The Commelina yellow mottle virus promoter drives companion-cell-specific gene expression in multiple organs of transgenic tobacco

Summary.  Previous work has demonstrated that some endogenous plant gene promoters are active in selective companion cells of the phloem, depending on organ types and developmental stages. Here we report that the Commelina yellow mottle virus (CoYMV) promoter is active in the companion cells of leaves, stems and roots of transgenic Nicotiana tabacum cv. Xanthi NN, using β-glucuronidase (GUS) as a reporter. Thus, the CoYMV promoter has a broad organ specificity. This promoter can be useful in molecular studies on the functions of companion cells in many aspects of phloem biology, such as regulation of long-distance transport, macromolecular traffic, plant development and interaction with pathogens. It may also be useful in engineering crops that produce specific gene products in the companion cells to block long-distance movement of pathogens. Received February 5, 2002; accepted March 27, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Department of Plant Biology and Plant Biotechnology Center, 207 Rightmire Hall, Ohio State University, 1060 Carmack Road, Columbus, OH 43210, U.S.A.     

Corticosterone acutely prolonged N-methyl-d-aspartate receptor-mediated Ca2+ elevation in cultured rat hippocampal neurons

This work reports the first demonstration that corticosterone (CORT) has a rapid and transient effect on NMDA receptor-mediated Ca2+ signaling in cultured rat hippocampal neurons. Using single cell Ca2+ imaging, CORT and agonists of glucocorticoid receptors were observed to modulate the NMDA receptor-mediated Ca2+ signals in a completely different fashion from pregnenolone sulfate. In the absence of steroids, 100 micro m NMDA induced a transient Ca2+ signal that lasted for 30-70 s in 86.1% of the neurons prepared from postnatal rats (3-5 days old). After pre-treatment with 0.1-100 micro m CORT for 10-20 min, NMDA induced extremely prolonged Ca2+ elevation. This prolonged Ca2+ elevation was terminated by the application of MK-801 and followed by washing out of CORT. The proportion of CORT-modulated neurons within the NMDA-responsive cells increased from 25.1 to 95.5% when the concentration of CORT was raised from 0.1 to 50 micro m. Substitution of BSA-conjugated CORT produced essentially the same results. When hippocampal neurons were preincubated with 10 micro m cortisol and 1 micro m dexamethasone for 20 min, a very prolonged Ca2+ elevation was also observed upon NMDA stimulation. The CORT-prolonged Ca2+ elevation caused a long-lasting depolarization of the mitochondrial membrane, as observed with rhodamine 123. In contrast, incubation with 100 micro m pregnenolone sulfate did not considerably alter the time duration of NMDA-induced transient Ca2+ elevation, but caused a significant increase in the peak amplitude of Ca2+ elevation in hippocampal neurons. These results imply that high levels of CORT induce a rapid and non-genomic prolongation of NMDA receptor-mediated Ca2+ elevation, probably via putative membrane surface receptors for CORT in the hippocampal neurons.     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.     

Apolipoprotein E and H polymorphisms in Mongolian Buryat: allele frequencies and relationship with plasma lipid levels

A Buryat population consisting of seven tribal groups in eastern Mongolia has been screened to determine the frequency distribution of different apolipoprotein E and H alleles (APOE and APOH, genes) coding for common isoforms and their association with quantitative plasma lipid levels. Allele frequencies at the APOE locus in 125 healthy Buryat aged 17 to 73 years were highest for APOE*3 (0.804), followed by APOE*4 (0.164) and APOE*2 (0.032). The APOH locus had high frequencies of APOH*2 (0.912) and APOH*3 (0.088). APOH*1 was not detected. No significant differences were observed in the overall APOE allele frequencies between the Buryat and the Siberian Evenki, Inuits, and Indians in Asia, or with some European whites. The frequency distribution of the overall APOH alleles of the Buryat was similar to that of the Japanese in Asia. Overall plasma lipid levels of the Buryat (males aged 20 to 73 years, females aged 21 to 64 years) were considerably lower, comparable to those of the Evenki. The APOE*4/E*3 males had significantly high total- and LDL-cholesterol levels compared with the APOE*3/E*3 males (p < 0.025 and p < 0.01, respectively). No significant effects of the APOH genotypes on any of the plasma lipid levels were observed. In particular, our data regarding APOE suggest that the Buryat are genetically close in allele frequencies to the Evenki and Inuits, but differ from them in the association of genotype APOE*4/E*3 with cholesterol levels.     

Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.     

Functional expression of Acetabularia acetabulum vacuolar H(+)-pyrophosphatase in a yeast VMA3-deficient strain

The function of the translation product of cDNA for Acetabularia vacuolar H(+)-pyrophosphatase was examined using the Saccharomyces cerevisiae VMA3-deficient strain. The open reading frame of Acetabularia H(+)-pyrophosphatase was revealed to encode 751 amino acids (721 or 751 amino acids in a previous paper). The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2. Immunoblot analysis also supported the localization of the translation product in the vacuolar-membrane-enriched fraction.     

Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.     

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.     

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.