An RNA-binding protein, AtRBP1, is expressed in actively proliferative regions in Arabidopsis thaliana

A cDNA clone, named XF41, that encodes an RNA-binding protein was isolated from Arabidopsis thaliana. The deduced protein, named AtRBP1, contains two conserved consensus sequence-type RNA-binding domains (CS-RBDs) in the N-terminal half, a putative PY motif (a target of a WW domain) in the center, and uncharacterized C-terminal domain. A binding assay demonstrated that the AtRBP1 can bind to single-stranded nucleic acids in vitro. Analysis of localization of the GUS activity of transgenic Arabidopsis thaliana plants that have the chimeric gene containing the upstream sequence of the AtRBP1 gene and GUS gene demonstrated that the AtRBP1 gene is expressed in meristematic tissues such as the vegetative shoot apex and root tips, developing organs such as floral buds and pistils of young flowers, abscission layers of immature siliques and junctions of pedicels. Considering the specificity of the expression, AtRBP1 may be required in the progress of cell proliferation.     

Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts

 When pumpkin (Cucurbita spp., cv. Ebisu Nankin) ascorbate oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow No. 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin ascorbate oxidase into the culture medium. These transgenic cells showed no morphological difference from wild-type cells. However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells. We propose that ascorbate oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall. Received: 28 October 1999 / Accepted: 18 January 2000     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

Interaction of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation. To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis. (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain. (iii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells. (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmunoprecipitated with HAX-1. (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus. (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay. Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1. These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1.     

Development of human T-cell leukemia virus type 1-transformed tumors in rats following suppression of T-cell immunity by CD80 and CD86 blockade

Host immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses. Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells. During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor regression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.     

A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression

We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the Paramyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 x 10(8) to 1. 0 x 10(8) cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.     

Y-27632 inhibits gastric motility in conscious rats

Y-27632, a highly selective inhibitor of p160ROCK, desensitizes the smooth muscle to Ca2+ and inhibits smooth muscle contraction. While this drug has the potential to become a novel drug for hypertension, it might also affect other smooth muscle, including that of gastrointestinal tract. We studied the effects of Y-27632 on gastric contractions in conscious rats. Strain gauge force transducers were sutured onto the serosal side of the gastric antrum and contractions were recorded before and after the intravenous injection of Y-27632. Doses of 1.0 mg/kg to 10 mg/kg significantly decreased contraction amplitude and the motility index in a dose dependent manner. With 10 mg/kg, the mean amplitude was decreased by up to 69 +/- 14% and the motility index by up to 81 +/- 7%. The change occurred immediately after drug infusion and lasted for 3.5h. Contraction frequency showed only a slight decrease. No signs of bowel obstruction were observed. These results indicate that Rho-mediated Ca sensitization has a role in the physiologic contractions of gastric smooth muscle in rats. Y-27632 is useful to investigate the physiology of gastrointestinal motility.     

Induced Response of Tomato Plants to Injury by Green and Red Strains of Tetranychus Urticae

We studied the induced response of tomato plants to the green strain and the red strain of the spider mite Tetranychus urticae. We focused on the olfactory response of the predatory mite Phytoseiulus persimilis to volatiles from T. urticae-infested tomato leaves in a Y-tube olfactometer. Tomato leaves attracted the predatory mites when slightly infested with the red strain, or moderately or heavily infested with the green strain. In contrast, neither leaves that were slightly infested with green-strain mites, nor leaves that were moderately or heavily infested with the red strain attracted the predators. We discuss the specific defensive responses of tomato plants to each of the two strains.     

Trend of adenovirus type 7 infection, an emerging disease in Japan. A report of the National Epidemiological Surveillance of Infectious Agents in Japan

The program of the National Epidemiological Surveillance of Infectious Diseases under the auspices of the Ministry of Health and Welfare started in 1981 apprehended in 1995 emergence of adenovirus type 7 in Japan. We analyzed the reported data of type 7 comparing with those of type 3, both belonging to the same subgenus B, and the following results were obtained: After 1981, the main serotypes in the reports of adenovirus isolation/detection were types 3, 2, and 1 in this order. The reports of isolation of adenovirus type 7 used to be extremely few, however, suddenly increased in 1995. In 1997, reports of isolation of adenovirus type 3 decreased and those of type 7 acquired the third place after those of types 2 and 3. Type 7 infection occur almost every month, but most frequently during May-September. The ages of cases from which type 7 was isolated were 0-4 years accounting for 55%, 5-9 years 35%, teens 6.3% and adults 4.0%, being similar proportions to those yielding type 3. Clinical diagnoses of cases yielding adenovirus type 7 were pharyngo-conjunctival fever (PCF) and influenza-like illness, these two accounting for half. The symptoms were severe, being characterized by higher maximum body temperature during the feverish period and severe pneumonia. Encephalitis and arthro-muscular pain were seen in only type 7-infected cases, although such cases were few.