β-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring β-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better β-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters K_m, V_max and K_cat/K_m were 0.81 mmol, 291.99 μmol/(mg·min), and 387.50 s^-1 mmol^-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.
beta-Glucosidase ; Sodium Chloride ; Temperature ; Glucose ; Ethanol/chemistry* ; Ions ; Hydrogen-Ion Concentration ; Enzyme Stability ; Substrate Specificity

The aim of the present study was to establish a cell model of volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.
Animals ; Anions ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; Ion Transport ; Membrane Proteins ; physiology ; Microscopy, Fluorescence ; Rats ; Rats, Inbred F344 ; Thyroid Gland ; cytology ; Transfection

PURPOSE: The purpose of this study was to evaluate the effectiveness of conventional sandblasted, large-grit, acid-etched (SLA) surface coated with a pH buffering solution based on surface wettability, blood protein adhesion, osteoblast affinity, and platelet adhesion and activation.METHODS: Titanium discs and implants with conventional SLA surface (SA), SLA surface in an aqueous calcium chloride solution (CA), and SLA surface with a pH buffering agent (SOI) were prepared. The wetting velocity was measured by the number of threads wetted by blood over an interval of time. Serum albumin adsorption was tested using the bicinchoninic acid assay and by measuring fluorescence intensity. Osteoblast activity assays (osteoblast adhesion, proliferation, differentiation, mineralization, and migration) were also performed, and platelet adhesion and activation assays were conducted.RESULTS: In both the wetting velocity test and the serum albumin adsorption assay, the SOI surface displayed a significantly higher wetting velocity than the SA surface (P=0.000 and P=0.000, respectively). In the osteoblast adhesion, proliferation, differentiation, and mineralization tests, the mean values for SOI were all higher than those for SA and CA. On the osteoblast migration, platelet adhesion, and activation tests, SOI also showed significantly higher values than SA (P=0.040, P=0.000, and P=0.000, respectively).CONCLUSIONS: SOI exhibited higher hydrophilicity and affinity for proteins, cells, and platelets than SA. Within the limits of this study, it may be concluded that coating an implant with a pH buffering agent can induce the attachment of platelets, proteins, and cells to the implant surface. Further studies should be conducted to directly compare SOI with other conventional surfaces with regard to its safety and effectiveness in clinical settings.
Adsorption ; Blood Platelets ; Calcium Chloride ; Coated Materials, Biocompatible ; Dental Implants ; Fluorescence ; Hydrogen-Ion Concentration ; Hydrophobic and Hydrophilic Interactions ; Immunoassay ; In Vitro Techniques ; Miners ; Osteoblasts ; Serum Albumin ; Surface Properties ; Titanium ; Wettability

PURPOSE: Surface treatment with pH buffering agent has been developed to achieve higher and faster osseointegration. The aim of this study was to evaluate its influence by measuring removal torque and analyzing histological characteristics. MATERIALS AND METHODS: Titanium implants with following surfaces were used in this study: sand-blasted acid-etched (SA) surface (SA group as control I group), SA surface in calcium chloride aqueous solution (CA group as control II group) and SA surface coated with pH buffering agent (pH group as test group). Removal torque test after 2 weeks and bone-to-implant contact and bone area analyses at 2 and 4 weeks were performed. RESULT: The rotational torque values at 2 weeks were significantly higher in pH group (107.5±6.2 Ncm, P < 0.05). The mean values of bone-to-implant contact at 2 and 4 weeks were both higher in pH group (93.0%±6.4% at 2 weeks, 88.6%±5.5% at 4 weeks) than in SA group (49.7%±9.7% at 2 weeks, 47.3%±20.1% at 4 weeks) and CA group (73.7%±12.4% at 2 weeks, 72.5%±10.9% at 4 weeks) with significances (P < 0.05). The means of bone area showed significantly higher numbers in pH group (39.5%±11.3% at 2 weeks, 71.9%±10.9% at 4 weeks, P < 0.05). CONCLUSION: Our findings demonstrated that surface modification with pH buffering agent improved early osseointegration with superior biomechanical property.
Calcium Chloride ; Dental Implants ; Hydrogen-Ion Concentration ; Hydrophobic and Hydrophilic Interactions ; Osseointegration ; Statistics as Topic ; Surface Properties ; Titanium ; Torque

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

BACKGROUND: Wound healing is an interaction of a complex signaling cascade of cellular events, including inflammation, proliferation, and maturation. K+ channels modulate the mitogen-activated protein kinase (MAPK) signaling pathway. Here, we investigated whether K+ channel-activated MAPK signaling directs collagen synthesis and angiogenesis in wound healing. METHODS: The human skin fibroblast HS27 cell line was used to examine cell viability and collagen synthesis after potassium chloride (KCl) treatment by Cell Counting Kit-8 (CCK-8) and western blotting. To investigate whether K+ ion channels function upstream of MAPK signaling, thus affecting collagen synthesis and angiogenesis, we examined alteration of MAPK expression after treatment with KCl (channel inhibitor), NS1619 (channel activator), or kinase inhibitors. To research the effect of KCl on angiogenesis, angiogenesis-related proteins such as thrombospondin 1 (TSP1), anti-angiogenic factor, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), pro-angiogenic factor were assayed by western blot. RESULTS: The viability of HS27 cells was not affected by 25 mM KCl. Collagen synthesis increased dependent on time and concentration of KCl exposure. The phosphorylations of MAPK proteins such as extracellular-signal-regulated kinase (ERK) and p38 increased about 2.5-3 fold in the KCl treatment cells and were inhibited by treatment of NS1619. TSP1 expression increased by 100%, bFGF expression decreased by 40%, and there is no significant differences in the VEGF level by KCl treatment, TSP1 was inhibited by NS1619 or kinase inhibitors. CONCLUSIONS: Our results suggest that KCl may function as a therapeutic agent for wound healing in the skin through MAPK signaling mediated by the K+ ion channel.
Blotting, Western ; Cell Count ; Cell Line ; Cell Survival ; Collagen ; Fibroblast Growth Factor 2 ; Fibroblasts ; Humans ; Inflammation ; Ion Channels ; Mitogen-Activated Protein Kinases* ; Phosphorylation ; Phosphotransferases ; Potassium Channels ; Potassium Chloride ; Protein Kinases ; Skin ; Thrombospondin 1 ; Vascular Endothelial Growth Factor A ; Wound Healing*

BACKGROUND: The purpose of this study was to elucidate the effects of fluid on the acid-base and electrolytes status and renal function after kidney transplantation (KT). METHODS: We retrospectively analyzed 103 patients who underwent KT. Analyses were performed separately according to the donor type (living, 52; deceased, 51). In the living donor KT group, 28 patients received normal saline solution (NS) and 24 patients received Hartmann's solution (HS). In the deceased donor KT group, 27 patients received NS, and 24 received HS. The acid-base and electrolyte status, urine volume, and renal function between patients receiving NS and patients receiving HS were compared in each group. RESULTS: Regardless of donor type, there were no differences in potassium, pH, base excess, PCO2 and HCO3 between HS and NS on immediate postoperative and postoperative day 1. However, changes to neutral acid-base balance in terms of pH, HCO3, and base excess were significantly higher in HS than in NS. In living donor KT, NS increased serum potassium and chloride significantly during fluid therapy. On postoperative day 7, renal function showed no difference between two groups but urine volume was significantly larger in NS than in HS. CONCLUSIONS: HS does not increase the incidence of hyperkalemia after KT. The use of HS resulted in less metabolic acidosis than the use of NS. Renal function was similar but polyuria was more severe in patients who received NS than in those who received HS.
Acid-Base Equilibrium ; Acidosis ; Electrolytes* ; Fluid Therapy ; Humans ; Hydrogen-Ion Concentration ; Hyperkalemia ; Incidence ; Kidney Transplantation* ; Kidney* ; Living Donors ; Polyuria ; Potassium ; Retrospective Studies ; Sodium Chloride* ; Tissue Donors

PURPOSE: This study was aimed to compare the effect between wet gauze with cold normal saline and wet gauze with cold water on thirst, oral cavity condition, and saliva pH among postoperative patients. METHODS: This study was a quasi-experimental study with 56 participants. The experimental group received wet gauze with cold normal saline, and control group received wet gauze with cold water for three times at 15 minute intervals. Data were collected using visual analog scale for thirst, oral assessment guide for oral cavity condition and acidity tape for saliva pH. Measurement were made before applying the gauze and at 15min, 30min, and 45min after applying gauze. Data were analyzed using repeated measured ANOVA. RESULTS: Thirst, oral cavity condition, and saliva pH improved in both groups with increasing number of gauze application. Thirst for the experimental group improved more than for the control group (F=4.29, p=.009), oral cavity condition except saliva, and saliva pH were not significantly different between the groups. CONCLUSION: This study results indicated that nurses can apply wet gauze with cold normal saline to reduce thirst and saliva acidity, and to improve the oral cavity condition for postoperative patients.
Humans ; Hydrogen-Ion Concentration* ; Mouth* ; Saliva* ; Sodium Chloride ; Thirst* ; Visual Analog Scale ; Water* ; Xerostomia

The regulated transport of salt and water is essential to the integrated function of many organ systems, including the respiratory, reproductive, and digestive tracts. Airway epithelial fluid secretion is a passive process that is driven by osmotic forces, which are generated by ion transport. The main determinant of a luminally-directed osmotic gradient is the mucosal transport of chloride ions (Cl(-)) into the lumen. As with many epithelial cells, a number of classic signal transduction cascades are involved in the regulation of ion transport. There are two well-known intracellular signaling systems: an increase in intracellular Ca(2+) concentration ([Ca(2+)]i) and an increase in the rate of synthesis of cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP). Therefore, Cl(-) secretion is primarily activated via the opening of apical Ca(2+)- or cAMP-dependent Cl(-) channels at the apical membrane. The opening of basolateral Ca(2+)- or cAMP-activated K(+) channels, which hyperpolarizes the cell to maintain the driving force for Cl(-) exit through apical Cl(-) channels that are constitutively open, is also important in regulating transepithelial ion transport. P2Y receptors are expressed in the apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Human airway epithelial cells express multiple nucleotide receptors. Extracellular nucleotides, such as UTP and ATP, are calcium-mobilizing secretagogues. They are released into the extracellular space from airway epithelial cells and act on the same cell in an autocrine fashion to stimulate transepithelial ion transport. In addition, recent data support the role of P2Y receptors in releasing inflammatory cytokines in the bronchial epithelium and other immune cells.
Biological Transport ; Cell Membrane ; physiology ; Chloride Channels ; physiology ; Cyclic AMP ; physiology ; Cytokines ; immunology ; Epithelial Cells ; physiology ; Epithelium ; immunology ; physiology ; Humans ; Ion Transport ; Receptors, Purinergic P2Y ; immunology ; physiology ; Signal Transduction

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the spine (ASC-SP and PSC-SP) and skull (ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were (2.47 ± 0.39)%, (5.62 ± 0.82)%, (3.57 ± 0.40)%, and (6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly (330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH (1-5) and lost their solubilities when the NaCl concentration was above 2% (W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.
Acids ; chemistry ; Amino Acids ; analysis ; Animals ; Collagen ; chemistry ; isolation & purification ; Collagen Type I ; chemistry ; isolation & purification ; Hydrogen-Ion Concentration ; Molecular Structure ; Molecular Weight ; Pepsin A ; chemistry ; Skull ; chemistry ; Sodium Chloride ; Solubility ; Spine ; chemistry ; Temperature ; Tuna