OBJECTIVETo examine the effect of SH2A gene in cell signal transduction and its subcellular localization.

METHODSRT-PCR method was used to amplify the coding sequence of SH2A gene. Eukaryotic recombined expression vector pcDNA 3.1-SH2A was constructed, and then Bel7402 cell and COS7 cell transfected by liposome. Multiple kinase assay was performed to examine the activity of protein kinase (PKC), mitogen activated protein kinase (MAPK), tyrosine protein kinase (TPK) in the transfected cells. Meantime, pEGFP-SH2A vector was also constructed and the cells transfected with it were examined by fluorescent microscopy.

RESULTSRecombined expression vector pcDNA3.2-SH2A and pEGFP-SH2A contained the coding sequence of SH2A cDNA. In both cell lines expressing SH2A gene, the cytoplasm PKC activity decreased by 40% or so, but no apparent alteration was found in MAPK and TPK activity. SH2A gene was found localized in the cytoplasm of transfected cells under fluorescent microscope.

CONCLUSIONSH2A gene may act as an inhibiting factor in PKC signal transduction, and it is localized in cytoplasm.

Animals ; COS Cells ; Cytoplasm ; chemistry ; Humans ; Membrane Proteins ; analysis ; genetics ; physiology ; Mitogen-Activated Protein Kinases ; metabolism ; Protein Kinase C ; physiology ; Signal Transduction ; physiology ; Transfection

Profile hidden Markov models (HMMs) based on classical HMMs have been widely applied for protein sequence identification. The formulation of the forward and backward variables in profile HMMs is made under statistical independence assumption of the probability theory. We propose a fuzzy profile HMM to overcome the limitations of that assumption and to achieve an improved alignment for protein sequences belonging to a given family. The proposed model fuzzifies the forward and backward variables by incorporating Sugeno fuzzy measures and Choquet integrals, thus further extends the generalized HMM. Based on the fuzzified forward and backward variables, we propose a fuzzy Baum-Welch parameter estimation algorithm for profiles. The strong correlations and the sequence preference involved in the protein structures make this fuzzy architecture based model as a suitable candidate for building profiles of a given family, since the fuzzy set can handle uncertainties better than classical methods.
Algorithms ; Animals ; Computational Biology ; Databases, Protein ; Fuzzy Logic ; Globins ; chemistry ; genetics ; Humans ; Markov Chains ; Models, Statistical ; Probability Theory ; Protein Kinases ; chemistry ; genetics ; Sequence Alignment ; statistics & numerical data ; Sequence Analysis, Protein ; statistics & numerical data

BACKGROUNDRecepteur d'originenantais (RON) is a receptor tyrosine kinase (RTK) that belongs to the MET proto-oncogene family. The aim of this study was to investigate the expression of RON receptor tyrosine kinase in human non-small cell lung cancer (NSCLC) and its relationship with clinical pathology of NSCLC and prognosis.

METHODSRON protein expression by immunohistochemistry (IHC) in 96 NSCLC specimens was evaluated and compared with the clinical pathology and prognosis, and 20 para-neoplastic tissues were included as controls. RON mRNA and protein expression in 25 fresh tissue samples of lung cancer and 10 normal lung tissues were also analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSThe rate of positive RON expression differed significantly between NSCLC tissues (55.2%, 53/96) and para-neoplastic tissues (5%, 1/20) (P < 0.001). RON protein expression was not found to be associated with gender or age. However, RON expression positively correlated with clinical TNM stage (P = 0.004), histological types (P = 0.001), lymph node metastasis (P = 0.012) and differentiation (P = 0.035). RT-PCR and Western blotting analysis also confirmed that the expression of RON mRNA and protein was significantly increased in the NSCLC tissues versus normal tissues. In addition, RON expression was associated with a poor prognosis for patients with NSCLC (P = 0.045).

CONCLUSIONSThe expression of RON protein and mRNA is significant in human NSCLC and low in para-neoplastic and normal tissues. Elevated RON expression may contribute to the occurrence, progression and metastasis of NSCLC, inferring that it could be useful as a new prognostic indicator for patients with NSCLC.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; chemistry ; mortality ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; chemistry ; mortality ; Male ; Middle Aged ; Prognosis ; RNA, Messenger ; analysis ; Receptor Protein-Tyrosine Kinases ; analysis ; genetics ; physiology ; Retrospective Studies

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.
Amino Acid Sequence ; Cloning, Molecular ; Conserved Sequence ; Escherichia coli/genetics ; Gene Expression ; Gene Expression Profiling ; Microscopy, Confocal ; Microscopy, Fluorescence ; Molecular Sequence Data ; Molecular Weight ; Plasmodium vivax/chemistry/*genetics ; Protein Kinases/analysis/chemistry/*genetics ; Protein Structure, Tertiary ; Protozoan Proteins/analysis/chemistry/*genetics ; Sequence Alignment

OBJECTIVETo clarify the patients' pathogenic mechanism in an achondroplasia family not according with the genetic law of autosomal dominant inheritance disease at gene level.

METHODSGenomic DNA from peripheral blood of all members in this family was used for amplification of the exon 10 of fibroblast growth factor receptor 3(FGFR3) gene by PCR; mutation was detected by DNA sequencing and identified by restriction endonuclease MaeIII.

RESULTSA new mutation of A to T at nucleotide 1180 was found in patients but not in unaffected members.

CONCLUSIONCombined with pedigree analysis, it was summarized that achondroplasia patients in this family might result from this new mutation.

Achondroplasia ; genetics ; pathology ; Base Sequence ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Family Health ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Protein-Tyrosine Kinases ; Receptor, Fibroblast Growth Factor, Type 3 ; Receptors, Fibroblast Growth Factor ; genetics

OBJECTIVE[corrected] To investigate the mutation at the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) nucleotide 1138 site for identifying the major pathologic mechanism of achondroplasia (ACH) and to evaluate the efficacy of denaturing gradient gel electrophoresis(DGGE) method for screening the point mutations.

METHODSThe genomic DNA from 17 clinically diagnosed ACH patients where analysed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) with Sfc I and Msp I restriction endonucleases and by PCR-DGGE technique for screening.

RESULTSG to A transition mutation at nucleotide 1138 was detected in 14/17 of the ACH patients as heterozygotes by PCR-RFLP with Sfc I digestion. No 1138 G to C transition was detected by Msp I digestion. All of the 14 samples with G to A mutation were also found to be positive for point mutation by PCR-DGGE. No mutation was detected in 3 negative samples by PCR-RFLP, implying that there was actually no point mutation in this amplified region.

CONCLUSIONNucleotide 1138 in transmembrane domain of FGFR3 gene is the hot point for mutation in ACH and hence its major pathologic cause. PCR-DGGE is a sensitive and reliable technique for point mutation screening, especially for the heterozygotes.

Achondroplasia ; genetics ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Male ; Point Mutation ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single-Stranded Conformational ; Protein-Tyrosine Kinases ; Receptor, Fibroblast Growth Factor, Type 3 ; Receptors, Fibroblast Growth Factor ; genetics

The research of p53 is being conducted to find the mechanisms of tumorigenesis and to treat various cancers. Homeodomain-interacting protein kinase2 (HIPK2) is an important factor to regulate p53 and to increase the stability of p53. Activation of HIPK2 leads to the selective phosphorylation of p53, resulting in growth arrest and the enhancement of apoptosis. In this study, the canine HIPK2 cDNA fragments were obtained, and their overlapping regions were aligned to give a total sequence of 3489 bp. The canine HIPK2 cDNA (GenBank accession number; AY800385) shares 93% and 90% sequence identity with those of human and mouse HIPK2, respectively. The canine HIPK2 cDNA contains an open reading frame encoding 1163 amino acid residues and the predicted amino acid sequence has 98% and 96% identity with those of human and mouse, respectively. The deduced amino acid sequence of canine HIPK2 has also all domains' sites compared with human and mouse HIPK2. Therefore, these structural similarities suggested that the canine HIPK2 shares the basic biological functions that HIPK2 exhibit in other species.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary/chemistry/genetics ; Dogs/metabolism/*physiology ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; Protein-Serine-Threonine Kinases/*genetics ; Sequence Alignment ; Sequence Analysis, DNA

To investigate the expression and function of the receptors of early-acting cytokines on cord blood CD34(+) hematopoietic stem/progenitor cells, the studies of Flt3 and c-kit were undertaken at the gene and protein levels. Fresh and cultured cord blood CD34(+) stem/progenitor cells were analyzed by flow cytometric two-color direct immunofluorescence methods and RT-PCR, while function of receptors was studied in vitro. It was found that (68.8 +/- 15.4)% of CD34(+) cells expressed Flt3, (50.6 +/- 12.7)% of CD34(+) cells expressed c-kit, the proportion of CD34(+) cells expressing Flt3 and c-kit decreased as in vitro culture time extended. It was concluded that cord blood CD34(+) stem/progenitor cells are capable of expressing Flt3 and c-kit for as long as 2 - 3 weeks in liquid medium, during the first week of culture, SCF and FL enhanced the generation of cells and progenitors notably.
Antigens, CD34 ; blood ; Cells, Cultured ; Fetal Blood ; cytology ; Flow Cytometry ; Hematopoietic Stem Cells ; chemistry ; Humans ; Proto-Oncogene Proteins ; analysis ; genetics ; physiology ; Proto-Oncogene Proteins c-kit ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptor Protein-Tyrosine Kinases ; analysis ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; fms-Like Tyrosine Kinase 3

Activin Receptors, Type I ; analysis ; Animals ; Arcuate Nucleus of Hypothalamus ; chemistry ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Immunohistochemistry ; Liver Regeneration ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; analysis ; Rats ; Receptors, Transforming Growth Factor beta ; analysis ; Sodium Glutamate ; Transforming Growth Factor alpha ; analysis ; genetics ; Transforming Growth Factor beta ; analysis ; genetics

We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolatioin and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed.
Sequence Homology, Amino Acid ; Sequence Analysis, DNA ; Rats ; Protein Isoforms/genetics ; Phosphorylation ; Phosphoproteins/genetics/*metabolism ; Molecular Sequence Data ; Mice ; Male ; Humans ; Gene Expression Profiling ; Female ; DNA, Complementary/chemistry/genetics ; Cyclic AMP-Dependent Protein Kinases/*physiology ; Cloning, Molecular ; Cell Line ; Base Sequence ; Animals ; Amino Acid Sequence ; Adult