Biomedical subjects

Y Jin

Publications and source records attributed to Y Jin.

At least 73 records · Page 4Linked to original sources

PubMed2001

Species-specific differences in the operational RNA code for aminoacylation of tRNA(Trp).

Identity elements play essential roles in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase. An operational RNA code relates amino acids to specific sequences and structural features of tRNA acceptor stems. In this study, a series of tRNA(Trp) variants was prepared by in vitro transcription and their efficiencies of aminoacylation by tryptophan (k(cat)/K(m)) were measured with the aid of Bacillus subtilis and human tryptophanyl-tRNA synthetases (TrpRS). The identity elements in the operational RNA code of human tRNA(Trp) were found to be: major element, discriminator base A73; minor elements, G1/C72 and U5/G68. From the cross-species aminoacylation assays, we conclude that the identity elements in tRNA(Trp) from B.subtilis and human all contribute to species-specific aminoacylation by TrpRS. Analyses of 22 TrpRS sequences covering three taxonomic domains (bacteria, eukarya and archaea) reveal that the sequences are divided into two evolutionarily distant groups. The same partition is also observed in the analyses of tRNA(Trp) acceptor stem sequences. Our data suggest that the two TrpRS groups may reflect co-adaptations needed to accommodate changes in the operational RNA code for tryptophan.

Acylation↗

PubMed2001

Exposure-response relationship between paternal smoking and children's pulmonary function.

We conducted a cross-sectional study to investigate paternal smoking and children's pulmonary function in rural communities of Anqing, China. Our analysis included 1,718 children 8 to 15 yr of age whose mothers were never-smokers. Multiple linear regression models were used to estimate the effect of paternal smoking on children's pulmonary function, with adjustment for children's age, sex, weight, height, square of height, asthma, and father's education. When compared with children of never-smoking fathers, children of smoking fathers had small, but detectable deficits in FEV(1) (-36 ml, SE = 20) and FVC (-37 ml, SE = 22). When children of smoking fathers were subdivided into two subgroups, father smoked < 30 cigarettes/day and >/= 30 cigarettes/day, we found that children whose fathers smoked >/= 30 cigarettes/day had the largest deficits in both FEV(1) (-79 ml, SE = 30) and FVC (-71 ml, SE = 34). This monotonic exposure-response relationship remained in all strata when we further stratified our analysis by children's sex and asthma status. Our data also suggested that the relationship was greatest among nonasthmatic girls, although neither sex nor asthma interaction terms were statistically significant. We conclude that there is a monotonic exposure-response relationship between paternal smoking and decline of pulmonary function in children in this rural Chinese population.

Adolescent↗

PubMed2001

Conserved function of Caenorhabditis elegans UNC-30 and mouse Pitx2 in controlling GABAergic neuron differentiation.

We are taking a cross-species approach to identify genes that are required for mammalian GABAergic neuron differentiation. On the basis of homeodomain similarity, the vertebrate Pitx genes appear to be orthologs of unc-30, a Caenorhabditis elegans gene necessary for differentiation of the GABAergic phenotype of type D neurons. One of the Pitx genes, Pitx2, is expressed in regions of GABAergic neurogenesis in the mammalian brain. These observations led us to test the functional conservation of the mouse Pitx2 and worm unc-30 genes using a rescue assay. Pitx2 rescues the GABAergic differentiation defect and partially rescues the axon guidance and behavioral phenotypes of unc-30 mutants, indicating a high degree of functional conservation between these evolutionarily related genes. Previous studies show that UNC-30 directly regulates the unc-25/glutamate decarboxylase gene that encodes the enzyme for GABA synthesis. We find that the promoter regions of the mouse and human genes coding for the 67 kDa glutamate decarboxylase (Gad1) also contain binding sites matching the UNC-30/Pitx2 consensus binding site sequence. We show that these sites specifically bind to Pitx2 protein in vitro and that in transfected neuroblastoma cells, the Pitx2 binding sites contribute to the basal activity of the Gad1 promoter. Furthermore, in cotransfection experiments, we find that Pitx2 strongly activates the Gad1 promoter. These results indicate that Pitx2 may regulate Gad1 expression in mammals, suggesting a new role for this key developmental transcription factor as a regulator of GABAergic differentiation during mammalian neural development. Our results suggest that some of the mechanisms regulating GABAergic differentiation are evolutionarily conserved.

Animals↗

PubMed2001

Crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP(+) and ursodeoxycholate.

The crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP(+) and 3alpha,7beta-dihydroxy-5beta-cholanic acid (ursodeoxycholate) at 3.0 A resolution is presented. Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily. AKR1C2 is implicated in the prostatic production of the potent androgen 5alpha-dihydrotestosterone and the hepatic transport of bile acids. It also catalyzes the formation of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria. Like other members of the superfamily, AKR1C2 folds into an alpha/beta-barrel and binds NADP(+) in an extended conformation. The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site. More interestingly, the orientation of ursodeoxycholate is essentially "backwards" and "upside-down" from that observed for testosterone in the related rat 3alpha-HSD.NADP(+).testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate. The orientation of ursodeoxycholate is thus similar to that expected of a 17beta-HSD substrate. The ternary structure explains the ability of AKR1C2 to catalyze 3alpha-, 17beta-, and 20alpha-HSD reactions. Comparison of the steroid binding pocket of AKR1C2 with that of rat 3alpha-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3alpha-HSD isoforms but not in the rat isoform.

Allosteric Regulation↗

PubMed2001

Parallel solid-phase synthesis of nucleoside phosphoramidate libraries.

Combinatorial chemistry is playing an increasingly prominent role in the process of drug discovery. A nucleic acid-based (NAB) scaffold can be engineered to create functional group and topological diversity in a library. Described herein is the parallel solid-phase synthesis of combinatorial libraries of nucleoside phosphoramidates, and the first evaluation of antiviral activity against hepatitis B virus (HBV).

Amides↗

PubMed2001

Identification of a new form of death-associated protein kinase that promotes cell survival.

In this study, two alternatively spliced forms of the mouse death-associated protein kinase (DAPK) have been identified and their roles in apoptosis examined. The mouse DAPK-alpha sequence is 95% identical to the previously described human DAPK, and it has a kinase domain and calmodulin-binding region closely related to the 130-150 kDa myosin light chain kinases. A 12-residue extension of the carboxyl terminus of DAPK-beta distinguishes it from the human and mouse DAPK-alpha. DAPK phosphorylates at least one substrate in vitro and in vivo, the myosin II regulatory light chain. This phosphorylation occurs preferentially at Ser-19 and is stimulated by calcium and calmodulin. The mRNA encoding DAPK is widely distributed and detected in mouse embryos and most adult tissues, although the expression of the encoded 160-kDa DAPK protein is more restricted. Overexpression of DAPK-alpha, the mouse homolog of human DAPK has a negligible effect on tumor necrosis factor (TNF)-induced apoptosis. Overexpression of DAPK-beta has a strong cytoprotective effect on TNF-treated cells. Biochemical analysis of TNF-treated cell lines expressing mouse DAPK-beta suggests that the cytoprotective effect of DAPK is mediated through both intrinsic and extrinsic apoptotic signaling pathways and results in the inhibition of cytochrome c release from the mitochondria as well as inhibition of caspase-3 and caspase-9 activity. These results suggest that the mouse DAPK-beta is a negative regulator of TNF-induced apoptosis.

Alternative Splicing↗

PubMed2001

Altering the DNA-binding specificity of the yeast Matalpha 2 homeodomain protein.

Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism. The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins. To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1. We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein. We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein. Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain. Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.

Amino Acid Substitution↗

PubMed2001

Controlled nucleation and growth of surface-confined gold nanoparticles on a (3-aminopropyl)trimethoxysilane-modified glass slide: a strategy for SPR substrates.

The thickness of the gold film and its morphology, including the surface roughness, are very important for getting a good, reproducible response in the SPR technique. Here, we report a novel alternative approach for preparing SPR-active substrates that is completely solution-based. Our strategy is based on self-assembly of the gold colloid monolayer on a (3-aminopropyl)trimethoxysilane-modified glass slide, followed by electroless gold plating. Using this method, the thickness of films can be easily controlled at the nanometer scale by setting the plating time in the same conditions. Surface roughness and morphology of gold films can be modified by both tuning the size of gold nanoparticles and agitation during the plating. Surface evolution of the Au film was followed in real time by UV-vis spectroscopy and in situ SPRS. To assess the surface roughness and electrochemical stability of the Au films, atomic force microscopy and cyclic voltammetry were used. In addition, the stability of the gold adhesion is demonstrated by three methods. The as-prepared Au films on substrates are reproducible and stable, which allows them to be used as electrodes for electrochemical experiments and as platforms for studying SAMs.

Journal Article↗

PubMed2001

Production and characterization of monoclonal antibodies against amphiphysins.

Amphiphysin I and II, proteins enriched in nerve terminals, form heterodimers and interact with dynamin and synaptojanin through their Src homology 3 (SH3) domain. In order to study the expression profile of Amphs in cells and tissues and the interaction state with other cellular molecules, we have prepared specific monoclonal antibodies (mAbs) designed to bait N-terminus, middle part, and C-terminus domains of Amph I, respectively by immunizing with the expressed smaller domain molecules using the GST gene fusion system. The expression of Amphs was found to be most abundant in PC12 cells, followed by B103 cells and vascular smooth muscle cells. Western blot analysis showed a relatively high level expression of Amphs that were found in both mouse and rat brain. There appeared to be some species difference in the expression pattern, i.e. Amphs are present more in the testis than in the lungs in rats, however, they are reversed in mice. Characterization of the mAbs revealed that clone 14-23 precipitated Amph I and II, whereas clone 8-2 could only precipitate Amph I. In addition, clathrin and dynamin in a complex with Amph were captured in the precipitate formed by mAbs and identified by the Western blot analysis. Cellular distribution of Amph was visualized with confocal immunofluorescence microscopy performed using the labeled-mAbs. Taken together, these results demonstrated that mAbs provided an excellent measure for studying Amphs' expression profile and their interacting proteins.

Animals↗

PubMed2001

Recyclable characteristics of hyaluronate-polyhydroxyethyl acrylate blend hydrogel for controlled releases.

Hyaluronate-hydroxyethyl acrylate blend hydrogels which have a wide range of composition and characteristics were investigated. Glycidyl methacrylate derivatized hyaluronate (GMA-HA) were synthesized by coupling GMA to hyaluronate (HA) in the presence of a photoinduced initiator for polymerization. By copolymerizing radically GMA-HA and hydroxyethyl acrylate (HEA) under various compositions (weight ratios of HEA and GMA-HA: 1-20), GMA-HA hydrogels could be prepared in wide ranges of characteristics. These HA-PHEA gels possessed the feature that the dried ones recovered completely to the original swelling states on repeated runs, i.e. recyclable gels. The water contents of these hydrogels in equilibrium swellings in water (W(w)) were 0.99-0.86, and their viscoelastic properties were measured by a creep. The spontaneous elastic moduli were 1.05x10(5)-1.94x10(5) N m(-2), and they were mechanically tough. Their effective charge densities were estimated from the partition coefficients of sodium benzoate (NaBA) and decreased from -0.033 to -0.044 mol dm(-3) with increasing contents of HEA. Release of NaBA was studied, and the diffusion coefficients were found to be from 6.95x10(-10) to 0.12x10(-10) m(2) s(-1) with increasing the ratio of HEA. Their diffusion coefficients were found to be much less than the values estimated from the lattice model.

Acrylates↗

PubMed2001

Myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1.

Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-alpha (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.

Animals↗

PubMed2001

The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila.

To analyze the function of the chromosomal kinase JIL-1, we generated an allelic series of hypomorphic and null mutations. JIL-1 is an essential kinase for viability, and reduced levels of JIL-1 kinase activity lead to a global change in chromatin structure. In JIL-1 hypomorphs, euchromatic regions of polytene chromosomes are severely reduced and the chromosome arms condensed. This is correlated with decreased levels of histone H3 Ser10 phosphorylation. These levels can be restored by a JIL-1 transgene placing JIL-1 directly in the pathway mediating histone H3 phosphorylation. We propose a model where JIL-1 kinase activity is required for maintaining the structure of the more open chromatin regions that facilitate gene transcription.

Alleles↗

PubMed2001

Sensitivity at the degenerate points of energy levels in a quantum system with nonintegrable perturbation.

The behavior of quantum states and observables of a quasi-integrable quantum system is studied. Sensitivity of the quantum states and nonperiodicity of the average value of observables at the degenerate points of energy levels are observed. The behavior demonstrates the quantum properties corresponding to the classical resonant tori with rational frequency ratio, where the global structure is easily destroyed by a small perturbation. Our result is helpful for further understanding the avoided crossing in hard chaotic quantum systems.

Journal Article↗

PubMed2001

Ribozyme-mediated inhibition of caspase-3 activity reduces apoptosis induced by 6-hydroxydopamine in PC12 cells.

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and PC12 cells, which are derived from rat pheochromocytoma tumors and have many properties similar to dopamine neurons. Biochemical and molecular approaches have shown that low doses of 6-OHDA induce apoptosis in PC12 cells and, in the processing of apoptosis, caspases are crucial mediators, and caspase inhibition is sufficient to rescue PC12 cells from apoptosis induced by 6-OHDA. However, because this caspase inhibition targets multiple caspases, it is not known whether a single caspase is primarily responsible for effecting cell death in this model. To assess the particular member (caspase-3) of the ced-3 family relevant to cell death and to position their activation within the apoptotic pathway, we constructed a hammerhead ribozyme directed against rat caspase-3, which could downregulate the expression of caspase-3 in vitro and in vivo, and transfer to PC12 cells. The results show that the ribozymes against caspase-3 could protect PC12 cells from apoptosis induced by low doses of 6-OHDA. The PC12 cell transfected with the ribozymes shows a significant decrease in caspase-3 activity compared with control cells at various time points. Parallel to the reduced caspase-3 protease activity, similar decreased levels of apoptotic cells and DNA fragmentation were also assessed by staining with Hoechst 33258 and ELISA, respectively. Overexpression of p35, a general caspase inhibitor, also protected PC12 cells from apoptosis. These results confirm that caspases play an important role in 6-OHDA-induced PC12 cell apoptosis and indicate that caspase-3 itself is one of the crucial mediators of neurotoxin-induced PC12 cell apoptosis.

Adrenergic Agents↗

PubMed2001

Expression profiling suggested a regulatory role of liver-enriched transcription factors in human hepatocellular carcinoma.

By using a cDNA array representing 14,000 cDNA clusters, we studied the expression profiles in paired clinical hepatocellular carcinoma (HCC) samples and the distal nontumorous liver tissues from the same patients. Despite the significant heterogeneity among the clinical samples, 72 genes (including 30 novel genes) were down-regulated and 84 genes (including 48 novel genes) were up-regulated in >50% of the cancer samples that were identified. The alterations in gene expression levels were confirmed by Northern blot and reverse-transcription PCR in all of 4 randomly selected genes. It was conspicuous that 21 of 38 hepatocarcinoma (HCC) down-regulated genes studied previously were reportedly regulated by a group of liver-enriched transcription factors (LETFs), and 12 of 36 HCC up-regulated genes studied previously were involved in protein translation. Reexamination of the cDNA array data further revealed that most of the genes known to be regulated by LETFs were down-regulated in at least a portion of the HCC samples. Among the LETFs, the expression level of CCAAT/enhancer-binding protein (C/EBP) alpha was down-regulated in cancer, whereas hepatocyte nuclear factor 1 (HNF-1), HNF-3beta, HNF-4alpha, and HNF-4gamma were up-regulated. The expression profiling thus suggested multiple regulatory pathways involved in HCC, especially that related to LETFs.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2001

Plant tissue-based chemiluminescence flow biosensor for glycolic acid.

A novel plant tissue-based chemiluminescence (CL) biosensor for glycolic acid combined with flow injection analysis is proposed in this paper. The spinach tissue acts as the molecular recognition element. Glycolic acid is oxidized by oxygen under the catalysis of glycolate oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of peroxidase of spinach tissue to generate a CL signal. The CL emission intensity was linear with glycolic acid concentration in the range of 4 x 10(-3)-4 x 10(-6) mol/L and the detection limit was 1.3 x 10(-6) mol/L. The biosensor was stable for about 3 weeks. A complete analysis, including sampling and washing, could be performed in 1.5 min with a relative standard deviation of 1.7%.

Biosensing Techniques↗

PubMed2001

A member of the nuclear factor-1 family is involved in the pituitary repression of the human placental growth hormone genes.

The human growth hormone (GH) gene family consists of five tandemly arranged and highly related genes, including the chorionic somatomammotropins (CSs), at a single locus on chromosome 17. Despite striking homologies in promoter and flanking DNA sequences, the genes within this locus have different tissue-specific patterns of expression: GH-N is expressed almost exclusively in the somatotrophs of the anterior pituitary; the remaining genes, including CS-A, are expressed in placental syncytiotrophoblast. Previously we proposed that active repression of the placental gene promoters in pituitary GC cells is mediated by upstream 'P' sequences and, specifically, a 263 bp region containing two 'P' sequence elements (PSE-A and PSE-B) and corresponding factors (PSF-A and PSF-B). We have now examined the possibility that PSF-A and PSF-B are members of the nuclear factor (NF)-1 family. Transcripts of NF-1A, NF-1C and NF-1X, but not of NF-1B, were readily detected in GC cells. High-affinity binding of NF-1 to PSE-B, but not to PSE-A, was confirmed by competition of DNA-protein interactions by using NF-1 DNA elements and antibodies. Functionally, a NF-1 element was able to substitute for PSE-B as a promoter-specific repressor in GC cells after gene transfer. However, there was a difference in the magnitude of repression exerted by the NF-1 and PSF-B elements on the CS-A promoter and, with the use of mutations, this difference was shown to be consistent with variations in NF-1-binding sequences. These results indicate that PSF-B, but not PSF-A, is a member of the NF-1 family, which participates in the PSF complex and in the repression of the CS-A promoter in pituitary GC cells.

Animals↗

PubMed2001

Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells.

We have compared the anti-proliferative effects of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA) and their derivatives, HS-1183, HS-1199 and HS-1200, on MCF-7 (wild-type p53) and MDA-MB-231 (mutant p53) cells. While UDCA and CDCA exhibited no significant effect, their novel derivatives inhibited the proliferation of both cell lines in a concentration-dependent manner, concomitant with apoptotic nuclear changes and the increase of a sub-G1 population and DNA fragmentation. Furthermore, we also observed an increase in the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 and cleavages of lamin B and poly(ADP-ribose) polymerase (PARP) in MCF-7 and MDA-MB-231 cells. Cell cycle related proteins, cyclin D1 and D3, as well as retinoblastoma protein (pRb) were down-regulated, while the level of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) was increased in both cancer cells after treatment with novel bile acids. These findings suggest that these cytotoxic effects of novel bile acid derivatives on human breast carcinoma cells were mediated via apoptosis through a p53-independent pathway.

Apoptosis↗