Cardiovascular complications such as hypertension are a continuous concern in patients with autosomal dominant polycystic kidney disease (ADPKD). The PKD2 encoding for polycystin-2 is mutated in ≈15% of ADPKD patients. Here, we show that polycystin-2 is localized to the cilia of mouse and human vascular endothelial cells. We demonstrate that the normal expression level and localization of polycystin-2 to cilia is required for the endothelial cilia to sense fluid shear stress through a complex biochemical cascade, involving calcium, calmodulin, Akt/PKB, and protein kinase C. In response to fluid shear stress, mouse endothelial cells with knockdown or knockout of Pkd2 lose the ability to generate nitric oxide (NO). Consistent with mouse data, endothelial cells generated from ADPKD patients do not show polycystin-2 in the cilia and are unable to sense fluid flow. In the isolated artery, we further show that ciliary polycystin-2 responds specifically to shear stress and not to mechanical stretch, a pressurized biomechanical force that involves purinergic receptor activation. We propose a new role for polycystin-2 in transmitting extracellular shear stress to intracellular NO biosynthesis. Thus, aberrant expression or localization of polycystin-2 to cilia could promote high blood pressure because of inability to synthesize NO in response to an increase in shear stress (blood flow).

We previously showed that blind rats whose vision was restored by gene transfer of Chlamydomonas channelrhodopsin-2 (ChR2) could only detect wavelengths less than 540 nm because of the action spectrum of the transgene product. Volvox-derived channelrhodopsin-1, VChR1, has a broader spectrum than ChR2. However, the VChR1 protein was mainly localized in the cytoplasm and showed weak ion channel properties when the VChR1 gene was transfected into HEK293 cells. We generated modified Volvox channelrhodopsin-1 (mVChR1), which is a chimera of Volvox channelrhodopsin-1 and Chlamydomonas channelrhodopsin-1 and demonstrated increased plasma membrane integration and dramatic improvement in its channel properties. Under whole-cell patch clamp, mVChR1-expressing cells showed a photo-induced current upon stimulation at 468–640 nm. The evoked currents in mVChR1-expressing cells were ~30 times larger than those in VChR1-expressing cells. Genetically, blind rats expressing mVChR1 via an adeno-associated virus vector regained their visual responses to light with wavelengths between 468 and 640 nm and their recovered visual responses were maintained for a year. Thus, mVChR1 is a candidate gene for gene therapy for restoring vision, and gene delivery of mVChR1 may provide blind patients access to the majority of the visible light spectrum.

During hemodialysis, platelets are activated across a dialyzer. Soluble P-selectin (sP-selectin) is a form of P-selectin which is a glycoprotein relocated from secretory granules to the surfaces of platelets and endothelial cells after these cells have been physiologically activated. To investigate whether sP-selectin is useful as a marker of platelet activation during hemodialysis, we measured the plasma concentration of sP-selectin by enzyme-linked immunosorbent assay in 6 patients hemodialyzed in our institute using regenerated cellulose (RC) membranes and thereafter polysulfone membranes. Concomitantly, we also measured the plasma concentration of platelet factor 4 and β-thromboglobulin which are released from α-granules of activated platelets. During hemodialysis with RC membranes, the β-thromboglobulin level was significantly increased 15 min (p < 0.05) and the sP-selectin level 15 (p < 0.05) and 180 min (p < 0.05) after initiation of dialysis on the venous side as compared with the arterial side of the hemodialyzer. During hemodialysis with polysulfone membranes, no significant variation in plasma β-thromboglobulin and sP-selectin levels was detected. The platelet factor 4 level increased more significantly across a dialyzer 180 min after initiation of dialysis with RC than with polysulfone membranes (p < 0.01). The changes in plasma platelet factor 4 and β-thromboglobulin levels demonstrated that platelets are more activated during hemodialysis with RC than with polysulfone membranes. The changes in plasma sP-selectin levels during hemodialysis with RC confirm that the release of P-selectin purely from activated platelets was detected by enzyme-linked immunosorbent assay. sP-selectin may be a marker of platelet activation during hemodialysis.

Bovine ceruloplasmin underwent fragmentation following non-enzymatic glycosylation. Western blot and ELISA analyses indicated that a polyclonal rabbit antiserum to hexitolysine reacted with bovine ceruloplasmin after incubation with 0.1 M glucose. The same fragmentation was seen upon exposure of the protein to a hydrogen peroxide bolus. Both catalase and EDTA blocked peroxide-dependent fragmentation. Incubation with glucose resulted in a time-dependent release of Cu2+. The released Cu2+ appeared to participate in a Fenton-type reaction to produce hydroxyl radicals, which effected the fragmentation. Hydroxyl radical scavengers such as thiourea, mannitol, methionine, and formate inhibited this cleavage. ESR spectral studies also supported participation of hydroxyl radicals. Inhibition by EDTA of the fragmentation induced by an H2O2 bolus also supports a role for copper in a Fenton-type reaction. Taken together these results suggest that reactive oxygen species, such as superoxide anion and H2O2, were formed by the Maillard reaction which led to hydroxyl radicals being produced by a copper-dependent Fenton-type reaction. Both processes are likely to be involved in the fragmentation of ceruloplasmin.

Chronic obstructive pulmonary disease (COPD) is a major global health problem that is predicted to become the third most common cause of death by 2020.1,2) Nearly 90% of COPD patients are smokers.3) COPD is a condition characterized by airway obstruction due to narrowing of the small airways that is not fully reversible, airway inflammation, and emphysema-related loss of the elastic recoil of the lung. [4][5][6] Pulmonary emphysema is a major component of COPD and is defined by a destructive change of the pulmonary alveoli and enlargement of the respiratory region of the lung distal to the terminal bronchioles. In addition, residual volume (RV) and functional residual capacity (FRC) are characteristically increased in COPD and are related to the degree of hyperinflation of the lungs, especially when there is predominately emphysema. 7,8) Still, the detailed sequence of events leading to the onset of pulmonary emphysema is not yet fully elucidated.On the other hand, it has been suggested that the morbidity and mortality of COPD are associated with acute exacerbations.9,10) Some exacerbations of COPD are undoubtedly related to respiratory bacteria, which infect the lower airways and increase overall airway inflammation. 11,12) In addition, meta-analyses of antibiotic therapy have indicated an advantage for the use of such interventions, which confirms that bacteria are responsible for the exacerbations in COPD. 13,14) In studies using guinea pigs and mice, it has been reported that repeated exposure of the animals to lipopolysaccharide (LPS) results in persistent chronic pulmonary inflammation observed in human subjects with COPD. 15,16) The use of appropriate experimental COPD animal models is indispensable for the development of new therapeutic drugs and for the analysis of the pathogenesis of the disease. By exposing animals to 3 or more months of cigarette smoke, various experimental COPD models have been developed in guinea pigs, rats and mice. [17][18][19][20][21][22][23][24] Although all of these models have emphysematous airspace enlargement, studies on the clinical pathogenesis of COPD would be much more efficient if the emphysematous condition could be experimentally reproduced within a much shorter time period.In order to shorten the period of development of an experimental COPD model, a more efficient delivery system of the constituents of cigarette smoke into the lung is required. To bypass the problem of using inhaled smoke, which is spontaneously exhaled, we attempted to intratracheally instill a cigarette smoke solution (CSS) over several weeks. Since respiratory infections are assumed to be the main risk factors for exacerbation of COPD, we decided to add LPS to the protocol of the multiple CSS administrations. Through the use of these multiple intratracheal administrations of CSS and LPS, we succeeded in inducing airway obstruction, bronchitis and emphysema over a considerably shorter term as compared to that seen for the other models that are currently in use. On the other hand, theophylline is ...

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone HMab-77 (IgG, kappa) was selected. Finally, immunohistochemical analyses with HMab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of HMab-77 to detect HER2 in pathological analyses of breast cancers.

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