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Biomedical subjects

S L Fong

Publications and source records attributed to S L Fong.

At least 19 recordsLinked to original sources
PubMed2000

Retinal degeneration in cone photoreceptor cell-ablated transgenic mice.

PURPOSE: To examine the effect of loss of cone photoreceptor cells on retinal degeneration. METHODS: We previously identified a cone photoreceptor cell-specific promoter of human cone transducin a-subunit (GNAT2) gene. In this report, a minigene, Trc-Tox176, that contains the GNAT2 promoter, an attenuated diphtheria toxin A-chain gene, and an enhancer element from human interphotoreceptor retinoid-binding protein (IRBP) was used to generate coneless transgenic mice. Transgenic mice were identified by PCR and the copy number of the transgene was determined by Southern hybridization, and examined by histology. RESULTS: The results of immunostaining with anti-mouse GNAT2 antibodies and reverse transcription-PCR (RT-PCR) analysis with mRNA from the retinas of transgenic mice showed that cone photoreceptor cells were ablated in one of four transgenic mouse lines. The ablation of cone cells began at postnatal day 8, at the same time as the expression of endogenous GNAT2. An age-related rod degeneration was also found in this cone-ablated mouse line, beginning at postnatal day 9, proceeding from the central retina to the peripheral retina. CONCLUSIONS: Cone photoreceptor cells may play an important role in the survival of rod photoreceptor cells during mouse retina development.

Animals↗
PubMed2000

Repair of muscle and musculotendinous junction injuries with an autogenous fascial patch.

We have used an autogenous fascia lata patch to repair muscle and musculotendinous junction injuries affecting 99 individual muscles in 23 patients with multiple incised wounds to the upper limbs. All patients were followed up for at least 2 years. Two patients with severe dystrophic changes following nerve injuries were excluded from the final analysis. Satisfactory grip strength was restored 6 months following the repair in 19 of the 21 patients we evaluated. Complete active and passive range of wrist and digital motion was seen in 18 of these 21 patients. Tightness of the flexor pollicis longus was seen in 2 patients, one of whom had a deformity of the interphalangeal joint of the thumb that was passively stretched and subsequently reduced. Scar adhesions were noted in 4 other patients, but this did not affect wrist or finger motion, except in one. The autogenous fascial patch repair technique provides strong and good apposition of muscle fibres simply by distributing the force and tension over a large area of the muscle, thereby allowing early mobilization and functional restoration of the extremity.

Arm Injuries↗
PubMed1999

Elements regulating the transcription of human interstitial retinoid-binding protein (IRBP) gene in cultured retinoblastoma cells.

PURPOSE: To identify cis-acting elements and trans-acting factors involved in the expression of human IRBP gene. METHODS: Transient transfection of WERI-Rb1 and HeLa cells, DNase 1 footprinting, gel mobility-shift assay and yeast one-hybrid system were used to study the regulatory elements that are involved in the expression of human IRBP gene. RESULTS: A region between -1620 and -1411 was shown to have enhancer properties. Using nuclear extracts from WERI-Rb1 and HeLa cells, four footprints were identified in the proximal promoter region (-206 to +68). The core promoter element IP1 binds to OTX2 in the yeast one-hybrid system. By cotransfecting HeLa cells, OTX2 could transactivate the irbp promoter. The functions of IP2 (from -119 to -86) and IP3 (from -183 to -147) remain to be determined. The region containing the HeLa cell-specific footprint IP4 (from -202 to -180) could silence the OTX2 transactivation of the irbp promoter. CONCLUSION: The 5'-flanking region of irbp contains an enhancer sequence. The possible silencer upstream from the core promoter may serve to suppress expression of irbp in HeLa cells. When the proximal promoter is used to identify binding proteins in a human retina library by the yeast one hybrid system, nine of the identified clones contained the cDNA sequence for the homeodomain protein OTX2. Since no clones for the homeodomain protein CRX were found, and since OTX2 can transcriptionally activate irbp in normally non-expressing HeLa cells, it is possible that OTX2 rather than CRX is the transcriptional activator for irbp in human photoreceptors.

Amino Acid Sequence↗
PubMed1998

Repression of Stat3 activity by activation of mitogen-activated protein kinase (MAPK).

STAT proteins are activated by phosphorylation at specific tyrosine residue at the carboxy-terminus which is required for dimer-formation, nuclear translocation, DNA binding and transcriptional activity in cells treated with cytokines and growth factors. Recent studies have indicated that STATs are also phosphorylated by MAPK, or extracellular signal-regulated kinase (ERK) on serine. We investigated the role of ERK on the regulation of STAT activity. Here, we report that ERK2 activated by its upstream kinase, MEK1, represses Stat3 transcriptional activity induced by Src or Jak-2. To unravel the mechanism of repression, we further showed that Stat3 DNA binding activity and its tyrosine phosphorylation are also inhibited under the same conditions. ERK2 phosphorylates Stat3 on three serine-containing peptides and decreases its tyrosine phosphorylation induced by EGF treatment. We also detected an association of ERK2 and Stat3 in vivo which is modulated positively by activation of ERK2, but negatively by Jak2. We propose that MAP kinase cascade may negatively regulate Stat3 activities by decreasing its tyrosine phosphorylation and also possibly by association.

3T3 Cells↗
PubMed1998

A CAT reporter construct containing 277bp GNAT2 promoter and 214bp IRBP enhancer is specifically expressed by cone photoreceptor cells in transgenic mice.

PURPOSE: The alpha-subunit of human cone transducin plays an important role in interacting with visual pigment and activating the cGMP-dependent phosphodiesterase (cGMP-PDE). The human GNAT2 gene (cone transducin alpha-subunit) has been cloned and characterized by Fong et al. In this report, we describe the use of transgenic mice to characterize the tissue specificity of the GNAT2 promoter. METHODS: A chimeric reporter gene construct which consists of a 277 bp 5'-flanking fragment of the GNAT2 gene at 5' end of the chloramphenicol acetyltransferase (CAT) gene and a 214 bp enhancer region from the human interphotoreceptor retinoid-binding protein (IRBP) gene at the 3' end of the CAT gene was used to generate transgenic mice. Transgenic mice were identified by Southern blot hybridization and polymerase chain reaction (PCR) analysis using tail DNA from experimental animals. Immunostaining was used to study the developmental expression of CAT and the endogenous GNAT2 gene. RESULTS: Analysis of four transgenic mouse lines revealed that three lines had low CAT activity in the retina. The CAT gene, along with the endogenous GNAT2 gene, was expressed at high levels in cone photoreceptor cells in the fourth transgenic mouse line as determined by CAT enzyme assays and immunostaining. CONCLUSION: The results show that the 277 bp 5'-flanking sequence from the human GNAT2 gene coupled with the 214 bp IRBP enhancer can direct a tissue-specific expression pattern of CAT reporter gene in mouse retina, which parallels the expression pattern of endogenous GNAT2.

Animals↗
PubMed1997

Resurfacing of thumb-pulp loss with a heterodigital neurovascular island flap using a nerve disconnection/reconnection technique.

The problems of late deterioration of sensation and a "double sensibility" phenomenon, either in the thumb or donor finger, are a known deficiency of the conventional heterodigital neurovascular island flap. This is probably related to unsatisfactory cortical reorientation following flap transfer. To obviate this problem, the authors have used a disconnection/reconnection technique for heterodigital island flaps in 17 patients, to resurface defects in the skin of the thumb. All patients were followed-up for 1 year, while 15 were followed-up for 2 years or more. There were no flap complications or failures, and the length of the thumb tip was preserved in all cases. The classic two-point discrimination was less than 6 mm in eight of these patients, and the remaining nine patients had two-point discrimination of between 6 and 8 mm. This technique was found to provide sensitive, supple, and well-vascularized skin with proper cortical representation, to replace the loss of the tactile pump of the thumb tip in one operative stage.

Adult↗
PubMed1997

Localization of upstream silencer elements involved in the expression of cone transducin alpha-subunit (GNAT2).

PURPOSE: To localize cis-acting elements involved in the expression of the cone-specific G-protein, cone transducin alpha-subunit (GNAT2). METHODS: In this study, the authors used a genomic clone, HGLG3, to sequence 3139 base pairs of the upstream region of the GNAT2 gene and to localize cis-acting elements involved in the expression of GNAT2. Upstream elements were localized functionally by transfection of chloramphenicol acetyltransferase gene constructs containing nested deletions of this upstream region into WERI-Rb1 cells. Cell specificity of the localized elements was determined by transfection of the HeLa cells. Trans-acting factor-binding sites to functional cis-acting elements were determined by DNasel footprinting. Cell specificity of protein interaction with footprinted regions was tested by electrophoretic mobility shifts with nuclear extracts from WERI-Rb1 and HeLa cells. RESULTS: Transfection of WERI-Rb1 and HeLa cells revealed the presence of a strong, noncell-specific silencer region between -1130 and -23, a weak, cell-specific promoter between -151 and -10, and a stronger, noncell-specific element between +143 and +167. DNaseI footprinting showed three major footprints (S1, S2, and S3) between -807 and -176, indicating the binding sites for putative negative trans-acting factors. Individual footprinted sequences had similar electrophoretic mobility shifts when they were incubated with nuclear extracts from either WERI-Rb1 or HeLa cells, suggesting that these cells express the same negative factors. CONCLUSIONS: The expression of the GNAT2 gene is controlled by a strong silencer region, a weak upstream cell-specific promoter, and a strong downstream element. The silencer region interacts with similar proteins from retina- and nonretina-derived cell lines.

Base Sequence↗
PubMed1995

Mapping of the human cone transducin alpha-subunit (GNAT2) gene to 1p13 and negative mutation analysis in patients with Stargardt disease.

We report localization of the human cone transducin (GNAT2) gene using fluorescence in situ hybridization on chromosome 1 in band p13. The recent assignment of a gene for Stargardt disease to the same chromosomal region by linkage analysis prompted us to investigate the possible role of GNAT2 in the pathogenesis of this disease. We investigated 66 unrelated patients for mutations in the coding region of the GNAT2 gene using polymerase chain reaction-single strand conformation polymorphism analysis (SSCP) and direct sequencing. No disease-specific mutations were found, indicating that GNAT2 is probably not involved in the pathogenesis of most cases of Stargardt disease.

Base Sequence↗
PubMed1993

Characterization of the gene encoding human cone transducin alpha-subunit (GNAT2).

The human cone transducin alpha-subunit (GNAT2) gene has been completely characterized. The human GNAT2 transcription unit is 9967-bp in length and consists of eight exons with seven introns. The eight exons are identical to the reported cDNA sequence (Lerea et al., 1989). Northern blot analysis of RNA from human retinas and a retinoblastoma cell line, WERI-RB1, reveals a 1.7-kb transcript for GNAT2. Multiple transcription initiation sites were mapped for human retina and WERI-RB1 RNA by primer extension and S1 nuclease protection assays. This gene has seven initiation sites spanning 31 bp. The sequence upstream of the GNAT2 gene shows a TATA box consensus sequence at -29, a CCAAT box consensus sequence at -58 (reverse orientation), and a sequence (CCATAT) similar to the CCAAT box consensus at -76. The GNAT2 upstream sequence shows no significant identity with the upstream region of the human rod transducin alpha-subunit gene (GNAT1) or with the upstream regions of the color visual pigment genes, indicating that the expression of GNAT2 may be regulated differently than these other rod- and cone-specific proteins.

Amino Acid Sequence↗
PubMed1992

Characterization of the human rod transducin alpha-subunit gene.

The human rod transducin alpha subunit (Tr alpha) gene has been cloned. A cDNA clone, HG14, contained a 1.1 kb insertion when compared with the human Tr alpha cDNA published by Van Dop et al. (1). Based on two overlapping clones isolated from a human genomic library, the human Tr alpha gene is 4.9 kb in length and consists of nine exons interrupted by eight introns. Northern blots of human retina total RNA showed that the gene is transcribed by rod photoreceptors into two species of mRNA, 1.3 kb and 2.4 kb in size. Apparently, this is the result of alternative splicing. Two putative transcription initiation sites were determined by primer extension and S1 nuclease protection assays. The putative promoter regions of the human and mouse Tr alpha genes have an identity of 78.1%. As found in the mouse gene (2), no TATA consensus sequence is present in the human gene.

Amino Acid Sequence↗
PubMed1992

Antigen-specific suppressor cells in experimental autoimmune uveitis.

Anti-I-A antibodies, administered in vivo at the time of S-antigen injection, suppress development of experimental autoimmune uveitis (EAU) in Lewis rats. While the effects of anti-I-A are profound, the exact mechanism for this suppression is unknown. We attempted adoptive transfer of this form of suppression by injecting lymphocytes from anti-I-A-treated animals into syngeneic recipients which were later injected with S-antigen. Histologically, globes of 75% of the anti-I-A-treated animals showed no inflammation while 25% of these animals developed mild uveitis. In the group of animals which were injected with S-antigen and also received spleen cells from anti-I-A-treated rats, only 1 showed mild uveitis while the remaining 7 had no inflammation. The animals undergoing adoptive transfer of spleen cells and which were primed with an irrelevant antigen, readily developed uveitis. Suppression of S-antigen-induced EAU was abrogated by pretreatment of donor animals with cyclophosphamide. In vitro studies revealed that spleen cells of S-antigen-primed, anti-I-A-treated donors specifically suppressed lymphocyte responses to S-antigen. These in vivo and in vitro results suggest that generation of antigen-specific suppressor cells play a role in the anti-I-A immunotherapy of EAU.

Animals↗
PubMed1990

Characterization and comparative structural features of the gene for human interstitial retinol-binding protein.

We have cloned the gene for human interstitial retinol-binding protein (IRBP) and compared its nucleotide sequence with that of the corresponding cloned cDNA. The human IRBP gene is approximately 9.5 kilobase pairs (kbp) in length and consists of four exons separated by three introns. The introns are 1.6-1.9 kbp long. The gene is transcribed by photoreceptor and retinoblastoma cells into an approximately 4.3-kilobase mRNA that is translated and processed into a glycosylated protein of 135,000 Da. The amino acid sequence of human IRBP can be divided into four contiguous homology domains with 33-38% identity, suggesting a series of gene duplication events. In the gene, the boundaries of these domains are not defined by exon-intron junctions, as might have been expected. The first three homology domains and part of the fourth are all encoded by the first large exon, which is 3,180 base pairs long. The remainder of the fourth domain is encoded in the last three exons, which are 191, 143, and approximately 740 base pairs long, respectively. This unusual structure is shared with the bovine IRBP gene. A large (1.7 kbp) fragment appears to have been lost from the 3'-noncoding region of the last human exon. We conclude that the human and bovine genes have similar evolutionary histories.

Amino Acid Sequence↗
PubMed1990

IRBP: preparation and characterization of site-specific monoclonal antibodies.

Interstitial retinoid binding protein (IRBP) is a 136,000 molecular weight photoreceptor cell protein which is a highly pathogenic autoantigen for the induction of experimental autoimmune uveitis (EAU). In this study we produced a series of monoclonal antibodies (MAbs) which define different epitopes in the native molecule. These MAbs were further subdivided into three distinct groups based on a radioimmunoassay, and by ELISA assay using native IRBP and synthetic peptides corresponding to its entire amino acid sequence. Group I MAbs (MAbD7-B1 and MAbC6-B4) bound to native IRBP but not to any synthetic peptides, suggesting that their antigenic epitopes are strictly conformation dependent. Group II MAbs (MAbC7-D3 and MAbG8-H4) bound weakly to multiple peptides which shared amino acid sequence similarity located within each of four homology domains indicating that these epitopes are also conformation dependent. In group III (MAbH3-B5, MAbH7-A5, and MAbB6-D12) MAb binding was localized to a specific peptide. The MAbH3-B5 binding site was further refined to amino acid positions 361 to 367 in the native molecule. MAbH3-B5 was also useful in localizing IRBP in the mouse retina by immunohistochemical techniques. The application of these MAbs in the study of EAU and interphotoreceptor transport mechanisms is discussed.

Amino Acid Sequence↗
PubMed1989

Human interstitial retinoid binding protein. A potent uveitopathogenic agent for the induction of experimental autoimmune uveitis.

Human interstitial retinoid binding protein (HIRBP) is a 136,000 m.w. photoreceptor cell protein which transports retinoids between the retina and the retinal pigment epithelium of the eye. The amino acid sequence of HIRBP suggests that the molecule consists of four continuous homology domains which arose by several gene duplications some 600 to 800 million years ago. When injected into susceptible animal species, including primates, it induces an experimental autoimmune uveitis (EAU), a predominantly T cell-mediated autoimmune disease of the retina and uveal tract of the eye, and the pineal gland. In order to further refine specific sites in HIRBP responsible for its uveitopathogenicity, we synthesized 120 overlapping peptide corresponding to its entire 1262 amino acid sequence, and tested each peptide for its ability to induce an EAU in Lewis rats. Five peptides with extensive amino acid sequence homology, designated HIRBP 715, HIRBP greater than 730 and HIRBP 745, HIRBP 778, and HIRBP 808 were uveitopathogenic when used at a 50 micrograms immunizing dose. The most potent peptide for the induction of EAU was HIRBP 715 (amino acid positions 521 to 540). In dose response studies as little as 0.1 microgram/animal was capable of inducing an inflammatory response. In addition, peptide HIRBP 946 which corresponds to the mid portion of peptide HIRBP 715 and contains only eight amino acids (RTATAAEE) was uveitopathogenic under our experimental conditions. Our study identifies multiple uveitopathogenic sites in HIRBP and further defines the amino acids necessary for the induction of EAU in one of these sites.

Amino Acid Sequence↗
PubMed1989

Retinoids bound to interstitial retinol-binding protein during light and dark-adaptation.

High-performance liquid chromatography was used to determine the types and amounts of retinoids bound to interstitial retinol-binding protein (IRBP) during light- and dark-adaptation in frogs. IRBP was separated from CRBP and CRA1BP by ion-exchange chromatography and quantitated by determining the amount of Serva Blue R dye bound to it in stained sodium dodecyl sulfate polyacrylamide gels. The amount of IRBP was not significantly different in light- and dark-adapted eyes (0.15 +/- 0.05 nmol/eye compared with 0.18 +/- 0.08 nmol/eye). In the dark-adapted state, IRBP bound mainly 11-cis retinol and 11-cis retinal in quantities that summed to about 1 mol/mol IRBP. After the onset of light-adaptation, all-trans retinol increased from its very low dark-adapted level, peaked at 0.2 mol/mol IRBP and then declined to the dark-adapted level again. Concomitantly, the total retinoid bound to IRBP fell, mainly because there was a drop in the amount of 11-cis retinal. During dark-adaptation, the amount of 11-cis retinal increased. No significant changes were seen in the amount of 11-cis retinol in light and darkness. These findings support the hypothesis that when rhodopsin is bleached IRBP transports all-trans retinol from the retina to the pigment epithelium and that it delivers 11-cis retinal to the rod outer segments for rhodopsin regeneration.

Animals↗