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T Y Lin

Publications and source records attributed to T Y Lin.

At least 19 recordsLinked to original sources
PubMed1992

Construction and expression of nonsense suppressor tRNAs which function in plant cells.

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.

Anticodon↗
PubMed1992

Smooth-pursuit eye tracking in first-episode psychotic patients and their relatives.

We wished to determine the specificity of smooth-pursuit eye tracking dysfunction to schizophrenia and the prevalences of dysfunction among functionally psychotic and normal individuals. Therefore, we investigated pursuit tracking in a large sample of psychotic patients, normal subjects, and first-degree relatives (N = 482). Patients were recruited as part of an epidemiological study of first-episode psychosis that used a broadly based referral network to identify all cases in a major metropolitan area over a 2 1/2-year period. Patients received diagnoses of schizophrenia, schizophreniform disorder, psychotic mood disorder, and paranoid or other psychotic disorder based on the third edition of the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 1980). The distribution of tracking performance was bimodal for the schizophrenic patients and their relatives, perhaps reflecting major gene action. Moreover, poor tracking ran in families. Pursuit tracking dysfunction was relatively specific to schizophrenic patients and their relatives and occurred infrequently in other psychotic patients and normal subjects.

Acute Disease↗
PubMed1991

Evaluating the effects of a single amino acid substitution on both the native and denatured states of a protein.

For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange. We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols. We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein. Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state. The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same. By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms.

Amino Acid Sequence↗
PubMed1991

Refined X-ray crystal structures of the reactive site modified ovomucoid inhibitor third domains from silver pheasant (OMSVP3*) and from Japanese quail (OMJPQ3*).

Tetragonal and triclinic crystals of two ovomucoid inhibitor third domains from silver pheasant and Japanese quail, modified at their reactive site bonds Met18-Glu19 (OMSVP3*) and Lys18-Asp19 (OMJPQ3*), respectively, were obtained. Their molecular and crystal structures were solved using X-ray data to 2.5 A and 1.55 A by means of Patterson search methods using truncated models of the intact (virgin) inhibitors as search models. Both structures were crystallographically refined to R-values of 0.185 and 0.192, respectively, applying an energy restraint reciprocal space refinement procedure. Both modified inhibitors show large deviations from the intact derivatives only in the proteinase binding loops (Pro14 to Arg21) and in the amino-terminal segments (Leu1 to Val6). In the modified inhibitors the residues immediately adjacent to the cleavage site (in particular P2, P1, P1') are mobile and able to adapt to varying crystal environments. The charged end-groups, i.e. Met18 COO- and Glu19 NH3+ in OMSVP3*, and Lys18 COO- and Asp19 NH3+ in OMJPQ3*, do not form ion pairs with one another. The hydrogen bond connecting the side-chains of Thr17 and Glu19 (i.e. residues on either side of the scissile peptide bond) in OMSVP3 is broken in the modified form, and the hydrogen-bond interactions observed in the intact molecules between the Asn33 side-chain and the carbonyl groups of loop residues P2 and P1' are absent or weak in the modified inhibitors. The reactive site cleavage, however, has little effect on specific interactions within the protein scaffold such as the side-chain hydrogen bond between Asp27 and Tyr31 or the side-chain stacking of Tyr20 and Pro22. The conformational differences in the amino-terminal segment Leu1 to Val6 are explained by their ability to move freely, either to associate with segments of symmetry-related molecules under formation of a four-stranded beta-barrel (OMSVP3* and OMJPQ3) or to bind to surrounding molecules. Together with the results given in the accompanying paper, these findings probably explain why Khyd of small protein inhibitors of serine proteinases is generally found to be so small.

Amino Acid Sequence↗
PubMed1991

Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo.

The asymmetry of the dorsal-ventral pattern of the Drosophila embryo appears to depend on the ventral activation of the transmembrane Toll protein. The Toll protein is found around the entire dorsal-ventral circumference of the embryo, and it appears to act as a receptor for a ventral, extracellular signal and to then relay that signal to the cytoplasm in ventral regions of the embryo. Three of five recessive loss-of-function alleles of Toll are caused by point mutations in the region of the cytoplasmic domain of Toll that is similar to the mammalian interleukin-1 receptor, supporting the hypothesis that Toll acts as a signal-transducing receptor. Nine dominant gain-of-function alleles that cause Toll to be active in dorsal, as well as ventral, regions of the embryo are caused by mutations in the extracellular domain. Three of the dominant alleles appear to cause the protein to be constitutively active and are caused by cysteine-to-tyrosine changes immediately outside the transmembrane domain. All six of the remaining dominant alleles require the presence of a wild-type transmembrane Toll protein for their ventralizing effect and all encode truncated proteins that lack the transmembrane and cytoplasmic domains.

Alleles↗
PubMed1991

Inactivation of human fibroblast collagenase by chloroacetyl N-hydroxypeptide derivatives.

When human fibroblast collagenase was incubated with ClCH2CO-(N-OH)Leu-Ala-Gly-NH2 (2-5 mM) in Tris buffer, pH 7.4 at 25 degrees C, a slow, time-dependent inhibition of the enzyme was observed. Dialysis against a buffer to remove free inhibitor did not reactivate the enzyme. A reversible competitive inhibitor, phthaloyl-GlyP-Ile-Trp-NHBzl (50 microM) partially protected the enzyme from inactivation by the compound. From the concentration dependent rates of inactivation Ki = 0.5 +/- 0.1 mM and k3, the rate constant for inactivation = 3.4 +/- 0.3 x 10(-3) min-1 were determined. The inactivation followed the pH optimum (6.5-7.0) for the enzyme activity, suggesting direct involvement of the same active site residue(s). The reaction mode of the inhibitor may be analogous to that of the inactivation of Pseudomonas aeruginosa elastase [Nishino, N. and Powers, J. (1980) J. Biol. Chem., 255, 3482] in which the catalytic glutamate carboxyl was alkylated by the inhibitor after its binding to enzyme through the hydroxamic Zn2+ ligand. All carboxyl groups in the inactivated collagenase were modified with 0.1 M ethyl dimethylaminopropyl carbodiimide/0.5 M glycinamide in 4 M guanidine at pH 5. The inactivator-affected carboxyl group was then regenerated with 1 M imidazole at pH 8.9, 37 degrees C for 12 h and the protein was radiolabeled with 3H-glycine methyl ester and carbodiimide to incorporate 0.9 residue glycine per mol enzyme.

Amino Acid Sequence↗
PubMed1991

[Bacteremia in febrile children].

Bacteremia in children under 2 years of age is not uncommon. Several studies indicated that a small proportion of young children with fever who do not appear to be seriously ill may have bacteremia, and a small proportion of this subgroup may go on to develop serious illness such as meningitis. The rationale for this study was to assess both the incidence of bacteremia in febrile children in Taipei area and the predictive value of the white blood cell count and degree of body temperature for bacteremia. Three hundred febrile children (B. T. greater than or equal to 39 degrees C), without underlying disease or readily recognizable viral illness, who visited the emergency service of Chang Gung Memorial Hospital, were enrolled. Blood cultures and complete blood counts were obtained. Clinically important pathogens were identified in 6 of 300 cases (2%). The incidence and pathogens of various age group were: (1) Neonate, 2/15 (13%), one each of Group D streptococcus & streptococcus viridans; (2) 1 month to 1 year old, 3/66 (5%), 2 Escherichia coli and 1 Salmonella Group D; (3) 1 to 2 years old, 0/49; (4) 2 to 5 years old, 0/72; (5) 5 to 10 years old, 0/67; (6) 10 to 15 years old, 1/26 (4%), Salmonella typhosa. Neither the white blood cell count, the absolute segmented or band neutrophil count, nor the degree of body temperature had predictive value for bacteremia.

Adolescent↗
PubMed1990

Gestational ethanol consumption on tissue amino acid levels: decreased free histidine and tryptophan in fetal tissues with concomitant increase in urinary histamine excretion.

The effects of ethanol consumption during pregnancy on maternal, placental, and fetal tissue amino acid levels and metabolism were investigated. Pregnant Sprague-Dawley rats were given 35% ethanol-calorie liquid diet, ad libitum, from gestation day 7 to 21. Control rats were pair-fed with isocaloric sucrose substituted for ethanol. Ethanol consumption decreased fetal body weight and increased placental weight. Twenty-four amino acids were determined in six tissues (maternal plasma and liver, placenta, fetal plasma, liver, and brain) by HPLC with orthophthalaldehyde derivatization. The effects of ethanol on free amino acid levels differed from tissue to tissue. In general, ethanol affected more amino acids in maternal plasma, fetal plasma, and liver. Maternal liver, placenta, and fetal brain amino acids were more resistant to ethanol effect. Two essential amino acids, histidine and tryptophan, were consistently decreased in fetal tissues by maternal ethanol consumption. The values (ethanol vs. control, nmole/ml or g, mean +/- SEM, N = 20) of fetal plasma, liver, and brain for histidine were 51.8 +/- 6.0 vs. 85.3 +/- 4.5 (p = 0.001), 269.0 +/- 26.4 vs. 503.7 +/- 47.3 (p = 0.0004), and 117.9 +/- 7.7 vs. 154.6 +/- 8.7 (p = 0.0055), respectively; and for tryptophan were 105.7 +/- 3.1 vs. 132.2 +/- 4.1 (p = 0.0001), 128.8 +/- 3.7 vs. 144.3 +/- 6.0 (p = 0.0407), and 83.4 +/- 7.2 vs. 103.6 +/- 3.2 (p = 0.0198), respectively. Histidine was also decreased in placenta by ethanol (138.1 +/- 6.6 vs. 189.1 +/- 11.8 nmole/g, p = 0.0014).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1990

Production and purification of prostromelysin and procollagenase from IL-1 beta-stimulated human gingival fibroblasts.

Conditions were established to stimulate human gingival fibroblast explant cultures to synthesize milligram quantities of the metalloproteinase proenzymes, prostromelysin and procollagenase. To stimulate enzyme production, cells were treated with 1 nM recombinant human IL-1 beta for approximately 7 days under serum free conditions. Using a combination of rapid column chromatography steps, approximately 10 milligrams of prostromelysin and 5 milligrams of procollagenase were purified from 1 liter of conditioned media. Prostromelysin electrophoresed as a doublet with molecular weights of 55,57 kD, whereas, procollagenase migrated with slightly lower molecular weights of 52, 54 kD. Both proenzymes were treated with trypsin or aminophenylmercuric acetate to generate active species. The molecular weights of the active enzymes were approximately 10 kD smaller than the proenzymes. Active enzymes were inhibited by metal chelators and the natural metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP), but not by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). Activated stromelysin degraded a number of substrates including transferrin, proteoglycan monomer, proteoglycan aggregated with hyaluronic acid, and substance P. By contrast, collagenase degraded interstitial type I collagen and the peptide thioester, Ac-Pro-Leu-Gly-SCH(iBu)Co-Leu-GlyOEt. Identity of both enzymes were confirmed by amino-terminal protein sequence analysis as well as by immunoblot analysis using monoclonal antibodies.

Amino Acid Sequence↗
PubMed1989

Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure.

Thioredoxin contains a single disulfide bond that can be reduced without perturbing significantly the structure of the enzyme. Upon reduction of the disulfide, protein stability decreases. We have experimentally tested the expected linkage relationship between disulfide bond formation and protein stability for thioredoxin. In order to do this, it is necessary to measure the equilibrium constant for disulfide bond formation in both the folded and unfolded states of the protein. Using glutathione as a reference species, we have measured the equilibrium constant for forming the disulfide bond (effective concentration) in thioredoxin as a function of urea concentration. As a control, we show that urea per se does not interfere with our measurements of thiol-disulfide equilibrium constants. Comparison of the values obtained for disulfide bond formation in the folded and unfolded states with the free energies for unfolding oxidized and reduced thioredoxin using circular dichroism confirms the expected linkage relationship. The urea dependence of thiol-disulfide equilibria provides a sensitive assay for folded structure in peptides or proteins. The method should also be useful to evaluate the stabilizing or destabilizing effect of natural or genetically engineered disulfides in proteins. In future work, the effects of amino acid substitutions on disulfide bond formation could be evaluated individually in the native and unfolded states of a protein.

Bacterial Proteins↗
PubMed1989

Synthesis of a high specific activity 125I-labeled analog of PK 11195, potential agent for SPECT imaging of the peripheral benzodiazepine binding site.

The peripheral benzodiazepine binding site ligand PK 11195 has been 125I-labeled by direct displacement of aromatic chlorine under solid-state conditions in 50-76% radiochemical yield and greater than 94% radiochemical purity. Purification by high pressure liquid chromatography increased the specific activity of the product from an initial 15-17 Ci/mmol to a final activity of 260-910 Ci/mmol. To determine the affinity of this [125I]PK 11195 analog for human glioma cells, saturation experiments were performed on monolayers of U251 human glioblastoma cells. Scatchard analysis of saturation data demonstrated that the [125I]PK 11195 analog binds to a single class of sites with a KD of 8.0 +/- 1.7 nM and maximal binding of 3.8 +/- 0.1 pmol/mg protein. These values are similar to those obtained when [3H]PK 11195 was assayed in U251 cells (KD = 14 +/- 3.4, Bmax = 4.1 +/- 1.3) suggesting that iodination does not appreciably alter the binding of PK 11195 to human glioma cells. In vivo autoradiographic studies of brain in C6 glioma bearing rats demonstrate selective binding of the radioligand to the tumor. These results suggest that this [125I]PK 11195 analog may be a useful radiotracer for the study of peripheral benzodiazepine binding sites.

Animals↗
PubMed1989

Structure of human neutrophil elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84-A resolution.

Human neutrophil elastase (HNE) has been implicated as a major contributor to tissue destruction in various disease states, including emphysema. The structure of HNE, at neutral pH, in complex with methoxysuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone (MSACK), has been solved and refined to an R factor of 16.4% at 1.84-A resolution. Results are consistent with the currently accepted mechanism of peptide chloromethyl ketone inhibition of serine proteases, in that MSACK cross-links the catalytic residues His-57 and Ser-195. The structure of the HNE-MSACK complex is compared with that of porcine pancreatic elastase in complex with L-647,957, a beta-lactam inhibitor of both elastases. The distribution of positively charged residues on HNE is highly asymmetric and may play a role in its specific association with the underlying negatively charged proteoglycan matrix of the neutrophil granules in which the enzyme is stored.

Amino Acid Chloromethyl Ketones↗
PubMed1989

The role of social relationships in the course of first-episode schizophrenia and affective psychosis.

The Markers and Predictors of Psychosis study at the University of British Columbia addresses the role of psychosocial factors, such as social relationships, in predicting the short-term course of first-episode schizophrenia. Before their first episode of illness, schizophrenic subjects had fewer and less satisfactory social relationships than subjects with affective psychosis and a matched, normal comparison group. Nonfamily social resources were positively associated with good prognosis for both psychotic groups. While involvement with family members also predicted good prognosis among subjects with affective psychosis, family involvement had a negative association with outcome among schizophrenic subjects.

Adolescent↗