New functions of histamine found in histidine decarboxylase gene knockout mice

Gene targeting techniques have revolutionized the investigation of the effects of bioactive substances in pathological and physiological conditions. Histamine synthesis is uniquely catalyzed by L-histidine decarboxylase. The knockout mice of this gene express no histamine-producing activity and lack histamine. These mice have been used to examine the mechanisms of histamine in several known phenotypes, e.g., gastric acid secretion, contraction of smooth muscles, vascular permeability, and awakening, and have also been used to explore unreported effects of histamine in the whole body. First, we will review the former mechanisms and then move to the latter, new effects. Especially, in the latter mechanisms, we focus on several important roles of histamine in angiogenesis, neutrophil and eosinophil recruitment, bacterial infection, and systemic anaphylaxis in this review. Moreover, to our surprise, the morphology of mast cells in the knockout mice was severely affected by the absence of histamine in terms of their granules.     

Role of water-soluble polysaccharides in bacterial cellulose production

Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC) and a water-soluble polysaccharide called acetan in corn steep liquor-fructose medium. Acetobacter xylinum EP1, which is incapable of acetan production was derived by disrupting the aceA gene of BPR2001. The BC production by EP1 (2.88 g/L) was lower than that by BPR2001 (4.6 g/L) in baffled-flask culture. When purified acetan or agar was added to the medium from the start of cultivation, the BC production by EP1 was enhanced and the final BC yield of EP1 was almost the same as that of BPR2001. A similar improvement of BC production by EP1 by the addition of agar was also confirmed by cultivation in a 50-L airlift reactor. From these results, the role of acetan in BC production is associated with the increase in the viscosity of the culture medium which may hinder coagulation of BC and cells in the culture, thereby accelerating the growth of BPR2001 and BC production by BPR2001.     

Trichothecene nonproducer Gibberella species have both functional and nonfunctional 3-O-acetyltransferase genes

The trichothecene 3-O-acetyltransferase gene (FgTri101) required for trichothecene production by Fusarium graminearum is located between the phosphate permease gene (pho5) and the UTP-ammonia ligase gene (ura7). We have cloned and sequenced the pho5-to-ura7 regions from three trichothecene nonproducing Fusarium (i.e., F. oxysporum, F. moniliforme, and Fusarium species IFO 7772) that belong to the teleomorph genus Gibberella. BLASTX analysis of these sequences revealed portions of predicted polypeptides with high similarities to the TRI101 polypeptide. While FspTri101 (Fusarium species Tri101) coded for a functional 3-O-acetyltransferase, FoTri101 (F. oxysporum Tri101) and FmTri101 (F. moniliforme Tri101) were pseudogenes. Nevertheless, F. oxysporum and F. moniliforme were able to acetylate C-3 of trichothecenes, indicating that these nonproducers possess another as yet unidentified 3-O-acetyltransferase gene. By means of cDNA expression cloning using fission yeast, we isolated the responsible FoTri201 gene from F. oxysporum; on the basis of this sequence, FmTri201 has been cloned from F. moniliforme by PCR techniques. Both Tri201 showed only a limited level of nucleotide sequence similarity to FgTri101 and FspTri101. The existence of Tri101 in a trichothecene nonproducer suggests that this gene existed in the fungal genome before the divergence of producers from nonproducers in the evolution of Fusarium species.     

Molecular structural and functional characterization of tumor suppressive anti-ErbB-2 monoclonal antibody by phage display system

To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.     

A temperature-sensitive mutant of the mammalian RNA helicase,DEAD-BOX X isoform,DBX, defective in the transition from G1 to S phase

ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.     

Bacterial Distribution and Phylogenetic Diversity in the Changjiang Estuary before the Construction of the Three Gorges Dam

The bacterial community structure in the Changjiang estuary was studied for comparison with future changes, related to the construction of the Three Gorges Dam. Population densities of bacteria in the surface water at station C1 estimated by CFU on marine agar plates and by DAPI direct count, were 2.8 x 10(4) ml(-1) and 4.2 x 10(5) ml(-1), respectively. Physicochemical properties of water, such as temperature and salinity, suggested that station C1 was affected by freshwater from the Changjiang River. Cluster analysis of the PCR-RFLP patterns obtained from 9 samples showed that the bacterial community structure at station C1 was different from the structure at the other stations. Bacterial diversity in the surface water at station C1 was studied based on the genotypes of the 250 clones of 16S rRNA, and on the phenotypes generated on Biolog GN plates for 70 isolates. Sequences of bacteria from two common marine groups, alpha- and gamma-Proteobacteria, were frequently observed. Some other divisions, including the beta-Proteobacteria, C/F/B group, low G+C gram positive, high G+C gram positive, chloroplasts, and relatives of Verrucomicrobia were also observed. The putative dominant species based on both genotype and phenotype analyses were close relatives of Alteromonas macleodii or Roseobacter spp. These results reflected the nutrient-rich environment at station C1.     

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd.     

Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.     

Characterization of mouse cysteinyl leukotriene receptors mCysLT1 and mCysLT2: differential pharmacological properties and tissue distribution

Cysteinyl leukotrienes (LTs) are important proinflammatory mediators. Their precise roles in mice need to be elucidated to interpret mouse models of inflammatory diseases. For this purpose, we cloned and characterized mouse receptors for cysteinyl LTs, mCysLT(1) and mCysLT(2). mCysLT(1) and mCysLT(2) were composed of 339 amino acids with 87.3% identity and 309 amino acids with 73.4% identity to human orthologues, respectively. A pharmacological difference was noted between mouse and human CysLT(2). Pranlukast, a specific inhibitor for human CysLT(1), antagonized mCysLT(2) responses as determined by Ca(2+) elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT(1) mRNA was expressed mainly in skin, lung, and small intestine. mCysLT(2) was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT(1) and mCysLT(2) were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT(2) between human and mouse and the different distributions of CysLTs between mouse strains suggest that careful choice and interpretation are necessary for a study of CysLTs using animal models.