The Bi-Functional Organization of Human Basement Membranes

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.     

Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper-immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3x10(8) Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen-specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction-neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 microg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17.     

Living cell positioning on the surface of gold film for SPR analysis

Living cell reactions are detected as changes of the angle of resonance (AR) for surface plasmon resonance (SPR). Since SPR reflects the events in the field of evanescence, cells need to be fixed on the sensor chip. In this study, we developed methods to fix living cells on a gold surface and to recover adherent cells from the culture dish, preserving their functions to be analyzed by SPR. Human basophils and B cells were fixed to the sensor chip by a biocompatible anchor for cell membranes (alpha-succinimidyloxysuccinyl omega-oleyloxy polyoxyethylene), aminoalkanethiol (cyteamine, 8-amino octanethiol) or an amino-reactive cross-linker (dithiobis [succinimidylpropionate]). They showed an increase of AR in response to various stimuli. RBL-2H3 cells, which firmly adhered to the culture dish, were cultured/recovered with HydroCell/simple pipetting, with RepCell/pipetting at 4 degrees C, or on normal plastic culture dishes with trypsinization or by scraping at 4 degrees C, respectively. The exocytosis of RBL-2H3 cells was largely impaired by scraping, but only slightly by the treatment with pipetting on HydroCell, on RepCell, or with trypsin. The membrane ruffling of the cells prepared by the last three treatments induced by antigens appeared the same. However, the change of AR with cells prepared by trypsin and those by scraping at 4 degrees C were lower than those by HydroCell or RepCell, suggesting that trypsin may harm molecules involved in cellular reactions. Thus, the methods of cell fixation and removal with HydroCell or RepCell should enable us to analyze various reactions in either adherent or non-adherent cells by SPR.     

Characterization of the Testis-specific Proteasome Subunit α4s in Mammals

The 26 S proteasome is responsible for regulated proteolysis in eukaryotic cells. It is composed of one 20 S core particle (CP) flanked by one or two 19 S regulatory particles. The CP is composed of seven different α-type subunits (α1-α7) and seven different β-type subunits, three of which are catalytic. Vertebrates encode four additional catalytic β subunits that are expressed predominantly in immune tissues and produce distinct subtypes of CPs particularly well suited for the acquired immune system. In contrast, the diversity of α subunits remains poorly understood. Recently, another α subunit, referred to as α4s, was reported. However, little is known about α4s. Here we provide a detailed characterization of α4s and the α4s-containing CP. α4s is exclusively expressed in germ cells that enter the meiotic prophase and is incorporated into the CP in place of α4. A comparison of structural models revealed that the differences in the primary sequences between α4 and α4s are located on the outer surface of the CP, suggesting that α4s interacts with specific molecules via these unique regions. α4s-containing CPs account for the majority of the CPs in mouse sperm. The catalytic β subunits in the α4s-containing CP are β1, β2, and β5, and immunosubunits are not included in the α4s-containing CP. α4s-containing CPs have a set of peptidase activities almost identical to those of α4-containing CPs. Our results provide a basis for understanding the role of α4s and male germ cell-specific proteasomes in mammals.     

Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.     

Effective molecular detection of Diaphorina citri Kuwayama (Hemiptera: Liviidae) in bulk insect samples from sticky traps

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the principal vector of citrus greening (huanglongbing) disease. Invasion of new areas by the vector increases the risk of further spread of the disease and has economic impacts on the global citrus industry. Effective implementation of vector surveys is essential to contain disease outbreaks. This is especially true in countries such as Japan, where most of the major citrus‐producing areas are free from citrus greening. Recently, vector surveys have been routinely conducted to maintain ‘disease‐free’ and ‘disease‐ and vector‐free’ areas in Japan, and improvement of methods that can detect D. citri in native insect populations is imperative. Here, we developed a method of using conventional and real‐time PCR to detect D. citri among bulk insects captured in sticky traps without the need for preliminary differentiation steps based on morphology. DNA fragments of D. citri were specifically detected by both conventional and real‐time PCR in a mixture of a 10^?3 dilution (ca. 0.008–0.009 ng/μl) of D. citriDNA and 100 ng/μl of bulk insect DNA, indicating that small body parts such as pieces of leg or parts of wings of D. citri were detectable in the bulk insect samples. No misleading amplification of fragments from the other psyllid species and citrus pests we used occurred under our PCR conditions. Our results suggest that the technique is applicable to extensive surveys of D. citri in early warning programmes.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Repetitive transient depolarizations of the inner mitochondrial membrane induced by proton pumping

Single mitochondria show the spontaneous fluctuations of DeltaPsim. In this study, to examine the mechanism of the fluctuations, we observed DeltaPsim in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K(+) from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca(2+), CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of DeltaPsim are discussed in relation to the matrix pH and the permeability transitions.     

A new Asphondylia species (Diptera: Cecidomyiidae) and a eulophid wasp (Hymenoptera) inducing similar galls on leaf buds of Schoepfia jasminodora (Schoepfiaceae), with reference to their ecological traits and a description of the new gall midge

Different gall inducers belonging to distinct insect orders are rarely known to induce similarly shaped galls on the same host plant organs. We report that Asphondylia tojoi Elsayed & Tokuda sp. nov. (Diptera: Cecidomyiidae) and Ceratoneura sp. (Hymenoptera: Eulophidae) induce galls on leaf buds of Schoepfia jasminodora Sieb. et Zucc. (Schoepfiaceae). We describe the gall midge species as new to science and report a phylogenetic analysis for known Japanese Asphondylia species. We also describe life histories of the two species, based on monthly surveys during 2015–2017: although both species are multivoltine, A. tojoi overwinters as first instars in galls, whereas Ceratoneura sp. possibly does so as adults outside the galls. In addition, the internal structure of galls differed between the two species. Galls containing A. tojoi consist of a single chamber with inner walls clearly covered with whitish fungal mycelia after the gall midges develop into second instars. Those containing the Ceratoneura sp. have multiple chambers with hard black inner walls. Although some eulophids are known to be inquilines of galls induced by Asphondylia species, we consider that the Ceratoneura sp. is probably a true gall inducer because of the different gall structure and absence of fungal mycelia in their galls. This is the first report detailing the annual life history of a Ceratoneura species. Asphondylia tojoi represents the first example of monophagous Asphondylia species with a multivoltine life history on a deciduous tree.     

Hydrolysable tannins and proanthocyanidins from green tea

Two hydrolysable tannins were isolated from green tea, and their structures were characterized by chemical and spectral means as 1,4,6-tri-O -galloyl-β-d-glucose and 1-O-galloyl-4,6-(?)-hexahydroxydiphenoyl-β-d-glucose. In addition, a new proanthocyanidin gallate was isolated, together with the known procyanidins B-2, B-4 and C-1. The structure of the proanthocyanidin was established as epigallocatechin-(4β → 8)-3-O-galloylepicatechin.