Conjugation of both on-axis and off-axis light in Nipkow disk confocal microscope to increase availability of incoherent light source

Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.     

Design and evaluation of a 2-(2,3,6-trifluorophenyl)acetamide derivative as an agonist of the GPR119 receptor

The design and synthesis of a GPR119 agonist bearing a 2-(2,3,6-trifluorophenyl)acetamide group is described. The design capitalized on the conformational restriction found in N-β-fluoroethylamide derivatives to help maintain good levels of potency while driving down both lipophilicity and oxidative metabolism in human liver microsomes. The chemical stability and bioactivation potential are discussed.     

Phosphorylation of a heme-regulated eukaryotic initiation factor 2α kinase enhances the interaction with heat-shock protein 90 and substantially upregulates kinase activity

Heme-regulated eukaryotic initiation factor 2α kinase (HRI) functions under conditions of heme shortage caused by blood diseases such as erythropoietic protoporphyria and β-thalassemia, and retains the heme:globin ratio at 1:1 by sensing the heme concentration in reticulocytes. This HRI function is regulated by various factors including autophosphorylation and protein-protein interactions. A heat-shock protein controls HRI function, however, the molecular mechanism of catalytic regulation of HRI by the heat-shock protein is unclear. In the present study, we examined the interactions of HRI with a heat-shock protein, Hsp90, under various conditions, using a pull-down assay and measuring catalytic activity. It was found that [1] an interaction between Hsp90 and phosphorylated HRI was evident, whereas no interaction was observed between Hsp90 and HRI dephosphorylated by treatment with λ protein phosphatase; [2] Hsp90 enhanced the kinase activity of phosphorylated HRI but not dephosphorylated HRI, but this enhancement was not observed in the presence of heme; and, [3] autophosphorylation of HRI was not influenced by Hsp90. Therefore, we propose that autophosphorylation of HRI is critical for catalytic regulation by Hsp90 under heme-shortage conditions.     

IFN-γ plays an essential role in the pathogenesis of gastric lymphoid follicles formation caused by Helicobacter suis infection

In this study, we aimed to assess the role of helper T cells in the development of gastric lymphoid follicles induced by Helicobacter suis infection. C57BL/6J mice were orally inoculated with H. suis. Six weeks after infection, gastric lymphoid follicles were observed in the gastric mucosa by hematoxylin and eosin staining, and the number of follicles was increased throughout the infection period. An immunohistological examination showed that the lymphoid follicles were composed of B cells, CD4-positive helper T cells, and dendritic cells (DC). It was also revealed that the mRNA expression level of interferon-γ (IFN-γ) in the gastric mucosa was significantly increased at 12 weeks after infection. No gastric lymphoid follicles were detected in IFN-γ-deficient mice that had been infected with H. suis at 12 weeks after infection, although the development of lymphoid follicles in IL-4-deficient mice infected with H. suis was similar to that seen in the wild-type mice. In conclusion, IFN-γ, a Th1 cytokine, is deeply involved in the pathogenesis of gastric lymphoid follicles induced by H. suis infection, and it is suggested that CD4-positive T cells and DC aid in the expansion of gastric lymphoid follicles.     

Genetic Diversity and Geographic Distribution of Genetically Distinct Rabies Viruses in the Philippines

Background

Rabies continues to be a major public health problem in the Philippines, where 200–300 human cases were reported annually between 2001 and 2011. Understanding the phylogeography of rabies viruses is important for establishing a more effective and feasible control strategy.

Methods

We performed a molecular analysis of rabies viruses in the Philippines using rabied animal brain samples. The samples were collected from 11 of 17 regions, which covered three island groups (Luzon, Visayas, and Mindanao). Partial nucleoprotein (N) gene sequencing was performed on 57 samples and complete glycoprotein (G) gene sequencing was performed on 235 samples collected between 2004 and 2010.

Results

The Philippine strains of rabies viruses were included in a distinct phylogenetic cluster, previously named Asian 2b, which appeared to have diverged from the Chinese strain named Asian 2a. The Philippine strains were further divided into three major clades, which were found exclusively in different island groups: clades L, V, and M in Luzon, Visayas, and Mindanao, respectively. Clade L was subdivided into nine subclades (L1–L9) and clade V was subdivided into two subclades (V1 and V2). With a few exceptions, most strains in each subclade were distributed in specific geographic areas. There were also four strains that were divided into two genogroups but were not classified into any of the three major clades, and all four strains were found in the island group of Luzon.

Conclusion

We detected three major clades and two distinct genogroups of rabies viruses in the Philippines. Our data suggest that viruses of each clade and subclade evolved independently in each area without frequent introduction into other areas. An important implication of these data is that geographically targeted dog vaccination using the island group approach may effectively control rabies in the Philippines.     

Suppression of type III effector secretion by polymers

Bacteria secrete effector proteins required for successful infection and expression of toxicity into host cells. The type III secretion apparatus is involved in these processes. Previously, we showed that the viscous polymer polyethylene glycol (PEG) 8000 suppressed effector secretion by Pseudomonas aeruginosa. We thus considered that other viscous polymers might also suppress secretion. We initially showed that PEG200 (formed from the same monomer (ethylene glycol) as PEG8000, but which forms solutions of lower viscosity than the latter compound) did not decrease effector secretion. By contrast, alginate, a high-viscous polymer formed from mannuronic and guluronic acid, unlike PEG8000, effectively inhibited secretion. The effectiveness of PEG8000 and alginate in this regard was closely associated with polymer viscosity, but the nature of viscosity dependence differed between the two polymers. Moreover, not only the natural polymer alginate, but also mucin, which protects against infection, suppressed secretion. We thus confirmed that polymer viscosity contributes to the suppression of effector secretion, but other factors (e.g. electrostatic interaction) may also be involved. Moreover, the results suggest that regulation of bacterial secretion by polymers may occur naturally via the action of components of biofilm or mucin layer.     

RNA-Seq-based DNA marker analysis of the genetics and molecular evolution of Triticeae species

Functional & Integrative Genomics - The release of high-quality chromosome-level genome sequences of members of the Triticeae tribe has greatly facilitated genetic and genomic analyses of...     

Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.     

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.