cDNA cloning of a novel cdc2+/CDC28-related protein kinase from rice

A cDNA clone, named R2, has been isolated by screening a rice cell cDNA library with a redundant oligonucleotide probe derived from the conserved ATP binding site of cdc2+/CDC28 protein kinases. The cDNA contained the entire coding sequence for a 424 amino acid polypeptide with a molecular mass of 47.6 kDa. The R2 mRNA, 2.1 kb in size, was expressed in both cultured rice cells and rice seedlings at similar levels. The predicted R2 protein has canonical motifs for ATP binding and catalysis, and is significantly homologous (up to 47%) to members of the cdc2+/CDC28 subfamily of serine/threonine protein kinase. The R2 protein is a novel member of the subfamily.     

Localization of BAI-associated protein1/membrane-associated guanylate kinase-1 at adherens junctions in normal rat kidney cells: polarized targeting mediated by the carboxyl-terminal PDZ domains

Brain-specific angiogenesis inhibitor (BAI)-associated protein (BAP)1 (also called membrane-associated guanylate kinase [MAGI]-1) is composed of six PSD-95/Dlg-A/ZO-1 (PDZ) domains, two WW domains, and one guanylate kinase (GK) domain. We previously reported that BAP1 is localized at tight junctions in Madine Darby canine kidney (MDCK) cells and intestinal epithelial cells. Here, we have determined the localization of BAP1 in normal rat kidney (NRK) cells that do not form tight junctions. BAP1 was colocalized with E-cadherin along the lateral membrane, suggesting its localization at adherens junctions. Green fluorescent protein (GFP)-BAP1 was distributed in the cytosol in separate NRK cells, and accumulated to the cell-cell contacts when NRK cells have contact with each other. The GFP-BAP1 mutant containing either the first PDZ and GK domains or the WW and second PDZ domains was localized in the cytosol and the nucleus. The GFP-BAP1 mutant containing the second to fourth PDZ domains was distributed in the cytosol. The construct containing the fifth and sixth PDZ domains was localized at the cell-cell contacts along the lateral membrane and slightly in the nucleus, whereas the construct lacking the fifth and sixth PDZ domains was localized in the cytosol and in the nucleus. BAP1 was tyrosine-phosphorylated in vivo, but the tyrosine phosphorylation of BAP1 was not correlated with its localization. These results suggest that the signal in the carboxyl-terminal PDZ domains functions dominantly in vivo to target BAP1 to the lateral membrane, although potential nuclear localization signals exist in the N-terminal region of BAP1.     

Axotomy induces axonogenesis in hippocampal neurons through STAT3

After axotomy of embryonic hippocampal neurons in vitro, some of the axotomized axons lose their identity, and new axons arise and grow. This axotomy-induced axonogenesis requires importin, suggesting that some injury-induced signals are transported via axons to elicit axonogenesis after axotomy. In this study, we show that STAT3 is activated in response to axotomy. Because STAT3 was co-immunoprecipitated with importin β in the axotomized neurons, we suggest that STAT3 is retrogradely transported as molecular cargo of importin α/β heterodimers. Indeed, inhibition of importin α binding with STAT3 resulted in the attenuation of axonogenesis. Silencing STAT3 blocked the axonogenesis, demonstrating that STAT3 is necessary for axotomy-induced axonogenesis. Furthermore, the overexpression of STAT3 enhanced axotomy-induced axonogenesis. Taken together, these results demonstrate that activation and retrograde transport of STAT3 in injured axons have key roles in the axotomy-induced axonogenesis of hippocampal neurons.     

Expression of a soybean nodule‐enhanced phosphoenolpyruvate carboxylase gene that shows striking similarity to another gene for a house‐keeping isoform

Three different cDNAs for phosphoenolpyruvate carboxylase (PEPC) were isolated from soybean root nodules. The full-length cDNA of the most abundant isoform (GmPEPC7) was very similar to another one (GmPEPC15), the nucleotide sequence of which is identical to that of a reported clone (gmppc1) (Vazquez-Tello, A., Whittier, R.F., Kawasaki, T., Sugimoto, T., Kawamura, Y., Shibata, D. (1993) Plant Physiol. 103, 1025–1026). In the coding region, the newly isolated GmPEPC7 and the previously reported were gmppc1 99% and 98% identical at the amino acid and nucleotide levels, respectively. In contrast, they exhibited only 39% identity in the 3′ non-coding region, indicating that they are encoded by distinct genes. Northern blot analysis with 3′ non-coding regions as isoform-specific probes showed that GmPEPC7 is nodule-enhanced whereas GmPEPC15 (gmppc1) is expressed in most soybean tissues. The third clone (GmPEPC4) was much less homologous to the above two clones and thus was not further characterized. It was also shown by in situ hybridization that the nodule-enhanced isoform is expressed in all cell types in nodules, including in Bradyrhizobium-infected and uninfected cells and cortical cells. A relatively strong hybridization signal was detected in the vascular bundle pericycle. Southern blot analysis indicated that there are only two PEPC genes exhibiting a high degree of similarity in the soybean genome, one for the nodule-enhanced GmPEPC7 and the other for the constitutively expressed gmppc1. A phylogenetic tree based on the amino acid sequences of soybean PEPCs and nodule-enhanced PEPCs of alfalfa and pea suggested that the soybean nodule-enhanced isoform evolved from the housekeeping PEPC gene after the ureid-translocating and amide-translocating legumes diverged from each other.     

Isolation of endophytic fungi from leaves of Pasania edulis and their within-leaf distributions

 Endophytic fungi were isolated from leaves of Pasania edulis, one of the most important trees of the warm temperate forests in southern Kyushu, by the surface sterilization method using H₂O₂ as a sterilizing agent. From a tree in the Experimental Nursery of Kagoshima University, located at the city of Kagoshima, Phyllosticta sp. and Colletotrichum spp. were frequently isolated. From a stand in a laurel forest in Mt. Takakuma, an ascomycetous fungus (Ascomycete sp. 1) and Phomopsis sp. were frequently isolated. Phyllosticta sp. was isolated more frequently from petiole segments and leaf segments with midrib and Phomopsis sp. from petiole segments and leaf-base segments with midrib than other segments. Colletotrichum spp. were isolated less frequently from petioles and Ascomycete sp. 1 from petiole segments and leaf-base segments with midrib than other segments. As possible causes of such biases in within-leaf distributions of the endophytes, differences in infection modes and negative interactions of major endophytes within leaves are suggested. Received: December 13, 2001 / Accepted: June 7, 2002 Acknowledgments The authors thank the staff members of the Experimental Forests of Kagoshima University for enabling the present study. Correspondence to:K. Hata     

Crystal structure of the antigen-binding fragment of apoptosis-inducing mouse anti-human Fas monoclonal antibody HFE7A.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A), which induces apoptosis, has been humanized based on a structure predicted by homology modeling. A version of humanized HFE7A is currently under development for the treatment of autoimmune diseases such as rheumatoid arthritis. For a deeper understanding of the protein engineering aspect of antibody humanization, for which information on the three-dimensional structure is essential, we determined the crystal structure of the m-HFE7A antigen-binding fragment (Fab) by X-ray crystallography at 2.5 A resolution. The main-chain conformation of the five loops in the six complementarity-determining regions (CDRs) was correctly predicted with root-mean-square deviations of 0.30-1.04 A based on a comparison of the crystal structure with the predicted structure. The CDR-H3 conformation of the crystal structure, which was not classified as one of the canonical structures, was completely different from that of the predicted structure but adopted the conformation which followed the "H3-rules." The results of charge distribution analysis of the antigen-binding site suggest that electrostatic interactions may be important for its binding to Fas.     

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Pyrindicin,a New Alkaloid from a Streptomyces Strain

In the course of our examination for the alkaloid productivities of Streptomyces strains, Streptomyces sp. NA–15 was found to produce a new alkaloid, pyrindicin, in the culture medium. The strain NA–15 was found to be a variant of Streptomyces griseoflavus and was designated as S. griseoflavus var. pyr indie us nov. var.

After the culture conditions for pyrindicin production were studied, pyrindicin was obtained as its hydrochloride (mp 145°C, decomp.) from the cultured broth. The compound was shown to possess weak antimicrobial and several pharmacological activities. The LD₅₀ of the hydrochloride (ip, in mice) was 87 mg/kg.