Typical three-domain cry proteins of Bacillus thuringiensis strain A1462 exhibit cytocidal activity on limited human cancer cells

Bacillus thuringiensis strain A1462 produced two parasporal inclusion proteins with a molecular mass of 88 kDa that were converted to 64-kDa toxins when activated by proteinase K digestion. Both toxins exhibited strong cytocidal activity against two human cancer cell lines, HL60 (myeloid leukemia cells) and HepG2 (liver cancer cells), while low or no toxicities were observed against 11 human and three mammalian cell lines, including four non-cancer cell lines. The cytotoxicity of both toxins on susceptible cells was characterized by rapid cell swelling. Gene cloning experiments provided two novel genes encoding 88-kDa Cry proteins, Cry41Aa and Cry41Ab. The amino acid sequences of the two proteins contain five block regions commonly conserved in B. thuringiensis insecticidal Cry proteins. This is the first report of the occurrence of typical three-domain Cry proteins with cytocidal activity preferential for cancer cells.     

Expression cloning of a variety of novel protein kinases in Lotus japonicus

To investigate protein kinases expressed in Lotus japonicus, a cDNA expression library of the root-nodule of L. japonicus was immunologically screened with monoclonal antibodies directed to a highly conserved region in protein serine/threonine kinases (Ser/Thr kinases). Among 178 positive clones obtained from the lambdaZAPII cDNA library, 164 clones were found to encode novel proteins possessing the subdomain VIB sequences characteristic of Ser/Thr kinases. By phylogenetic analysis on the basis of deduced amino acid sequences, the isolated clones could be classified into five different families of Ser/Thr kinases : the SnRK family, GSK-3 family, Ndr kinase family, Ark family, and receptor kinase family. These results suggest that this expression cloning using the kinase-specific antibodies will provide new clues for investigations of a wide variety of known and novel protein kinases in higher plants.     

Identification of receptors and signaling pathways for orphan bone morphogenetic protein/growth differentiation factor ligands based on genomic analyses

There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.     

Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.     

Thermolabile Variant, PHOX-S, of Prophenol Oxidase in Drosophila melanogaster

The Phox(S) strain of Drosophila melanogaster is an electrophoretically slow variant found in a wild population at Victoria, Australia. Prophenol oxidase isoform A(1) from PHOX-S was purified and characterized biochemically and genetically. The purified fraction of A(1) from PHOX-S showed a homodimer with a molecular weight of the subunit of approximately 77 kDa. The Phox(S) strain was temperature sensitive in vivo in culture, and the purified protein was thermolabile in vitro. By the deletion mapping method, the Phox(S) locus was cytologically estimated to be at the location 55-A on the right arm of the second chromosome and 79.6 genetically. These data show that PHOX-S is an electrophoretic variant of MOX and that PHOX-S is the first thermolabile protein found in invertebrate prophenol oxidase.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix

Mammalian sperm-borne oocyte activating factor (SOAF) induces oocyte activation from a compartment that engages the oocyte cytoplasm, but it is not known how. A SOAF-containing extract (SE) was solubilized from the submembrane perinuclear matrix, a domain that enters the egg. SE initiated activation sufficient for full development. Microinjection coupled to tandem mass spectrometry enabled functional correlation profiling of fractionated SE without a priori assumptions about its chemical nature. Phospholipase C-zeta (PLCzeta) correlated absolutely with activating ability. Immunoblotting confirmed this and showed that the perinuclear matrix is the major site of 72-kDa PLCzeta. Oocyte activation was efficiently induced by 1.25 fg of sperm PLCzeta, corresponding to a fraction of one sperm equivalent (approximately 0.03). Immunofluorescence microscopy localized sperm head PLCzeta to a post-acrosomal region that becomes rapidly exposed to the ooplasm following gamete fusion. This multifaceted approach suggests a mechanism by which PLCzeta originates from an oocyte-penetrating assembly--the sperm perinuclear matrix--to induce mammalian oocyte activation at fertilization.     

Construction of a novel human artificial chromosome vector for gene delivery

Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.     

In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.