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P Xu

Publications and source records attributed to P Xu.

At least 37 records · Page 2Linked to original sources
PubMed2001

Optimal design features of camelized human single-domain antibody libraries.

We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.

Amino Acid Sequence↗
PubMed2001

Estimation of divergence times from multiprotein sequences for a few mammalian species and several distantly related organisms.

When many protein sequences are available for estimating the time of divergence between two species, it is customary to estimate the time for each protein separately and then use the average for all proteins as the final estimate. However, it can be shown that this estimate generally has an upward bias, and that an unbiased estimate is obtained by using distances based on concatenated sequences. We have shown that two concatenation-based distances, i.e., average gamma distance weighted with sequence length (d(2)) and multiprotein gamma distance (d(3)), generally give more satisfactory results than other concatenation-based distances. Using these two distance measures for 104 protein sequences, we estimated the time of divergence between mice and rats to be approximately 33 million years ago. Similarly, the time of divergence between humans and rodents was estimated to be approximately 96 million years ago. We also investigated the dependency of time estimates on statistical methods and various assumptions made by using sequence data from eubacteria, protists, plants, fungi, and animals. Our best estimates of the times of divergence between eubacteria and eukaryotes, between protists and other eukaryotes, and between plants, fungi, and animals were 3, 1.7, and 1.3 billion years ago, respectively. However, estimates of ancient divergence times are subject to a substantial amount of error caused by uncertainty of the molecular clock, horizontal gene transfer, errors in sequence alignments, etc.

Animals↗
PubMed2001

Identification of a spliced leader RNA binding protein from Trypanosoma cruzi.

Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.

Amino Acid Sequence↗
PubMed2001

Identification of a high frequency of mutation at exon 8 of the ATP7B gene in a Chinese population with Wilson disease by fluorescent PCR.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder of copper transport. Mutation analysis has led to the discovery of more than 100 mutations at ATP7B, and most of them are population specific. OBJECTIVES: To verify the high frequency of mutation at exon 8 of ATP7B in Chinese patients with WD and to establish a DNA diagnostic method for WD. SETTING: University medical centers. PATIENTS AND METHODS: Screening for mutations at exon 8 of ATP7B by fluorescent polymerase chain reaction analysis and restriction analysis was conducted in 106 unrelated Chinese patients with WD and in 55 individuals from 10 Chinese families with WD. RESULTS: Five homozygotes and 32 heterozygotes were identified. Sequence analysis showed a missense mutation (2273G-->T) and a nonsense mutation (2250C-->G) together at exon 8. The rate of gene mutation in 106 patients was 35% (5% homozygous and 30% heterozygous). Samples of DNA from 55 individuals from 10 Chinese families with WD were examined by fluorescent polymerase chain reaction. We found that 13 siblings were carriers (24%). CONCLUSIONS: A high frequency of mutation at exon 8 of the ATP7B gene exists in the Chinese population, and fluorescent polymerase chain reaction analysis may be an effective and accurate assay in detection of the WD gene.

Adenosine Triphosphatases↗
PubMed2001

Trypanosoma cruzi: exogenously regulated gene expression.

A regulated expression vector would provide a strong tool for the dissection of gene function in Trypanosoma cruzi. Herein, we establish a system in which genes in T. cruzi expression vectors can be exogenously regulated by tetracycline. We first generated strains of T. cruzi that stably express the repressor of the bacterial tetracycline resistance gene and T7 RNA polymerase. Based on these strains, we developed two T. cruzi expression systems regulated by tetracycline--the first by use of a regulated rRNA promoter and the second by use of a regulated T7 promoter. In the former, we constructed an expression vector in which tetracycline resistance gene operators flank the transcription start point of the T. cruzi rRNA gene promoter. Reporter gene activity from this modified promoter was regulated up to 20-fold in the presence of different concentrations of tetracycline. In the T7 system, tetracycline resistance gene operators flank the transcription start point of the T7 promoter. Reporter gene activity from this modified promoter was regulated up to 150-fold in the presence of different concentrations of tetracycline. Expression in these systems was repressed when tetracycline was removed even after full induction for extended periods in the presence of tetracycline. We are now using these two systems to test protein function in T. cruzi.

Animals↗
PubMed2001

Purification and in vitro folding of recombinant human thrombopoietin receptor expressed in Escherichia coli.

Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells. TPOR plays an important role in regulating platelet production. Due to its low expression level in human tissue, studies on the biochemical and biophysical properties of TPOR have been limited. In the present study, an extracellular domain of recombinant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies. Purification was achieved by metal chelated chromatography under denaturing condition and was refolded by gel filtration chromatography. Far UV circular dichroism spectroscopy and surface plasmon resonance experiments were performed to demonstrate that the protein was in a refolded state and could bind with its ligand. Thus, a production and purification scheme was developed by which sufficient quantities of rh TPOR-EN can be made available for biochemical and biophysical characterizations.

Binding Sites↗
PubMed2001

In vivo modification of Na(+),K(+)-ATPase activity in Drosophila.

We have constructed and characterized transgenic Drosophila lines with modified Na(+),K(+)-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type alpha subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na(+),K(+)-ATPase alpha subunit hypomorphic mutant, 2206. In contrast, a mutant alpha subunit (alpha(D369N)) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled alpha transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of alpha subunit transgene. Using the Gal4-UAS system we have limited expression of alpha transgenes to cell types that normally express a particular Drosophila Na(+),K(+)-ATPase beta (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.

Animals↗
PubMed2001

Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.

Mice carrying mutations in the fatty liver dystrophy (fld) gene have features of human lipodystrophy, a genetically heterogeneous group of disorders characterized by loss of body fat, fatty liver, hypertriglyceridemia and insulin resistance. Through positional cloning, we have isolated the gene responsible and characterized two independent mutant alleles, fld and fld(2J). The gene (Lpin1) encodes a novel nuclear protein which we have named lipin. Consistent with the observed reduction of adipose tissue mass in fld and fld(2J)mice, wild-type Lpin1 mRNA is expressed at high levels in adipose tissue and is induced during differentiation of 3T3-L1 pre-adipocytes. Our results indicate that lipin is required for normal adipose tissue development, and provide a candidate gene for human lipodystrophy. Lipin defines a novel family of nuclear proteins containing at least three members in mammalian species, and homologs in distantly related organisms from human to yeast.

3T3 Cells↗
PubMed2001

Effects of matrix proteins on the expression of matrix metalloproteinase-2, -9, and -14 and tissue inhibitors of metalloproteinases in human cytotrophoblast cells during the first trimester.

The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins alpha(1), alpha(5), alpha(6), beta(1), and beta(4). Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function.

Adult↗
PubMed2001

pH-induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: implications for zymogen activation.

The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N-terminal, 81-residue inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep 'autocatalytic' removal of the prodomain at the C-terminal side of the two consensus sites, R(78)-T-K-R(81) approximately and R(44)-G-V-T-K-R(49) approximately. The furin-mediated cleavage at R(44)-G-V-T-K-R(49) approximately, in particular, is significantly accelerated in an 'acidic' environment. Here, we show that under neutral pH conditions, the inhibitory prodomain of furin is partially folded and undergoes conformational exchanges as indicated by extensive broadening of the NMR spectra. Presence of many ring-current shifted methyl resonances suggests that the partially folded state of the prodomain may still possess a 'semirigid' protein core with specific packing interactions among amino acid side chains. Measurements of the hydrodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformational stability of the prodomain appears to be pH sensitive, in that the prodomain undergoes an unfolding transition towards acidic conditions. Our NMR analyses establish that the acid-induced unfolding is mainly experienced by the residues from the C-terminal half of the prodomain (residues R(44)-R(81)) that contains the two furin cleavage sites. A 38-residue peptide fragment derived from the entire pH-sensitive C-terminal region (residues R(44)-R(81)) does not exhibit any exchange-induced line broadening and adopts flexible conformations. We propose that at neutral pH, the cleavage site R(44)-G-V-T-K-R(49) approximately is buried within the protein core that is formed in part by residues from the N-terminal region, and that the cleavage site becomes exposed under acidic conditions, leading to a facile cleavage by the furin enzyme.

Amino Acid Sequence↗
PubMed2001

A peptide derived from the C-terminal part of a plant cysteine protease folds into a stack of two beta-hairpins, a scaffold present in the emerging family of granulin-like growth factors.

A 35 amino acid residue peptide corresponding to the N-terminal subdomain of the granulin-like repeat from rice oryzain beta was synthesized and regioselectively oxidized to produce a species with a [1-3, 2-4] disulfide-pairing pattern. The resulting peptide was studied in solution using NMR and was shown to adopt the tertiary topology of a stack of two beta-hairpins found in the emerging family of granulin-like growth factors. Because of the longer second beta-hairpin, the overall conformation of the peptide is somewhat more flexible than that of its well-structured carp granulin-1 analog. Except for the cysteine alignment, there is very little sequence homology between granulin-like growth factors from the animal kingdom and the granulin-like repeats at the C-termini of plant cysteine proteases. Therefore, the stack of two beta-hairpins may be a conserved three-dimensional organization of the granulin-like repeats from evolutionary distant sources with a significant role in specific protein-protein interactions.

Amino Acid Sequence↗
PubMed2001

[Using fluorescence PCR analysis for early diagnosis and carriers detection of Chinese Wilson's disease].

OBJECTIVE: To diagnose the pre-symptomatic cases of Chinese Wilson's disease(WD) and detect the potential carriers by using fluorescence PCR technique. METHODS: Screening the high frequency spot Arg778Leu of WD gene mutation in 66 Chinese WD patients, 55 healthy family members and 30 controls, and selecting 3 random samples (2 from WD patients, 1 from controls) for DNA sequencing to testify the accuracy of fluorescence PCR. RESULTS: Among 66 Chinese WD cases, 5 were found homozygous for mutation of Arg778Leu and 21 were compound heterozygous; the gene mutation rate was totally 39.4%. Out of 55 healthy family members, 12 were found heterozygous, and it was confirmed that 11 of these 12 individuals were WD gene carriers but not pre-symptomatic patients. The results of direct DNA sequencing consisted with those results detected by fluorescence PCR. CONCLUSION: The Arg778Leu mutation in exon 8 is the high frequency spot of Chinese Wilson's disease gene. Fluorescence PCR analysis is a rapid, accurate gene diagnostic method and demonstrates a high detecting rate.

Adolescent↗
PubMed2001

[A study of gene products encoded by Wilson disease gene].

OBJECTIVE: To investigate the pathogenesis of Wilson disease (WD) by detecting its gene products encoded by WD gene (ATP7B). METHODS: Patients diagnosed as WD were analyzed by SDS-PAGE in conjunction with Western blot. Two antibodies were used, which are specially against the sixth copper binding domain (Anti-CuBD) and ATP binding domain (Anti-ABD) of WD protein. RESULTS: The WD proteins were not expressed in two patients when using anti-CuBD, and poorly expressed when using anti-ABD. CONCLUSIONS: WD is highly heterogeneous in clinical manifestations and inheritance pattern. Two WD patients might simultaneously have exon 5 mutations and exon 8 mutations. The study of WD gene products would probe into the pathogenesis of WD.

Adenosine Triphosphatases↗
PubMed2001

[A study of Wilson's disease gene encoded products and gene mutations].

OBJECTIVE: To investigate the pathogenesis of Wilson's disease(WD). METHODS: Hepatocytes were isolated from WD patients' bioptic hepatic samples and cultured in vitro; WD proteins, the gene putative encoded products, were detected by SDS-PAGE in conjunction with Western blotting in liver samples of three patients and two controls. Their genomic DNAs were analyzed by means of direct DNA sequencing of WD gene (ATP7B) on exon 8. RESULTS: The WD proteins lanes from two WD patients were found to be much weaker than that from the control, from which one WD patient was proven as heterozygote of 778 position CGG-->CTG(Arg778Leu) and 770 position CTC-->CTG change of ATP7B. CONCLUSION: WD is highly heterogeneous in clinical manifestations and inheritance pattern. Abnormally expressed putative WD proteins in WD patients might be the results of ATP7B mutations, and the study of ATP7B products would help to probe into the pathogenesis of WD.

Adenosine Triphosphatases↗
PubMed2001

Technical-economic modelling of integrated water management: wastewater reuse in a French island.

An integrated technical-economic model is used to address water management issues in the French island of Noirmoutier. The model simulates potable water production and supply, potable and non potable water demand and consumption, wastewater collection, treatment and disposal, water storage, transportation and reuse. A variety of water management scenarios is assessed through technical, economic and environmental evaluation. The scenarios include wastewater reclamation and reuse for agricultural and landscape irrigation as well as domestic non potable application, desalination of seawater and brackish groundwater for potable water supply. The study shows that, in Noirmoutier, wastewater reclamation and reuse for crop irrigation is the most cost-effective solution to the lack of water resources and the protection of sensitive environment. Some water management projects which are regarded as having less economic benefit in the short-term may become competitive in the future, as a result of tightened environmental policy, changed public attitudes and advanced water treatment technologies. The model provides an appropriate tool for water resources planning and management.

Agriculture↗
PubMed2001

[Preparation of green fluorescent protein retrovirus and its application in mediating gene transfer into retinal pigment epithelial cells].

OBJECTIVE: To determine whether human retinal pigment epithelial (RPE) cells can be infected by retrovirus and modified by retrovirus-mediated gene transfer. METHODS: Recombination retroviral vector pLNCX-GFP (green fluorescent protein, GFP) was generated by inserting 780 bp GFP cDNA fragment into the MCS site of pLNCX. pLNCX-GFP was transfected into ecotropic packaging cell line PhiX-Eco and amphotropic packaging cell line PhiX-Ampho and PA317. Retroviral titer was tested by counting GFP expression of NIH3T3 cells. Then RPE cells were infected by using GFP retrovirus-containing supernatant. RESULTS: GFP was expressed and retrovirus was produced upon pLNCX-GFP being transfected into packaging cell line. The GFP retrovirus was able to infect primary cultured human RPE cells and immortalized RPE cell line. CONCLUSION: The retrovirus can introduce a foreign gene into RPE cells efficiently, thereby it can be used as an important tool to deliver gene into RPE for therapy of fundus diseases.

Cells, Cultured↗
PubMed2001

[Establishment of human neuroectodermal tumor cell line (PFSK) with stable expression of xanthine oxidoreductase].

Xanthine Oxidoreductase (XOR) is the key enzyme in purine metabolism and also produces oxygen free radicals. As a continuation of our previous work, in this study, we constructed a retrovirus expression vector (pLNCX2-XOR) containing full length of XOR cDNA. Retrovirus was produced by the virus package cell line PT-67 following the transfection of PT-67 with pLNCX2-XOR and used to infect the human primitive neuroectodermal tumor cell line (PFSK). Infected PFSK cells were selected by G418 to establish cell line with stable expression of XOR. The expression of XOR in the cell line we established was confirmed by RT-PCR, Immunocytochemistry and XOR activity assay.

Genetic Vectors↗