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Biomedical subjects

L Y Lin

Publications and source records attributed to L Y Lin.

At least 55 records · Page 3Linked to original sources
PubMed1994

Functional integrity of metallothionein genes in testicular cell lines.

The presence and inducibility of the major cadmium (Cd) chelating protein metallothionein (MT) in testicular cells has been controversial. In this study, the induction and production of MT in testicular cells were studied using mouse Leydig and Sertoli cell lines. Metal accumulation was studied by subjecting the cells to increasing levels of Cd. The presence of transcription factors for MT synthesis was analyzed by transfecting the cells with a reporter gene under the control of the MT promoter. The dose- and time-dependent induction of MT were conducted by Northern analyses. Expression of MT genes occurred in both Leydig and Sertoli cells. To avoid cross hybridization of the MT probe with mRNAs encoding testicular metal binding proteins and to investigate the integrity of MT mRNA, isoMT mRNA identification and primer extension experiments were performed. Those studies show that the induced mRNA indeed encodes MT. The biosynthesis of MT was confirmed by following 35S-cysteine incorporation into the protein. Finally, cadmium tolerance of testicular cells is compared with that of fibroblast cells. By these studies, we conclude that the MT genes are functional and inducible in testicular cells.

Animals↗
PubMed1994

Participation of cytochrome P450-2B and -2D isozymes in the demethylenation of methylenedioxymethamphetamine enantiomers by rats.

The cytochrome P450 isozymes in rat liver microsomes that catalyze the demethylenation of methylenedioxymethamphetamine enantiomers to the corresponding dihydroxymethamphetamine were characterized. Dihydroxymethamphetamine formation in liver microsomes from male Sprague-Dawley rats exhibited multienzyme kinetics, with Km values in the micromolar/millimolar range. The stereoselectivity [(+)-isomer versus (-)-isomer] varied from 0.78 to 1.94 after pretreatment of the rats with phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, or pyrazole, suggesting that different isozymes participate in the reaction. The low-Km demethylenation was not induced by these compounds and was not inhibited by antibodies raised against CYP2C11. Liver microsomes from female Dark-Agouti rats, a strain genetically deficient in CYP2D1, exhibited demethylenation activities that were 9% of those in microsomes from male Sprague-Dawley rats. The low-Km demethylenation was also inhibited by CYP2D substrates such as sparteine, bufuralol, or desipramine and was almost completely inhibited by antibodies against P450 BTL, which belongs to the CYP2D family. The higg-Km demethylation activity was induced by phenobarbital and pregnenolone-16 alpha-carbonitrile and the activity in both untreated and phenobarbital-induced microsomes was suppressed by anti-CYP2B1 IgG. Experiments with IgG raised against cytochrome b5 suggested that the hemoprotein contributed to the low-Km activity but not the high-Km activity. These results indicate that cytochrome P450 isozymes belonging to the CYP2D subfamily catalyze demethylenation with low Km values and that the reaction occurring with high Km values is likely to be mediated by members of the CYP2B family, but with the possible participation of other phenobarbital-inducible isoforms.

3,4-Methylenedioxyamphetamine↗
PubMed1993

A long-term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443.

Although technically possible, limb allotransplantation has not been applied clinically. The skin component is especially antigenic, requiring high immunosuppressant doses with an unacceptable toxicity profile. RS-61443, an experimental mycophenolic acid ester that inhibits lymphocyte proliferation without major systemic toxicity, was tested as an immunosuppressant to prevent rejection of rat hindlimb allotransplants. Utilizing Brown-Norway donors and F344 recipients to provide a major mismatch at the MHC, midfemur orthotopic limb transfer was performed with microsurgical repair of femoral vessels and sciatic nerve. Four primary groups were studied: autografts (n = 4); untreated allografts (n = 6); allografts receiving CsA 10 mg/kg for 20 days, then twice per week (n = 6); and allografts receiving RS-61443 30 mg/kg/day (n = 6). Skin and soft tissues were biopsied to assess rejection. Autografts had indefinite limb survival, while untreated allografts had complete acute rejection within 10-12 days. Five of the six CsA rats developed delayed mild-moderate acute rejection within 6 months. In contrast, 5 of the 6 RS-61443 rats had no rejection after at least 32 weeks, while the sixth rat developed only slight rejection on skin biopsy. All animals regained full sensation and partial functional return. RS-61443 is highly effective as a primary immunosuppressant for hindlimb allotransplantation. The disturbing moderate rejection observed in CsA animals, which was absent with RS-61443, may significantly hamper function of transplanted limbs.

Animals↗
PubMed1992

Regiochemical differences in cytochrome P450 isozymes responsible for the oxidation of methylenedioxyphenyl groups by rabbit liver.

The cytochrome P450 isozymes catalyzing the oxidation of the methylenedioxyphenyl compounds methylenedioxybenzene (MDB) and methylenedioxyamphetamine (MDA) have been investigated in rabbit liver preparations. The aromatic ring in MDB undergoes both demethylenation to catechol and aromatic hydroxylation to sesamol, whereas that in MDA undergoes only demethylenation to dihydroxyamphetamine. Formation of catechol and sesamol from MDB in microsomal incubation mixtures was enhanced about 5- and 3-fold, respectively, by pretreatment of the rabbits with phenobarbital, which induced CYP2B4 and CYP4B1. The cytochrome P450 isozyme responsible for aromatic hydroxylation of MDB was induced by beta-naphthoflavone and was inhibited by alpha-naphthoflavone. Microsomal demethylenation of MDA was minimally sensitive to pretreatment of the rabbits with phenobarbital, beta-naphthoflavone, pyrazole, or rifampicin. However, MDA competitively inhibited the N-demethylation of erythromycin. Antibodies against CYP2B4, but not those against CYP4B1, caused a marked inhibition of the demethylenation and aromatic hydroxylation of MDB. Antibodies against CYP2C3 did not inhibit the demethylenation of MDA, nor did substrates or inhibitors of the CYP2D family except for bufuralol. MDB and MDA were both capable of forming metabolic intermediate complexes, and the rate of complex formation was accelerated by phenobarbital induction. Reconstitution experiments with CYP2B4 suggested that phenobarbital-inducible complex formation from MDA was not due to the carbene pathway involving the methylenedioxy group but was due to oxidation of the amino group. These results indicate that CYP2B4 oxidizes different regions of methylenedioxyphenyl compounds depending on their structure. MDB undergoes oxidation at the methylenedioxy group (major) and the benzene ring (minor). MDA is oxidized at the alkylamino side chain at the nitrogen and alpha-carbon. The results suggested that one or more constitutive isoforms (probably unknown) of cytochrome P450 present in rabbit liver microsomes are primarily responsible for MDA demethylenation but that CYP3A6 contributes slightly.

3,4-Methylenedioxyamphetamine↗
PubMed1991

Molecular cloning of Chinese hamster metallothionein II gene and its 5' flanking region.

A genomic DNA clone containing Chinese hamster metallothionein II (MTII) gene and its 5' flanking region was isolated from Cd resistant Chinese hamster ovary (CHO) cells. DNA sequence analysis showed that there are three exons and two introns in the structure of the MTII gene. Further characterization of the 5' flanking region reveals the possible transcription initiation site, metal responsive element and basal-level enhancer sequence. Putatively, this is the promoter region of CHO MTII gene.

Animals↗
PubMed1991

[Stress analysis of post-reinforced maxillary anterior teeth].

This study used plane stress analysis to survey the stress distribution of maxillary anterior teeth reinforced with posts of various materials, and maxillary anterior teeth without posts. Two-dimensional finite element models of the maxillary central incisor were subjected to a simulated biting force of 200N applied under various occlusal contact conditions. The results of this study suggest that posts can transfer stress to the dentin and supporting alveolar bone, and that there is a more even stress distribution than in teeth without posts. Also, with post reinforcement the relative stress magnitudes and concentrations at the lingual surface of the post cervical area indicates that conservative enlargement of the root canal of the lingual surface is necessary. Finally, it seems that posts material with the same elastic modulous as the dentin may be the best choice.

Dental Stress Analysis↗
PubMed1990

Primary sequence of duck metallothionein.

Only one metallothionein appears in domesticated duck upon zinc induction. The complete amino acid sequence has been elucidated. This metallothionein has the same sequence as the chicken metallothionein, as determined by chemical sequencing of overlapping peptides produced by selective proteinase digestion and confirmed by mass spectrometry. The observation that animals of divergent origins share a common gene product presents an example of extreme conservation of a stress-inducible protein.

Amino Acid Sequence↗
PubMed1990

Complete homology in metallothionein from two genera of ducks and their hybrids.

Metallothionein purified from two genera of domesticated duck, Anas platyrhnchos and Cairina muschata, and their hybrid were analyzed and shown to consist of one identical amino acid sequence. Since most vertebrates carry two or more isospecies of this inducible, thiol rich, metal sequestering protein, this finding suggests that duck metallothionein is evolutionary primitive.

Amino Acid Sequence↗
PubMed1990

Maternal transmission of mitochondrial DNA in ducks.

Maternal transmission of mitochondrial DNA (mtDNA) has been studied in amphibians, insects and mammals, but little is known about mtDNA inheritance in the ovaripirous avian species. In this study, we have constructed the physical maps of mitochondrial genomes from two different genera of ducks (Cairina and Anas) and taken advantage of the availability of their hybrids to demonstrate that mtDNA is maternally inherited.

Animals↗
PubMed1990

Pigeon metallothionein consists of two species.

Two isospecies of metallothionein, a cysteine-rich protein that binds metals, exist in all mammals examined, but only one in some invertebrates and lower animals. Lower vertebrates such as fish and birds have one or two metallothionein genes depending upon the organism. In this study, we show by amino acid sequence determinations that two isospecies of metallothionein, 75% homologous to each other, can be induced by zinc to accumulate in pigeon livers. This is in contrast to single isospecies found in chicken and duck. Each of these two sequences consists of 63 amino acids, with all 20 cysteines in positions held invariant in most if not all class I mammalian metallothioneins. One of these two pigeon isometallothioneins is terminated with histidine at the carboxyl end, which is apparently unique to avians. Its sequence differs from that of duck and chicken by only four substitutions and is the predominant isospecies that accumulates upon induction. The other pigeon metallothionein has lysine at its carboxyl terminus and is devoid of arginine. None of these isospecies carries any aromatic amino acid, which is also characteristic of all higher metallothioneins. As this is the first demonstration with sequence data that two isospecies of metallothionein indeed exist in birds, these results suggest that pigeon metallothionein genes evolved from an ancestral form through duplication and mutation upon specification.

Amino Acid Sequence↗
PubMed1990

Heavy metal accumulation and transport through detention ponds receiving highway runoff.

Sediment accumulations and associated heavy metal concentrations into retention/detention ponds receiving highway runoff from several ponds and metal concentrations in various sediment layers were analyzed to determine the extent of vertical migration. Extraction of heavy metals had been evaluated using various solutions including the United States Environmental Protection Agency Toxicity Characteristics Leaching Procedures (TCLP). Also, sediments were incubated under various conditions of redox potential and pH to investigate the effects of changes in sediment conditions on the stability of metal-sediment associations.

Florida↗
PubMed1990

[Cloning and sequencing of hemolysin gene from clinical Aeromonas hydrophila].

Aerolysin is one of the important virulence factors for Aeromonas hydrophila infection. To understand the characteristics of the aerolysin gene in clinical A. hydrophila, a genomic library was constructed by using pUC19 as cloning vector. The positive clone containing the aerolysin gene was selected by the colony hybridization method, using the aerolysin probe (an 48 mer oligonucleotide) of aquatic A. hydrophila. Four positive out of 1,200 transformants were obtained from the primarily screened genomic library, and were further identified for specific binding capability by the dot blot hybridization method. One of three positive clones, designated as pAH-1, was further analyzed by the restriction mapping technique. For further sequencing the aerolysin gene nucleotides, the 3.0 kb fragment of A. hydrophila isolated from pAH-1 was cloned into pBluescriptII, pKS+, resulting in plasmid pKAH-1. Subclones of pKAH-1 were constructed and determined for the presence of 48 mer aerolysin gene sequence by dot blot hybridization method. These (subclones pKS+SS and pSK+SS) were selected for nucleotide sequencing by single-stranded dideoxy sequencing method. The nucleotide sequence similarity coefficient between clinical and aquatic strains of A. hydrophila was 76%; whereas between clinical strains of A. hydrophila and A. sobria was 71%. Since the length of the A. hydrophila DNA fragment cloned into pAH-1 is 3.0 kb, a larger size than the aerolysin gene of aquatic A. hydrophila (1.45 kb) and, also, includes 3' region of aerolysin gene in aquatic A. hydrophila, the indication is that the clone, pKAH-1, contains an entire aerolysin gene of A. hydrophila. The similarity between a small region (containing 8 amino acids) of the aerolysin in the clinical A. hydrophila and the alpha toxin of Staphylococcus aureus suggests that there is functional significance in this region.

Aeromonas↗
PubMed1989

Metalloforms of metallothionein induced by parenteral copper: the influence of route of administration.

Previous results regarding the two metalloforms of MT which accumulate in chick liver following the parenteral administration (ip) of copper were discussed. One metalloform, which is exclusively zinc, was suggested to reflect the marked accumulation of hepatic zinc following copper injection. The present report shows that there is a marked difference in hepatic zinc accumulation if copper is administered iv. Under these conditions there is virtually no change in hepatic zinc and thus MT produced under these conditions appears to contain only copper. We suggest the the changes in zinc metabolism as effected by copper when given intraperitoneally reflect a secondary response analogous to that observed when iron is similarly administered.

Animals↗
PubMed1989

[Determination of protein concentration in human saliva].

Seven colorimetric methods (Biuret, Lowry, a modified Lowry technique using bicinchoninic acid, Coomassie brilliant blue G (CBBG) dye-binding in phosphoric acid, perchloric acid or hydrochloric acid, and bromophenol blue dye-binding) were evaluated for determination of protein concentration in human whole saliva. Using bovine serum albumin (BSA), mucin, and thyroglobulin as protein standards to calculate salivary protein concentration gave different results among the assays used and even with the different standards of the same method. Among the seven methods used, the CBBG dye-binding assay in hydrochloric acid appeared to provide the most accurate estimation of protein concentration in human saliva. Using BSA as a standard, the mean values of human salivary proteins from 39 healthy individuals ranged from 0.72 to 2.45 mg/ml. The optimum conditions for the CBBG dye-binding assay in hydrochloric acid are: (1) the absorbance at 595 nm was measured between 15 to 30 minutes after addition of reagent. (2) the optimum concentration of dye in hydrochloric acid was 0.06-0.12% (w/v) for the assay. (3) the dye reagent was stable within one month.

Colorimetry↗