Arabidopsis TOBAMOVIRUS MULTIPLICATION (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1

The tom2-1 mutation of Arabidopsis thaliana reduces the efficiency of intracellular multiplication of tobamoviruses. The tom2-1 mutant was derived from fast-neutron-irradiated seeds, and the original mutant line also carries ttm1, a dominant modifier that increases tobamovirus multiplication efficiency in a tobamovirus-strain-specific manner in the tom2-1 genetic background. Here, we show that the tom2-1 mutation involved a deletion of approximately 20 kb in the nuclear genome. The deleted region included two genes named TOM2A and TOM2B that were both associated with the tom2-1 phenotype, whereas ttm1 corresponded to the translocation of part of the deleted region that included intact TOM2B but not TOM2A. TOM2A encodes a 280 amino acid putative four-pass transmembrane protein with a C-terminal farnesylation signal, while TOM2B encodes a 122 amino acid basic protein. The split-ubiquitin assay demonstrated an interaction of TOM2A both with itself and with TOM1, an integral membrane protein of A.thaliana presumed to be an essential constituent of tobamovirus replication complex. The data presented here suggest that TOM2A is also an integral part of the tobamovirus replication complex.     

New approach for the establishment of an hepatocyte cell line derived from rat early embryonic stem cells

A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and 1 ng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a bio-hybrid typed liver apparatus.     

Asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine: preparation,resolution and biological activities toward insects and their nerve preparations

The asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine, was prepared, and the absolute configurations of the enantiomers were determined by an X-ray analysis. The insecticidal activity against the housefly measured with metabolic inhibitors showed the (S) enantiomer to be slightly more active than the (R) isomer. Electrophysiological measurements on the American cockroach central nerve cord showed the compounds to elicite the impulses and subsequently blocked them. The neuroblocking potency of the (S) isomer was 5.9 microM, while that of the (R) isomer was as high as 73 microM. The molar concentrations required for 50% inhibition of the specific binding of [3H]imidacloprid to the housefly head membrane preparation were respectively 0.19 microM and 0.95 microM for the (S) and (R) isomers. This enatioselectivity ratio was smaller than 35 for nicotine isomers but greater than 2 for epibatidine isomers.     

Sequence database of 1172 T-DNA insertion sites in Arabidopsis activation-tagging lines that showed phenotypes in T1 generation

Plant genomic resources harbouring gain-of-function mutations remain rare, even though this type of mutation is believed to be one of the most useful for elucidating the function of unknown genes that have redundant partners in the genome. An activation-tagging T-DNA was introduced into the genome of Arabidopsis creating 55,431 independent transformed lines. Of these T1 lines, 1,262 showed phenotypes different from those of wild-type plants. We called these lines 'AT1Ps' (activation T1 putants). The phenotypes observed include abnormalities in morphology, growth rate, plant colour, flowering time and fertility. Similar phenotypes re-appeared either in dominant or semi-dominant fashion in 17% of 177 AT2P plants tested. Plasmid rescue or an adaptor-PCR method was used to identify 1172 independent genomic loci of T-DNA integration sites in the AT1P plants. Mapping of the integration sites revealed that the chromosomal distribution of these sites is similar to that observed in conventional T-DNA knock-out lines, except that the intragenic type of integration is slightly lower (27%) in the AT1P plants compared to that observed in other random knock-out populations (30-35%). Ten AT2P lines that showed dominant phenotypes were chosen to monitor expression levels of genes adjacent to the T-DNA integration sites by RT-PCR. Activation was observed in 7 out of 17 of the adjacent genes detected. Genes located up to 8.2 kb away from the enhancer sequence were activated. One of the seven activated genes was located close to the left-border sequence of the T-DNA, having an estimated distance of 5.7 kb from the enhancer. Surprisingly, one gene, the first ATG of which is located 12 kb away from the enhancer, showed reduced mRNA accumulation in the tagged line. Application of the database generated to Arabidopsis functional genomics research is discussed. The sequence database of the 1172 loci from the AT1P plants is available (http://pfgweb.gsc.riken.go.jp/index.html).     

Solid-liquid phase behavior of binary fatty acid mixtures. 2. Mixtures of oleic acid with lauric acid, myristic acid, and palmitic acid

Solid-liquid phase behavior was investigated for binary fatty acid mixtures composed of oleic acid (OA; cis-9-octadecenoic acid) and saturated fatty acids, lauric acid (LA; dodecanoic acid), myristic acid (MA; tetradecanoic acid), and palmitic acid (PA; hexadecanoic acid), by means of differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). When the mixture was heated immediately after the solidification from the melt, the heat effect due to the gamma-to-alpha transformation of OA varied depending on the composition of the mixture. However, the mixture subjected to an annealing at the temperature slightly below the melting temperature provided the transformation at constant temperature which corresponds to the gamma-to-alpha transformation temperature of pure OA. This suggests that a solid phase formed by cooling of the melt of the mixture is not in an equilibrium state, but it relaxes to a stable solid during the annealing process. The T-X phase diagrams of these mixtures constructed from the DSC measurements demonstrate that the two fatty acid species are completely immiscible in a solid phase regardless of the type of polymorphs of OA, alpha- or gamma-form. According to a thermodynamic analysis of liquidus line basing on the regular solution model for the melt, the non-ideality of mixing tends to increase with the decrease in the acyl chain length of the saturated fatty acid, although the mixing is rather close to ideal.     

Egg yolk phosvitin inhibits hydroxyl radical formation from the fenton reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical ((.-)OH) formation from H(2)O(2) in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited (.-)OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the (.-)OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H(2)O(2). These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Neuropeptide Y enhances permeability across a rat aortic endothelial cell monolayer

Previously, in vivo studies showed that neuropeptide Y (NPY) elevates vascular permeability in isolated lung perfusion preparations, possibly through binding to the NPY Y(3) receptor. The present study used monolayers in a double-chamber culture method under conditions of normoxia (5% CO(2)-20% O(2)-75% N(2)) or hypoxia (5% CO(2)-5% O(2)-90% N(2)) to test the hypothesis that NPY directly affects rat aortic endothelial cells (RAECs). RAECs were cultured on the base of the upper chamber, into which FITC-labeled albumin was introduced, and permeation into the lower chamber was measured. The RAEC monolayer was treated with 10(-8)-3 x 10(-7) M NPY for 2 h in normoxia or hypoxia. In hypoxia, NPY concentration dependently increased the permeability of the RAEC monolayer, whereas in normoxia no significant change was observed. Peptide YY, NPY Y(1), and NPY Y(2) receptor agonists and NPY Y(1) receptor antagonist exerted no significant effects under hypoxic conditions. NPY-(18-36), an NPY Y(3) receptor antagonist, elicited an inhibitory action on the NPY-induced increase in monolayer permeability. Furthermore, neither N-monomethyl-l-arginine, a nitric oxide synthase inhibitor, the bradykinin B(2) receptor antagonist FK-3657, nor the vascular endothelial growth factor receptor-coupled tyrosine kinase inhibitor tyrphostin SU-1498, injected into the medium of the upper chamber, affected the NPY-induced permeability changes under hypoxic conditions. The results suggest that the NPY-induced increase in permeability across the RAEC monolayer is closely related to low O(2) tension, possibly mediated by direct action on the NPY Y(3) receptor expressed on the endothelial cell membrane. Furthermore, this NPY-induced increase is not likely due to nitric oxide, bradykinin, or vascular endothelial growth factor.     

Translocation of caveolin regulates stretch-induced ERK activity in vascular smooth muscle cells

Mechanical stress contributes to vascular disease related to hypertension. Activation of ERK is key to mediating cellular proliferation and vascular remodeling in response to stretch stress. However, the mechanism by which stretch mediates ERK activation in the vascular tissue is still unclear. Caveolin, a major component of a flasklike invaginated caveolae, acts as an adaptor protein for an integrin-mediated signaling pathway. We found that cyclic stretch transiently induced translocation of caveolin from caveolae to noncaveolar membrane sites in vascular smooth muscle cells (VSMCs). This translocation of caveolin was determined by detergent solubility, sucrose gradient fractionation, and immunocytochemistry. Cyclic stretch induced ERK activation; the activity peaked at 5 min (the early phase), decreased gradually, but persisted up to 120 min (the late phase). Disruption of caveolae by methyl-beta-cyclodextrin, decreasing the caveolar caveolin and accumulating the noncaveolar caveolin, enhanced ERK activation in both the early and late phases. When endogenous caveolins were downregulated, however, the late-phase ERK activation was subsided completely. Caveolin, which was translocated to noncaveolar sites in response to stretch, is associated with beta1-integrins as well as with Fyn and Shc, components required for ERK activation. Taken together, caveolin in caveolae may keep ERK inactive, but when caveolin is translocated to noncaveolar sites in response to stretch stress, caveolin mediates stretch-induced ERK activation through an association with beta1-integrins/Fyn/Shc. We suggest that stretch-induced translocation of caveolin to noncaveolar sites plays an important role in mediating stretch-induced ERK activation in VSMCs.     

Characterization of in vitro gutlike organ formed from mouse embryonic stem cells

Using an embryoid body (EB) culture system, we have made a functional organlike cluster: the "gut" from embryonic stem (ES) cells (ES gut). There are many types of ES clusters, because ES cells have a pluripotent ability to develop into a wide range of cell types. Before inducing specific differentiation by exogenously added factors, we characterized comprehensive physiological and morphological properties of ES guts. Each ES gut has a hemispherical (or cystic) structure and exhibits spontaneous contractions [mean frequency: 13.5 ± 8.8 cycles per min (cpm)]. A dense distribution of interstitial cells of Cajal (ICC) was identified by c-Kit immunoreactivity, and specific subcellular structures of ICC and smooth muscle cells were identified with electron microscopy. ICC frequently formed close contacts with the neighboring smooth muscle cells and occasionally formed gap junctions with other ICC. Widely propagating intracellular Ca²⁺ concentration oscillations were generated in the ES gut from the aggregates of c-Kit immunopositive cells. Plateau potentials, possibly pacemaker potentials in ICC, and electrical slow waves were recorded for the first time. These events were nifedipine insensitive, as in the mouse gut. Our present results indicate that the rhythmic pacemaker activity generated in ICC efficiently spreads to smooth muscle cells and drives spontaneous rhythmic contractions of the ES gut. The present characterization of physiological and morphological properties of ES gut paves the way for making appropriate models to investigate the origin of rhythmicity in the gut. intracellular calcium concentration oscillation; interstitial cells of Cajal; peristalsis