Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation.

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.

[Linkage analysis of susceptibility loci in 2 target chromosomes in pedigrees with paranoid schizophrenia and undifferentiated schizophrenia].

OBJECTIVE: To investigate the relationship of susceptibility loci in chromosomes 1q21-25 and 6p21-25 and schizophrenia subtypes in Chinese population. METHODS: A genomic scan and parametric and non-parametric analyses were performed on 242 individuals from 36 schizophrenia pedigrees, including 19 paranoid schizophrenia and 17 undifferentiated schizophrenia pedigrees, from Henan province of China using 5 microsatellite markers in the chromosome region 1q21-25 and 8 microsatellite markers in the chromosome region 6p21-25, which were the candidates of previous studies. All affected subjects were diagnosed and typed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR; American Psychiatric Association, 2000). All subjects signed informed consent. RESULTS: In chromosome 1, parametric analysis under the dominant inheritance mode of all 36 pedigrees showed that the maximum multi-point heterogeneity Log of odds score method (HLOD) score was 1.33 (α = 0.38). The non-parametric analysis and the single point and multi-point nonparametric linkage (NPL) scores suggested linkage at D1S484, D1S2878, and D1S196. In the 19 paranoid schizophrenias pedigrees, linkage was not observed for any of the 5 markers. In the 17 undifferentiated schizophrenia pedigrees, the multi-point NPL score was 1.60 (P= 0.0367) at D1S484. The single point NPL score was 1.95(P= 0.0145) and the multi-point NPL score was 2.39 (P= 0.0041) at D1S2878. Additionally, the multi-point NPL score was 1.74 (P= 0.0255) at D1S196. These same three loci showed suggestive linkage during the integrative analysis of all 36 pedigrees. In chromosome 6, parametric linkage analysis under the dominant and recessive inheritance and the non-parametric linkage analysis of all 36 pedigrees and the 17 undifferentiated schizophrenia pedigrees, linkage was not observed for any of the 8 markers. In the 19 paranoid schizophrenias pedigrees, parametric analysis showed that under recessive inheritance mode the maximum single-point HLOD score was 1.26 (α = 0.40) and the multi-point HLOD was 1.12 (α = 0.38) at D6S289 in the chromosome 6p23. In nonparametric analysis, the single-point NPL score was 1.52 (P= 0.0402) and the multi-point NPL score was 1.92 (P= 0.0206) at D6S289. CONCLUSION: Susceptibility genes correlated with undifferentiated schizophrenia pedigrees from D1S484, D1S2878, D1S196 loci, and those correlated with paranoid schizophrenia pedigrees from D6S289 locus are likely present in chromosome regions 1q23.3 and 1q24.2, and chromosome region 6p23, respectively.

Screening of LRRK2 interactants by yeast 2-hybrid analysis.

OBJECTIVE: To isolate and identify the potential binding partners of LRRK2, a gene linked to both dominant familial form and sporadic form of Parkinson's disease, thus to further our knowledge of its function. METHODS: We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait. The bait amplified by polymerase chain reaction (PCR) was then cloned into a yeast expression plasmid pGBKT7. After being sequenced and analyzed, pGBKT7-bait was transformed into the yeast strain AH109. Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain. Then human fetal brain cDNA library was transformed into that yeast strain, which could express pGBKT7-bait fusion protein. The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade) containing X-alpha-gal. We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7, respectively. At last, these plasmids isolated from these true positive colonies were analyzed by bioinformatics. RESULTS: We obtained 9 true positive colonies, these colonies were sequenced, and we performed sequence Blast in GenBank. Three colonies of the 9 positive colonies were not in open reading-frames. Among other 6 colonies, there were known proteins including spermatid perinuclear RNA-binding protein (STRBP) and BCL2-associated athanogene 5 isoform b (BAG5), as well as unknown proteins including tyrosine phosphatase non-receptor type (PTPN23), l(3)mbt-like 3 isoform b (L3MBTL3), RALY RNA binding protein-like isoform 1 (RALYL), and Homo sapiens mRNA for KIAA1783 protein, partial cds (KIAA1783). CONCLUSION: True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid. Our screened proteins may provide a new research clue for revealing biological functions of LRRK2, pathogenesis of Parkinson's disease, and other neurodegerations.

Application of methyl parathion hydrolase (MPH) as a labeling enzyme.

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 microL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. [figure: see text]

Efficient protein expression from the endogenous RNA polymerase I promoter using a human ribosomal DNA targeting vector.

Vector systems to deliver, integrate and express therapeutic genes in host cells are essential for gene therapy. In the present study, we investigated a novel vector system for integration and expression of a transgene. In this system, the transgene expression was driven by an endogenous RNA polymerase I (Pol I) promoter after being integrated into the ribosomal DNA (rDNA) locus. Human coagulation factor IX coding sequence (FIX), with an internal ribosome entry sites element at its leader region, was targeted into the 18S rDNA locus via homologous recombination. FIX protein expression, which was under the control of the endogenous Pol I promoter, was found to be similar to that of a moderate Pol II promoter. The average FIX expression level of the rDNA recombinants was additionally enhanced to that from a strong Pol II promoter as a result of elimination of position effects. Our data suggest the possibility of applying this system in gene therapy for hereditary diseases.

[Mapping of pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris].

OBJECTIVE: To elucidate the pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris (IV). METHODS: Linkage analysis was performed by using STR markers in chromosome 1, and mutation detection was used to screen for FLG gene mutation. RESULTS: A maximum two-point Lod score of 3.46 (theta=0) was obtained at D1S2696. Haplotype analysis placed the critical region in a 15-CM interval defined by D1S2726 and D1S305, but no mutation of FLG was found in our IV patients. CONCLUSION: The pathologic gene of the IV family locates near D1S2696, and the FLG gene may not ruled out from the pathologic genes.

[Biological characteristics and safety evaluation of endothelial progenitor cells from the umbilical cord blood].

OBJECTIVE: To investigate the biological characteristics of endothelial progenitor cells (EPCs) from the umbilical cord blood (UCB), and to evaluate their oncogenicity after long-term culture in vitro. METHODS: The mononuclear cells (MNCs) were isolated from the UCB and cultured in MCDB131 medium supplemented with 20% FBS, VEGF and other growth factors. Morphology of the EPCs was observed, and the growth curve of the EPCs was investigated. Surface antigens of the EPCs were analyzed by the flow-cytometer. The capability of intaking the acetylated low-density lipoprotein (acLDL) of the EPCs was detected using fluoresencent chemical method. The vasoformative capability and genetic stability of EPCs were cultured in matrigel, and examined by karyotype analysis. The oncogenicity of EPCs was verified by the tumorigenesis test in athymic mouse and soft agar. RESULTS: EPCs were successfully derived from the UCB, and could be passaged to at least 42(nd) generation and had strong abilities of proliferation, acLDL intake and vasoformation, but there was not oncogenicity. They expressed endothelial cell-surface antigens and maintained normal karyotype. CONCLUSION: The EPCs with proliferative potential can be isolated from the UCB. They can be passaged in long-term cultures without oncogenicity, and can maintain normal karyotype. The EPCs can be served as a new type of cells in cell and gene therapy.

[Azoospermia factor microdeletion on Y chromosome in patients with idiopathic azoospermia or severe oligozoospermia].

OBJECTIVE: To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions, and to establish a reliable genetic diagnosis in idiopathic infertile male patients with azoospermia or severe oligozoospermia. METHODS: Multiplex PCR amplification of 6 sequence-tagged sites in AZF regions of the Y chromosome was examined among 100 normal karyotype male patients with azoospermia or oligozoospermia. RESULTS: Four patients (4%) had Y chromosome microdeletions, the microdeletions of 3 patients were idiopathic azoospermic and those of the other 1 patient were secretory azoospermia. CONCLUSION: The PCR-based Y chromosome microdeletion screening is simple and effective in the diagnosis of patients with severe male infertility. Microdeletion of Y chromosome is one of the major causes of severe dyszooospermia.

[Preimplantation genetic diagnosis of Duchenne muscular dystrophy by single cell triplex PCR].

OBJECTIVE: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation. METHODS: Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status. RESULTS: In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0. CONCLUSION: The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.

[Identification of the origin of marker chromosome by comparative genomic hybridization].

OBJECTIVE: To identify the origin of the marker chromosome in a patient with chromosome aberration, and to provide the precise genetic diagnosis. METHODS: Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) were performed to detect the known small marker chromosome in this patient. RESULTS: The small marker chromosome originated from chromosome 13 pter->q12. CONCLUSION: CGH and FISH can be used to detect the small marker chromosome, which is convenient and quick in detecting the origin of small marker chromosome.

[Expression of single chain fragment variable P1D3 antibody against shrimp white spot syndrome virus in Pichia pastoris].

White spot syndrome virus (WSSV) is a major pathogen in aquaculture penaeid shrimp, which caused catastrophic economic losses in the worldwide. No adequate treatments against WSSV are available. In order to study infection mechanism of WSSV, a phage display scFv cDNA library against WSSV was constructed and a neutralizing antibody of scFv P1D3 was selected in our lab previously. In this study, scFv P1D3 was expressed successfully in yeast. Firstly, the original expression vector of P1D3, M13 phagmid, was used as a template to design primers with restriction sites of SnaB I and EcoR I . Then the gene of P1D3 was amplified by PCR. After digested by SnaB I and EcoR I , the fragment of scFv P1D3 with E-tag was inserted into yeast and E. coli shuttle plasmid pPIC9k. The recombinant plasmid pPIC9k-scFv P1D3-Etag was linearized with Bgl II and then transformed into Pichia pastoris GS115 by electroporation. Positive clones were selected and verified by PCR and DNA sequencing. The scFv PID3 was induced to express in yeast by methanol. The results of ELISA demonstrate that scFv P1D3 expressed in yeast still has high specificity to bind on WSSV and the binding activity is higher than that expressed in E. coli TG1. After several optimizing experiments, the results show that the expression amount of scFv P1D3 can reach to 302 mg/L in yeast culture supernatant. This experiment has offered a new source of antibody for the researches on passive immunology for shrimp.

[A mutation 1633-26(C-->A) in EXT1 gene causes multiple exostoses].

OBJECTIVE: To study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation. METHODS: Polymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation. RESULTS: By DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before. Mismatch primer amplification and restriction fragment length polymorphism analysis suggested that this mutation was not detected in the normal control. CONCLUSION: The mutation 1633-26(C-->A) may be the disease-causing mutation in this patient with multiple exostoses.

[Hotspot of the mutations of keratin 9 gene in a diffuse palmoplantar keratoderma family].

OBJECTIVE: To identify the gene causing diffuse palmoplantar keratoderma in a Chinese pedigree. METHODS: Four normal individuals and 3 patients in a diffuse palmoplantar keratoderma family and 10 unrelated control samples were recruited. The hotspot of the mutations of keratin 9 gene was analyzed by polymerase chain reaction and direct sequencing. RESULTS: We found a G485A transition in ke ratin 9 gene, resulting in the substitution of glutamine for arginine at codon 162 in this diffuse palmoplantar keratoderma family. The mutation was not found in the 10 unrelated control samples and 4 normal individuals. CONCLUSION: The mutation G485A found in keratin 9 gene is the disease-causing mutation in the diffuse palmoplantar keratoderma family.

A minidystrophin-EGFP fusion gene expressed in Cos-7 cells mediated by human source vector.

OBJECTIVE: To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells. METHODS: The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine. RESULTS: Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane. CONCLUSION: The minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.

[Prenatal diagnosis of prelingual deafness by determination of SLC26A4 gene mutation].

OBJECTIVE: To identify deafness related gene and provide its prenatal diagnosis to avoid deaf fetus delivery. METHODS: DNA was extracted from amniotic cells in a pregnant woman close to 21 weeks' gestation, as well as from peripheral blood cells of the pregnant woman, her husband and their two sons. Screening for GJB2 and SLC26A4 gene mutations was firstly performed in the deafness proband (the first son of the couple), and then it was carried out in the fetus and the rest family members. RESULTS: The first child of the family, i.e., the proband, was homozygous in the IVS7-2A > G mutation of SLC26A4, the parents and the second child were carriers of the same mutation, while the fetus had a wild-type form. CONCLUSION: It is feasible to identify deafness related genes by screening for GJB2 and SLC26A4 mutation, thus providing correct prenatal diagnosis and avoiding deaf delivery of baby.

Molecular analysis of SLC26A4 gene in a Chinese deafness family.

OBJECTIVE: To identify the pathogenic gene for a non-syndromic hearing loss family. METHODS: Mutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons of SLC26A4 (solute carrier family 26, member 4) gene. RESULTS: Compound heterozygous mutations N392Y and S448X were detected in the proband of the family, heterozygous mutation S448X was detected in the father, heterozygous mutation N392Y was detected in the mother. CONCLUSION: The proband's hearing loss resulted from the compound heterozygous mutations N392Y and S448X for SLC26A4 gene.

Intracellular distribution, assembly and effect of disease-associated connexin 31 mutants in HeLa cells.

Mutations in connexin 31 (Cx31) are associated with erythrokeratodermia variabilis (EKV), hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant (L34P) and four hearing impairment-associated mutants (66delD, 141delI, R180X and E183K), significantly reduced plaque formation in the cells with five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) and no obvious change in the cells with two other mutants (I141V and 652del12). Immunoblotting analysis showed that 12 mutated Cx31s, like WT-Cx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) caused cell death in HeLa cells. However, the five hearing impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, disease-associated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants.