C-terminus of PIEZO1 governs Ca2+ influx and intracellular ERK1/2 signaling pathway in mechanotransduction.

Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca2+ influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca2+ influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs.

Aquaporin 3 inhibition suppresses the mitochondrial respiration rate and viability of multiple myeloma cells.

Aquaporin 3 (AQP3) is a member of the aquaporin water channel family expressed by numerous cell types, including some cancer cells. Accumulating evidence suggests that AQP3 inhibition may impede cancer progression, but drugs targeting AQP3 are still in the early pre-clinical stage of development. Here, we examined the effect of AQP3 inhibition on multiple myeloma (MM), an incurable plasma cell malignancy. Four MM cell lines were cultured in the presence of an anti-AQP3 monoclonal antibody (mAb), the AQP3 inhibitor DFP00173, or corresponding controls, and the effects on cell viability, proliferation, apoptosis, and mitochondrial respiration capacity were compared. Both anti-AQP3 mAb and DFP00173 reduced cell growth, mitochondrial respiration rate, and electron transport chain complex I activity. Both agents also potentiated the antiproliferative efficacy of the anticancer drug venetoclax. Administration of the anti-AQP3 mAb to immunodeficient mice inoculated with RPMI8226 or KMS-11 MM cells significantly suppressed tumor growth. These data provide evidence that AQP3 blockade can suppress MM cell growth in vitro and tumor growth in mice. Thus, AQP3 inhibition may be an effective therapeutic strategy for MM.

Assessing Gluten-Free Soy Bread Quality and Amino Acid Content.

The nutritional and palatability relevance of bread prepared with soy flour was examined. There are a few effective nutritional measures that combine palatability, convenience, and functionality in the suppression of muscle loss (contributing to the improvement and prevention of sarcopenia). Therefore, in the present study, we attempted to produce bread using soybeans, which are rich in amino acids involved in the synthesis and degradation of skeletal muscle proteins. Rice flour was also used to avoid gluten intolerance. The bread was baked in an automatic bread maker, and the rheological properties of its breadcrumbs were determined using a creep meter. We found that a 70 g slice of soy bread satisfied approximately one-fifth of the daily nutritional requirement for leucine. Although soy decreased the specific volume of bread by preventing starch construction, the use of preprocessed rice flour recovered the volume, and corn starch improved the taste. We propose that the addition of soy bread to the daily diet may be an effective protein source.

Anti-tumor effect of aquaporin 3 monoclonal antibody on syngeneic mouse tumor model.

Aquaporin-3 (AQP3), a water channel protein, has been found to be involved in cancer progression via water and small molecule transport function. However, drug development targeting AQP3 has not yet begun. Here, we showed that a recently established anti-AQP3 monoclonal antibody (mAb) suppresses tumor growth in allograft mouse colorectal tumor models produced using CT26 or MC38 cancer cells. Administration of the anti-AQP3 mAb to BALB/c mice with transplanted CT26 cells increased the M1/M2 ratio of tumor-associated macrophages (TAM) and improved the mitochondrial function of T cells in the tumor microenvironment (TME). Administration of anti-AQP3 mAb also restored the TAM-induced decrease in T cell proliferation. Macrophage depletion in wild-type mice counteracted the antitumor effect of anti-AQP3 mAb in the mouse tumor model, suggesting that one of the primary targets of anti-AQP3 mAb is macrophages. In in vitro studies using mice bone marrow monocytes and human monocyte THP-1 cells, anti-AQP3 mAb attenuated carcinoma cell-mediated polarization of monocytes into M2-like TAMs. These data suggest that anti-AQP3 mAb suppresses tumor growth by attenuating immunosuppressive M2-like TAMs, which in turn maintains the antitumor function of T cells in the TME. Thus, the anti-AQP3 mAb is a potential cancer therapy that functions by targeting TAMs.

Effect of topical application of lipopolysaccharide on contact hypersensitivity.

Lipopolysaccharide (LPS) is the principal component of the outer membrane of gram-negative bacteria. The prior oral administration of LPS attenuates inflammatory responses, such as intestinal injury and atopic dermatitis, in mouse models; however, the underlying mechanism remains unclear. Here, we examined the effect of topical LPS application on allergic contact dermatitis and its mechanism of action using a murine contact hypersensitivity (CHS) model. Prolonged LPS application to the skin significantly suppressed 2,4-dinitrofluorobenzene (DNFB)-induced CHS. LPS application to the skin also reduced the phagocytosis of fluorescein isothiocyanate (FITC)-dextran by Langerhans and dendritic cells. Cutaneous cell migration into the skin-draining lymph nodes (LNs) induced by FITC painting was reduced by LPS application. During the CHS response, DNFB application induced T-cell proliferation and inflammatory cytokine production in skin-draining LNs, whereas prolonged LPS application inhibited DNFB-induced T-cell growth and interferon gamma production, indicating suppression of DNFB-induced sensitization. These results suggest that prolonged LPS application suppressed DNFB-induced sensitization and subsequently CHS response. Our findings imply that topical application of LPS may prevent allergic dermatitis such as CHS.

Inhibition of aquaporin-3 in macrophages by a monoclonal antibody as potential therapy for liver injury.

Aquaporin 3 (AQP3) is a transporter of water, glycerol and hydrogen peroxide (H2O2) that is expressed in various epithelial cells and in macrophages. Here, we developed an anti-AQP3 monoclonal antibody (mAb) that inhibited AQP3-facilitated H2O2 and glycerol transport, and prevented liver injury in experimental animal models. Using AQP3 knockout mice in a model of liver injury and fibrosis produced by CCl4, we obtained evidence for involvement of AQP3 expression in nuclear factor-κB (NF-κB) cell signaling, hepatic oxidative stress and inflammation in macrophages during liver injury. The activated macrophages caused stellate cell activation, leading to liver injury, by a mechanism involving AQP3-mediated H2O2 transport. Administration of an anti-AQP3 mAb, which targeted an extracellular epitope on AQP3, prevented liver injury by inhibition of AQP3-mediated H2O2 transport and macrophage activation. These findings implicate the involvement of macrophage AQP3 in liver injury, and provide evidence for mAb inhibition of AQP3-mediated H2O2 transport as therapy for macrophage-dependent liver injury.

Assessment of right ventricular function by isovolumic acceleration of pulmonary and tricuspid annulus in surgically repaired tetralogy of Fallot.

Assessment of right ventricular (RV) function is quite important in patients with surgically corrected tetralogy of Fallot (TOF). However, quantitative assessment of RV function remains challenging, mainly because of the complex RV geometry. This prospective study investigated isovolumic acceleration (IVA), a parameter of myocardial systolic function not influenced by either preload or afterload, using tissue Doppler imaging. We evaluated IVA measured on pulmonary annulus (PA-IVA) and tricuspid annulus (TA-IVA), because we considered that PA-IVA and TA-IVA correspond with systolic function of the RV outflow tract (RVOT) and RV basal function, respectively. Thirty-nine patients with surgically repaired TOF (TOF group) and 40 age-matched healthy children (control group) were enrolled in this study. No significant difference was seen between TA-IVA (2.5 ±â€…0.8 m/s2) and PA-IVA (2.4 ±â€…0.8 m/s2) in the control group. In the TOF group, PA-IVA (1.0 ±â€…0.5 m/s2) was significantly lower than TA-IVA (1.3 ±â€…0.6 m/s2, p < 0.05). Both TA-IVA and PA-IVA were significantly lower in the TOF group than in the control group (p < 0.05 each). We concluded that PA-IVA offers a useful index to assess RVOT function in TOF patients. J. Med. Invest. 67 : 145-150, February, 2020.

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes.

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined. Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity. In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin. Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.

Effects of acute kidney injury after liver resection on long-term outcomes.

BACKGROUND: To investigate the effects of acute kidney injury (AKI) after liver resection on the long-term outcome, including mortality and renal dysfunction after hospital discharge. METHODS: We conducted a historical cohort study of patients who underwent liver resection for hepatocellular carcinoma with sevoflurane anesthesia between January 2004 and October 2011, survived the hospital stay, and were followed for at least 3 years or died within 3 years after hospital discharge. AKI was diagnosed based on the Acute Kidney Injury Network classification within 72 hours postoperatively. In addition to the data obtained during hospitalization, serum creatinine concentration data were collected and the glomerular filtration rate (GFR) was estimated after hospital discharge. RESULTS: AKI patients (63%, P = 0.002) were more likely to reach the threshold of an estimated GFR (eGFR) of 45 ml/min/1.73 m2 within 3 years than non-AKI patients (31%) although there was no significant difference in mortality (33% vs. 29%). Cox proportional hazard regression analysis showed that postoperative AKI was significantly associated with the composite outcome of mortality or an eGFR of 45 ml/min/1.73 m2 (95% CI of hazard ratio, 1.05-2.96, P = 0.033), but not with mortality (P = 0.699), the composite outcome of mortality or an eGFR of 60 ml/min/1.73 m2 (P =0.347). CONCLUSIONS: After liver resection, AKI patients may be at higher risk of mortality or moderate renal dysfunction within 3 years. These findings suggest that even after discharge from the hospital, patients who suffered AKI after liver resection may need to be followed-up regarding renal function in the long term.

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed.

KEY MESSAGE: Prolamin-GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin-GFP fusion proteins were gradually digested from outside by pepsin digestion. In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin-GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin-GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin-GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin-GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin-GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

Unusual O-alkylation of 2-hydroxy-1,4-naphthoquinone utilizing alkoxymethyl chlorides.

A new type of O-alkylation of 2-hydroxy-1,4-naphthoquinone with alkoxymethyl chlorides is described. The reaction course can be controlled by the choice of base and yields O-alkylated or O-alkoxymethylated products in high yield with high selectivity.

Expression and localization of aquaporin-4 in sensory ganglia.

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4(-/-) mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4(-/-) mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system.

Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols.

Enhanced skin delivery of quercetin by microemulsion.

OBJECTIVES: For topical application of quercetin it is necessary to improve the low efficiency of its intradermal delivery as well as its low solubility in aqueous and organic vesicles. The aim of this study was to determine the usefulness of a microemulsion for that purpose. METHODS: A microemulsion consisting of isopropyl myristate, 150 mM NaCl solution, Tween 80 and ethanol was prepared. The skin delivery of quercetin by microemulsion using excised guinea-pig and Yucatan micropig skin in Franz diffusion cells was examined. Lipid peroxidation in skin was also tested using iron(II) and citrate. KEY FINDINGS: Using a w/o microemulsion as a vehicle, intradermal delivery of quercetin was significantly increased, as was its solubility. Quercetin penetrated deep into the skin, but no transfer was observed into the receptor compartment. It was confirmed that quercetin retained in the skin dose-dependently inhibited lipid peroxidation. CONCLUSIONS: The findings indicate the potential use of microemulsions for the skin delivery of quercetin, where it exerts antioxidative effects.

The stability and aggregation properties of the GTPase domain from human SEPT4.

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTP. At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease.

An intermediate structure in the thermal unfolding of the GTPase domain of human septin 4 (SEPT4/Bradeion-beta) forms amyloid-like filaments in vitro.

SEPT4 is a member of the mammalian septin family of GTPases. Mammalian septins are conserved proteins which form heteropolymers in vivo and which are implicated in a variety of cellular functions such as cytokinesis, exocytosis, and vesicle trafficking. However, their structural properties and modes of action are largely unknown. There is a limited, but as yet inconclusive, amount of experimental data suggesting that SEPT4 may accumulate in tau-based filamentous deposits and cytoplasmic inclusions in Alzheimer's and Parkinson's disease, respectively. Here we report an intermediate structure of the GTPase domain of human SEPT4 (SEPT4-G) during unfolding transitions induced by temperature. This partially unfolded intermediate, which is rich in beta-sheet and free of bound nucleotide, was plagued by irreversible aggregation. The aggregates have the ability to bind specific dyes such as Congo red and thioflavin-T, suggesting they are amyloid in nature. Under electron microscopy, fibers of variable diameter extending for several micrometers in length can be visualized. This is the first report of amyloid formation by a septin or domain thereof, and the capacity of SEPT4-G to form such fibrillar aggregates may shed some light on the current discussion concerning the formation of homo- and heteropolymers of septins in vitro.

Dissection of a human septin: definition and characterization of distinct domains within human SEPT4.

The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melanoma, as well as being involved in neurodegenerative disorders. However, many of the details of the modes of action of septins in general remain unclear, and little is known of their detailed molecular architecture. Here, we define explicitly and characterize the domains of human SEPT4. Regions corresponding to the N-terminal, GTPase, and C-terminal domains as well as the latter two together were successfully expressed in Escherichia coli in soluble form and purified by affinity and size-exclusion chromatographies. The purified domains were analyzed by circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering, and small-angle X-ray scattering, as well as with bioinformatics tools. Of the three major domains that comprise SEPT4, the N-terminal domain contains little regular secondary structure and may be intrinsically unstructured. The central GTPase domain is a mixed alpha/beta structure, probably based on an open beta sheet. As defined here, it is catalytically active and forms stable homodimers in vitro. The C-terminal domain also forms homodimers and can be divided into two regions, the second of which is alpha-helical and consistent with a coiled-coil structure. These studies should provide a useful basis for future biophysical studies of SEPT4, including the structural basis for their involvement in diseases such as cancer and neurodegenerative disorders.

In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis.

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.

Structure of the cytosolic Cu,Zn superoxide dismutase from Schistosoma mansoni.

Cu,Zn superoxide dismutase (Cu,Zn SOD) is an essential enzyme for protecting cells from the toxic effects of reactive oxygen species. In humans, two distinct Cu,Zn SOD genes are located on chromosomes 4 and 21 and mutations in the latter have been associated with familial amyotrophic lateral sclerosis. Similarly, schistosomes (trematode parasites responsible for the chronically debilitating disease schistosomiasis) also produce two distinct Cu,Zn SODs, in this case one cytosolic and one bearing a signal peptide. The crystal structure of the cytosolic form of the enzyme from the human trematode Schistosoma mansoni (SmCtSOD) was solved and refined to a resolution of 2.2 A (space group P2(1)2(1)2(1), R = 17.6% and R(free) = 24.1%) and 1.55 A (space group P2(1), R = 15.7% and R(free) = 17.1%). This is the first report of a crystal structure of a Cu,Zn superoxide dismutase derived from a human parasite. Alternate positions for the catalytic copper and its water ligand were refined for the 1.55 A SmCtSOD model, but the most interesting structural differences between SmCtSOD and the human homologue reside in the loops used for electrostatic guidance of the substrate to the enzyme active site.