Biomedical subjects

S Lin

Publications and source records attributed to S Lin.

At least 253 records · Page 14Linked to original sources

PubMed1998

Does paternal occupational lead exposure increase the risks of low birth weight or prematurity?

The relation between paternal occupational lead exposure and low birth weight/prematurity was examined in a retrospective cohort study. Birth weight and gestational age (1981-1992), obtained from New York State birth certificates, were compared between lead-exposed and nonexposed workers. The exposed group (n = 4,256) consisted of births to male workers of reproductive age reported to the New York State Heavy Metals Registry. The control group (n = 2,318) consisted of the offspring of a random sample of male bus drivers, frequency matched by age and residence. There were no statistically significant differences in birth weight or gestational age between the exposed and the control groups. However, workers who had elevated blood lead levels for more than 5 years had a higher risk of fathering a child with low birth weight (risk ratio = 3.85, 95% confidence interval 1.5-9.88) or prematurity (risk ratio = 2.45, 95% confidence interval 1.03-5.84) than did controls after adjustment for paternal age, low maternal education, race, residence, gravidity, maternal spontaneous abortion history, perinatal complications, adequacy of prenatal care, and infant gender. The risks of low birth weight and prematurity increased with the duration of exposure to lead. Our results were limited by the inability to control for some potential confounders, such as pregravid underweight and maternal nutrition status.

Adult↗

PubMed1998

Mutations in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells.

The composition of the plasma membrane domains of epithelial cells is maintained by biosynthetic pathways that can sort both proteins and lipids into transport vesicles destined for either the apical or basolateral surface. In MDCK cells, the influenza virus hemagglutinin is sorted in the trans-Golgi network into detergent-insoluble, glycosphingolipid-enriched membrane domains that are proposed to be necessary for sorting hemagglutinin to the apical cell surface. Site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal. The region of the transmembrane domain required for apical transport included the residues most conserved among hemagglutinin subtypes. Several mutants were found to enter detergent-insoluble membranes but were not properly sorted. Replacement of transmembrane residues 520 and 521 with alanines converted the 2A520 mutant hemagglutinin into a basolateral protein. Depleting cell cholesterol reduced the ability of wild-type hemagglutinin to partition into detergent-insoluble membranes but had no effect on apical or basolateral sorting. In contrast, cholesterol depletion allowed random transport of the 2A520 mutant. The mutant appeared to lack sorting information but was prevented from reaching the apical surface when detergent-insoluble membranes were present. Apical sorting of hemagglutinin may require binding of either protein or lipids at the middle of the transmembrane domain and this normally occurs in detergent-insoluble membrane domains. Entry into these domains appears necessary, but not sufficient, for apical sorting.

Amino Acid Sequence↗

PubMed1998

T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function.

T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Th1 and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing interleukin (IL)-4, IL-5, and/or IL-10, but not interferon-gamma. One such gene, T1/ST2, is expressed stably on both Th2 clones and Th2-polarized cells activated in vivo or in vitro. T1/ST2 expression is independent of induction by IL-4, IL-5, or IL-10. T1/ST2 plays a critical role in Th2 effector function. Administration of either a mAb against T1/ST2 or recombinant T1/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 production in vivo following adoptive transfer of Th2 cells.

Animals↗

PubMed1998

Weight-value convergence of the SOM algorithm for discrete input.

Some insights on the convergence of the weight values of the self-organizing map (SOM) to a stationary state in the case of discrete input are provided. The convergence result is obtained by applying the Robbins-Monro algorithm and is applicable to input-output maps of any dimension.

Algorithms↗

PubMed1998

Toward a resolution of the introns early/late debate: only phase zero introns are correlated with the structure of ancient proteins.

We present evidence that a well defined subset of intron positions shows a non-random distribution in ancient genes. We analyze a database of ancient conserved regions drawn from GenBank 101 to retest two predictions of the theory that the first genes were constructed by exon shuffling. These predictions are that there should be an excess of symmetric exons (and sets of exons) flanked by introns of the same phase (positions within the codon) and that intron positions in ancient proteins should correlate with the boundaries of compact protein modules. Both these predictions are supported by the data, with considerable statistical force (P values < 0.0001). Intron positions correlate to modules of diameters around 21, 27, and 33 A, and this correlation is due to phase zero introns. We suggest that 30-40% of present day intron positions in ancient genes correspond to phase zero introns originally present in the progenote, while almost all of the remaining intron positions correspond to introns added, or moved, appearing equally in all three intron phases. This proposal provides a resolution for many of the arguments of the introns-early/introns-late debate.

Animals↗

PubMed1998

Modification of bacterial artificial chromosomes through chi-stimulated homologous recombination and its application in zebrafish transgenesis.

The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.

Animals↗

PubMed1998

Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

Mechanism of microsomal epoxide hydrolase. Semifunctional site-specific mutants affecting the alkylation half-reaction.

Microsomal epoxide hydrolase (MEH) catalyzes the addition of water to epoxides in a two-step reaction involving initial attack of an active site carboxylate on the oxirane to give an ester intermediate followed by hydrolysis of the ester. An efficient bacterial expression system for the enzyme from rat that facilitates the production of native and mutant enzymes for mechanistic analysis is described. Pre-steady-state kinetics of the native enzyme toward glycidyl-4-nitrobenzoates, 1, indicate the rate-limiting step in the reaction is hydrolysis of the alkyl-enzyme intermediate. The enzyme is enantioselective, turning over (2R)-1 about 10-fold more efficiently than (2S)-1, and regiospecific toward both substrates with exclusive attack at the least hindered oxirane carbon. Facile isomerization of the monoglyceride product is observed and complicates the regiochemical analysis. The D226E and D226N mutants of the protein are catalytically inactive, behavior that is consistent with the role of D226 as the active-site nucleophile as suggested by sequence alignments with other alpha/beta-hydrolase fold enzymes. The D226N mutant undergoes hydrolytic autoactivation with a half-life of 9.3 days at 37 degreesC, suggesting that the mutant is still capable of catalyzing the hydrolytic half-reaction (in this instance an amidase reaction) and confirming that D226 is in the active site. The indoylyl side chain of W227, which is in or near the active site, is not required for efficient alkylation of the enzyme or for hydrolysis of the intermediate. However, the W227F mutant does exhibit altered stereoselectivity toward (2R)-1, (2S)-1, and phenanthrene-9,10-oxide, suggesting that modifications at this position might be used to manipulate the stereo- and regioselectivity of the enzyme.

Alkylation↗

PubMed1998

Transdermal testosterone delivery in castrated Yucatan minipigs: pharmacokinetics and metabolism.

The feasibility of using the castrated Yucatan minipig as a hypogonadal animal model to investigate the transdermal controlled systemic delivery of testosterone was studied. During a 24 h application of a testosterone transdermal delivery device (T-TDD), serial blood samples were withdrawn from the minipigs, without anesthesia, at predetermined time intervals and the plasma concentrations of testosterone as well as its major metabolites, dihydrotestosterone and estradiol, were assayed by radioimmunoassay. The compartmental pharmacokinetic modeling analysis of the plasma profiles of total testosterone indicated that as much as 92% of the total testosterone dose released from the T-TDD had been delivered transdermally into the systemic circulation during the initial rapid input period (the first 11 h of the application), while only 8% was delivered during the slow input period (up to 23h). Good correlation was observed between the in vivo input doses [1.9 (+/- 0.2), 4.8 (+/- 0.2) and 6.4 (+/- 0.5) mg/day], determined by the Wagner-Nelson equation, and the daily doses released [1.9 +/- (0.2), 4.7 (+/- 0.2) and 6.6 (+/- 0.5) mg/day, respectively, for 1, 2, and 3 units of T-TDD]. While the in vivo rate of input in the castrated minipigs was observed to be similar to that in hypogonadal men treated with the T-TDD during the first 8 h period, the input rate was found to be slower during the last 12 h. The agreement could suggest that the mechanism for the transdermal systemic delivery of testosterone in the castrated minipig could be similar to that in the hypogonadal men. However, the plasma testosterone profiles attained in the castrated minipigs were observed to be similar to, but slightly lower than that in the hypogonadal men reported in the literature. The delta Cmax (baseline normalized peak plasma concentration) and delta Cavg (baseline normalized average plasma concentration) data in the castrated minipigs were 40 and 44%, respectively, of that in hypogonadal men. The approximately 2.4 fold lower values in delta Cmax and delta Cavg data could result from the difference in the clearance rate of testosterone which approximately 2.8 fold higher in minipigs than in the human. Despite the difference in clearance rate, the castrated minipigs could be a suitable large animal model for studying the pharmacokinetics of testosterone delivered transdermally in human with hypogonadism.

Administration, Cutaneous↗

PubMed1998

Photoaffinity labeling by 4-thiodideoxyuridine triphosphate of the HIV-1 reverse transcriptase active site during synthesis. Sequence of the unique labeled hexapeptide.

The active site of HIV-1 reverse transcriptase (HIV-1 RT) was investigated by photoaffinity labeling based on catalytic competence. A stable ternary elongation complex was assembled containing enzyme, DNA template (RT20), DNA primer molecule (P12), and the necessary dNTPs (one of which was alpha-32P-labeled) needed for primer elongation. The photoaffinity probe 4-thiodideoxyuridine triphosphate was incorporated uniquely at the 3' terminus of the 32P-labeled DNA product. Upon photolysis, the p66 subunit of a HIV-1 RT heterodimer (p66/p51) was uniquely cross-linked to the DNA product and subsequently digested by either trypsin or endoproteinase Lys-C. The labeled HIV-1 RT peptide was separated, purified, and finally subjected to Edman microsequencing. A unique radioactive hexapeptide (V276RQLCK281) was identified and sequenced. Our photoaffinity labeling results were positioned on the HIV-1 RT. DNA.Fab complex x-ray crystallography structure and compared with the suggested aspartic triad active site.

Binding Sites↗

PubMed1998

Delta9-THC training dose as a determinant for (R)-methanandamide generalization in rats.

The objective of this study was to examine if (R)-methanandamide, a metabolically stable chiral analog of the endogenous ligand anandamide, is a cannabimimetic with a lower efficacy than delta9-THC. Employing a two-lever choice drug discrimination procedure, rats were trained to discriminate between 1.8, 3.0, or 5.6 mg/kg delta9-tetrahydrocannabinol (delta9-THC) and vehicle. Different training doses were used in order to create assays with different efficacy demands. Generalization tests with 18 mg/kg (R)-methanandamide yielded around 90% delta9-THC responses in the two lower delta9-THC training dose conditions. However, only around 60% delta9-THC responses occurred in the 5.6 mg/kg delta9-THC training dose condition in tests with 18 mg/kg (R)-methanandamide; a higher dose (30 mg/kg) produced even fewer delta9-THC-appropriate responses in this group. Morphine did not substitute for delta9-THC. In conclusion, the data with delta9-THC and (R)-methanandamide indicate that cannabinoid agonists can have varying degrees of intrinsic activity at a receptor site, or may produce their behavioral actions through multiple mechanisms, or both.

Animals↗

PubMed1998

Pre- and postnatal exposure to 3,3',4,4'-tetrachlorobiphenyl: II. Effects on the reproductive capacity and fertilizing ability of eggs in female mice.

This study investigated the effects of 3,3',4,4'-tetrachlorobiphenyl (TCB) on the reproductive capacity of female mice. Female C57BL/6J mice (F-0) were fed diets containing 0, 3, or 30 ppm TCB for 2 weeks before pairing with nontreated C57BL/6J males for a 10-day breeding period. Females were continued on their treatment diet throughout mating, gestation, and lactation. Female offspring (F-1) were fed the same diet as their dams throughout the study. The reproductive capacity of F-1 females was examined by mating with non-treated B6D2-F1 males. In addition, the fertilizing ability of eggs from F-1 females was examined in vitro by insemination with sperm from nontreated B6D2-F1 males. Fecundity in F-0 females after mating was 80%, 71%, and 47% in the 0, 3, and 30 ppm treatment groups, respectively. Four-day and 21-day survival indices were lower for offspring of 30 ppm TCB-treated F-0 females than for offspring of the control females. Fecundity in F-1 females was the same among all treatment groups, however, all offspring born to 3- and 30-ppm TCB-treated F-1 females died before 4 days of age. Although the litter size at birth was not affected, the in vitro fertilizing ability of eggs in the 3- and 30-ppm treatment groups was lower than in the control group. This decrease in fertilizing ability was associated with an increase in degenerated eggs. F-0 females treated with 30 ppm TCB had enlarged livers during pregnancy and lactation. At 5 and 6 weeks, liver enlargement and thymus atrophy were apparent in F-1 females exposed to 30 ppm TCB. This study demonstrated impaired reproductive capacity and decreased egg fertilizing ability in TCB-treated female mice.

Animals↗

PubMed1998

Examination of ligand-induced conformational changes in the beta2 adrenergic receptor.

The environmentally sensitive and cysteine reactive fluorescent probe, IANBD, was used to monitor ligand-induced structural changes in the beta2 adrenergic receptor (beta2AR) by fluorescent spectroscopy. We found that agonists caused a dose-dependent and reversible decrease in fluorescence from the purified IANBD-labeled beta2AR. This suggested that agonists promote a conformational change in the receptor that leads to an increase in the polarity of the environment around one or more IANBD labeled cysteines. The wildtype receptor contains eight free cysteines and mutagenesis and peptide mapping experiments have indicated that several of these sites are accessible for chemical derivatization. Thus, to identify the cysteine(s) involved in the agonist-induced change in fluorescence and thereby map agonist-induced conformational changes in the beta2AR, we generated a series of mutant receptors having limited numbers of cysteines available for fluorescent labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants showed that IANBD labeled 125Cys and 285Cys are responsible for the observed changes in fluorescence consistent with movements of TM III and VI in response to agonist binding.

Adrenergic beta-Agonists↗

PubMed1998

Structural requirements for arachidonylethanolamide interaction with CB1 and CB2 cannabinoid receptors: pharmacology of the carbonyl and ethanolamide groups.

Analogs of arachidonylethanolamide (anandamide) were prepared to investigate the structural requirements for ligand binding to and activation of the CB1 and CB2 cannabinoid receptors. The importance of the presence and the placement of the carbonyl was examined with analogs lacking the carbonyl or with the carbonyl amide order reversed. The presence and location of the carbonyl is essential for high-affinity binding to both cannabinoid receptor subtypes, and for determination of signal transduction via G-proteins. Methyl groups were substituted on the 1'- and 2'-positions of arachidonylethanolamide and the significance of chirality was examined. Stereochemical differences in the ethanolamide group influence the affinity for both cannabinoid receptor subtypes and the signal transduction capabilities of the methanandamide derivatives.

Adenylyl Cyclases↗

PubMed1998

Allergy-associated FcRbeta is a molecular amplifier of IgE- and IgG-mediated in vivo responses.

A role for the Fc receptor beta chain (FcRbeta) in the pathogenesis of allergy has been suggested by genetic studies. FcRbeta is a subunit common to the high-affinity IgE (FcepsilonRI) and low-affinity IgG (FcgammaRIII) receptors, both of which contribute to the initiation of allergic reactions. Current in vitro data suggest that FcRbeta can function as either a positive or negative regulator, leaving a mechanistic explanation for its association with the development of atopy unclear. To address this controversy, we have generated novel mouse models relevant to human Fc receptor function. Analysis of FcepsilonRI- and FcgammaRIII-dependent responses in these mice provides unequivocal genetic evidence that FcRbeta functions as an amplifier of early and late mast cell responses and, remarkably, in vivo anaphylactic responses.

Anaphylaxis↗

PubMed1998

C-met activation is necessary but not sufficient for liver colonization by B16 murine melanoma cells.

Metastasis to the liver is a frequent event in clinical oncology, the molecular mechanisms of which are not fully understood. We have recently reported a consistent overexpression of c-met in B16 melanoma cells selected in vivo for enhanced liver metastatic ability. In this study we address the question as to whether constitutive activation of c-met is a necessary and sufficient condition for enhanced liver colonization by B16 melanoma cells. Different levels of c-met expression and/or activation in B16 cells were achieved by subcloning, or by c-DNA transfection with either HGF/SF or the oncogenic form of c-met (tpr-met). Metastatic ability of the different populations was then evaluated in vivo by the lung colonization (experimental metastasis) assay. Results indicate that c-met (but not tpr-met) activation in B16 melanoma cells may increase their liver colonizing potential, probably by enhancing motility and invasion in response to paracrine interactions with its ligand. C-met expression per se, however, is not able to change the organ specificity of the cells. C-met activation appears instead to be required at later stages of liver colonization by B16 melanoma cells, in order to enhance their site-specific metastatic ability.

Animals↗

PubMed1998

A zebrafish model for hepatoerythropoietic porphyria.

Defects in the enzymes involved in the haem biosynthetic pathway can lead to a group of human diseases known as the porphyrias. yquem (yqe(tp61)) is a zebrafish mutant with a photosensitive porphyria syndrome. Here we show that the porphyric phenotype is due to an inherited homozygous mutation in the gene encoding uroporphyrinogen decarboxylase (UROD); a homozygous deficiency of this enzyme causes hepatoerythropoietic porphyria (HEP) in humans. The zebrafish mutant represents the first genetically 'accurate' animal model of HEP, and should be useful for studying the pathogenesis of UROD deficiency and evaluating gene therapy vectors. We rescued the mutant phenotype by transient and germline expression of the wild-type allele.

Amino Acid Sequence↗

PubMed1998

Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen.

The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential.

Calcium Channel Blockers↗