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Biomedical subjects

Gary W Conrad

Publications and source records attributed to Gary W Conrad.

18 recordsLinked to original sources
PubMed2006

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry.

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.

Diiodothyronines↗
PubMed2006

Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry.

In this work two monoiodothyronines, 3-T1 and 3'-T1, have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS. MS2 spectra in either negative or positive ion mode can be used to differentiate 3-T1 and 3'-T1. Based on the relative abundance of fragment ions in MS2 spectra in the negative ion mode, quantification of the 3-T1 and 3'-T1 isomers in mixtures is achieved without prior separation. Solid-phase extraction in combination with ESI-MS/MS provides a practicable procedure that can be used to determine the molar ratio of 3-T1 and 3'-T1 in human serum with an error less than 3%. The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively.

Humans↗
PubMed2006

Thyroxine affects expression of KSPG-related genes, the carbonic anhydrase II gene, and KS sulfation in the embryonic chicken cornea.

PURPOSE: Opaque chick corneas become thin and transparent from embryonic day (E)9 to E20 of incubation. Thyroxine (T4) injected in ovo on E9 induces precocious transparency by E12. The present study was conducted to determine whether corneal cells differentially express genes for T4 regulation, keratan sulfate proteoglycan (KSPG) synthesis, crystallins, and endothelial cell ion transporters during transparency development and whether these expressions are altered when E9 embryos are treated with T4. METHODS: E9 eggs received T4 or buffer; corneas were dissected on E12. Corneal transparency was measured digitally and thickness was determined from cryostat cross sections. mRNA expressions were determined by real-time PCR using cDNA synthesized from whole-cell RNA, cells expressing T4 receptor mRNAs assessed by in situ hybridization, and KS disaccharide sulfation measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: All corneal layers expressed T4 receptor alpha (THRA) mRNA; keratocytes and endothelial cells expressed T4 receptor beta (THRB) mRNA. During normal development, THRB expression increased 20-fold from E12 to E20; THRA expression remained constant. Expressions of most genes involved in KS synthesis increased from E9 to E16, and then decreased from E16 to E20. From E9 to E20, expressions of crystallin genes increased; T4/3-deiodinase DIII (DIO3) increased 10-fold; and sodium-potassium ATPase transporter (ATP1A1), sodium-bicarbonate transporter (NBC), and carbonic anhydrase II (CA2) increased 5- to 10-fold. E9 T4 administration decreased corneal thickness by E12; increased DIO3, THRB, and CA2 expressions 5- to 20-fold; decreased KSPG core protein genes and galactose sulfotransferase CHST1 expressions 2-fold; and reduced KS disulfated/monosulfated disaccharide (DSD/MSD) ratios. CONCLUSIONS: Thyroxine modifies expressions of KSPG synthesis and carbonic anhydrase genes.

Animals↗
PubMed2006

Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas.

PURPOSE: To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas. METHODS: Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes. Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Concentrations of both monosulfated GlcNAc(6S)-beta-1,3-Gal (MSD2) and disulfated Gal (6S)-beta-1,4-GlcNAc(6S) (DSD) KS disaccharides from the LASIK interface scars were significantly lower than in normal control corneal stromas. No significant difference was found for the concentration of nonsulfated (NSD) KS disaccharides in LASIK interface scars compared with normal controls. The concentration of DeltaUA-beta-1,3-GalNAc(6S) (Deltadi-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of DeltaUA-beta-1,3-GalNAc(4S) (Deltadi-4S) and nonsulfated Deltadi-0S CS/DS disaccharides demonstrated no significant differences from normal corneas. CONCLUSIONS: The profiles of KS and CS/DS disaccharides in LASIK interface scars are significantly different from those in normal cornea stromal tissue, as revealed by LCM and ESI-MS/MS.

Chondroitin Sulfates↗
PubMed2005

Detection and quantification of 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine by electrospray ionization tandem mass spectrometry.

A novel and rapid method for identifying and quantifying 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3) has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). MS(2) spectra in either negative ionization mode or positive ionization mode can be used to differentiate T3 and rT3. Quantification of the T3 and rT3 isomers under the negative ionization mode is also achieved without prior separation by HPLC.

Algorithms↗
PubMed2005

Expression and localization of leucine-rich B7 protein in human ocular tissues.

PURPOSE: Using a web-based tool to screen the human proteome for potential mimecan/osteoglycin interacting proteins, we found that the leucine-rich B7 protein may have functional associations with several small leucine-rich proteoglycans (SLRP), including mimecan. The purpose of this study was to determine the expression of leucine-rich B7 protein in human eye tissues and its subcellular localization in MG-63 cells. METHODS: Primers were synthesized to amplify the two known differentially spliced B7 mRNA transcripts. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to determine the expression of B7 mRNAs in human ocular and nonocular tissues. A rabbit anti-human B7 antibody was generated that specifically immunostained B7 proteins. The expression of B7 proteins in the human eye was determined by immunohistochemistry (IHC). Intracellular localization of leucine-rich B7 and mimecan proteins were determined using transient co-transfections, cell immunostaining, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis showed moderate expression of B7, transcript variant 2, in human cornea, iris, sclera, and retina. In contrast, B7, transcript variant 1, was strongly expressed only in the cornea. The two B7 mRNAs were highly expressed in human brain, blood, peripheral mononuclear cells, and other human tissues. By IHC, immunostaining for leucine-rich B7 protein was found in epithelial and endothelial layers of the cornea, epithelial and fiber cells of the lens, in sclera, and in the rod and cone layer of the retina of adult human eye. Leucine-rich B7 protein was found to localize to both nucleus and cytoplasm of MG-63 cells, whereas mimecan was found only in the cytoplasm of these cells. Merged images obtained by confocal microscopy revealed certain cytoplasmic regions in MG-63 cells where B7 and mimecan proteins appeared to co-localize. CONCLUSIONS: The present work is the first to demonstrate the expression and localization of leucine-rich B7 protein in human eye and other human tissues. The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of leucine-rich B7 proteins in health and disease.

Animals↗
PubMed2005

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry.

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.

Acetylglucosamine↗
PubMed2005

Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS.

PURPOSE: To identify and quantify changes in keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) sulfated disaccharides in the developing chick cornea using electrospray ionization tandem mass spectrometry (ESI-MS/MS). METHODS: Cryostat sections of fresh nonfixed corneas were obtained from White Leghorn embryonic day (E)8 to E20 chicks, and from 4- and 70-week-old chickens. Tissue sections on glass slides were incubated with selected glycosidase enzymes. Digest solutions were analyzed directly by ESI-MS/MS. RESULTS: The concentration of KS monosulfated disaccharide (MSD) Gal-beta-1,4-GlcNAc(6S) in E8 cornea equaled that at E20, declined to its lowest level by E10, increased to a second peak by E14, decreased to a second low by E18, peaked again by E20, and remained high in adult corneas. A similar concentration profile was observed for KS disulfated disaccharide (DSD) Gal(6S)-beta-1,4-GlcNAc(6S), and thus also for total sulfated KS disaccharides. The molar percent of DSD was higher than that of MSD from E8 to E18, equivalent at E20, and less than that of MSD in adult corneas. In contrast, total concentration of CS/DS Deltadi-4S plus Deltadi-6S decreases as development progresses and is lowest in adult corneas. Concentration and molar percent of Deltadi-6S is highest at E8, then decreases through development as the concentration and molar percent of Deltadi-4S increases from E8 and exceeds that of Deltadi-6S after E14. CONCLUSIONS: New, rapid, direct chemical analysis of extracellular matrix components obtained from sections from embryonic and adult chick corneas reveals heretofore undetected changes in sulfation characteristics of KS and CS/DS disaccharides during corneal development.

Animals↗
PubMed2005

An x-ray diffraction study of corneal structure in mimecan-deficient mice.

PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea.

Animals↗
PubMed2004

Analysis of transcriptional regulation of the small leucine rich proteoglycans.

PURPOSE: Small leucine rich proteoglycans (SLRPs) constitute a family of secreted proteoglycans that are important for collagen fibrillogenesis, cellular growth, differentiation, and migration. Ten of the 13 known members of the SLRP gene family are arranged in tandem clusters on human chromosomes 1, 9, and 12. Their syntenic equivalents are on mouse chromosomes 1, 13, and 10, and rat chromosomes 13, 17, and 7. The purpose of this study was to determine whether there is evidence for control elements, which could regulate the expression of these clusters coordinately. METHODS: Promoters were identified using a comparative genomics approach and Genomatix software tools. For each gene a set of human, mouse, and rat orthologous promoters was extracted from genomic sequences. Transcription factor (TF) binding site analysis combined with a literature search was performed using MatInspector and Genomatix' BiblioSphere. Inspection for the presence of interspecies conserved scaffold/matrix attachment regions (S/MARs) was performed using ElDorado annotation lists. DNAseI hypersensitivity assay, chromatin immunoprecipitation (ChIP), and transient transfection experiments were used to validate the results from bioinformatics analysis. RESULTS: Transcription factor binding site analysis combined with a literature search revealed co-citations between several SLRPs and TFs Runx2 and IRF1, indicating that these TFs have potential roles in transcriptional regulation of the SLRP family members. We therefore inspected all of the SLRP promoter sets for matches to IRF factors and Runx factors. Positionally conserved binding sites for the Runt domain TFs were detected in the proximal promoters of chondroadherin (CHAD) and osteomodulin (OMD) genes. Two significant models (two or more transcription factor binding sites arranged in a defined order and orientation within a defined distance range) were derived from these initial promoter sets, the HOX-Runx (homeodomain-Runt domain), and the ETS-FKHD-STAT (erythroblast transformation specific-forkhead-signal transducers and activators of transcription) models. These models were used to scan the genomic sequences of all 13 SLRP genes. The HOX-Runx model was found within the proximal promoter, exon 1, or intron 1 sequences of 11 of the 13 SLRP genes. The ETS-FKHD-STAT model was found in only 5 of these genes. Transient transfections of MG-63 cells and bovine corneal keratocytes with Runx2 isoforms confirmed the relevance of these TFs to expression of several SLRP genes. Distribution of the HOX-Runx and ETS-FKHD-STAT models within 200 kb of genomic sequence on human chromosome 9 and 500 kb sequence on chromosome 12 also were analyzed. Two regions with 3 HOX-Runx matches within a 1,000 bp window were identified on human chromosome 9; one located between OMD and osteoglycin (OGN)/mimecan genes, and the second located upstream of the putative extracellular matrix protein 2 (ECM2) promoter. The intergenic region between OMD and mimecan was shown to coincide with different patterns of DNAse I hypersensitivity sites in MG-63 and U937 cells. ChiP analysis revealed that this region binds Runx2 in U937 cells (mimecan transcript note detectable), but binds Pitx3 in MG-63 cells (expressing high level of mimecan), thereby demonstrating its functional association with mimecan expression. Upon comparing the predictions of S/MARs on the relevant chromosomal context of human chromosomes 9 and 12 and their rodent equivalents, no convincing evidence was found that the tandemly arranged genes build a chromosomal loop. CONCLUSIONS: Twelve of 13 known SLRP genes have at least one HOX-Runx module match in their promoter, exon 1, intron 1, or intergenic region. Although these genes are located in different clusters on different chromosomes, the common HOX-Runx module could be the basis for co-regulated expression.

Animals↗
PubMed2004

Analysis of the expression of chondroadherin in mouse ocular and non-ocular tissues.

PURPOSE: Chondroadherin (Chad) is a unique member of the small leucine rich repeat proteoglycan gene family. It is expressed at high levels in certain zones of cartilage and also has been detected in bone, tendon, bone marrow, and chondrosarcoma cells. Recently, we demonstrated that Chad is expressed in human and mouse lens and that its expression is decreased in the absence of mimecan/osteoglycin. This finding prompted us to explore the expression of Chad in other ocular and non-ocular tissues. METHODS: Reverse transcription-polymerase chain reaction amplification (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used to determine the expression of Chad in human and mouse tissues. RESULTS: RT-PCR analysis showed strong expression of Chad in mouse brain, heart, lung, and embryo. Moderate expression was detected in mouse thymus, spleen, testis, and ovary, and very low expression in kidney and liver. Chad was highly expressed in human cornea, brain, and skeletal muscle, and moderately in human retina and lens. By ISH, Chad mRNA was detected in cornea, lens, and retina of postnatal day 21 mouse eyes. By IHC, immunostaining for Chad was seen in epithelial and endothelial layers of the cornea, as well as in lens, ciliary body, and retina of the adult mouse eye. Strong immunostaining was noted in retinal rod, cone, and plexiform layers. IHC analysis of tissue microarray demonstrated presence of Chad in brain (cerebellum), skeletal and cardiac muscles, lung, gastrointestinal tract, ovary, and cartilage. In most tissues, Chad expression was associated with either peripheral nerves and/or blood vessels. In the stomach and intestines, positive immunostaining was observed in Meissner's plexus and enteroendocrine cells. Intriguingly, positive immunostaining also was observed in Purkinje cells of the cerebellum and pancreatic islets. CONCLUSIONS: The present work establishes the expression and localization of Chad in several new locations including cornea, lens, ciliary body, retina, cerebellum, pancreatic islet, blood vessels, and peripheral nerves. The surprisingly broad expression pattern of Chad suggests potential roles for this protein in cell specific and/or tissues specific function(s). The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of Chad in health and disease.

Animals↗
PubMed2004

Differentially expressed genes in the lens of mimecan-null mice.

PURPOSE: Members of the small leucine-rich proteoglycans (SLRP) gene family are essential for normal collagen fibrillogenesis in various connective tissues and important regulators of cellular growth, differentiation, and tissue repair. Mimecan is a member of this gene family and is expressed in many connective tissues. We have previously reported that knockout of the mouse mimecan gene results in abnormal collagen fibrillogenesis, mainly in the cornea and skin. During the course of our studies on biological roles of mimecan in the eye, we found that this gene is expressed in the mouse lens. Here, we sought to identify gene expression changes in the lens that are associated with the absence of mimecan. METHODS: Reverse transcription-polymerase chain reaction amplification (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used to determine mimecan expression in human and mouse eyes. Microarray hybridization was used to determine gene expression differences between lenses isolated from mimecan-null and wild type mice. Relative quantitative RT-PCR was used to verify the expression levels of a subset of the identified genes. RESULTS: By ISH and IHC, mimecan mRNA was detected in cornea and lens at embryonic day 16.5 (E16.5) and postnatal day 10 (P10) mouse eyes. By RT-PCR, mimecan mRNA was detected in human cornea, lens, iris, and retina. In mimecan-null mice lenses, microarray analysis of 5,002 mouse genes demonstrated a more than two fold increase in expression of 65 genes and a more than two fold decrease in expression of 76 genes. Among genes with increased expression were cell adhesion molecules, G-protein coupled receptors, intracellular signaling molecules, genes involved in protein biosynthesis and degradation, and genes involved in immune function. Decreased expression was found in extracellular matrix molecules, calcium binding and transporting proteins, and genes known for their roles in regulating cellular motility. Intriguingly, decreased gene expression was observed with two SLRP family members, biglycan and condroadherin, as well as with several stress-response proteins, including gammaA-crystallin, hemoglobin alpha 1, and metallothionein 1. Quantitative RT-PCR confirmed changes in expression of 12 genes selected from the arrays. CONCLUSIONS: In this report we present the first demonstration that mimecan is constitutively expressed in the vertebrate lens. The results from gene expression profiling reveal the ability of mimecan to influence expression of biglycan and chondroadherin, thereby indicating possible novel regulatory interactions between these SLRP family members. As with mimecan, the expression of chondroadrein in vertebrate lens has not been reported previously. Our results provide insight into the function of mimecan in the lens and enable further characterization of molecular mechanisms by which this protein exerts its biological roles.

Animals↗
PubMed2004

Graded potential of neural crest to form cornea, sensory neurons and cartilage along the rostrocaudal axis.

Neural crest cells arising from different rostrocaudal axial levels form different sets of derivatives as diverse as ganglia, cartilage and cornea. These variations may be due to intrinsic properties of the cell populations, different environmental factors encountered during migration or some combination thereof. We test the relative roles of intrinsic versus extrinsic factors by challenging the developmental potential of cardiac and trunk neural crest cells via transplantation into an ectopic midbrain environment. We then assess long-term survival and differentiation into diverse derivatives, including cornea, trigeminal ganglion and branchial arch cartilage. Despite their ability to migrate to the periocular region, neither cardiac nor trunk neural crest contribute appropriately to the cornea, with cardiac crest cells often forming ectopic masses on the corneal surface. Similarly, the potential of trunk and cardiac neural crest to form somatosensory neurons in the trigeminal ganglion was significantly reduced compared with control midbrain grafts. Cardiac neural crest exhibited a reduced capacity to form cartilage, contributing only nominally to Meckle's cartilage, whereas trunk neural crest formed no cartilage after transplantation, even when grafted directly into the first branchial arch. These results suggest that neural crest cells along the rostrocaudal axis display a graded loss in developmental potential to form somatosensory neurons and cartilage even after transplantation to a permissive environment. Hox gene expression was transiently maintained in the cardiac neural tube and neural crest at 12 hours post-transplantation to the midbrain, but was subsequently downregulated. This suggests that long-term differences in Hox gene expression cannot account for rostrocaudal differences in developmental potential of neural crest populations in this case.

Animals↗
PubMed2003

Interferon-gamma regulation of the human mimecan promoter.

PURPOSE: The human mimecan/osteoglycin promoter contains multiple interferon-stimulated response elements (ISRE) and interferon-gamma-activation sites (GAS). ISRE and GAS motifs are present in a variety of interferon (IFN)-inducible genes. The purpose of this study was to investigate whether IFN-gamma affects mimecan gene expression and, if so, to determine the cis-elements and transcription factors that mediate its action. METHODS: Electrophoretic mobility shift assay (EMSA) was used to investigate whether nuclear proteins from IFN-gamma-treated cells bind to regions of the human mimecan promoter containing ISRE sites. Incubation of nuclear extracts with specific antibodies was used to identify transcription factors that bind to these sites. Transcriptional activity of the promoter was evaluated by transient transfections of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Co-transfection experiments were used to study the role of transcription factors that bind ISRE elements in the promoter and mediate the IFN-gamma response. Expression of mRNAs was analyzed by reverse transcription-polymerase chain reaction. RESULTS: Using probes that correspond to two ISRE sites located in the first intron of the human mimecan gene, we detected specific DNA-protein complexes with nuclear extracts from IFN-gamma-treated cells. Formation of DNA-protein complexes was abrogated by competition with unlabeled probe and one of the complexes was supershifted by the anti-interferon regulatory factor-1 (IRF-1) antibody. Interestingly, when probe that corresponds to a conserved E-box (CACATG) in the proximal promoter and nuclear extracts from IFN-gamma-treated cells were used in EMSA, increased binding of upstream stimulatory factor-1 (USF-1) was observed. Co-transfection of a mimecan promoter construct that contained the entire first intron with IRF-1, or with both IRF-1 and USF-1 expression plasmids, suppressed luciferase activity of the promoter in corneal keratocytes and T-47D cells. In contrast, co-transfection experiments with IRF-2, or with both IRF-2 and USF-1, led to increased luciferase activity of the same promoter construct. RT-PCR analyses demonstrate that IFN-gamma rapidly and transiently suppresses mimecan expression and induces IRF-1 and IRF-2 mRNAs in bovine corneal keratocytes. CONCLUSIONS: IRF-1 binds to ISRE sites, located in the first intron of the human mimecan gene, and negatively regulates mimecan expression at the level of transcription. Consistent with these observations, an inverse correlation between the expression of mimecan and IRF-1 in bovine corneal keratocytes and non-corneal cells was demonstrated. IRF-2 positively regulates mimecan transcription in corneal keratocytes. Because the intact E-box in the proximal promoter was required for IRF-1 and IRF-2 effects on mimecan transcription, potential direct or indirect interactions between USF-1 and IRF-1 and IRF-2 are likely.

Animals↗
PubMed2003

The UV responsive elements in the human mimecan promoter: a functional characterization.

PURPOSE: A major environmental stress encountered by humans is solar UV light, which can cause a spectrum of eye diseases, such as photokeratitis, cataract, pterygia, and ocular neoplasms. Mammalian defense mechanisms in response to adverse effects of UV light result in induction of a number of genes. Studies on the transcriptional regulation of genes that are expressed in the eye will increase understanding of both the physiological functions of these genes in the mammalian UV response, and the molecular bases for abnormalities associated with the above diseases. Mimecan is an extracellular matrix proteoglycan that is abundantly expressed in the cornea. The purpose of this study was to determine and characterize the UV responsive regulatory elements of the human mimecan promoter. METHODS: Transcriptional activity of the promoter was evaluated, before and after UV irradiation, using transient transfection of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Site directed mutagenesis and corresponding functional assays were used to determine the contribution of UV responsive regions to human mimecan transcription. Co-transfection experiments were used to investigate the role of transcription factors that bind these elements in the promoter and mediate the UV response. mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The shortest promoter construct that was strongly activated following UV irradiation contained three initiator elements, an E-box element that is conserved between species, and the entire first intron of the human mimecan gene. Deletion of the intronic p53 binding site from this construct considerably diminished transcription and the UV response of the promoter. Surprisingly, deletion of the E-box sequence from this construct completely abolished both transcription and UV response of the promoter. These results demonstrated that the E-box is essential to transcription of the human mimecan gene and also is required for activation by p53. The role of the E-box, and the E-box binding protein, USF-1, in transcription and UV responses of the human mimecan promoter were confirmed by co-transfection experiments using dominant negative transcription factor, A-USF. In addition to these positive regulators, we demonstrate that the region between nucleotides -1314 and -1907 contains a transcriptional repressor site that is active in a time dependent manner following UV irradiation. Finally, we show that UV irradiation results in changes in mimecan mRNA levels in bovine corneal keratocytes in a time-dependent manner. CONCLUSIONS: The human mimecan promoter contains several UV responsive regulatory elements that are conserved between human and bovine species and include the intronic p53 DNA binding site, the E-box in the proximal promoter, and the region between nucleotides -1314 and -1907. The E-box plays an important role in transcription and UV response of the human mimecan promoter. UV irradiation modulates expression of mimecan mRNA in bovine corneal keratocytes and non-corneal cells.

Animals↗
PubMed2003

The keratocan gene is expressed in both ocular and non-ocular tissues during early chick development.

Extracellular matrix (ECM) keratan sulfate proteoglycans (KSPGs) are core proteins with sulfated polylactosamine side chains (KS). The KSPG core protein keratocan gene (Kera) is expressed almost exclusively in adult vertebrate cornea, but its embryonic expression is little known. Embryonic chick in situ hybridization reveals Kera mRNA expression in corneal endothelium from embryonic day (E) 4.5, Hamburger-Hamilton (HH) 25, in stromal keratocytes from E6.5, HH30, and in iris distal surface cells from E8, HH34. As highly sulfated, antibody I22-positive KS increases extracellularly from posterior to anterior across the stroma, nerves enter and populate only anterior stroma and epithelium. RT-PCR and in situ hybridization demonstrate that developmentally regulated Kera mRNA expression initiates in midbrain and dorsolateral mesenchyme at E1, HH7, then spreads caudally in hindbrain and cranial and trunk mesenchyme flanking the neural tube through E2, HH20. Cranial expression extends ventrally through the developing head, and concentrates in mesenchyme surrounding eye anterior regions and cranial ganglia, and in subepidermal pharyngeal arch mesenchyme by E3.5, HH22. Kera expression in the trunk at E3.5, HH22 and E4.5, HH25, is strong in dorsolateral subepidermal, sclerotomal and nephrogenic mesenchymes, but absent in neural tube, dorsal root ganglia, nerve outgrowths, notochord, heart and gut. Early limb buds express Kera mRNA throughout their mesenchyme, then in restricted proximal and distal mesenchymes. I22-positive KS appears only in notochord in E3.5, HH22 and E4.5, HH25, embryos. Results suggest the hypothesis that keratocan, or keratocan with minimally sulfated KS chains, may play a role in structuring ECM for early embryonic cell and neuronal migrations.

Amino Acid Sequence↗
PubMed2002

Mimecan/osteoglycin-deficient mice have collagen fibril abnormalities.

PURPOSE: To study the role of mimecan, a member of the small leucine-rich proteoglycans (SLRPs) gene family and one of the major components of the cornea and other connective tissues, mice that lack a functional mimecan gene were generated and characterized. METHODS: Mimecan-deficient mice were generated by gene-targeting using standard techniques. Mice were genotyped by Southern blot analysis. The absence of mimecan transcripts was confirmed by Northern blot analysis. Corneal clarity was examined by slit lamp biomicroscopy. The strength of the skin was evaluated using a biomechanical skin fragility test. Collagen morphology in cornea and skin preparations from mimecan-null and control wild-type mice was analyzed by transmission electron microscopy. The diameter of collagen fibrils in these tissues was determined by morphometric analysis. RESULTS: Mice lacking mimecan appear to develop normally, are viable and fertile. In a controlled laboratory environment they do not display an evident pathological phenotype compared to wild type mice. Examination of corneal clarity and measurements of corneal thickness show no significant changes in the cornea. However, a skin fragility test revealed a moderate reduction in the tensile strength of skin from mutant mice. Ultrastructural analyses show, on average, thicker collagen fibrils in both corneal and skin preparations from mimecan-null mice. Collagen fibrils from the cornea of mutant mice show an average diameter of 31.84+/-0.322 nm, versus 22.40+/-0.296 nm in their wild type litter-mates. The most pronounced increase in collagen fibril diameter was found in the skin of mimecan-null mice, who demonstrated an average diameter of 130.33+/-1.769 nm, versus 78.82+/-1.157 nm in the wild type mice. In addition, size variability and altered collagen morphology was detected in dorsal and tail skin preparations from the mutant mice. CONCLUSIONS: The results of the present study demonstrate that mimecan, similar to other members of the SLRP gene family, has a role in regulating collagen fibrillogenesis in vivo. Further studies, such as functional challenges, an evaluation of potential compensation by other proteins (including members of the SLRP family), and generation of double-knockouts will be necessary to fully uncover physiological functions of mimecan in mice.

Animals↗