Planar Cell Polarity Enables Posterior Localization of Nodal Cilia and Left-Right Axis Determination during Mouse and Xenopus Embryogenesis

Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP) in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell''s apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP) is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2) in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.     

Conformational Change of a Natto Mucin in Solution

The mucin obtained from a natto sample was found to be composed of 58 % of γ-polyglutamic acid and 40% of polysaccharide. The ratio of l- and d-glutamic acid was determined to be 58:42 using l-glutamic acid decarboxylase. The weight- and z-average molecular weight were 2.08 × 10⁵ and 2.22 × 10⁵, respectively. The distribution curve of the sedimentation coefficient showed a small heterogeneity. The mucin molecule was considered to be randomly coiled at pH 5.0 ~ 8.8 and to be a rod-like molecule in the lower pH region.     

Identification of a novel type of CA19-9 carrier in human bile and sera of cancer patients: an implication of the involvement in nonsecretory exocytosis

Carbohydrate antigen 19-9 (CA19-9) is a well-known tumor marker for pancreatic cancer. Although the CA19-9 level is measured using anti-sialyl Lewis A antibodies, it remains unknown which molecules carry CA19-9 other than mucins. Here we report the identification and characterization of a novel type of CA19-9 carrier, BGM (bile globular membrane), which is thought to exist in normal bile and to be secreted into sera of patients with pancreatic cancer. We purified the BGM from bile juice using a β-casein column because surface plasmon resonance analysis could detect such carrier vesicles binding to β-casein in sera of patients with pancreatic cancer. We identified characteristic molecules for BGM such as AHNAK (desmoykoin) and a novel golgin family member, CABIN (CAsein Binding domain integral protein with golgIN motif) by mass spectrometry analysis. BGM was detected in the sera of patients with pancreatic cancer as well as athymic mice with transplanted pancreatic cancer cells. Down regulation of CABIN inhibited the secretion of CA19-9 on BGM in pancreatic cancer cell lines. We measured and visualized BGM in sera of patients with cancer. Thus, BGM might be another CA19-9 carrier (glyco-lipids on membrane vesicles) other than mucins and could be applied to the diagnosis of pancreatic cancer.     

A method of gentle hydration to prepare oil-free giant unilamellar vesicles that can confine enzymatic reactions

We report a new and improved method to prepare, by gentle hydration of lipid films, oil-free giant unilamellar vesicles (GUVs), in which enzymatic reactions can be encapsulated. The traditional method of gentle hydration requires very low concentrations of metal ions, whereas enzymatic reactions generally require mono- and divalent metal ions at physiological concentrations. In order to improve the production of oil-free GUVs that can confine enzymatic reactions, we developed a novel method also based on gentle hydration, but in which the precursor lipid film was doped with both 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEGylated lipid) and sugar. Close examination of the size, shape, and lamellarity of vesicles prepared in this manner demonstrated that the process improves the production of oil-free GUVs even at low temperatures and physiological salt concentrations. PEGylated lipid and sugar were found to synergistically improve GUV formation. Finally, we demonstrate the successful enzymatic synthesis of RNA within oil-free GUVs that were prepared on ice.     

Differentiation in wild-type allele of the sd1 locus concerning culm length between indica and japonica subspecies of Oryza sativa (rice)

A dwarfing gene (allele) sd1-d has been intensively utilized to develop short-culm indica varieties in southeast Asia up to now. Before the first sd1-d-carrying variety IR8 was released, rice researchers had recognized the general tendency that culm length is higher in indica varieties than in temperate-japonica ones. Inter-subspecific difference of the tall (wild-type) allele SD1 at the sd1 locus was examined on the common genetic background, using five isogenic lines developed by substituting sd1-d of the recurrent parent IR36 by SD1s of two indica varieties, two temperate-japonica varieties and one tropical-japonica variety. The two indica -donor isogenic lines had longer culms than the three japonica-donor isogenic lines consistently in two different environmental conditions. Moreover, nonsynonymous single-nucleotide polymorphism between the two subspecies was detected at two sites in Exon 1 and Exon 3 of the sd1 locus. It is demonstrated that the inter-subspecific differentiation of SD1 contributes height difference between indica and japonica. The indica-originating and japonica-originating alleles at the sd1 locus were designated as SD1-in(t) and SD1-ja(t), respectively.     

Proxy comparison in ancient peat sediments: pollen,macrofossil and plant DNA

We compared DNA, pollen and macrofossil data obtained from Weichselian interstadial (age more than 40 kyr) and Holocene (maximum age 8400 cal yr BP) peat sediments from northern Europe and used them to reconstruct contemporary floristic compositions at two sites. The majority of the samples provided plant DNA sequences of good quality with success amplification rates depending on age. DNA and sequencing analysis provided five plant taxa from the older site and nine taxa from the younger site, corresponding to 7% and 15% of the total number of taxa identified by the three proxies together. At both sites, pollen analysis detected the largest (54) and DNA the lowest (10) number of taxa, but five of the DNA taxa were not detected by pollen and macrofossils. The finding of a larger overlap between DNA and pollen than between DNA and macrofossils proxies seems to go against our previous suggestion based on lacustrine sediments that DNA originates principally from plant tissues and less from pollen. At both sites, we also detected Quercus spp. DNA, but few pollen grains were found in the record, and these are normally interpreted as long-distance dispersal. We confirm that in palaeoecological investigations, sedimentary DNA analysis is less comprehensive than classical morphological analysis, but is a complementary and important tool to obtain a more complete picture of past flora.     

Fluorescent detection of α-aminoadipic and γ-glutamic semialdehydes in oxidized proteins

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. α-Aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.     

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Cleavage of a model DNA replication fork by a Type I restriction endonuclease

Cleavage of a DNA replication fork leads to fork restoration by recombination repair. In prokaryote cells carrying restriction–modification systems, fork passage reduces genome methylation by the modification enzyme and exposes the chromosome to attack by the restriction enzyme. Various observations have suggested a relationship between the fork and Type I restriction enzymes, which cleave DNA at a distance from a recognition sequence. Here, we demonstrate that a Type I restriction enzyme preparation cleaves a model replication fork at its branch. The enzyme probably tracks along the DNA from an unmethylated recognition site on the daughter DNA and cuts the fork upon encountering the branch point. Our finding suggests that these restriction–modification systems contribute to genome maintenance through cell death and indicates that DNA replication fork cleavage represents a critical point in genome maintenance to choose between the restoration pathway and the destruction pathway.