Biomedical subjects

Zhen Guo

Publications and source records attributed to Zhen Guo.

13 recordsLinked to original sources

PubMed2026

Dual signal-enhanced immunochromatographic test strip based on Au@PtNPs: From sensitive detection of thiamethoxam to multiplex pesticide screening in vegetables.

Immunochromatographic test strip (ICTS) is a rapid analytical technique widely used in environmental and food detection owing to its merits of simple operation and short analysis time. Herein, three-dimensional nanoflower-structured gold‑platinum nanoparticles (Au@PtNPs) were synthesized via a seed-growth method. Compared with conventional gold nanoparticles (AuNPs), Au@PtNPs exhibited stronger signal intensity, excellent catalytic performance, and efficient antibody binding efficiency. Colorimetric Au@PtNPs-ICTS and catalytic colorimetric Au@PtNPs-ICTS were developed for the sensitive detection of thiamethoxam (THI) in vegetables. The limits of detection (LODs) for colorimetric Au@PtNPs-ICTS and catalytic colorimetric Au@PtNPs-ICTS quantitative analysis were 0.18 ng/mL and 0.093 ng/mL, respectively, representing approximately 3-fold and 6-fold improvement compared to AuNPs-ICTS (0.56 ng/mL). Furthermore, highly sensitive detection of multiple pesticide residues (chlorpyrifos, acetamiprid, and imidacloprid) was achieved by replacing the corresponding target antigens and antibodies, which further verified the universality of this immunochromatographic strategy.

Thiamethoxam↗

PubMed2006

Long-range multilocus haplotype phasing of the MHC.

Haplotypes are a powerful tool for identifying the genetic basis of common complex diseases. Disease-association mapping requires molecular methods for haplotyping biallelic SNP variation and highly complex polymorphisms. We developed a method for phasing HLA-A, HLA-B, and HLA-DRB1 alleles on chromosome 6 in unrelated individuals. This method uses the highly polymorphic HLA-B locus to discriminate the two HLA haplotypes in heterozygous individuals and its ideal location 1.4 Mbp telomeric to HLA-DRB1 and 1.2 Mbp centromeric to HLA-A to capture 2-Mbp-long genomic DNA. Genomic DNA representing a single HLA-B-captured haplotype is genotyped for HLA-A and HLA-DRB1 alleles and linkage to HLA-B is established. Proof of principle was established in a large blinded study of phase-known samples. Availability of an efficient method for MHC haplotype phase determination will facilitate the mapping of causative MHC-resident genes in many human diseases and has the potential to be broadened to other polymorphic gene complexes.

Genes, MHC Class I↗

PubMed2006

Automation of fluorescent differential display with digital readout.

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Most of previous DD work has taken a "shot-gun" approach of identifying one gene at a time, with a limited number of polymerase chain reaction (PCR) reactions set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a new platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

Automation↗

PubMed2005

PKA-mediated protein phosphorylation protects ezrin from calpain I cleavage.

Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase (PKA) activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies demonstrate the functional relevance of PKA-mediated phosphorylation of ezrin in parietal cell secretion [R. Zhou, X. Cao, C. Watson, Y. Miao, Z. Guo, J.G. Forte, X. Yao, Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651]. Here we show that activation of PKA protects ezrin from calpain I-mediated proteolysis without alteration of calpain I activation and fodrin breakdown. To determine whether phosphorylation of Ser66 by PKA affects the insensitivity to the calpain I-mediated cleavage, recombinant proteins of ezrin, both wild type and S66A/D mutants, were incubated with the purified calpain I. Indeed, phosphorylation-like S66D mutant ezrin is resistant to calpain I-mediated proteolysis while wild type and S66A mutant were sensitive. In fact, expression of phosphorylation-like S66D, but not S66A, mutant in parietal cells confers its resistance to calpain I-mediated proteolysis. Taken together, these results indicate that phosphorylation of ezrin by PKA modulates its sensitivity to calpain I cleavage.

Animals↗

PubMed2005

PALS1 specifies the localization of ezrin to the apical membrane of gastric parietal cells.

The ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells.

Animals↗

PubMed2004

Nek2A kinase regulates the localization of numatrin to centrosome in mitosis.

Chromosome segregation in mitosis is orchestrated by the kinetochore and spindle microtubules stemming from two centrosomes. Our recent studies demonstrated the importance of Nek2A in faithful chromosome segregation during mitosis. Here, we report that Nek2A regulates the function of numatrin in mitosis. The biochemical interaction between Nek2A and numatrin in mitotic cells was revealed by a set of reciprocal immunoprecipitation experiments using Nek2A and numatrin antibodies, respectively. The interaction is validated by a pull-down assay using recombinant Nek2A and numatrin proteins. Moreover, our immunofluorescence studies demonstrate that numatrin becomes centrosome-associated as the cell enters into mitosis and depart from the centrosome after sister chromatid separation in anaphase. The co-localization of numatrin and Nek2A to the centrosome suggests their interaction with and involvement in centrosome function. Indeed, elimination of Nek2A kinase by siRNA diminished its association with the centrosome. Furthermore, we show that numatrin is phosphorylated by wild type but not kinase-death Nek2A. Our studies suggest that the Nek2A kinase cascade is essential for the localization of numatrin to the centrosome.

Centrosome↗

PubMed2003

Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation.

Gastric ezrin was initially identified as a phosphoprotein associated with parietal cell activation. To explore the nature of ezrin phosphorylation, proteins from resting and secreting gastric glands were subjected to two-dimensional SDS-PAGE. Histamine triggers acid secretion and a series of acidic isoforms of ezrin on two-dimensional SDS-PAGE. Mass spectrometric analysis of these acidic ezrin spots induced by stimulation suggests that Ser66 is phosphorylated. To determine whether Ser66 is a substrate of protein kinase A (PKA), recombinant proteins of ezrin, both wild type and S66A mutant, were incubated with the catalytic subunit of PKA and [32P]ATP. Incorporation of 32P into wild type but not the mutant ezrin verified that Ser66 is a substrate of PKA. In addition, expression of S66A mutant ezrin in cultured parietal cells attenuates the dilation of apical vacuolar membrane associated with stimulation by histamine, indicating that PKA-mediated phosphorylation of ezrin is necessary for acid secretion. In fact, expression of phosphorylation-like S66D mutant in parietal cells mimics histamine-stimulated apical vacuole remodeling. Further examination of H,K-ATPase distribution revealed a blockade of stimulation-induced proton pump mobilization in S66A but not S66D ezrin-expressing parietal cells. These data suggest that PKA-mediated phosphorylation of ezrin plays an important role in mediating the remodeling of the apical membrane cytoskeleton associated with acid secretion in parietal cells.

Adenosine Triphosphate↗

PubMed2003

Determination of EDTA species in water by square-wave voltammetry using a chitosan-coated glassy carbon electrode.

The electrochemical behavior of Fe(EDTA)(-/2-) on a chitosan-coated glassy carbon electrode (GCE) and the determining method of the EDTA species in water samples by square-wave voltammetry (SWV) have been investigated. Fe(EDTA)(-) can be adsorbed on the positively charged chitosan membrane assembled on a GCE and be reduced at a potential of -0.095V vs. Ag/AgCl. On the chitosan-coated GCE, the cathodic stripping peak current is proportional to the concentration of EDTA after the operating parameters of SWV were optimized. The measuring range of EDTA was from 2.0x10(-6) to 8.0x10(-5)mol/L with a correlation coefficient of r=0.998 and a detection limit of 2.0x10(-7)mol/L. The relative standard deviation was less than 6.4% (n=5) and the recovery was in the range of 98-105% for the determination of practical samples. The result was consistent with that from the HPLC method.

Carbon↗

PubMed2003

Polarized distribution of IQGAP proteins in gastric parietal cells and their roles in regulated epithelial cell secretion.

Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.

Animals↗

PubMed2003

Determination of EDTA species in water by second-derivative square-wave voltammetry using a chitosan-coated glassy carbon electrode.

Based on the adsorption of Fe(EDTA)- on a chitosan-coated glassy carbon electrode, a second-derivative square-wave voltammetry for the determination of the EDTA species in water samples was investigated. The measuring range of EDTA was from 6.0 x 10(-7) to 5.0 x 10(-5) mol/L with a correlation coefficient of 0.998 and a detection limit of 2.8 x 10(-7) mol/L. The relative standard deviation was less than 6.2% (n = 5) and the recovery was in the range of 98-105% for the determination of practical samples. The result was consistent with that from the HPLC method.

Journal Article↗

PubMed2003

Triptolide inhibits the growth and metastasis of solid tumors.

Triptolide (TPL), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F, was tested for its antitumor properties in several model systems. In vitro, TPL inhibited the proliferation and colony formation of tumor cells at extremely low concentrations (2-10 ng/ml) and was more potent than Taxol. Likewise, in vivo, treatment of mice with TPL for 2-3 weeks inhibited the growth of xenografts formed by four different tumor cell lines (B16 melanoma, MDA-435 breast cancer, TSU bladder cancer, and MGC80-3 gastric carcinoma), indicating that TPL has a broad spectrum of activity against tumors that contain both wild-type and mutant forms of p53. In addition, TPL inhibited experimental metastasis of B16F10 cells to the lungs and spleens of mice. The antitumor effect of TPL was comparable or superior with that of conventional antitumor drugs, such as Adriamycin, mitomycin, and cisplatin. Importantly, tumor cells that were resistant to Taxol attributable to the overexpression of the multidrug resistant gene 1 were still sensitive to the effects of TPL. Studies on cultured tumor cells revealed that TPL induced apoptosis and reduced the expression of several molecules that regulate the cell cycle. Taken together, these results suggest that TPL has several attractive features as a new antitumor agent.

Animals↗

PubMed2002

Oligonucleotide arrays for high-throughput SNPs detection in the MHC class I genes: HLA-B as a model system.

A simple and efficient oligonucleotide array was developed to identify single nucleotide polymorphisms (SNPs) encoded within the highly polymorphic human major histocompatibility complex (MHC) using HLA-B as a model system. A total of 137 probes were designed to represent all known polymorphisms encoded in exons 2 and 3. PCR products were amplified from human genomic DNA and allowed to hybridize with the oligonucleotide array. Hybridization was detected by fluorescence scanning, and HLA-B alleles were assigned by quantitative analysis of the hybridization results. Variables known to influence the specificity of hybridization, such as oligonucleotide probe size, spacer length, surface density, hybridization conditions, and array uniformity and stability were studied. The efficiency and specificity of identifying HLA-B SNPs using the oligonucleotide arrays was evaluated by blinded analysis of 100 samples from unrelated individuals representing all HLA-B phenotypes. The oligonucleotide array method described in this paper provides unambiguous detection of complex heterozygous SNP combinations. This methodological approach may be applied to other highly polymorphic gene systems.

Alleles↗

PubMed2002

Osmoregulatory alterations in taurine uptake by cultured human and bovine lens epithelial cells.

PURPOSE: Comparative assessment of cultured human lens epithelial cells (HLECs) and bovine lens epithelial cells (BLECs) established the nature of the relationship between taurine-concentrating capability and intracellular polyol accumulation or extracellular hypertonicity. METHODS: The kinetic characteristics of active taurine accumulation based on the measurement of in vitro [3H]-taurine uptake were resolved by side-to-side review of cultured HLECs and BLECs pre-exposed to either galactose-supplemented medium or extracellular hypertonicity. Competitive RT-PCR was used to appraise variation in taurine transporter (TauT) mRNA abundance from cells maintained in hyperosmotic medium over a 72-hour exposure period. RESULTS: The capacity to accumulate [3H]-taurine was significantly lowered after prolonged (20-hour) incubation of cultured BLECs in 40 mM galactose in contrast to HLECs, the latter cells' velocity curve being indistinguishable from control cells in physiological medium. Inhibition of the intracellular taurine transport site appeared to be noncompetitive, in that there was a marked reduction in the V(max) without significant alteration in the K(m) to a high-affinity transport site. Galactitol content in BLECs exceeded five times that found in HLECs. The coadministration of the aldose reductase inhibitor, sorbinil, with 40 mM galactose completely prevented the inhibitory effect of galactose on [3H]-taurine uptake. Acute exposure (3 hours) of HLECs and BLECs to a range of 10 to 40 mM galactitol or 10 to 40 mM galactose plus sorbinil-supplemented medium suggested by Dixon plot that neither galactitol nor galactose interacted with the extracellular taurine transport site. In contrast, [3H]-taurine accumulation was markedly elevated in both HLECs and BLECs after prolonged exposure to galactose-free medium made hyperosmotic by supplementation with sodium chloride. The enhanced taurine uptake capacity involved increase in peak velocity (V(max)) without significant change in Michaelis-Menten constant (K(m)). Cultured HLECs and BLECs responded to hypertonicity with an inducible but transitory upregulation of TauT mRNA. CONCLUSIONS: These results demonstrate that lens epithelial cells express a high-affinity TauT protein capable of active uptake, but predisposed to inhibition by intracellular galactitol when the sugar alcohol is present in sufficiently high concentration to interfere with cell metabolism. Furthermore, lens epithelial cells respond to hypertonic stress by raising taurine transport activity. The increase in taurine uptake is due to an increase in the number of high-affinity TauTs expressed as a result of an increase in the manifestation of taurine mRNA stemming from exposure to hypertonic medium.

Aldehyde Reductase↗