing classical molecular dynamics simulation for parallel computers has enabled a simulation to be carried out on a protein with explicit representation of water an order of magnitude longer than previously reported and will soon enable such simulations to be carried into the microsecond time range. We have used this approach to study the folding of the villin headpiece subdomain, a 36-residue small protein consisting of three helices, from an unfolded structure to a molten globule state, which has a number of features of the native structure. The time development of the solvation free energy, the radius of gyration, and the mainchain rms difference from the native NMR structure showed that the process can be seen as a 60-nsec "burst" phase followed by a slow "conformational readjustment" phase. We found that the burial of the hydrophobic surface dominated the early phase of the folding process and appeared to be the primary driving force of the reduction in the radius of gyration in that phase.

Biophysical Phenomena↗

PubMed1998

Measurement of thrombomodulin mRNA expression in brain capillaries by polymerase chain reaction.

Thrombomodulin (TM), an endothelial integral membrane protein, is a potent activator of the protein C anticoagulant pathway. TM protein expression is limited and regionally distributed in the brain. Recent investigations have demonstrated low TM mRNA expression by brain endothelium, corresponding to its distribution at the protein level. To facilitate the study of TM expression at the transcriptional level, we measured TM mRNA by quantitative-competitive polymerase chain reaction (QC-PCR) and by standard densitometric analysis of reverse transcriptase-PCR products (RT-PCR) in different regions of bovine brain. QC-PCR demonstrated differential TM mRNA expression in the pons (100+/-9%), cerebellum (359+/-103%), and cortex (441+/-24%). We compared these results with those of RT-PCR and found similar differences in relative TM mRNA expression in the pons (100+/-44%), cerebellum (343+/-8%), and cortex (404+/-62%). Data derived by QC-PCR and RT-PCR were highly correlated (r=0.99, p<0.03). These findings indicate that either QC-PCR or RT-PCR can be used to accurately quantify TM mRNA.

Animals↗