Search PubMed⌕ Search

Biomedical subjects

Q Li

Publications and source records attributed to Q Li.

At least 361 records · Page 20Linked to original sources
PubMed1999

Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.

CpG island methylation has been linked to tumor suppressor gene inactivation in neoplasia and may serve as a useful marker to clone novel cancer-related genes. We have developed a novel PCR-based method, methylated CpG island amplification (MCA), which is useful for both methylation analysis and cloning differentially methylated genes. Using restriction enzymes that have differential sensitivity to 5-methyl-cytosine, followed by adaptor ligation and PCR amplification, methylated CpG rich sequences can be preferentially amplified. In a model experiment using a probe from exon 1 of the p16 gene, signal was detected from MCA products of a colorectal cancer cell line but not in normal colon mucosa. To identify novel CpG islands differentially methylated in colorectal cancer, we have applied MCA coupled with representational difference analysis to the colon cancer cell line Caco2 as a tester and normal colon mucosa as a driver. Using this strategy, we isolated 33 differentially methylated DNA sequences, including fragments identical to several known genes (PAX6, Versican, alpha-tubulin, CSX, OPT, and rRNA gene). The association of hypermethylation of the clones obtained and transcriptional suppression in colorectal cancer was confirmed by examining the Versican gene, which we found to be silenced in methylated cell lines and reactivated by the methylation inhibitor 5-aza-2'-deoxycytidine. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.

5-Methylcytosine↗
PubMed1999

Analysis of gamma-globin expression cassettes in retrovirus vectors.

With the goal of optimizing retrovirus vectors for human gamma-globin, we studied the effect of several globin gene expression elements on vector titer, stability, and expression. We found that all combinations tested were genetically stable, but that vectors with therapeutic titers (0.5 to 2 x 10(6) colony-forming units/ml) could be achieved only by either partially or fully deleting the second intron of the Agamma-globin gene. Efficient transfer and high-level expression was achieved only when an optimized beta-globin promoter was linked to an Agamma-globin cassette containing an intact intron 1 and a 714-bp internal deletion of intron 2. When flanked by two copies of the HS-40 enhancer core from the alpha-globin locus, this cassette expressed gamma-globin mRNA at 46 +/- 19% per copy of mouse alpha-globin in the murine erythroleukemia cell line MEL585. Complete deletion of the first or second intron diminished expression to < or = 2.0%, and deletion of the HS-40 enhancer diminished expression to 7 +/- 8%. High-level, uniform expression of gamma-globin protein was confirmed in MEL585 clones (n = 12) transduced with the optimized vector. Efficient but variable expression of the optimized vector was also observed in erythroid progenitor colonies (n = 6) grown from transduced mouse bone marrow. Taken together, these studies demonstrate the role of intronic, promoter, and enhancer sequences on retrovirus vectors for human gamma-globin, and the development of an optimized vector capable of efficient expression in a murine erythroid cell line and primary cultures.

3T3 Cells↗
PubMed1999

[A comparative study of HLA-A locus in northern and southern Chinese by means of PCR/SSOP typing]

OBJECTIVE: To compare the HLA-A locus in a population selected from Beijing (northern group) with that in a population selected from Guangzhou (southern group). METHODS: HLA typing was performed by using PCR/SSOP method. A pair of primer and 54 probes were used. RESULTS: In 18 alleles identified, the differences in gene frequency between northern and southern Chinese were found. In northern group, A*0205, 0210 and 2901 were absent, and the frequencies of A*2601, 3001 and 3101 were higher than those of southern group; while in southern group, A*3103, 3201 and 6801 were absent, the frequencies of A*0203 and 1101 were higher than those of northern group; and in addition, six subtypes of A2 serological specificity, namely A*0201, 0203, 0205, 0206, 0207 and 0210, were found, in which A*0201 predominated. CONCLUSION: There are some differences in the genetic background of northern and southern Chinese. A2 subtypes have important implications for unrelated-donor transplantation.

Journal Article↗
PubMed1999

Severe liver degeneration in mice lacking the IkappaB kinase 2 gene.

Phosphorylation of inhibitor of kappa B (IkappaB) proteins is an important step in the activation of the transcription nuclear factor kappa B (NF-kappaB) and requires two IkappaB kinases, IKK1 (IKKalpha) and IKK2 (IKKbeta). Mice that are devoid of the IKK2 gene had extensive liver damage from apoptosis and died as embryos, but these mice could be rescued by the inactivation of the gene encoding tumor necrosis factor receptor 1. Mouse embryonic fibroblast cells that were isolated from IKK2-/- embryos showed a marked reduction in tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1alpha-induced NF-kappaB activity and an enhanced apoptosis in response to TNF-alpha. IKK1 associated with NF-kappaB essential modulator (IKKgamma/IKKAP1), another component of the IKK complex. These results show that IKK2 is essential for mouse development and cannot be substituted with IKK1.

Animals↗
PubMed1999

Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette.

Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.

Anemia, Sickle Cell↗
PubMed1999

Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus.

Human UDP-glucuronosyltransferases (UGTs) are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues [Strassburg, Manns and Tukey (1998) J. Biol. Chem. 273, 8719-8726]. Previous work suggests that these enzymes play a protective role in chemical carcinogenesis [Strassburg, Manns and Tukey (1997) Cancer Res. 57, 2979-2985]. In this study, UGT1 and UGT2 gene expression was investigated in human oesophageal epithelium and squamous-cell carcinoma in addition to the characterization of individual UGT isoforms using recombinant protein. UGT mRNA expression was characterized by duplex reverse transcriptase-PCR analysis and revealed the expression of UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mRNAs. UGT1A1, UGT1A3, UGT1A4, UGT1A5 and UGT1A6 transcripts were not detected. UGT2 expression included UGT2B7, UGT2B10 and UGT2B15, but UGT2B4 mRNA was absent. UGT2 mRNA was present at significantly lower levels than UGT1 transcripts. This observation was in agreement with the analysis of catalytic activities in oesophageal microsomal protein, which was characterized by high glucuronidation rates for phenolic xenobiotics, all of which are classical UGT1 substrates. Whereas UGT1A9 was not regulated, differential regulation of UGT1A7 and UGT1A10 mRNA was observed between normal oesophageal epithelium and squamous-cell carcinoma. Expression and analysis in vitro of recombinant UGT1A7, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 demonstrated that UGT1A7, UGT1A9 and UGT1A10 catalysed the glucuronidation of 7-hydroxybenzo(alpha)pyrene, as well as other environmental carcinogens, such as 2-hydroxyamino-1-methyl-6-phenylimidazo-(4, 5-beta)-pyridine. Although UGT1A9 was not regulated in the carcinoma tissue, the five-fold reduction in 7-hydroxybenzo(alpha)pyrene glucuronidation could be attributed to regulation of UGT1A7 and UGT1A10. These data elucidate an individual regulation of human UGT1A and UGT2B genes in human oesophagus and provide evidence for specific catalytic activities of individual human UGT isoforms towards environmental carcinogens that have been implicated in cellular carcinogenesis.

Aged↗
PubMed1999

Generation of the O630 photointermediate of bacteriorhodopsin is controlled by the state of protonation of several protein residues.

The last stages of the photocycle of the photosynthetic pigment all-trans bacteriorhodopsin (bR570), as well as its proton pump mechanism, are markedly pH dependent. We have measured the relative amount of the accumulated O630 intermediate (Phir), as well as its rise and decay rate constants (kr and kd, respectively), over a wide pH range. The experiments were carried out in deionized membrane suspensions to which varying concentrations of metal cations and of large organic cations were added. The observed pH dependencies, s-shaped curves in the case of Phir and bell-shaped curves for kr and kd, are interpreted in terms of the titration of three protein residues denoted as R1, R2, and R3. The R1 titration is responsible for the increase in Phir, kr, and kd upon lowering the pH from pH approximately 9.5 to 7. At low pH Phir exhibits a secondary rise which is attributed to the titration of a low pKa group, R2. After reaching a maximum at pH approximately 7, kr and kd undergo a decrease upon decreasing the pH, which is attributed to the titration of R3. All three titrations exhibit pKa values which decrease upon increasing the salt concentration. As in the case of the Purple (bR570) if Blue (bR605) equilibrium, divalent cations are substantially more effective than monovalent cations in shifting the pKa values. Moreover, bulky organic cations are as effective as small metal cations. It is concluded that analogously to the Purple if Blue equilibrium, the salt binding sites which control the pKa values of R1, R2, and R3 are located on, or close to, the membrane surface. Possible identifications of the three protein residues are considered. Experiments with the E204Q mutant show that the mutation has markedly affected the R2 (Phir) titration, suggesting that R2 should be identified with Glu-204 or with a group whose pKa is affected by Glu-204. The relation between the R1, R2 and R3 titrations and the proton pump mechanism is discussed. It is evident that the pH dependence of Phir is unrelated to the measured pKa of the group (XH) which releases the proton to the extracellular medium during the photocycle. However, since the same residue may exhibit different pKa values at different stages of the photocycle, it cannot be excluded that R2 or R3 may be identified with XH.

Amino Acids↗
PubMed1999

Modulation of excision repair cross complementation group 1 (ERCC-1) mRNA expression by pharmacological agents in human ovarian carcinoma cells.

Excision repair cross complementation group 1 (ERCC-1) is a DNA repair gene that is essential for life, and it appears to be a marker gene for nucleotide excision repair activity. Overexpression of ERCC-1 during cisplatin-based chemotherapy is associated with clinical and cellular drug resistance. We therefore began to assess the influence of various pharmacological agents on the induction of ERCC-1 mRNA in A2780/CP70 human ovarian carcinoma cells. Cisplatin exposure in culture resulted in a 4- to 6-fold induction for the steady-state level of ERCC-1 mRNA in A2780/CP70 cells. ERCC-1 mRNA induction was concentration and time dependent. Cyclosporin A and herbimycin A, which suppress c-fos and c-jun gene expressions, respectively, blocked the cisplatin-induced increase in ERCC-1 mRNA. This effect of cyclosporin A or herbimycin A on the down-regulation of ERCC-1 correlates with enhanced cytotoxicity of cisplatin in this system. The products of c-fos and c-jun are components of the transcription factor AP-1 (activator protein 1). 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known AP-1 agonist, induced ERCC-1 mRNA to the same extent as cisplatin, but did not synergize with cisplatin in this regard. The TPA effect was biphasic, with an initial increase during the first 1-6 hr, followed by decreasing mRNA levels at 24-72 hr. These data suggest that the effects of these pharmacological agents on ERCC-1 gene expression may be mediated through the modulation of AP-1 activities.

Benzoquinones↗
PubMed1999

Mitogenic activity of purified capsular polysaccharide A from Bacteroides fragilis: differential stimulatory effect on mouse and rat lymphocytes in vitro.

Bacteroides fragilis, a Gram-negative colonic bacterium, induces the formation of abscesses associated with intra-abdominal sepsis in humans. The singular ability of this organism to modulate abscess formation in experimental rodent models resides in the structurally distinct and ionically charged capsular polysaccharides A (PS A) and B (PS B). The regulation of abscess formation in animals is dependent on T lymphocytes. However, the manner in which PS A interacts with T cells remains unknown. We therefore tested the T cell stimulatory capacity of purified PS A on mouse and rat lymphocytes in cellular proliferation assays and found that the PS A molecule possesses mitogenic characteristics distinguishable from those of the polyclonal B cell activator LPS, the T cell mitogen Con A, and staphylococcal enterotoxin A superantigen. Further, PS A stimulated proliferation of normal mouse and rat lymphocytes differentially. Mouse B cells responded to PS A in a fashion that did not require exogenous APC function, while rat T lymphocyte responses to PS A required APC function derived from autologous or xenogenic feeder cells. Cellular depletion experiments showed that the CD4+ subset of rat spleen cells was the primary responder cell type to PS A in vitro. The differential stimulatory effects of PS A on mouse and rat lymphocytes may reflect its ability to stimulate different lymphocyte subsets in vivo through the activities of receptor/counter-receptor pairs present on responder lymphocytes and cognate APC.

Animals↗
PubMed1999

Protein kinase calpha regulates human monocyte O-2 production and low density lipoprotein lipid oxidation.

Our previous studies have shown that human native low density lipoprotein (LDL) can be oxidized by activated human monocytes. In this process, both activation of protein kinase C (PKC) and induction of superoxide anion (O-2) production are required. PKC is a family of isoenzymes, and the functional roles of individual PKC isoenzymes are believed to differ based on subcellular location and distinct responses to regulatory signals. We have shown that the PKC isoenzyme that is required for both monocyte O-2 production and oxidation of LDL is a member of the conventional PKC group of PKC isoenzymes (Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508-17515). The conventional PKC group includes PKCalpha, PKCbetaI, PKCbetaII, and PKCgamma. With the exception of PKCgamma, each of these isoenzymes was detected in human monocytes. In these studies, we investigated the requirement for select PKC isoenzymes in the process of monocyte-mediated LDL lipid oxidation. Our data indicate that PKC activity was rapidly induced upon monocyte activation with the majority of the activity residing in the membrane/particulate fraction. This enhanced PKC activity was sustained for up to 24 h after activation. PKCalpha, PKCbetaI, and PKCbetaII protein levels were induced upon monocyte activation, and PKCalpha and PKCbetaII substantially shifted their location from the cytosol to the particulate/membrane fraction. To distinguish between these isoenzymes for regulating monocyte O-2 production and LDL oxidation, PKCalpha or PKCbeta isoenzyme-specific antisense oligonucleotides were used to selectively suppress isoenzyme expression. We found that suppression of PKCalpha expression inhibited both monocyte-mediated O-2 production and LDL lipid oxidation by activated human monocytes. In contrast, inhibition of PKCbeta expression (including both PKCbetaI and PKCbetaII) did not affect O-2 production or LDL lipid oxidation. Further studies demonstrated that the respiratory burst oxidase responsible for O-2 production remained functionally intact in monocytes with depressed levels of PKCalpha because O-2 production could be restored by treating the monocytes with arachidonic acid. Taken together, our data reveal that PKCalpha, and not PKCbetaI or PKCbetaII, is the predominant isoenzyme required for O-2 production and maximal oxidation of LDL by activated human monocytes.

Base Sequence↗
PubMed1999

Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets.

The human serotonin transporter (5-HTT), encoded by a single gene on chromosome 17q11.2, is expressed in brain and blood cells. 5-HTT is implicated in mood and anxiety regulation, and is where antidepressant and antianxiety drugs initially act in the brain. A 5-HTT-linked promoter region (5-HTTLPR) insertion/deletion polymorphism with long (l) and short (s) forms affects transporter expression and function. The s variant reduced 5-HTT gene transcription in a reporter gene construct and human lymphoblasts, resulting in reduced transporter levels and 5-HT uptake, acting as a dominant allele. In this study, we investigated the expression and function of 5-HTT in platelets from healthy male volunteers. The l variant was associated with more rapid initial platelet 5-HT uptake (Vmax), the index of platelet 5-HTT function most clearly heritable, while the s allele was dominant. The 5-HTTLPR genotype had no effect on platelet [3H]paroxetine binding (Bmax), affinity for [3H]5-HT or [3H]paroxetine, or 5-HT content. The 5-HT uptake findings support a functional difference in the two 5-HTTLPR variants, reinforcing their attractiveness as candidate genes in neuropsychiatric research.

Adult↗
PubMed1999

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching.

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.

Animals↗
PubMed1999

Effect of optical digitizer selection on the application accuracy of a surgical localization system-a quantitative comparison between the OPTOTRAK and flashpoint tracking systems.

Application accuracy is a crucial factor for stereotactic surgical localization systems, in which space digitization camera systems are one of the most critical components. In this study we compared the effect of the OPTOTRAK 3020 space digitization system and the FlashPoint Model 3000 and 5000 3D digitizer systems on the application accuracy for interactive localization of intracranial lesions. A phantom was mounted with several implantable frameless markers which were randomly distributed on its surface. The target point was digitized and the coordinates were recorded and compared with reference points. The differences from the reference points represented the deviation from the "true point." The root mean square (RMS) was calculated to show the differences, and a paired t-test was used to analyze the results. The results with the phantom showed that, for 1-mm sections of CT scans, the RMS was 0.76 +/- 0. 54 mm for the OPTOTRAK system, 1.23 +/- 0.53 mm for the FlashPoint Model 3000 3D digitizer system, and 1.00 +/- 0.42 mm for the FlashPoint Model 5000 system. These preliminary results showed that there is no significant difference between the three tracking systems, and, from the quality point of view, they can all be used for image-guided surgery procedures.

Equipment Design↗
PubMed1999

Analysis of 9-fluorenylmethyloxycarbonyl derivatives of catecholamines by high performance liquid chromatography, liquid chromatography/mass spectrometry and tandem mass spectrometry.

9-Fluorenylmethyloxycarbonyl (FMOC) derivatives of catecholamines, including dopamine (DA; 3,4-dihydroxyphenethylamine), norepinephrine (NE; 2-amino-1-(3,4-dihydroxyphenyl)ethanol) and epinephrine (EPI; 1-phenyl-1-hydroxy-2-methylaminopropane) as well as 3, 4-dihydroxybenzylamine (DHBA) have been analyzed using high performance liquid chromatography coupled to fluorometric detection and atmospheric pressure chemical ionization mass spectrometry at low femtomole levels. Structures of the derivatives have also been characterized by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry and electrospray/tandem mass spectrometry. Protonated molecules of FMOC-DA and FMOC-DHBA as well as [M + H - H(2)O](+) ions of FMOC-NE and FMOC-EPI appear in their conventional mass spectra, and abundant fragments characteristic of catecholamines dominated the spectra. Structurally diagnostic ions using several hundred femtomoles of the FMOC derivatives of catecholamine were observed. Collision-induced dissociation (CID) of electrospray-generated protonated molecules of FMOC-DA and FMOC-DHBA as well as [M + H - H(2)O](+) ions of FMOC-NE and FMOC-EPI produced diagnostic product ion spectra. Liquid chromatography/atmospheric pressure chemical ionization mass spectrometric analysis of the FMOC derivatives of catecholamines and DHBA should prove useful in the separation and characterization of these compounds from biological materials.

Animals↗
PubMed1999

Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13.

Isolation of cell lines that display various degrees of transformed phenotypes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from human diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14-22 region, where the RB1 gene is located. By transfection with SV40 early genes and repeated passage, we succeeded in obtaining SV40-transfected mortal, immortalized, anchorage-independent, and tumorigenic RB cell lines. DNA fingerprinting showed that these cell lines were not contaminants, but derivatives of the original RB cells. The remaining RB1 allele may be wild-type even in the malignant cell lines, because the expression and the LT-binding ability were normal. Furthermore, we did not find any homozygous loss in 16 polymorphic markers located in the 13q14-22 region in the transformed cell lines. However, introduction of a copy of a normal chromosome 13 into the anchorage-independent cell line suppressed its anchorage-independent growth ability. All these data, together with the fact that the RB cells containing the deletion progressed to a tumorigenic state spontaneously, but normal fibroblasts did not, raise the possibility that a new tumor suppressor gene, located at 13q14-22, may play a critical role in neoplastic transformation. We conclude that these RB cell lines provide an excellent system for identification of genes involved in malignant transformation of human cells. Genes Chromosomes Cancer 26:47-53, 1999.

Cell Adhesion↗
PubMed1999

Genetic analysis of the role of the drosophila fat facets gene in the ubiquitin pathway.

The Drosophila fat facets gene encodes a deubiquitinating enzyme required during eye development to limit the number of photoreceptors in each facet to eight. Ubiquitin is a small polypeptide that targets proteins for degradation by the proteasome. Deubiquitinating enzymes cleave ubiquitin-protein bonds. In order to investigate the role of FAT FACETS in the ubiquitin pathway, genetic interactions between fat facets and the Drosophila UbcD1 gene were assessed. In addition, three yeast deubiquitinating enzyme genes were tested for their ability to substitute for fat facets in the developing Drosophila eye and for their effects on eye morphology. The results of these experiments support the hypothesis that FAT FACETS activity antagonizes that of the proteolytic machinery. The implications of these results for the specificity of FAF and yeast UBPs are discussed as well.

Animals↗
PubMed1999

Cytokine-induced calgranulin C expression in keratocytes.

The authors have identified a corneal stromal protein (CO-Ag) that may be involved in the pathogenesis of Mooren's ulcer. The CO-Ag cDNA sequence is identical to that of human neutrophil calgranulin C (CaGC). This study sought to demonstrate expression of the CaGC gene in the human cornea and in corneal keratocytes after cytokine stimulation. In situ hybridization and immunohistochemistry were used to localize CaGC mRNA and protein in normal and diseased human corneas, including a specimen with Mooren's ulcer. Cultured bovine keratocytes were stimulated with IL-1 alpha or TNF-alpha, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify CaGC cDNA from cytokine-stimulated keratocytes and unstimulated controls. Southern blotting verified the specificity of the RT-PCR amplification products. In situ hybridization detected human CaGC mRNA in the stroma of corneas with Fuchs' dystrophy, postinfection corneas, and a cornea with Mooren's ulcer. In cultured bovine keratocytes, peak levels of CaGC mRNA were reached 6 h after cytokine stimulation. Southern blots with an oligonucleotide probe specific for CaGC detected the RT-PCR products of expected sizes (273 bp) and confirmed that the amplified CO-Ag sequence was identical to that of CaGC. These studies are the first to demonstrate the presence of CaGC in the human cornea and the ability of stromal keratocytes to produce CaGC (CO-Ag). The up-regulation of CaGC gene expression by corneal keratocytes due to proinflammatory cytokines from trauma or inflammation may induce autoimmunity that ultimately results in Mooren's ulceration.

Animals↗