Isolation of p10 gene fromBombyx mori nuclear polyhedrosis virus and study of its promoter activity in recombinant baculovirus vector system

A homologue ofAutographa californica NPV (AcNPV) p10 gene was identified and cloned fromBombyx mori NPV (BmNPV). BmNPV p10 gene encodes truncated protein of 70 amino acid residues that lacks carboxyl terminus comparing with the p10 protein encoded by AcNPV. The putative TATA box sequence and the ATAAG motif which is the consensus sequence of baculovirus very late promoter were conserved. A transfer vector, pBNT1, which includes the p10 promoter region of BmNPV for foreign gene expression was constructed. By using pBNT1, a recombinant BmNPV, Bmp10-Luc, in which the p10 gene was replaced by the firefly luciferase gene, was obtained. We also obtained another recombinant virus, BmPH-Luc, in which the polyhedrin gene was replaced by the luciferase gene. The luciferase activity detected in BoMo-15AIIc insect cells infected with Bmp10-Luc was approximately 50% of that infected with BmPH-Luc, suggesting that although both the p10 and polyhedrin promoters of BrnNPV are effective in high-level expression of foreign gene, the p10 promoter is not so strong as the polyhedrin promoter.     

Cloning and structure of the human immune interferon-gamma chromosomal gene.

Two clones containing the human immune interferon-gamma (IFN-gamma) chromosomal gene were isolated from a human DNA library present in lambda Charon4A phage. DNA from these clones specified biologically active interferon upon injection into the nuclei of Xenopus laevis oocytes. Analysis of the clones revealed that they were derived from the same chromosomal segment. Restriction fragments that hybridized with 32P-labeled cDNA probes were subcloned into plasmids and the complete sequence of the IFN-gamma gene was determined. Unlike IFNs-alpha and -beta, IFN-gamma does contain introns. Their presence was also revealed by electron microscopy. It is intriguing that the smallest of the three introns is located just in the middle of the Glu-Glu sequence which is conserved among all three forms of interferon at approximately the same position. The promoter region was found to contain a prototype TATA box, many palindromic structures and several repeating sequences and two symmetrical structures. Particularly interesting was the existence of two sequences homologous to those present in the chicken albumin and the human IFN-beta gene promoter region. A sequence GTGTTG common to several other genes was found in the region approximately 10 nucleotides downstream from the polyadenylation site.     

A novel meta-cleavage dioxygenase that cleaves a carboxyl-group-substituted 2-aminophenol. Purification and characterization of 4-amino-3-hydroxybenzoate 2,3-dioxygenase from Bordetella sp. strain 10d.

A bacterial strain that grew on 4-amino-3-hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4-amino-3-hydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110-fold increase in specific activity. The purified enzyme was characterized as a meta-cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4-amino-3-hydroxybenzoic acid, with the consumption of 1 mol of O2 per mol of substrate. The enzyme was therefore designated as 4-amino-3-hydroxybenzoate 2,3-dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21-kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4-amino-3-hydroxybenzoic acid. The Km and Vmax values for this substrate were 35 micro m and 12 micro mol.min-1.(mg protein)-1, respectively. Of the 2-aminophenols tested, only 4-aminoresorcinol and 6-amino-m-cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2-aminophenol 1,6-dioxygenase, in molecular mass, subunit structure and catalytic properties.     

Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.     

Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.     

Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces cerevisiae

In wild-type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His+ gene conversions that have a parental configuration of flanking markers, but approximately 22% of recombinants have associated reciprocal crossovers. In rad52 strains, gene conversion is reduced 75-fold and the majority of His+ recombinants are crossover associated, with the largest class being half-crossovers in which the other participating chromatid is lost. We report that UV irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in wild-type (WT) cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2n - 1 events is markedly reduced. Both spontaneous and UV-induced RAD52-independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52. The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59. Those outcomes also rely on the UV-inducible kinase Dun1 and Dun1's target, the repressor Crt1, whereas gene conversion events arising spontaneously depend on Rad59 and Crt1. Thus, there are at least two Rad52-independent recombination pathways in budding yeast.     

Probability distributions,vulnerability and sensitivity in Fagus crenata forests following predicted climate changes in Japan

Question: How much is the probability distribution of Fagus crenata forests predicted to change under a climate change scenario by the 2090s, and what are the potential impacts on these forests? What are the main factors inducing such changes? Location: The major islands of Japan. Methods: A predictive distribution model was developed with four climatic factors (summer precipitation, PRS; winter precipitation, PRW; minimum temperature of the coldest month, TMC; and warmth index, WI) and five non‐climatic factors (topography, surface geology, soil, slope aspect and inclination). A climate change scenario was applied to the model. Results: Areas with high probability (> 0.5) were predicted to decrease by 91%, retreating from the southwest, shrinking in central regions, and expanding northeastwards beyond their current northern limits. A vulnerability index (the reciprocal of the predicted probability) suggests that Kyushu, Shikoku, the Pacific Ocean side of Honshu and southwest Hokkaido will have high numbers of many vulnerable F. crenata forests. The forests with high negative sensitivity indices (the difference between simulated probabilities of occurrence under current and predicted climates) mainly occur in southwest Hokkaido and the Sea of Japan side of northern Honshu. Conclusion: F. crenata forest distributions may retreat from some islands due to a high WI. The predicted northeastward shift in northern Hokkaido is associated with increased TMC and PRS. High vulnerability and negative sensitivity of the forests in southern Hokkaido are due to increased WI.     

Characterization of microsatellite loci in red wood ants Formica (s. str.) spp. and the related genus Polyergus

Ants are interesting subjects for studies of evolution of altruism. We developed 13 microsatellite loci in a red wood ant Formica (s. str.) yessensis from random amplified polymorphic DNA fragments to study genetic structure within populations and colonies. Five loci bore two to five alleles in both F. (s. str.) yessensis and F. (s. str.) truncorum and two were also polymorphic in a related species, Polyergus samurai. Results suggest that the loci will be useful in evolutionary studies on Formica and Polyergus species.     

Mechanism of dimerization and structural features of human LI-cadherin

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca²⁺-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion–free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin–dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.