Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

Analysis of the transgenic tobacco plants expressing Panicum miliaceum aspartate aminotransferase genes

 Expression of Panicum miliaceum L. (proso millet) mitochondrial and cytosolic aspartate aminotransferase (mAspAT and cAspAT, respectively) genes in transgenic tobacco plants (Nicotiana tabacum) and their influences on protein synthesis were examined. The mAspAT- or cAspAT-transformed plants had about threefold or 3.5-fold higher AspAT activity in the leaf than non-transformed plants, respectively. Interestingly, the leaves of both transformed plants had increased levels of phosphoenolpyruvate carboxylase (PEPC) and transformed plants with cAspAT also had increased levels of mAspAT in the leaf. These results suggest that the increased expression of Panicum cAspAT in transgenic tobacco enhances the expression of its endogenous mAspAT and PEPC, and the increased expression of Panicum mAspAT enhances the expression of its endogenous PEPC. Received: 6 February 1998 / Revision received: 6 August 1999 · Accepted: 6 September 1999     

Bundle sheath chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts

We investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants. After the seedlings were grown in a glasshouse for 1 month, they were treated for drought stress using two methods. One drought treatment was imposed by reducing the water supply to the plants for 1 month. The other was imposed by withholding water for 2 weeks to examine the withering process of leaves by drought stress. The ultrastructural changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll cells under both drought stress treatments. Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content in bundle sheath chloroplasts reduced more dramatically than in mesophyll chloroplasts by drought stress. Although a slight swelling of thylakoids was sometimes observed in bundle sheath chloroplasts in moderate stress for 1 month, the thylakoids were less affected by drought stress than chloroplast envelope. These results suggest that chloroplasts in bundle sheath cells were more sensitive to drought stress than those in mesophyll cells and the thylakoids were less damaged by drought stress compared with chloroplast envelope.     

Abnormal Localization of STK17A in Bile Canaliculi in Liver Allografts: An Early Sign of Chronic Rejection

The biological significance of STK17A, a serine/threonine kinase, in the liver is not known. We analyzed STK17A expression in HepG2 cells and human liver tissue. Accordingly, we investigated whether STK17A could help in identifying earlier changes during the evolution of chronic rejection (CR) after liver transplantation. RT-PCR and immunofluorescence were used to analyze STK17A expression in HepG2 cells. Antibody microarray was performed using human liver samples from CR and healthy donors. Immunohistochemistry was used to verify the clinical utility of STK17A on sequential biopsies for the subsequent development of CR. A novel short isoform of STK17A was found in HepG2 cells. STK17A was localized in the nuclei and bile canaliculi in HepG2 cells and human livers. Microarray of STK17A revealed its decrease in failed liver allografts by CR. During the evolution of CR, the staining pattern of bile canalicular STK17A gradually changed from diffuse linear to focal intermittent. The focal intermittent staining pattern was observed before the definite diagnosis of CR. In conclusion, the present study was the first to find localization of STK17A in normal bile canaliculi. Abnormal expression and localization of STK17A were associated with CR of liver allografts since the early stage of the rejection process.     

Enhancement of hypocotyl elongation by LOV KELCH PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

Auxin and two phytochrome-interacting factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in the enhancement of hypocotyl elongation in transgenic Arabidopsis thaliana plants that overproduce LOV KELCH PROTEIN2 (LKP2).

Abstract

LOV KELCH PROTEIN2 (LKP2) is a positive regulator of hypocotyl elongation under white light in Arabidopsis thaliana. In this study, using microarray analysis, we compared the gene expression profiles of hypocotyls of wild-type Arabidopsis (Columbia accession), a transgenic line that produces green fluorescent protein (GFP), and two lines that produce GFP-tagged LKP2 (GFP-LKP2). We found that, in GFP-LKP2 hypocotyls, 775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, and 21 genes involved in responses to red or far-red light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Hypocotyls of GFP-LKP2 seedlings, but not cotyledons or roots, contained a higher level of indole-3-acetic acid (IAA) than those of control seedlings. Auxin inhibitors reduced the enhancement of hypocotyl elongation in GFP-LKP2 seedlings by inhibiting the increase in cortical cell number and elongation of the epidermal and cortical cells. The enhancement of hypocotyl elongation was completely suppressed in progeny of the crosses between GFP-LKP2 lines and dominant gain-of-function auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function mutants pif4, pif5, and pif4 pif5. Our results suggest that the enhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the up-regulated expression of PIF4 and PIF5 in hypocotyls.

     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.     

Correspondence between food consistency and suprahyoid muscle activity, tongue pressure, and bolus transit times during the oropharyngeal phase of swallowing.

This study aimed to evaluate the effects of food texture and viscosity on the swallowing function by measuring tongue pressure and performing a videofluorographic (VF) examination. Eleven normal adults were recruited for this study. Test foods with different consistencies and liquid contents, i.e., a half-solid nutrient made of 0.8 and 1.5% agar powder, syrup, and a liquid containing 40 wt/vol% barium sulfate, were swallowed, and the anterior (AT) and posterior tongue pressures (PT) and electromyographic (EMG) activity of the suprahyoid muscles were recorded, together with VF images. The timing of each event obtained from EMG, tongue pressure, and VF recordings was measured and then compared. We found that the AT and PT activity patterns were similar and showed a single peak. The peak, area, and time duration of all of the variables for AT and PT and EMG burst increased with increasing hardness of the bolus. The onset of the EMG burst always preceded those of the AT and PT activities, while there were no significant differences in peak and offset times among EMG burst, AT, and PT. Total swallowing time and oral ejection time were significantly longer during the swallowing of 1.5% agar than any other boluses, while pharyngeal transit time and clearance time were significantly longer during the swallowing of syrup, which was as hard as the liquid, but showed a higher viscosity than the liquid. The results suggested that the major effects of food hardness were to delay oral ejection time, which strongly delays total swallowing time. In addition, pharyngeal bolus transit is not dependent on the hardness of food but on its viscosity.     

Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.     

Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody: the case of an anti-melanoma antibody,M2590, to GM3-ganglioside

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside G_M3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of G_M3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of G_M3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.