Coordination of cell polarity during Xenopus gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and na?ve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity.     

Reevaluation of <Emphasis Type="Italic">Pholiota squarrosa</Emphasis> lectin-reactive haptoglobin as a pancreatic cancer biomarker using an improved ELISA system

An increase in Lewis- and core-type fucosylation of haptoglobin has been reported in patients with pancreatic cancer (PC), suggesting that fucosylated haptoglobin is a candidate PC biomarker. Previously, we developed a Pholiota squarrosa lectin antibody enzyme-linked immunosorbent assay (PhoSL-ELISA) system for the detection of core-fucosylated haptoglobin. However, with this methodology, positive results were only obtained for some patients with PC, demonstrating the need for a more sensitive detection system. In the current study, we developed an improved PhoSL-ELISA system with higher sensitivity to detect core-fucosylated haptoglobin using high-concentration urea as a denaturing agent with lectin to facilitate detection. We then reevaluated the performance of PhoSL reactive-core-fucosylated haptoglobin (PhoSL-HP) as a PC biomarker using the improved PhoSL-ELISA system. PhoSL-HP levels in the sera of patients with PC were significantly higher than those in healthy volunteers, with an area under the curve (AUC) value of 0.753. Furthermore, the AUC value of CA19–9 improved from 0.793 to 0.907 when combined with PhoSL-HP. Additionally, several CA19–9-negative cases among the patients with PC were diagnosed as positive for PhoSL-HP. In conclusion, PhoSL-HP detection using our improved ELISA system might allow PhoSL-HP to serve as a potential biomarker for PC and thus might be useful to complement the detection of CA19–9 in PC diagnosis.     

Variety in excitation energy transfer processes from phycobilisomes to photosystems I and II

Photosynthesis Research - The light-harvesting antennas of oxygenic photosynthetic organisms capture light energy and transfer it to the reaction centers of their photosystems. The light-harvesting...     

Distribution of the tryptophan pathway-derived defensive secondary metabolites gramine and benzoxazinones in Poaceae

The Poaceae is a large taxonomic group consisting of approximately 12,000 species and is classified into 12 subfamilies. Gramine and benzoxazinones (Bxs), which are biosynthesized from the tryptophan pathway, are well-known defensive secondary metabolites in the Poaceae. We analyzed the presence or absence of garamine and Bxs in 64 species in the Poaceae by LC-MS/MS. We found that Hordeum brachyantherum and Hakonechloa macra accumulated gramine, but the presence of gramine was limited to small groups of species. We also detected Bxs in four species in the Pooideae and six species in the Panicoideae. In particular, four species in the Paniceae tribe in Panicoideae accumulaed Bxs, indicating that this tribe is a center of the Bx distribution. Bxs were absent in the subfamilies other than Pooideae and Panicoideae. These findings provide an overview of biased distribution of gramine and Bxs in Poaceae species.     

Muscle-specific exonic splicing silencer for exon exclusion in human ATP synthase gamma-subunit pre-mRNA.

Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.     

Ultrastructural localization of photosynthetic and photorespiratory enzymes in epidermal, mesophyll, bundle sheath, and vascular bundle cells of the C4 dicot Amaranthus viridis.

In the leaves of the NAD-malic enzyme (NAD-ME)-type C4 dicot Amaranthus viridis L., there are chloroplasts in the vascular parenchyma cells (VPC), companion cells (CC), ordinary epidermal cells (EC), and guard cells (GC), as well as in the mesophyll cells (MC) and the bundle sheath cells (BSC). However, the chloroplasts of the VPC, CC, EC, and GC are smaller than those of the MC and BSC. In this study, the accumulation of photosynthetic and photorespiratory enzymes in these leaf cell types was investigated by immunogold labelling and electron microscopy. Strong labelling for phosphoenolpyruvate carboxylase was found in the MC cytosol. Weak labelling was observed in the CC and GC cytosol. Labelling for pyruvate, Pi dikinase occurred to varying degrees in the chloroplasts of all cell types except CC. Labelling for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase was detected in the chloroplasts of all cell types except MC. For both NAD-ME and the P-protein of glycine decarboxylase, intense labelling was found in the BSC mitochondria; weaker labelling was recognized in the VPC mitochondria. These data indicate that when not only the MC and BSC but also the other leaf cell types are included, the cell-specific expression of the enzymes in C4 leaves becomes more complex than has been known previously. These findings are discussed in relation to the metabolic function of epidermal and vascular bundle cells.     

Preparation of phosphopeptide thioesters by Fmoc- and Fmoc(2-F)-solid phase synthesis

Summary Efficient methods for the preparation of phosphopeptide thioesters were examined, using Fmoc-based solid-phase method. Phosphopeptide thioesters were obtained in good yields by the use of 1-methylpyrrolidine hexamethyl-eneimine and 1-hydroxybenzotriazole in a DMSO-DMF (1∶1, v/v) solution for deblocking the Fmoc groups. Epimerization, which is often observed at the C-terminal amino acid, was effectively suppressed by shortening the time of deblocking process via the use of highly base sensitive Fmoc(2-F) groups for α-amino protection.     

Purification and partial characterization of a mitogenic factor from bovine liver: structural homology with basic fibroblast growth factor

Two mitogenic peptides in bovine liver extract were purified to apparent homogeneity by monitoring the purification steps with two in vitro bioassays; one based on stimulation of adult bovine aortic arch endothelial cell proliferation and the other incorporation of [3H]thymidine to mouse fibroblast 3T3 cells. The purification procedure involved cation-exchange chromatography followed by affinity chromatography on heparin-Sepharose and two steps of reversed-phase HPLC. The purified material showed the same biological activity as pituitary basic fibroblast growth factor (FGF). Amino acid analyses of the purified mitogen yielded a similar, but not identical composition to that of bovine pituitary basic FGF(1-146) reported previously. Gas-phase microsequencing identified two sequences in equal amounts in the purified preparation. Furthermore, the sequencing results are in accord with the theoretical data obtained when two truncated forms of basic FGF, corresponding to FGF(12-146) and (16-146), are being sequenced simultaneously. Basic FGF(12-146) is a novel truncated form of basic FGF which has not been isolated before although the (16-146) fragment has been found previously in kidney, corpus luteum, and adrenal. SDS-PAGE analysis could not separate the two forms and showed that both migrated as a protein of about 15,100 daltons, which is slightly smaller than intact basic FGF(1-146) (16,200 daltons). These results, taken together, indicate that at least some of the mitogenic activity in liver may be derived from basic FGF-related polypeptides.     

Site-specific integration of a transgene mediated by a hybrid adenovirus/adeno-associated virus vector using the Cre/loxP-expression-switching system

As vectors, adenoviruses (Ads) have many attractive advantages for in vivo gene therapy. However, Ads do not usually integrate into the host genome and gene expression is, thus, transient. Adeno-associated virus (AAV) integrates into a specific locus (AAVS1) on the human host's chromosome 19, while conventional recombinant AAV (rAAV) vectors do not possess this property because such vectors lack the rep gene. AAV vectors carrying the rep gene do not have enough space for insertion of a transgene. We have constructed a hybrid adenovirus/adeno-associated virus (Ad/AAV) vector which has the advantages of both Ads and AAVs. Given that the rep gene products inhibit propagation of Ads, we used the Cre/loxP-expression-switching system to regulate the expression of the rep gene. The Ad/AAV vector easily propagates, can efficiently infect a broad range of cell types, and can integrate into a specific locus on host chromosomes.