Biomedical subjects

R Zhao

Publications and source records attributed to R Zhao.

At least 37 records · Page 2Linked to original sources

PubMed2001

High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography.

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.

Amino Acid Sequence↗

PubMed2001

Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis.

The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.

Animals↗

PubMed2001

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry.

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

Chromatography, Liquid↗

PubMed2001

An efficient method to successively introduce transgenes into a given genomic locus in the mouse.

BACKGROUND: Expression of transgenes in mice requires transcriptional regulatory elements that direct expression in a chosen cell type. Unfortunately, the availability of well-characterized promoters that direct bona-fide expression of transgenes in transgenic mice is limited. Here we described a method that allows highly efficient targeting of transgenes to a preselected locus in ES cells. RESULTS: A pgk-LoxP-Neo cassette was introduced into a desired genomic locus by homologous recombination in ES cells. The pgk promoter was then removed from the targeted ES cells by Cre recombinase thereby restoring the ES cells' sensitivity to G418. We demonstrated that transgenes could be efficiently introduced into this genomic locus by reconstituting a functional Neo gene. CONCLUSION: This approach is simple and extremely efficient in facilitating the introduction of single-copy transgenes into defined genomic loci. The availability of such an approach greatly enhances the ease of using endogenous regulatory elements to control transgene expression and, in turn, expands the repertoire of elements available for transgene expression.

5' Untranslated Regions↗

PubMed2001

Association of tyrosine phosphatase SHP-2 with F-actin at low cell densities.

SHP-2 is an intracellular SH2 domain-containing protein-tyrosine phosphatase with an essential role in cell signaling. Here we demonstrate that localization of SHP-2 is regulated by cell density in a cell adhesion-dependent manner. When cells were plated at low densities, SHP-2 was distributed in Triton X-100-insoluble fractions, whereas it was totally soluble when cells were plated at high densities or when low density cells approached confluency. In all cases, the total protein level of SHP-2 was not changed. Fluorescent cell staining revealed that SHP-2 was co-localized with actin stress fibers to the cell peripheral at low cell densities but was diffused in the entire cytoplasm at high cell densities. Transient transfection of cells with truncated forms of SHP-2 demonstrated that the catalytic domain of the enzyme was responsible for the density-regulated distribution of SHP-2, but the catalytic activity was not required. An in vitro co-sedimentation study demonstrated direct binding of full-length and SH2 domain-truncated forms of SHP-2 to F-actin. The data indicate that SHP-2 is regulated by cell density and that it may have a role in assembling and disassembling of the actin network.

3T3 Cells↗

PubMed2001

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.

Oligopeptides↗

PubMed2001

FYVE-DSP2, a FYVE domain-containing dual specificity protein phosphatase that dephosphorylates phosphotidylinositol 3-phosphate.

We have recently isolated FYVE-DSP1, a FYVE domain-containing dual specificity protein phosphatase (R. Zhao, Y. Qi, and Z. J. Zhao, Biochem. Biophys. Res. Commun. 270, 222--229 (2000)). Here, we report a novel isozyme that we designated FYVE-DSP2. FYVE-2 contains a single FYVE domain at the C-terminus, and it shares approximately 47% overall sequence identity with FYBE-DSP1. Genomic sequence analyses revealed that the FYVE-DSP1 and FYVE-DSP2 genes share similar intron/exon organization. They are localizedon human chromosome 22q12 and chromosome 17, respectively. Like FYVE-DSP1, recombinant FYVE-DSP2 dephosphorylated low-molecular-weight phosphatase substrate para-nitrophenylphosphate, and its activity was inhibited by sodium vanadate. More importantly, our study also revealed that both FYVE-DSP1 and FYVE-DSP2 efficiently and specifically dephosphorylated phosphotidylinositol 3-phosphate. Subcellular fractionation demonstrated partition of FYVE-DSP1 and FYVE-DSP2 in membrane fractions, and immunofluorescent cell staining showed perinuclear localization of the enzymes. FYVE-DSP2 is expressed in many human tissues with an alternatively spliced isoform expressed in the kidney. Together with two homologous hypothetical proteins found in Caenorhabditis elegans and Drosophila, FYVE-DSP1 and FYVE-DSP2 form a subfamilyof phosphatases that may have an importantrole in cellular processes.

Adenoviridae↗

PubMed2001

Derivatization and fluorescence detection of amino acids and peptides with 9-fluorenylmethyl chloroformate on the surface of a solid adsorbent.

An approach that exploits the surface of a solid adsorbent is proposed for precolumn FMOC derivatization of amino acids and peptides. Amino acids (Ser, Glu, GABA, Val, Phe, Lys) and two neuropeptides (substance P and Leuenkephalin) were adsorbed on alkaline silica gel cartridges. After drying, they were reacted with 9-fluorenyl-methyl chloroformate (FMOC-Cl) in toluene. After washing off the excess FMOC-Cl with ethyl acetate, the derivatives were eluted with aqueous eluant. The eluates were separated and detected by means of HPLC with fluorescence detection. Compared with the traditional derivatization in the liquid phase, the extent of formation of byproducts of FMOC-Cl with water was greatly decreased, and the excess FMOC-Cl was eliminated completely.

Amino Acids↗

PubMed2001

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics.

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.

Chromatography, Liquid↗

PubMed2001

Study on the degeneracy of antisense peptides using affinity chromatography.

The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. Similar result was obtained from another experiment that Arg-substituted antisense peptide of fusion peptide (1-11) of influenza virus A was also shown the highest affinity binding to immobilized fusion peptide.

Amino Acid Sequence↗

PubMed2001

Marked suppression of the activity of some, but not all, antifolate compounds by augmentation of folate cofactor pools within tumor cells.

Folates have been co-administered with some antifolates to diminish host toxicity; however, the extent to which this will reduce antitumor activity is not known. To further clarify this issue, studies were undertaken to characterize and quantitate the impact of alterations in intracellular folate levels on the activities of a variety of antifolates in L1210 murine leukemia cells. Intracellular folate cofactor levels increased almost in proportion to the increase in extracellular 5-formyltetrahydrofolate (5-CHO-THF) over a concentration range that encompassed physiological levels of 5-methyltetrahydrofolate. This resulted in a spectrum of increases in the ic50 values of antifolates upon continuous exposure to drugs [Lometrexol (DDATHF) (70x) > trimetrexate (TMQ) (30x), multitargeted antifolate, LY231514 (ALIMTA) (30x) > Raltitrexed, Tomudex (ZD1694) (10x), 6R-2',5'-thienyl-5,10-dideazatetrahydrofolic acid (LY309887) (10x) > methotrexate (MTX) (6x) > (2S)-2-[o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido]-4-(tetrazol-5-yl) butyric acid (ZD9331) (3x), N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-l-ornithine (PT523) (3x)]. Upon a 4-hr pulse exposure to drug, the ic50 values for DDATHF and ALIMTA were increased > 180- and 5-fold, respectively, with only a 2.5-fold increase in the extracellular 5-CHO-THF level within the physiological range. The reductions in drug sensitivities could be attributed to decreases in accumulation of polyglutamate derivatives of ALIMTA and DDATHF. Hence, in these studies, natural folates diminished the activity of agents that undergo polyglutamation by suppression of the formation of these active congeners at the level of folylpolyglutamate synthetase. For inhibitors of dihydrofolate reductase, the suppressive effect of endogenous folates appears to be due to competition between the antifolate and dihydrofolate at the level of the target enzyme. These data should be carefully considered in the design of regimens with antifolates, which incorporate co-administration of folates.

Animals↗

PubMed2001

Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs.

The reduced folate carrier (RFC1) is an important route by which the major blood folate, 5-methyltetrahydrofolate, is transported into mammalian cells. In this study we determined the consequences of inactivation of RFC1 in mice by homologous recombination. While RFC1-null embryos died in utero before embryonic day 9.5 (E9.5), near-normal development could be sustained in RFC1(-)/- embryos examined at E18.5 by supplementation of pregnant RFC1(+/-) dams with 1-mg daily subcutaneous doses of folic acid. About 10% of these animals went on to live birth but died within 12 days. These RFC1(-)/- mice showed a marked absence of erythropoiesis in bone marrow, spleen, and liver along with lymphoid depletion in the splenic white pulp and thymus. In addition, there was some impairment of renal and seminiferous tubule development. These data indicate that in the absence of RFC1 function, neonatal animals die due to failure of hematopoietic organs.

Alleles↗

PubMed2001

New method for high-performance liquid chromatographic separation and fluorescence detection of ginsenosides.

A novel pre-column derivatization method for the quantitative determination of ginsenosides by HPLC with fluorescence detection was established. The double bond at the C24-C25 position of ginsenoside was converted into an aldehyde group by means of ozonolysis. Then the aldehyde group reacts with FMOC-hydrazine forming the ginsenoside FMOC-hydrazone. The derivatized products were separated by RP-HPLC with gradient elution. The detection limits of ginsenosides Rg1 and Rb1 were 2.0 ng (about 2.5 pmol) and 1.0 ng (about 0.9 pmol), respectively. This method can be used for all ginsenosides having the C24-C25 double bond.

Chromatography, High Pressure Liquid↗

PubMed2001

Molecular cloning and characterization of SPAP1, an inhibitory receptor.

We have cloned a novel cell-surface protein designated SPAP1a for SH2 domain-containing phosphatase anchor protein 1a. SPAP1a belongs to the group of type I transmembrane proteins. Its extracellular domain contains a single immunoglobulin-like domain, and its intracellular segment has two immunoreceptor tyrosine-based inhibition motifs (ITIMs). We also identified two alternatively spliced products that were named SPAP1b and SPAP1c. SPAP1b contains a short intracellular part without ITIMs, while SPAP1c lacks the transmembrane segment and represents a potential soluble protein. Sequence alignment with the genomic database revealed that the SPAP1 gene contains seven exons and is localized at chromosome 1q21. PCR analyses demonstrated that SPAP1a mRNA is specifically expressed in human hematopoietic tissues including spleen, peripheral blood, and bone marrow, and it may be restricted to expression in B cells. Recombinant SPAP1a is tyrosine phosphorylated in cells upon pervanadate stimulation and tyrosine-phosphorylated SPAP1a recruits the SH2 domain containing phosphatase SHP-1, but not SHP-2. As a specific anchor protein of SHP-1, SPAP1a may have an important role in hematopoietic cell signaling.

Alternative Splicing↗

PubMed2001

Molecular cloning of human thiamin pyrophosphokinase.

Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) catalyses phosphorylation of thiamin to thiamin pyrophosphate, an active enzyme cofactor. Here we describe the cloning of complete human TPK1 cDNA from an adult liver library. Human TPK1 is 89% identical to murine TPK1 at the protein level. The gene maps to chromosome 7q34-36, consists of at least eight exons, and spans a distance at least of 420 kb. The mRNA of human TPK1 is highly expressed in testis, small intestine and kidney with lesser but detectable expression in brain, liver, placenta and spleen. The availability of the human TPK1 gene will provide another useful tool for studying the role of this enzyme in human thiamin metabolism and deficiency state.

Amino Acid Sequence↗

PubMed2001

Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells.

The thiamin transporter encoded by SLC19A2 and the reduced folate carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. This conclusion was supported by the following observations. (i) Efflux of intracellular TPP was increased in cells with high expression of RFC1. (ii) Methotrexate inhibits TPP influx. (iii) TPP competitively inhibits methotrexate influx. (iv) Loading cells, which overexpress RFC1 to high levels of methotrexate to inhibit competitively RFC1-mediated TPP efflux, augment TPP accumulation. (v) There was an inverse correlation between thiamin accumulation and RFC1 activity in cells grown at a physiological concentration of thiamin. The modulation of thiamin accumulation by RFC1 in murine leukemia cells suggests that this carrier may play a role in thiamin homeostasis and could serve as a modifying factor in thiamin nutritional deficiency as well as when the high affinity thiamin transporter is mutated.

Animals↗

PubMed2001

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma.

Novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin-PGMA-I was prepared by directly coupling amino-groups of heparin with PGMA. Heparin-PGMA-II and III were prepared by the coupling of heparin to amino-PGMA, which was obtained by amination of PGMA. Heparin-PGMA-II was prepared by coupling the carboxyl groups of heparin to amino-PGMA by using water-soluble carbodiimide as coupling reagent, and heparin-PGMA-III was prepared by the reductive amination of heparin and amino-PGMA with sodium cyanoborohydride. The heparin contents of heparin-PGMA-I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin-PGMA-I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non-specific interaction with other proteins and can be used in a wide pH range. All the three heparin-PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography.

Antithrombin III↗

PubMed2001

Capillary electrophoresis of catecholamines with laser-induced fluorescence intensified charge-coupled device detection.

A capillary electrophoresis-laser-induced fluorescence-intensified charge-coupled device system was used for the separation and determination of catecholamines. Optimization of derivatization and separation conditions was investigated in order to reach a high separation efficiency and sensitivity. All fluorecein isothiocyanate derivatives of catecholamines were satisfactorily separated within 12 min. The detection limits were in attomole ranges. This method allows determination of catecholamines with high separation efficiencies and sensitivity.

Benzoates↗