Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation.

The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR).

A carboxy-terminally truncated form of the Vpr protein of human immunodeficiency virus type 1 retards cell proliferation independently of G(2) arrest of the cell cycle.

Vpr, one of the accessory gene products of HIV-1, is a 96-residue protein with several functions. It is involved in import of the HIV-1 preintegration complex into the nucleus of nondividing cells, in cellular differentiation, inducing cell cycle arrest at the G(2)/M phase, in immune suppression, and in enhancement of replication of the virus. We found recently that Vpr interferes with the proliferation of mouse NIH3T3 fibroblasts but fails to arrest these cells in the G(2) phase.

MAP kinase cascades in Arabidopsis: their roles in stress and hormone responses.

Mitogen-activated protein kinase (MAPK) cascades have essential roles in diverse intracellular signaling processes in plants, animals and yeasts. In plants, MAPK and MAPK-like kinase activities are transiently activated in response to environmental stresses and plant hormones. In addition, transcription of genes encoding protein kinases involved in MAPK cascades in upregulated by environmental stresses.

Relationship between amino acid properties and protein stability: buried mutations.

In order to understand the mechanism of protein stability and to develop a simple method for predicting mutation-induced stability changes, we analyzed the relationship between stability changes caused by buried mutations and changes in 48 amino acid properties. As expected from the importance of hydrophobicity, properties reflecting hydrophobicity are strongly correlated with the stability of proteins. We found that subgroup classification based on secondary structure increased correlations significantly, and mutations within beta-strand segments correlated better than did those in alpha-helical segments, which may result from stronger hydrophobicity of the beta-strands.

Transient expression of the beta-glucuronidase gene in tissues of Arabidopsis thaliana by bombardment-mediated transformation.

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the beta-glucuronidase (GUS) gene in cultured plant cells and tissues and the stable transformation of various plants using a pneumatic particle gun. In this chapter, we describe transient expression of the GUS gene in Arabidopsis thaliana leaves and roots using the pneumatic particle gun.

Identification of human APC10/Doc1 as a subunit of anaphase promoting complex.

Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B. Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human. It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13.

Evaluation of a cDNA scanning method concerning the fidelity and efficiency of cDNA selection using the YAC CIC3B1-S region of Arabidopsis thaliana chromosome 5.

We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155 161). We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1-S DNA were concentrated.

Characterization of a novel kinetochore protein, CENP-H.

Macromolecular centromere-kinetochore complex plays a critical role in sister chromatid separation, but its complete protein composition as well as its precise dynamic function during mitosis has not yet been clearly determined. Here we report the isolation of a novel mouse kinetochore protein, CENP-H. The CENP-H, with an apparent molecular mass of 33 kDa, was found to contain a coiled-coil structure and a nuclear localization signal.

Regional insertional mutagenesis of specific genes on the CIC5F11/CIC2B9 locus of Arabidopsis thaliana chromosome 5 using the Ac/Ds transposon in combination with the cDNA scanning method.

We have recently developed a novel cDNA selection method (the cDNA scanning method) to select cDNAs for expressed genes in specific regions of the genome [Hayashida et al. (1995) Gene 165: 155, Seki et al. (1997) Plant J.

The coactivators p300 and CBP have different functions during the differentiation of F9 cells.

We have characterized an element (differentiation response element, DRE) in the promoter region of the c-jun gene that is both necessary and sufficient for retinoic acid (RA) and adenovirus early region (E1A) mediated up-regulation of c-jun gene expression during the differentiation of F9 cells. The DRF complex, which binds specifically to DRE, is composed of the E1A-associated protein p300 and the activation transcription factor-2 (ATF-2) as a DNA-binding subunit of the DRF. The molecular association of p300 and ATF-2 enhances the transcription of the c-jun gene, which requires protein kinase C alpha mediated phosphorylation of Ser-121 of ATF-2 within its p300 interaction domain.

Regulation of APC activity by phosphorylation and regulatory factors.

Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase-anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated.

The DNA-binding and transcriptional activities of MAZ, a myc-associated zinc finger protein, are regulated by casein kinase II.

Myc-associated zinc finger protein (MAZ) is a transcription factor that contains proline-rich, alanine repeats and six C(2)H(2)-type zinc finger motifs, as well as five putative sites of phosphorylation by casein kinase II (CKII). Site-specific mutagenesis of MAZ revealed that the serine residue at position 480 was the major site of phosphorylation by CKII both in vitro and in vivo. Phosphorylation of MAZ by CKII at this serine residue was required for maximum binding of MAZ to the pyrimidine-rich DNA of the nuclease-hypersensitive element (NHE) in the 5'-end promoter region of the c-myc gene.

Characterization of a novel member of the FGF family, XFGF-20, in Xenopus laevis.

The cDNA for a novel member of the FGF family (XFGF-20) was isolated from a Xenopus cDNA library prepared at the tailbud stage using as a probe the product of degenerate PCR performed with primers based on mammalian FGF-9s. This cDNA was 1860 bp long, and contained a single open reading frame that encoded 208 amino acid residues. The deduced amino acid sequence contained a motif characteristic of the FGF family and it was similar (73.

A simple method for predicting transmembrane alpha helices with better accuracy.

The prediction of a protein's structure from its amino acid sequence has been a long-standing goal of molecular biology. In this work, a new set of conformational parameters for membrane spanning alpha helices was developed using the information from the topology of 70 membrane proteins. Based on these conformational parameters, a simple algorithm has been formulated to predict the transmembrane alpha helices in membrane proteins.

Role of structural and sequence information in the prediction of protein stability changes: comparison between buried and partially buried mutations.

Predicting mutation-induced changes in protein stability is one of the greatest challenges in molecular biology. In this work, we analyzed the correlation between stability changes caused by buried and partially buried mutations and changes in 48 physicochemical, energetic and conformational properties. We found that properties reflecting hydrophobicity strongly correlated with stability of buried mutations, and there was a direct relation between the property values and the number of carbon atoms.

Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis.

C-Jun amino terminal kinase/stress-activated protein kinases (JNK/SAPK) and p38 subgroups of mitogen-activated protein kinases have been suggested to play a critical role in apoptosis, cell growth, and/or differentiation. We found that a short exposure of SKT6 cells, which respond to erythropoietin (Epo) and induce erythroid differentiation, to osmotic or heat shock induced transient activation of JNK/SAPK and p38 and inactivation of ERK and resulted in erythroid differentiation without Epo, whereas long exposure of the cells to these stresses induced prolonged activation/inactivation of the same kinases and caused apoptosis. Inhibition of JNK/SAPK and p38 resulted in inhibition of stress-induced erythroid differentiation and apoptosis.

H-NMR and mass spectral study of a D-enriched acetylenic norlignan, asparenyol, from cultured cells of Asparagus officinalis.

A suspension of cultured cells of Asparagus officinalis fed L-phenyl-D5-alanine-2,3,3-D3 (D5-phe) and DL-phenylalanine-3,3-D2 (D2-phe) yielded D-enriched asparenyol, 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyllphenol (1). A H-NMR spectral study indicated that incorporation of deuterium atoms into the chains of the products was restricted to the C-9 position. The molecular ion regions of the MS of D-enriched metabolites, 1(D8-200), 1(D8-100) and 1(D8-50), obtained from feeding experiments using 200 mg, 100 mg and 50 mg of Ds-phe, respectively, coincided with the theoretical spectra.

Mouse ATF-2 null mutants display features of a severe type of meconium aspiration syndrome.

Mouse null mutants of transcription factor ATF-2 were generated by the gene targeting method. They died shortly after birth and displayed symptoms of severe respiratory distress with lungs filled with meconium. These features are similar to those of a severe type of human meconium aspiration syndrome.

Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana.

Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), in plants.

Physical interaction between retinoic acid receptor and Sp1: mechanism for induction of urokinase by retinoic acid.

Induction of urokinase plasminogen activator (uPA) by retinoic acid (RA) is the initial event preceding certain subsequent biological changes in vascular endothelial cells. We investigated the molecular mechanism by which RA stimulates the expression of uPA, which lacks a canonical RA receptor (RAR)-responsive element, in bovine and human aortic endothelial cells. Upon stimulation with RA, mRNA levels of RARalpha and beta transiently increased in parallel with the induction of uPA, and this increase was inhibited by cycloheximide.

Application of the RLGS image analysis tool (RAT) to the construction of a genetic linkage map of recombinant inbred strain SMXA.

The construction of a genetic linkage map is the first, fundamental step to analyze the genetic properties of any organism. For this purpose, the restriction landmark genome scanning method (RLGS) can be used and has been shown to have high productivity in various genetic analyses. However, construction of a genetic linkage map by the RLGS method is laborious, because hundreds of spots must be scored, usually by visual observation.

Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron.

Synaptotagmin I or II (Syt I/II) is involved in Ca2+-regulated exocytosis of secretory vesicles, probably serving as a Ca2+-sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca2+-regulated exocytosis.

Automated filtration-based high-throughput plasmid preparation system.

Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method.

Activation of hematopoietic progenitor kinase-1 by erythropoietin.

Hematopoietic progenitor kinase-1 (HPK1), which is expressed predominantly in hematopoietic cells, was identified as a mammalian Ste20 homologue that, upon transfection, leads to activation of JNK/SAPK in nonhematopoietic cells. The JNK/SAPK pathway is activated by various environmental stresses and proinflammatory and hematopoietic cytokines. Upstream activators of HPK1 currently remain elusive, and its precise role in hematopoiesis has yet to be defined.