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Biomedical subjects

Jun Yin

Publications and source records attributed to Jun Yin.

At least 19 recordsLinked to original sources
PubMed2006

Deletion of the V2 vasopressin receptor gene in two Chinese patients with nephrogenic diabetes insipidus.

BACKGROUND: Congenital nephrogenic diabetes insipidus (NDI) is a rare X-linked inherited disorder characterized by the excretion of large volumes of diluted urine and caused by mutations in arginine vasopressin receptor 2 (AVPR2) gene. To investigate the mutation of AVPR2 gene in a Chinese family with congenital NDI, we screened AVPR2 gene in two NDI patients and eight family members by PCR amplification and direct sequencing. RESULTS: Five specific fragments, covering entire coding sequence and their flanking intronic sequences of AVPR2 gene, were not observed in both patients, while those fragments were all detected in the control subjects. Several different fragments around the AVPR2 locus were amplified step by step. It was revealed that a genomic fragment of 5,995-bp, which contained the entire AVPR2 gene and the last exon (exon 22) of the C1 gene, was deleted and a 3-bp (GAG) was inserted. Examination of the other family members showed that the mothers and the grandmother were carriers for this deletion. CONCLUSION: Our findings suggest that the two patients in a Chinese family suffering from congenital NDI had a 5,995-bp deletion and 3-bp (GAG) insertion at Xq28. The deletion contained the entire AVPR2 gene and exon 22 of the C1 gene.

Adult↗
PubMed2006

5-HT(1B) receptors play a prominent role in the proliferation of T-lymphocytes.

Serotonin plays a role in T cell activation, but there is no clear consensus of which of the 14 serotonergic receptors control this activations pathway. We have used a broad range of serotonergic receptor antagonists to define the functional involvement of these receptors governing the proliferation of primary T cells as well as in T cell lines. Our data shows that antagonism of the 5-HT(1B) receptor inhibits the proliferation of both human and murine primary helper T cells and of human helper T cell lines. As a whole, our data suggest that other serotonergic receptors may contribute to the proliferative signals, but the 5-HT(1B) receptor plays the most dominant role.

Animals↗
PubMed2006

Trimethylamine (TMA) biofiltration and transformation in biofilters.

Bioremoval of trimethylamine (TMA) in two three-stage biofilters packed with compost (A) and sludge (B), respectively, was investigated. Both biofilters were operated with an influent TMA concentration of 19.2-57.2mgm(-3) for 67 days. Results showed that all of the inlet TMA could be removed by both biofilters. However, removal efficiency and transformation of TMA in each section of both biofilters was different. In the Introduction section, TMA removal efficiency and maximum elimination capacity of the compost medium were greater than those of sludge medium under higher inlet TMA concentration. In comparison with biofilter A, considerably higher NH(3) concentrations in effluent of all three sections in biofilter B were observed after day 19. Although, NO(2)(-)-N concentration in each section of biofilter A was relatively lower, NO(3)(-)-N content in each section of biofilter A increased after day 26, especially in the Materials and method section which increased remarkably due to a lesser amount of TMA and higher ammonia oxidation and nitrification in compost medium. In contrast, neither NO(2)(-)-N nor NO(3)(-)-N were detected in either section of biofilter B at any time throughout the course of the experiment. The cumulative results indicated that compost is more favorable for the growth of TMA-degrading and nitrifying bacteria as compared to the sludge and could be a highly suitable packing material for biodegradation and transformation of TMA.

Ammonia↗
PubMed2006

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells.

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Caco-2 Cells↗
PubMed2006

Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g., G-to-T, G-to-A, -1 frameshifts, etc.) via its major adduct [+ta]-B[a]P-N2-dG. We recently showed that the dominant G-to-T mutation depends on DNA polymerase V (DNAP V), but not DNAPs IV or II, when studied in a 5'-TG sequence in E. coli. Herein we investigate what DNAPs are responsible for non-mutagenic bypass with [+ta]-B[a]P-N2-dG, along with its mirror image adduct [-ta]-B[a]P-N2-dG. Each adduct is built into a 5'-TG sequence in a single stranded M13 phage vector, which is then transformed into eight different E. coli strains containing all combinations of proficiency and deficiency in the three lesion-bypass DNAPs II, IV and V. Based on M13 progeny output, non-mutagenic bypass with [-ta]-B[a]P-N2-dG depends on DNAP IV. In contrast, non-mutagenic bypass with [+ta]-B[a]P-N2-dG depends on both DNAPs IV and V, where arguments suggest that DNAP IV is involved in dCTP insertion, while DNAP V is involved in extension of the adduct-G:C base pair. Numerous findings indicate that DNAP II has a slight inhibitory effect on the bypass of [+ta]- and [-ta]-B[a]P-N2-dG in the case of both DNAPs IV and V. In conclusion, for efficient non-mutagenic bypass (dCTP insertion) in E. coli, [+ta]-B[a]P-N2-dG requires DNAPs IV and V, [-ta]-B[a]P-N2-dG requires only DNAP IV, while DNAP II is inhibitory to both, and experiments to investigate these differences should provide insights into the mechanism and purpose of these lesion-bypass DNAPs.

Bacteriophage M13↗
PubMed2006

Peroxynitrite induces Alzheimer-like tau modifications and accumulation in rat brain and its underlying mechanisms.

To investigate the upstream effector that led to tau hyperphosphorylation, nitration, and accumulation as seen in Alzheimer's disease brain, and the underlying mechanisms, we bilaterally injected SIN-1, a recognized peroxynitrite donor, into the hippocampus of rat brain. We observed that the level of nitrated and hyperphosphorylated tau was markedly increased in rat hippocampus 24 h after drug administration, and these alterations were prevented by preinjection of uric acid, a natural scavenger of peroxynitrite. Concomitantly, we detected a significant activation in glycogen synthase kinase-3beta (GSK-3beta) and p38 MAPKs, including p38alpha, p38beta, and p38delta, but no obvious change was measured in the activity of p38gamma, ERK, and c-Jun amino-terminal kinase (JNK). Both nitrated tau and hyperphosphorylated tau were aggregated in the hippocampus, in which the activity of 20S proteasome was significantly arrested in SIN-1-injected rats. Further studies demonstrated that the hyperphosphorylated tau was degraded as efficiently as normal tau by 20S proteasome, but the nitrated tau with an unorderly secondary structure became more resistant to the proteolysis. These results provide the first in vivo evidence showing that peroxynitrite simultaneously induces tau hyperphosphorylation, nitration, and accumulation, and that activation of GSK-3beta, p38alpha, p38beta, p38delta isoforms and the inhibition of proteasome activity are respectively responsible for the peroxynitrite-induced tau hyperphosphorylation and accumulation. Our findings reveal a common upstream stimulator and a potential therapeutic target for Alzheimer-like neurodegeneration.

Alzheimer Disease↗
PubMed2006

Melatonin arrests peroxynitrite-induced tau hyperphosphorylation and the overactivation of protein kinases in rat brain.

The purpose of this study was to examine the in vivo effect of melatonin (MEL) on peroxynitrite-induced tau hyperphosphorylation and the involvement of glycogen synthase kinase-3beta (GSK-3beta) and mitogen-activated protein kinase (MAPK) families. Melatonin was injected into the right cerebroventricle of the rats 1 hr before the bilateral hippocampal injection of 3-morpholino-sydnonimine chloride (SIN-1), the recognized donor of peroxynitrite. Thereafter, the phosphorylation level of tau and the activity of the kinases were analyzed. The injection of SIN-1 induced hyperphosphorylation of tau at pS396 epitope with a concomitant activation of GSK-3beta and selective MAPK isoforms including p38alpha, p38beta, and p38delta but not p38gamma. The effect of peroxynitrite was confirmed using uric acid, a recognized scavenger of peroxynitrite. Preinjection of MEL significantly arrested the peroxynitrite-induced hyperphosphorylation of tau and the activation of GSK-3beta and MAPKs. Melatonin also ameliorated peroxynitrite-induced oxidative stress. We conclude that MEL can efficiently arrest peroxynitrite-induced tau hyperphosphorylation, and the underlying mechanism may involve scavenging the reactive species and suppressing the activated GSK-3beta and p38 MAPK family.

Animals↗
PubMed2006

[Screening of botulinum neurotoxin type A-binding peptides by phage display peptide library].

To seek effective inhibitor against BoNT/A, in this study, BoNT/A-binding peptides were screened from phage display peptide library using synthesized and identified mimicry peptides that contained antigenic epitopes as targets. According to the homology of the amino acid sequences of displayed peptides, most had same motifs respectively. ELISA assay confirmed that identified positive clones respectively against P4 and P5 could specifically bind BoNT/A. The mice assay showed the specific BoNT/A-binding peptides could partially protect against challenge of BoNT/A. The results from the study may aid in the future identification of more potent small molecule inhibitors against BoNT/A.

Amino Acid Sequence↗
PubMed2006

[Secretory expression of sCD14 in Pichia pastoris and its LPS binding activity].

AIM: To achieve secretory expression of mature sCD14 gene in Pichia pastoris and to analyze its LPS binding activity. METHODS: sCD14 gene was cloned into yeast expression vector pPIC9K. Recombinant plasmid was lineared by Sac I and transformed into Pichia pastoris GS115. Positive integrated clones were screened and expressed by methanol inducing. RESULTS: The mature sCD14 was expressed in BMMY with pH 6.5 after optimization of inducing expression condition. Western blot and ELISA data revealed recombinant products combined with specific antibody. The results from FACS showed recombinant product had biological activity of binding with LPS. CONCLUSION: The recombinant mature sCD14 molecules with favorable LPS-binding activity could be used for further study on its structure and functions.

Bodily Secretions↗
PubMed2006

[Study on molecular identification and pre-harvest sprouting characteristic of the transgenic anti-trxs-gene wheat line 00T89].

Pre-harvest sprouting of wheat is caused by a series of enzymes, in which alpha-amylase plays a key role. Thioredoxin (trx) is an important protein capable of reducing S-S to -SH. The authors transferred the anti-trxs gene into wheat ( Triticum aestivum L.) cultivar Wanmai 48 in 2000 in order to obtain a transgenic line with low activities of trx and alpha-amylase in wheat seeds to enhance their resistance to pre-harvest sprouting. In this work, a pair of specific anti-trxs primers were used in PCR test to validate the transgenic plants in T4 lines. A pair of primers of a wheat actin gene cDNA and a pair of trxs gene primers were used in the reverse transcription PCR test to measure the relative content of trx mRNA in the transgenic positive lines. The experiments of pre-harvest sprouting were carried out to test the resistance to pre-harvest sprouting. The results showed that there were 13 positive lines with the anti-trxs gene among 18 transgenic lines. In the maturity stage, the 8 relative contents of the trx mRNA in the 13 positive lines were reduced evidently (P < 0.01). The trx mRNA was correlated with the sprouting parameters significantly (r = 0.7181) . 6 lines with low trx mRNA showed stronger resistance to pre-harvest sprouting during the period from 30 days after anthesis to 10 days after maturity. Compared with non-transgenic plant (Wanmai 48, CK), the average sprouting time of these lines was postponed by 2.7 days (P < 0.01), the sprouting ratio of seeds on spikes and the spike sprouting degree were reduced by 35.5% (P < 0.01) and 47.5% (P < 0.01), respectively. However, 25 days after maturity, the sprouting capacity of the positive lines recovered gradually, and no significant differences were found between them (P > 0.05) . Thus, it can be concluded that the wheat pre-sprouting was controlled effectively in some 00T89 transgenic lines as a result of the expression of anti-trxs gene.

Genes, Plant↗
PubMed2006

[Screening of short peptides binding to StxB by phage-display library].

Under induction with 41 degrees C, pBV220-stxb/DH5alpha expressed the recombinant protein Shiga Toxin B Subunit (StxB), which was purified by centrifugation, salting out and ion exchange chromatography. As a target, the purified-protein-coated ELISA plate was used to screen phages able to bind onto it from a random 12-mer peptide library. After 4 rounds of affinity screening, a group of clones were isolated from the peptide library. ELISA assay detected their binding activity with the target. 27 clones showed the specific binding activity. The peptide sequences of these positive phage clones were analyzed. 16 of them had the same sequence named A6, 2 clones had the A9 sequence, and 3 clones had the A3 sequence. To evaluate the neutralization effect of A6 phage, animal test was carried out. The Shiga Toxin was incubated with A6 phage clone, then used to attack the Balb/C mice. As a control, the toxin was incubated with negative phage. In the control group, no mice survived. Comparing with it, the survival rate of the mice in neutralization group could reach to a level of 33.3%. It showed that the toxicity of Shiga Toxin was partly inhibited. The A6 peptide could be developed as an inhibitor of Shiga Toxin to cure the diseases caused by Shiga Toxin.

Animals↗
PubMed2005

Efficient synthesis and characterization of novel bibenzimidazole oligomers and polymers as potential conjugated chelating ligands.

[Structure: see text]. A simple and mild condensation route for the synthesis of novel bibenzimidazole oligomers and polymers is reported here using methyl 2,2,2-trichloroacetimidate as a key starting material. The dimer, trimer, tetramer, and polymers of bibenzimidazole were synthesized as a new series of potential conjugated chelating ligands for metallopolymer studies. The polymers show a maximum absorption at around 400 nm. The optical band gap of the polymer was estimated to be 2.68 eV.

Journal Article↗
PubMed2005

Chemoenzymatic approaches for streamlined detection of active site modifications on thiotemplate assembly lines using mass spectrometry.

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

Acylation↗
PubMed2005

Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase.

An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl transferase-catalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an alpha-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.

Amino Acid Sequence↗
PubMed2005

Nitric oxide induces tau hyperphosphorylation via glycogen synthase kinase-3beta activation.

Nitric oxide is associated with neurofibrillary tangle, which is composed mainly of hyperphosphorylated tau in the brain of Alzheimer's disease (AD). However, the role of nitric oxide in tau hyperphosphorylation is unclear. Here we show that nitric oxide produced by sodium nitroprusside (SNP), a recognized donor of nitric oxide, induces tau hyperphosphorylation at Ser396/404 and Ser262 in HEK293/tau441 cells with a simultaneous activation of glycogen synthase kinase-3beta (GSK-3beta). Pretreatment of the cells with 10 mM lithium chloride (LiCl), an inhibitor of GSK-3, 1 h before SNP administration inhibits GSK-3beta activation and prevents tau from hyperphosphorylation. This is the first direct evidence demonstrating that nitric oxide induces AD-like tau hyperphosphorylation in vitro, and GSK-3beta activation is partially responsible for the nitric oxide-induced tau hyperphosphorylation. It is suggested that nitric oxide may be an upstream element of tau abnormal hyperphosphorylation in AD.

Cells, Cultured↗
PubMed2005

Paraquat induces selective dopaminergic nigrostriatal degeneration in aging C57BL/6 mice.

BACKGROUND: Paraquat (PQ; 1, 1'-dimethyl-4, 4'-bipyridinium), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPTP (1-methyl-1, 2, 3, 6-tetrahydropyridine), has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). Aging is an accepted risk factor for idiopathic Parkinson's disease. The aim of this study was to test the hypothesis that paraquat could induce PD-like nigrostriatal dopaminergic degeneration in aging C57BL/6 mice. METHODS: Senile male C57BL/6 mice were intraperitoneally injected with either saline or PQ at 2-day intervals for a total of 10 doses. Locomotor activity and performance on the pole test were measured 7 days after the last injection and animals were sacrificed one day later. Level of dopamine (DA) and its metabolites levels in the striatum were measured by high-performance liquid chromatography with an electrochemical detector (HPLC-ECD), and numbers of tyrosine hydroxylase (TH) positive neurons were estimated using immunohistochemistry. RESULTS: Locomotor activities were significantly decreased and the behavioral performance on the pole test were significantly impaired in the PQ treated group. Level of DA and its metabolites levels in the striatum were declined by 8 days after the last injection. Immunohistochemical analyses showed that PQ was associated with a reduction in numbers of tyrosine hydroxylase positive neurons. CONCLUSIONS: Long-term repeated exposes to PQ can selectively impair the nigrostriatal dopaminergic system of senile mice, suggesting that PQ could play an important role in the pathogenesis of Parkinson's disease (PD). Our results also validate a novel model of PD induced by exposure to a toxic environmental agent.

Aging↗
PubMed2005

SyrB2 in syringomycin E biosynthesis is a nonheme FeII alpha-ketoglutarate- and O2-dependent halogenase.

The nine-residue lipodepsipeptide syringomycin E, elaborated as a phytotoxin by Pseudomonas syringae pv. syringae B301D contains a 4-Cl-L-Thr-9 moiety where failure to chlorinate results in a 3-fold drop in biological activity. The proteins SyrB1 and SyrB2 encoded by the biosynthetic cluster are shown to act as a substrate and enzyme pair for SyrB2-mediated chlorination of the aminoacyl-S-enzyme L-Thr-S-SyrB1. SyrB2 is a member of the nonheme Fe(II) alpha-ketoglutarate-dependent enzyme superfamily, and requires O2 and alpha-ketoglutarate as well as chloride ion to carry out monochlorination of the -CH3 group of L-Thr-S-SyrB1. Chlorination of L-Thr-S-SyrB1 was validated by thioesterase-mediated release of L-Thr and 4-Cl-L-Thr, N-derivatization as fluorescent isoindoles, and HPLC separation compared with authentic standards. Incubations with L-[14C]Thr and [36Cl-] as well as MS of the released products further validated identification. Enzymatic oxidative halogenation is a previously uncharacterized reaction type for nonheme Fe(II) enzymes and may be the general mode for biosynthetic halogenation of aliphatic carbons of natural products.

Chlorides↗