Biophysical and biochemical characterization of recombinant human Pop2 deadenylase

Pop2, a component of the Ccr4–Not complex, functions as a deadenylase both in vitro and in vivo. In this research, we found that the recombinant human Pop2 (hPop2) mainly existed in a compact monomeric state with a α + β tertiary structure type. The percentages of the secondary structures evaluated from the CD spectrum were about 37% α-helix, 14% β-sheet, and 19% β-turns. The optimal condition for hPop2 catalysis was pH 7–8 at 37 °C. Mg²⁺, Mn²⁺, and Co²⁺ had similar effects on the deadenylation activity of hPop2, and the optimal concentration was 0.3–0.5 mM. The deadenylase activity of hPop2 was, at least partially, specific when coordinated with divalent metal ions. The enzyme was not inhibited much by the nucleotide analogs, and the product 5′-AMP was the most efficient inhibitor. The dissimilarity in the metal ion dependence and inhibitory effects of the nucleotide analogs suggested that various deadenylases might have differential regulation mechanisms.     

Molecular and genetic characterization of the TonB2-cluster TtpC protein in pathogenic vibrios

TtpC is a fourth required protein in the TonB2 energy transduction system in Vibrio anguillarum. TtpC is necessary for iron transport mediated by the TonB2 system and is highly conserved in all pathogenic vibrio species studied to date as well as several marine organisms. We show here that the TtpC proteins from selected pathogenic vibrio species can function with the TonB2 system of V. anguillarum to allow iron transport mediated by a chimeric TonB2 system where the native ExbB2, ExbD2 and TonB2 function with an episomally expressed TtpC in trans from a different species. The discovery that inter-species complementation occurs can be used to identify the functional regions of the TtpC proteins and will lead to an investigation of the mechanism of interaction between the TtpC protein and other members of the TonB2 system.     

Comparative genome characterization of Achromobacter members reveals potential genetic determinants facilitating the adaptation to a pathogenic lifestyle

Members of the Achromobacter genus are Gram-negative bacteria including both environmental and clinical isolates, which are increasingly recovered from patients with cystic fibrosis (CF) as emerging pathogens. To better understand the features of the genus and its potential pathogenic mechanisms, six available Achromobacter genomes were compared in this study. The results revealed that: (1) Achromobacter had a pan-genome size of 10,750 genes with 3,398 core genes and a similar global classification of protein functions; (2) the Achromobacter genomes underwent a relatively low recombination that introduced nearly twice nucleotide substitutions less than the point mutation in genome evolution; (3) phylogenomic analysis based on 436 conserved proteins and average nucleotide identity both indicated that the Achromobacter genus had the closest relationship to the human/animal pathogen Bordetella rather than to Alcaligenes. The entire group of Achromobacter clustered with Bordetella in phylogeny, strongly suggesting a common origin, which therefore highlighted the potentially pathogenic nature of Achromobacter from the phylogenetic perspective, and (4) the CF clinical isolate possessed markedly unique genomic features discriminated from the environmental isolate and was equipped with numerous factors that facilitate its adaptation to a pathogenic lifestyle, such as a type III secretion system, a “polysaccharide island” (36.0 kb) of capsular/cellulose synthesis, adhesion-related proteins, alcaligin biogenesis, and several putative toxins. This study provided the first comprehensive genomic comparative analysis for Achromobacter, revealed information to better understand this far less-known genus on the genomic scale, and, importantly, identified potential virulence factors of the Achromobacter pathogen.     

Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein-protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.     

Generation and characterization of human insulin-releasing cell lines

Background  

The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines.     

Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization

Nineteen frog virus 3 temperature-sensitive mutants were isolated after mutagenesis with nitrosoguanidine and assayed for viral DNA, RNA, and protein synthesis, as well as assembly site formation at permissive (25 degrees C) and nonpermissive (30 degrees C) temperatures. In addition, mutants were characterized for complementation by both quantitative and qualitative assays. Based on the genetic and biochemical data, the 19 mutants, along with 9 mutants isolated earlier, were ordered into four phenotypic classes which define defects in virion morphogenesis (class I), late mRNA synthesis (class II), viral assembly site formation (class III), and viral DNA synthesis (class IV). In addition, we used two-factor crosses to order 11 mutants, comprising 7 complementation groups, onto a linkage map spanning 77 recombination units.     

Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans.

We have used the two PFK genes of Saccharomyces cerevisiae encoding the alpha and beta-subunit of the enzyme phosphofructokinase (Pfk) as heterologous probes to isolate fragments of the respective genes from the dimorphic pathogenic fungus Candida albicans. The complete coding sequences were obtained by combining sequences of chromosomal fragments and fragments obtained by inverse polymerase chain reaction (PCR). The CaPFK1 and CaPFK2 comprise open reading frames of 2961 bp and 2838 bp, respectively, encoding Pfk subunits with deduced molecular masses of 109 kDa and 104 kDa. The genes presumably evolved by a duplication event from a prokaryotic type ancestor, followed by another duplication. Heterologous expression in S. cerevisiae revealed that each gene alone was able to complement the glucose-negative phenotype of a pfk1 pfk2 double mutant. In vitro Pfk activity in S. cerevisiae was not only obtained after coexpression of both genes, but also in conjunction with the respective complementary subunits from S. cerevisiae. This indicates the formation of functional hetero-oligomers consisting of C. albicans and S. cerevisiae Pfk subunits. In C. albicans, specific Pfk activity was shown to decrease twofold upon induction of hyphal growth. CaPfk cross-reacts with a polyclonal antiserum raised against ScPfk and displays similar allosteric properties, i.e. inhibition by ATP and activation by AMP and fructose 2,6-bisphosphate.     

Structural and biochemical characterization of a human adenovirus 2/12 penton base chimera

The vertex of the adenoviral capsid is formed by the penton, a complex of two proteins, the pentameric penton base and the trimeric fiber protein. The penton contains all necessary components for viral attachment and entry into the host cell. After initial attachment via the head domain of the fiber protein, the penton base interacts with cellular integrins through an Arg-Gly-Asp (RGD) motif located in a hypervariable surface loop, triggering virus internalization. In order to investigate the structural and functional role of this region, we replaced the hypervariable loop of serotype 2 with the corresponding, but much shorter, loop of serotype 12 and compared it to the wild type. Here, we report the 3.6 A crystal structure of a human adenovirus 2/12 penton base chimera crystallized as a dodecamer. The structure is generally similar to human adenovirus 2 penton base, with the main differences localized to the fiber protein-binding site. Fluorescence anisotropy assays using a trimeric fiber protein mimetic called the minifiber and wild-type human adenovirus 2 and chimeric penton base demonstrate that fiber protein binding is independent of the hypervariable loop, with a K(d) for fiber binding estimated in the 1-2 microm range. Interestingly, competition assays using labeled and unlabeled minifiber demonstrated virtually irreversible binding to the penton base, which we ascribe to a conformational change, on the basis of comparisons of all available penton base structures.     

Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells

Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5-7 range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, which shows a large deletion in the lid domain. The presence of a full-length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.     

人胚胎干细胞建系和鉴定

人胚胎干细胞是一种取自人囊胚内细胞团且具有形成所有三个胚层细胞能力的全能细胞。建立一个理想的人胚胎干细胞培养系统是研究和利用这种具有巨大潜力细胞的首要条件。本文讨论了目前建立的人胚胎干细胞培养系统,阐述了其有利的和不利的一面,并着重讨论其体外培养方法和鉴定策略。     

Epstein-Barr virus-positive and -negative B-cell lines can be infected with human immunodeficiency virus types 1 and 2.

Human immunodeficiency virus type 1 (HIV-1) can infect CD4+ lymphocytes, monocytes-macrophages, and various other cell lines, including B-cell lines. To study the parameters of B-cell infections, we examined the susceptibility of 24 B-lymphoid cell lines to both HIV-1 and HIV-2 infections. These cell lines included a series of Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma cell lines and their EBV-converted counterparts. To infect these cells we used two HIV-1 isolates and one HIV-2 isolate. Infections were monitored with a cytoplasmic RNA dot-blot and a syncytium assay. HIV infection was also studied by a novel method based on electrophoresis of DNA liberated from cells that were lysed in situ in the well of an agarose gel. All human B-cell lines could be infected with HIV-1, regardless of the presence of EBV genomes; thus, EBV infection had no major effect on HIV susceptibility of B-cell lines. Integrated proviral HIV genomes could be detected by Southern blot analysis of DNA extracted from long-term, non-HIV-producing B-cell lines. This study suggests that B-lymphoid cells may serve as reservoirs for latent or persistent HIV infections in vivo, even in the absence of EBV infection.     

Establishment and characterization of five human myeloma cell lines

Since 1980 five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12-PE and KMS-12-BM, have been established in Kawasaki Medical School. Histologically, all the cell lines resembled plasma cells and were EBNA negative. KMM-1, KMS-11, KMS-12-PE and KMS-12-BM reacted with PCA-1, while KMM-1, KMS-12-PE and KMS-12-BM with CD38. KMM-1 and KMS-11 secreted immunoglobulins into culture medium. Karyologically, all the cell lines were abnormal. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Isolation and biochemical characterization of the S-layer protein from a pathogenic Aeromonas hydrophila strain.

The regular surface protein array (S layer) present on Aeromonas hydrophila TF7 is composed of a single species of protein of apparent molecular weight 52,000. This protein was extracted from whole cells by treatment with 0.2 M glycine hydrochloride (pH 3.0). The protein was purified to homogeneity by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. Amino acid composition analysis showed that the protein contained 520 residues per molecule, 41% of which were hydrophobic. Cysteine was absent. A pI of 4.6 was determined for the protein, and only a single isoelectric form was detected. The purified protein displayed the hydrophobic characteristic of binding to octyl-Sepharose gels, but the salt aggregation test showed that it did not confer hydrophobicity to the cell surface when present as an intact S layer. The molecule aggregated strongly in aqueous solution as determined by sedimentation equilibrium studies. Circular dichroism spectra showed that the S-layer protein was composed of a large amount of beta-sheet (approximately 44%), a limited amount of alpha-helix (19%), and 12% beta-turn, with the remainder of the molecule being aperiodic. No significant difference in secondary structure content was measured in the presence of the metal chelator EDTA. The N-terminal amino acid sequence was determined for the first 30 residues. No sequence homology with other S-layer proteins was found.     

Production and genetic characterization of near-isogenic lines in the bread-wheat cultivar Alpe

Two biotypes of the bread-wheat cultivar Alpe were shown to possess contrasting alleles at each of the glutenin (Glu-B1, Glu-D1, Glu-B3 and Glu-D3) and gliadin (Gli-B1 and Gli-D1) loci on chromosomes 1B and 1D. Fourteen near-isogenic lines (NILs) were produced by crossing these biotypes and used to determine the genetic control of both low-molecular-weight (LMW) glutenin subunits and gliadins by means of one-dimensional or two-dimensional electrophoresis. Genes coding for the B, C and D groups of EMW subunits were found to be inherited in clusters tightly linked with those controlling gliadins. Southern-blot analysis of total genomic DNAs hybridized to a -gliadin-specific cDNA clone revealed that seven NILs lack both the Gli-D1 and Glu-D3 loci on chromosome 1D. Segregation data indicated that these null alleles are normally inherited. Comparison of the null NILs with those possessing allele b at the Glu-D3 locus showed one B subunit, seven C subunits and two D subunits, as fractionated by two-dimensional A-PAGExSDS-PAGE, to be encoded by this allele. Alleles b and k at Glu-B3 were found to code for two C subunits plus eight and six B subunits respectively, whereas alleles b and k at Gli-B1 each controlled the synthesis of two -gliadins, one and two -gliadins. The novel Gli-B5 locus coding for two -gliadins was shown to recombine with the Gli-B1 locus on chromosome 1B. The two-dimensional map of glutenin subunits showed -gliadins encoded at the Gli-A2 locus on chromosome 6A. The use of Alpe NILs in the study of the individual and combined effects of glutenin subunits on dough properties is discussed.Research supported by a grant from the Commission of the European Communities, ECLAIR programme, Contract AGRE 0052     

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.     

Molecular and biochemical characterization of the human trk proto-oncogene.

Molecular analysis of the human trk oncogene, a transforming gene isolated from a colon carcinoma biopsy, revealed the existence of a novel member of the tyrosine kinase gene family. This locus, which we now designate the trk proto-oncogene, codes for a protein of 790 amino acid residues that has several features characteristic of cell surface receptors. They include (i) a 32-amino-acid-long putative signal peptide, (ii) an amino-terminal moiety (residues 33 to 407) rich in consensus sites for N-glycosylation, (iii) a transmembrane domain, (iv) a kinase catalytic region highly related to that of other tyrosine kinases, and (v) a very short (15 residue) carboxy-terminal tail. Residues 1 to 392 were absent in the trk oncogene, as they were replaced by tropomyosin sequences. However, no other differences were found between the transforming and nontransforming trk alleles (residues 392 to 790), suggesting that no additional mutations are required to activate the transforming potential of this gene. The human trk proto-oncogene codes for a 140,000-dalton glycoprotein, designated gp140proto-trk. However, its primary translational product is a 110,000-dalton glycoprotein which becomes immediately glycosylated, presumably during its translocation into the endoplasmic reticulum. This molecule, designated gp110proto-trk, is further glycosylated to yield the mature form, gp140proto-trk. Both gp110proto-trk and gp140proto-trk proteins possess in vitro kinase activity specific for tyrosine residues. Finally, iodination of intact NIH 3T3 cells expressing trk proto-oncogene products indicated that only the mature form, gp140proto-trk, cross the plasma membrane, becoming exposed to the outside of the cell. These results indicate that the product of the human trk locus is a novel tyrosine kinase cell surface receptor for an as yet unknown ligand.