Stereoselective synthesis of 5-O-carbamoylpolyoxamic acid (2-amino-5-O-carbamoyl-2-deoxy-l-xylonic acid

One of the constituents of polyoxin J, 2-amino-5-O-carbamoyl-2-deoxy-l-xylonic acid (3), has been synthesized stereoselectively from l-sorbopyranose. The amino acid function of 3 was formed in the final stage of the synthesis by reduction of the corresponding α-azido carboxylic acid.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

NDR2‐mediated Rabin8 phosphorylation is crucial for ciliogenesis by switching binding specificity from phosphatidylserine to Sec15

Primary cilia are antenna‐like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi‐derived vesicles to the pericentrosome, a process requiring Rab11‐mediated recruitment of Rabin8, a GDP–GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser‐272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8‐/Rab11‐containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP‐bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho‐mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2‐mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.     

Initial Contact of Glioblastoma Cells with Existing Normal Brain Endothelial Cells Strengthen the Barrier Function via Fibroblast Growth Factor 2 Secretion: A New In Vitro Blood–Brain Barrier Model

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood–brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.     

Controlling the secondary-structure-forming tendencies of proteins by a backbone torsion-energy term

We examined a new backbone torsion-energy term proposed by us in the force field for protein systems. This torsion-energy term is represented by a double Fourier series in two variables, namely the backbone dihedral angles φ and ψ. It gives a natural representation of the torsion energy in the Ramachandran space in the sense that any two-dimensional energy surface periodic in both φ and ψ can be expanded by the double Fourier series. We can then easily control secondary-structure-forming tendencies by modifying the torsion-energy surface. For instance, we can increase or decrease the α-helix-forming-tendencies by lowering or raising the torsion-energy surface in the α-helix region and likewise increase or decrease the β-sheet-forming tendencies by lowering or raising the surface in the β-sheet region in the Ramachandran space. We applied this torsion-energy modification method to six force fields, AMBER parm94, AMBER parm96, AMBER parm99, CHARMM27, OPLS-AA and OPLS-AA/L, and demonstrated that our modifications of the torsion-energy terms resulted in the expected changes of secondary-structure-forming tendencies by performing folding simulations of α-helical and β-hairpin peptides.     

Signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation

Cofilin and actin-depolymerizing factor (ADF) are actin-binding proteins that play an essential role in regulating actin filament dynamics and reorganization by stimulating the severance and depolymerization of actin filaments. Cofilin/ADF are inactivated by phosphorylation at the serine residue at position 3 by LIM-kinases (LIMKs) and testicular protein kinases (TESKs) and are reactivated by dephosphorylation by the slingshot (SSH) family of protein phosphatases and chronophin. This review describes recent advances in our understanding of the signaling mechanisms regulating LIMKs and SSHs and the functional roles of cofilin phospho-regulation in cell migration, tumor invasion, mitosis, neuronal development, and synaptic plasticity. Accumulating evidence demonstrates that the phospho-regulation of cofilin/ADF is a key convergence point of cell signaling networks that link extracellular stimuli to actin cytoskeletal dynamics and that spatiotemporal control of cofilin/ADF activity by LIMKs and SSHs plays a crucial role in a diverse array of cellular and physiological processes. Perturbations in the normal control of cofilin/ADF activity underlie many pathological conditions, including cancer metastasis and neurological and cardiovascular disorders.     

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl₂. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.     

Age-related effects of major and trace element concentrations in rat liver and their mutual relationships

The concentrations of 22 major and trace elements in livers from rats aging from 5 to 113 weeks old were determined. The rats investigated were the same rats previously reported with respect to 29 elements in bones (femur) and 26 elements in kidneys. The samples were decomposed with high-purity nitric acid and hydrogen peroxide. Seven elements (Na, Mg, P, K, Ca, Fe and Zn) were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), and 15 elements (Mn, Co, Cu, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Cs, Ba, Pb and Bi) were determined by inductively coupled plasma mass spectrometry (ICP-MS). Analysis of variance (ANOVA) for age variations indicated that the concentrations of many elements, such as Mg, P, K, Mn, Fe, Cu, Zn, Sr, Mo and Cd, were almost constant across the ages of the rats with the exception of 5 weeks old (p > 0.05). Arsenic, Pb and Bi showed significant increasing trends, while Na and Co showed decreasing trends (p < 0.01). Selenium showed a decreasing trend except at the initial stage of 5–9 weeks old. Calcium, Rb, Sn, Sb, Cs and Ba showed significant age-related variations, but their patterns were not monotonic. The liver clearly contrasts with the kidneys, in which many elements showed significant age-related variations with increasing trends. The concentration ranges of Mg, P, K, Mn, Cu, Zn, and Mo were controlled within 15% across all ages of rats. The homeostasis of the aforementioned elements may be well established in the liver. The toxic elements, such as Cd, Pb and Bi, showed a narrow concentration range among age-matched rats.     

Conversion of d-Glucose into d-Oxybiotin†

2,5-Anhydro-3-azido-3-deoxy-D-xylose dimethyl acetal (XI), the key intermediate for the stereospecific synthesis of d-oxybiotin, was prepared by methanolysis of 3-azido-3-deoxy-1,2-O-isopropylidene-5-O-p-tolylsulfonyl-α-d-xylofuranose (VIIIa) or of 3-azido-3-deoxy-l,2-O-cyclohexylidene-5-0-p-tolylsulfonyl-α-d-xylofuranose (VIIIb).