beta4 integrin and epidermal growth factor coordinately regulate electric field-mediated directional migration via Rac1

Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (beta)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), beta4 integrin-null human keratinocytes (beta4-), or those in which beta4 integrin was reexpressed (beta4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of beta4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding-defective beta4 (beta4+AD) or beta4 with a truncated cytoplasmic tail (beta4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the beta4 integrin ligand-binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus.     

Demonstration of osmotically dependent promotion of aerenchyma formation at different levels in the primary roots of rice using a 'sandwich' method and X-ray computed tomography

Background and Aims

The effect of environmental factors on the regulation of aerenchyma formation in rice roots has been discussed for a long time, because aerenchyma is constitutively formed under aerated conditions. To elucidate this problem, a unique method has been developed that enables sensitive detection of differences in the development of aerenchyma under two different environmental conditions. The method is tested to determine whether aerenchyma development in rice roots is affected by osmotic stress.

Methods

To examine aerenchyma formation both with and without mannitol treatment in the same root, germinating rice (Oryza sativa) caryopses were sandwiched between two agar slabs, one of which contained 270 mm of mannitol. The roots were grown touching both slabs and were thereby exposed unilaterally to osmotic stress. As a non-invasive approach, refraction contrast X-ray computed tomography (CT) using a third-generation synchrotron facility, SPring-8 (Super photon ring 8 GeV, Japan Synchrotron Radiation Research Institute), was used to visualize the three-dimensional (3-D) intact structure of aerenchyma and its formation in situ in rice roots. The effects of unilateral mannitol treatment on the development of aerenchyma were quantitatively examined using conventional light microscopy.

Key Results

Structural continuity of aerenchyma was clearly visualized in 3-D in the primary root of rice and in situ using X-ray CT. Light microscopy and X-ray CT showed that the development of aerenchyma was promoted on the mannitol-treated side of the root. Detailed light microscopic analysis of cross-sections cut along the root axis from the tip to the basal region demonstrated that aerenchyma developed significantly closer to the root tip on the mannitol-treated side of the root.

Conclusions

Continuity of the aerenchyma along the rice root axis was morphologically demonstrated using X-ray CT. By using this ‘sandwich’ method it was shown that mannitol promoted aerenchyma formation in the primary roots of rice.     

Protein recognition of hetero-/homoleptic ruthenium(II) tris(bipyridine)s for α-chymotrypsin and cytochrome c

We examined the relationship between the structures of hetero-/homoleptic ruthenium(II) tris(bipyridine) metal complexes (Ru(II)(bpy)(3)) and their binding properties for α-chymotrypsin (ChT) and cytochrome c (cyt c). Heteroleptic compound 1a binds to both ChT and cyt c in 1:1 ratio, whereas homoleptic 2 forms 1:2 protein complex with ChT but 1:1 complex with cyt c. These results suggest that the structure of the recognition cavity in Ru(II)(bpy)(3) can be designed for shape complementarity to the targeted proteins. In addition, Ru(II)(bpy)(3) complexes were found to be potent inhibitors of cyt c reduction and to permeate A549 cells.     

Effectiveness of Ureteroscopy-Assisted Retrograde Nephrostomy (UARN) for Percutaneous Nephrolithotomy (PCNL)

Objective

To determine the impact of ureteroscopy-assisted retrograde nephrostomy (UARN) during percutaneous nephrolithotomy (PCNL).

Materials and Methods

From April 2009 to September 2011, a total of 50 patients underwent PCNL for large renal stones (stone burden >2 cm). We performed UARN in the Galdakao-modified Valdivia position for 27 patients (UARN PCNL) and ultrasonography-assisted percutaneous nephrostomy in the prone position for 23 patients (prone PCNL).

Results

UARN PCNL significantly improved the stone-free rate (81.5% vs 52.2%) and the rate of residual stones (<4 mm, 92.6% vs 65.2%, P<0.05). The median length of the operation was significantly shorter for UARN PCNL, at 160 min, compared to 299 min for prone PCNL (P<0.001). There was one intraoperative complication in prone PCNL, namely a hemorrhage that resulted in stopping the initial treatment, but it was cured conservatively. The postoperative complications included a high grade fever that persisted for three days in two UARN PCNL patients (7.4%) and six prone PCNL patients (26.1%). The Clavien grading scores showed significantly lower postoperative complications for UARN PCNL compared to prone PCNL.

Conclusion

UARN is associated with a higher stone-free rate, shorter operation time, and fewer complications during PCNL than prone PCNL.     

Differences in Salivary Alpha-Amylase and Cortisol Responsiveness following Exposure to Electrical Stimulation versus the Trier Social Stress Tests

Background

Cortisol is an essential hormone in the regulation of the stress response along the HPA axis, and salivary cortisol has been used as a measure of free circulating cortisol levels. Recently, salivary alpha-amylase (sAA) has also emerged as a novel biomarker for psychosocial stress responsiveness within the sympathetic adrenomedullary (SAM) system.

Principal Findings

We measured sAA and salivary cortisol in healthy volunteers after exposure to the Trier Social Stress Test (TSST) and electric stimulation stress. One hundred forty-nine healthy volunteers participated in this study. All subjects were exposed to both the TSST and electric stimulation stress on separate days. We measured sAA and salivary cortisol levels three times immediately before, immediately after, and 20 min after the stress challenge. The State (STAI-S) and Trait (STAI-T) versions of the Spielberger Anxiety Inventory test and the Profile of Mood State (POMS) tests were administered to participants before the electrical stimulation and TSST protocols. We also measured HF, LF and LF/HF Heart Rate Variability ratio immediately after electrical stimulation and TSST exposure. Following TSST exposure or electrical stimulation, sAA levels displayed a rapid increase and recovery, returning to baseline levels 20 min after the stress challenge. Salivary cortisol responses showed a delayed increase, which remained significantly elevated from baseline levels 20 min after the stress challenge. Analyses revealed no differences between men and women with regard to their sAA response to the challenges (TSST or electric stimulations), while we found significantly higher salivary cortisol responses to the TSST in females. We also found that younger subjects tended to display higher sAA activity. Salivary cortisol levels were significantly correlated with the strength of the applied electrical stimulation.

Conclusions

These preliminary results suggest that the HPA axis (but not the SAM system) may show differential response patterns to distinct kinds of stressors.     

Prior immunization with severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein causes severe pneumonia in mice infected with SARS-CoV

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7-8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-gamma, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-beta), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.     

CRTH2 plays an essential role in the pathophysiology of Cry j 1-induced pollinosis in mice

PGD(2) is the major prostanoid produced during the acute phase of allergic reactions. Two PGD(2) receptors have been isolated, DP and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), but whether they participate in the pathophysiology of allergic diseases remains unclear. We investigated the role of CRTH2 in the initiation of allergic rhinitis in mice. First, we developed a novel murine model of pollinosis, a type of seasonal allergic rhinitis. Additionally, pathophysiological differences in the pollinosis were compared between wild-type and CRTH2 gene-deficient mice. An effect of treatment with ramatroban, a CRTH2/T-prostanoid receptor dual antagonist, was also determined. Repeated intranasal sensitization with Cry j 1, the major allergen of Cryptomeria japonica pollen, in the absence of adjuvants significantly exacerbated nasal hyperresponsive symptoms, Cry j 1-specific IgE and IgG1 production, nasal eosinophilia, and Cry j 1-induced in vitro production of IL-4 and IL-5 by submandibular lymph node cells. Additionally, CRTH2 mRNA in nasal mucosa was significantly elevated in Cry j 1-sensitized mice. Following repeated intranasal sensitization with Cry j 1, CRTH2 gene-deficient mice had significantly weaker Cry j 1-specific IgE/IgG1 production, nasal eosinophilia, and IL-4 production by submandibular lymph node cells than did wild-type mice. Similar results were found in mice treated with ramatroban. These results suggest that the PGD(2)-CRTH2 interaction is elevated following sensitization and plays a proinflammatory role in the pathophysiology of allergic rhinitis, especially pollinosis in mice.     

Localization of laminin α3B chain in vascular and epithelial basement membranes of normal human tissues and its down-regulation in skin cancers

The basement membrane (BM) proteins laminins, which consist of alpha, beta and gamma chains, play critical roles in the maintenance of tissue structures. One of laminin alpha chains, alpha3 has two isoforms, the truncated form alpha3A and the full-sized form alpha3B. In contrast to alpha3A laminins, little is known about alpha3B laminins. To show the histological distribution of the laminin alpha3B chain, we prepared alpha3B-specific monoclonal antibodies. Immunohistochemical analysis showed that the alpha3B chain was colocalized with the alpha3A, beta3 and gamma2 chains in the epithelial BMs of the skin, esophagus, breast and lung, suggesting the presence of laminin-3B32 (laminin-5B) and laminin-3A32 (laminin-5A). In the lung alveoli, laminin-3B32 was dominant over laminin-3A32, but vice versa in other epithelial BMs. In contrast, the BMs of blood vessels including capillaries were strongly positive for alpha3B, but almost or completely negative for alpha3A, beta3 and gamma2. alpha3B was colocalized with beta1 and gamma1 in these BMs. The alpha3B chain was scarcely detected in the vessels of malignant skin cancers, though the gamma2 and beta3 chains were highly expressed in the cancer cells. These results strongly suggest that the laminin alpha3B chain is widely expressed in vascular BMs of normal tissues, probably as laminin-3B11/3B21 (laminin-6B/7B).     

Airborne hyperspectral imaging for estimating acorn yield based on the PLS B-matrix calibration technique

Alternate bearing of acorn is a well-marked yield variability phenomenon in forest production. In Japan, this phenomenon is also related to wildlife management (e.g. of animals such as wild pigs, that rely on acorn as their major feed source). Effective management of animals dependent on acorn will require accurate estimation of acorn yield at an early stage. In this paper, we proposed a way to estimate acorn yield from the canopy reflectance values of individual trees. Using an Airborne Imaging Spectrometer for Application (AISA) Eagle System, hyperspectral images in 72 visible and near-infrared wavelengths (407–898 nm) were acquired over an acorn forest in Japan 10 times over three consecutive years (2003–2005) during the early acorn growing season. The canopy spectral reflectance values for individual trees at each wavelength were extracted from the images, and important wavelengths were determined as estimating factors by the B-matrix technique based on partial least squares (PLS) analysis. Yield-estimating models were then developed by multiple linear regression (MLR). Three models obtained from images acquired on June 27 in 2003, July 13 in 2004 and June 21 in 2005 estimated acorn yield well in comparison with ground truth, indicating that the procedure has considerable potential. The study also demonstrated the B-matrix technique based on PLS analysis to be reliable and efficient in identifying important wavelengths for determining suitable estimating factors that best contribute to the estimation model.