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Biomedical subjects

K O Tan

Publications and source records attributed to K O Tan.

7 recordsLinked to original sources
PubMed1999

F1Aalpha, a death receptor-binding protein homologous to the Caenorhabditis elegans sex-determining protein, FEM-1, is a caspase substrate that mediates apoptosis.

Apoptosis is an evolutionarily conserved process that is critical for tissue homeostasis and development including sex determination in essentially all multicellular organisms. Here, we report the cloning of an ankyrin repeat-containing protein, termed F1Aalpha, in a yeast two-hybrid screen using the cytoplasmic domain of Fas (CD95/APO-1) as bait. Amino acid sequence analysis indicates that F1Aalpha has extensive homology to the sex-determining protein FEM-1 of the Caenorhabditis elegans, which is required for the development of all aspects of the male phenotype. F1Aalpha associates with the cytoplasmic domains of Fas and tumor necrosis factor receptor 1, two prototype members of the "death receptor" family. The F1Aalpha protein also oligomerizes. Overexpression of F1Aalpha induces apoptosis in mammalian cells, and co-expression of Bcl-XL or the dominant negative mutants of either FADD or caspase-9 blocks this effect. Deletion analysis revealed the center region of F1Aalpha, including a cluster of five ankyrin repeats to be necessary and sufficient for maximum apoptotic activity, and the N-terminal region appears to regulate negatively this activity. Furthermore, F1Aalpha is cleaved by a caspase-3-like protease at Asp(342), and the cleavage-resistant mutant is unable to induce apoptosis upon overexpression. F1Aalpha is therefore a member of a growing family of death receptor-associated proteins that mediates apoptosis.

Amino Acid Sequence↗
PubMed1999

A novel BH3-like domain in BID is required for intramolecular interaction and autoinhibition of pro-apoptotic activity.

Upon activation of the Fas apoptotic signaling pathway, Bid, a "BH3 domain-only" pro-apoptotic molecule, is cleaved by caspase-8 into a 6.5-kDa N-terminal and a 15-kDa BH3 domain-containing C-terminal fragment, referred to as t(n)-Bid and t(c)-Bid, respectively. t(c)-Bid is a more potent inducer of apoptosis than full-length Bid, suggesting that the N-terminal region of Bid has an inhibitory effect on its pro-apoptotic activity. Here, we report the identification of a novel BH3-like motif (amino acid residues 35-43) in t(n)-Bid. Although Bid does not homodimerize, t(n)-Bid is able to associate avidly with t(c)-Bid. Site-directed mutagenesis revealed that both the novel BH3-like and BH3 domains are necessary for direct binding between t(n)-Bid and t(c)-Bid. While full-length Bid does not associate with t(n)-Bid, substitution of Leu(35), a critical residue in mediating t(n)-Bid/t(c)-Bid interaction, with Ala in full-length Bid is sufficient to establish Bid/t(n)-Bid interaction. Interestingly, the L35A Bid mutant is as effective as t(c)-Bid in inducing apoptosis and binding Bcl-X(L). We propose that the intramolecular interaction involving the BH3-like and BH3 domains serves to regulate the pro-apoptotic potential of Bid.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of avian muscle titin.

Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils. To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts. Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils. Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction. Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin. Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site. This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule.

Amino Acid Sequence↗
PubMed1993

Temporal sequence of changes in tear film composition during sleep.

Overnight eye closure induces a shift in the nature and composition of the tear film, from a dynamic reflex tear-rich to a stagnant secretory IgA-rich layer. This is accompanied by the induction of a state of sub-clinical inflammation, as evidenced by increases in albumin levels, plasminogen activation, conversion of complement C3 to C3c, and the recruitment of polymorphonuclear (PMN) cells into the tear film. To determine the time course and functional relationship between these potentially interdependent processes, tear samples were collected from ten non-contact lens wearers after 1, 2, 3 and 5 hours of sleep. A subgroup of 6 subjects also self-collected tear samples after 8 hours of sleep. Tear samples were analysed for albumin by quantitative immunofixation assay, secretory IgA (sIgA) by radial immunodiffusion assay, plasmin-like activity using a chromogenic substrate, and complement C3 to C3c conversion by immunoblot assay. Epithelial and PMN cells in the precorneal tear film were recovered from corneal washings from the same subjects after 1, 3, 5 and 8 hours of sleep, and quantified. Results revealed that, unlike epithelial cells which exhibited a slow progressive accumulation as a function of the period of sleep, PMN cell concentration exhibited a lag phase, with recruitment occurring after between 3 and 5 hours of eye closure. This was preceded by plasminogen activation, increases in albumin and sIgA levels, and complement C3 to C3c conversion, all of which occurred within 1 to 3 hours after eye closure. Plasmin-like activity appeared to plateau after 3 hours and then decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid.

To investigate the tear film in the closed eye, microliter tear samples were collected without overt reflex stimulation throughout the diurnal cycle, with closed eye samples recovered immediately upon eye opening. Samples were subjected to agarose, polyacrylamide, and two-dimensional electrophoresis, coupled with immunofixation, immunoblot, and lectin blot assays. Major protein constituents were densitometrically and immunologically quantified. Results revealed a distinct progression in composition from reflex to open to closed eye tear samples. Total protein increased from 6.0 to 9.0 to 18.0 mg/ml, secretory IgA increased from less than 0.23 to 0.85 to 8.40 mg/ml, and serum albumin increased from 0.02 to 0.06 to 1.10 mg/ml. In contrast, concentrations of the major reflex tear components (lysozyme, lactoferrin, and tear specific prealbumin) remained essentially static. Immunoblot assay for complement C3 and C3c revealed that eye closure was associated with C3 activation. Results indicate that: (1) the reflex and closed eye tear layers represent opposite extremes in composition and likely origins, with open eye tear film suggesting an intermediate origin; (2) reflex tears are derived from a neurologically inducible lacrimal or accessory gland secretion composed almost exclusively of lysozyme, lactoferrin, tear specific prealbumin, and a minor mixed alpha to beta globulin fraction; (3) upon eye closure, reflex secretion ceases or greatly diminishes, with ongoing slower flow maintained by a constitutive secretion composed almost exclusively of secretory IgA; (4) the closed eye environment induces a subclinical inflammation, accounting in part for the marked rise in albumin concentration. This increase, coupled with that of secretory IgA, may play a critical role in protecting the closed eye environment from pathogens. However, this may render the closed eye environment particularly vulnerable to inflammatory and immune-mediated pathological processes, such as those seen with extended wear soft contact lenses.

Adult↗