Effects of Pronase Treatment on Flow Cytometric Crossmatching

BACKGROUND: Flow cytometric crossmatching (FCXM) is widely used in hospitals performing solid organ transplantation. Pronase treatment of lymphocytes can increase the sensitivity and specificity of B-cell FCXM. However, it can also affect human leukocyte antigen (HLA) expression and results of FCXM. We treated lymphocytes with various concentrations of pronase and analysed the effect of the treatment on the FCXM results. METHODS: The peripheral blood mononuclear cells isolated from 10 renal transplant donors were treated with three different concentrations of pronase (0.5, 1.0, and 2.0 mg/mL). The effects of pronase on median fluorescence intensity (MFI) values of AB serum (Fcγ receptor), HLA class I and II, and on the MFI ratio of HLA class I and II were analysed. RESULTS: In B-cell FCXM, the MFI values of AB serum (Fcγ receptor) and HLA class I were significantly decreased by the pronase treatment. The MFI ratio of HLA class II was significantly increased upon treatment with 0.5, 1.0, and 2.0 mg/mL pronase (P<0.05, P<0.01, and P<0.01, respectively). In T-cell FCXM, the MFI ratio of HLA class I was significantly decreased by the pronase treatment (all P<0.01). CONCLUSIONS: When performing FCXM, it is recommended that B-lymphocytes should be treated with 1.0 or 2.0 mg/mL pronase. In the case of T-lymphocytes, pronase treatment should be adopted with caution.

Mucolytics as Adjuvant Agent to Improve Helicobacter pylori Eradication Rate: Still Long and Winding Road to Positive Results

No abstract available.

Additive Effect of Pronase on the Eradication Rate of First-Line Therapy for Helicobacter pylori Infection

BACKGROUND/AIMS: Helicobacter pylori colonizes on the apical surface of gastric surface mucosal cells and the surface mucous gel layer. Pronase is a premedication enzyme for endoscopy that can disrupt the gastric mucus layer. We evaluated the additive effects of pronase combined with standard triple therapy for H. pylori eradication. METHODS: This prospective, single-blinded, randomized, controlled study was conducted between June and October 2012. A total of 116 patients with H. pylori infection were enrolled in the study (n=112 patients, excluding four patients who failed to meet the inclusion criteria) and were assigned to receive either the standard triple therapy, which consists of a proton pump inhibitor with amoxicillin and clarithromycin twice a day for 7 days (PAC), or pronase (20,000 tyrosine units) combined with the standard triple therapy twice a day for 7 days (PACE). RESULTS: In the intention-to-treat analysis, the eradication rates of PAC versus PACE were 76.4% versus 56.1% (p=0.029). In the per-protocol analysis, the eradication rates were 87.5% versus 68.1% (p=0.027). There were no significant differences concerning adverse reactions between the two groups. CONCLUSIONS: According to the interim analysis of the trial, pronase does not have an additive effect on the eradication of H. pylori infection (ClinicalTrial.gov: NCT01645761).

A Comparative Study on Mechanical and Biochemical Properties of Bovine Pericardium After Single or Double Crosslinking Treatment

BACKGROUND AND OBJECTIVES: Glutaraldehyde (GA) has been used as a representative method of tissue preservation in cardiovascular surgery. However, GA has showed limited durability including calcification, mechanical failure and toxicity. To overcome those unsolved problems, we analyzed the crosslinking differences of primary amines, GA and genipin in their mechanical and biochemical properties with a single or double crosslinking agent for clinical application. MATERIALS AND METHODS: Samples were divided into 3 groups; control, single crosslinking fixation and double crosslinking fixation after decellurarization using bovine pericardium. For analysis of the biochemical and mechanical properties of each crosslinking method, tensile strength, percentage strain, thermal stability, resistance to pronase, nynhydrin and cytotoxicity test were studied. RESULTS: Combined hexamethylene diamine and suberic acid in the carbodiimide hydrochloride/N-hydroxysucinimide solution (EDC/NHS) after decellurarization, tensile strength and strain percentage were not statistically significant compared to the single crosslinking treated groups (p>0.05). Tissue crosslinking stability was weak in single treatment of diphenylphosphoryl azide, suberic acid, low concentration of EDC, hexamethylene diamine and procyanidin groups, but thermal stability and resistance to the pronase and ninhydrin were markedly increased in concentrated EDC/NHS or after combined double treatment with low concentration of GA or genipin (p<0.001). CONCLUSION: Single or double crosslinking with low concentration of carbodiimide, diphenylphosphonyl azide, procyanidin, suberic acid and hexane diamine were not as effective in mechanical, biochemical, cytotoxic and crosslinking properties compared to GA or genipin fixation, but their mechanical and chemical properties were much improved when combined with low concentrations of GA or genipin in the double crosslinking process.

Effectiveness of Premedication with Pronase for Visualization of the Mucosa during Endoscopy: A Randomized, Controlled Trial

BACKGROUND/AIMS: Gastric mucus should be removed before endoscopic examination to increase visibility. In this study, the effectiveness of premedication with pronase for improving visibility during endoscopy was investigated. METHODS: From April 2010 to February 2011, 400 outpatients were randomly assigned to receive endoscopy with one of four premedications as follows: dimethylpolysiloxane (DMPS), pronase and sodium bicarbonate with 10 minutes premedication time (group A, n=100), DMPS and sodium bicarbonate with 10 minutes premedication time (group B, n=100), DMPS, pronase and sodium bicarbonate with 20 minutes premedication time (group C, n=100), and DMPS and sodium bicarbonate with 20 minute premedication time (group D, n=100). One endoscopist, who was unaware of the premedication types, calculated the visibility scores (range, 1 to 3) of the antrum, lower gastric body, upper gastric body and fundus. The sum of the scores from the four locations was defined as the total visibility score. RESULTS: Group C showed significantly lower scores than other groups (p=0.002). Group C also had the lowest frequency of flushing, which was significantly lower than that of group D. Groups C and D had significantly shorter durations of examination than groups A and B. CONCLUSIONS: Using pronase 20 minutes before endoscopy significantly improved endoscopic visualization and decreased the frequency of water flushing.

Effects of Cyclic Nucleotide-Gated Channels in Vestibular Nuclear Neurons / 전남의대학술지

This study was designed to investigate the effects an 8-Br-cGMP on the neuronal activity of rat vestibular nuclear cells. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated vestibular nuclear cells were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium currents were recorded by standard patch-clamp techniques under current and voltage-clamp modes. Twelve vestibular nuclear cells revealed excitatory responses to 1-5 microM of 8-Br-cGMP, and 3 neurons did not respond to 8-Br-cGMP. Whole potassium currents of vestibular nuclear cells were decreased by 8-Br-cGMP (n=12). After calcium-dependent potassium currents were blocked by tetraethylammonium, the potassium currents were not decreased by 8-Br-cGMP. These experimental results suggest that 8-Br-cGMP changes the neuronal activity of vestibular nuclear cells by blocking the calcium-dependent potassium currents that underlie the afterhyperpolarization.

Effects of Cyclic Nucleotide-Gated Channels in Vestibular Nuclear Neurons / 전남의대학술지

This study was designed to investigate the effects an 8-Br-cGMP on the neuronal activity of rat vestibular nuclear cells. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated vestibular nuclear cells were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium currents were recorded by standard patch-clamp techniques under current and voltage-clamp modes. Twelve vestibular nuclear cells revealed excitatory responses to 1-5 microM of 8-Br-cGMP, and 3 neurons did not respond to 8-Br-cGMP. Whole potassium currents of vestibular nuclear cells were decreased by 8-Br-cGMP (n=12). After calcium-dependent potassium currents were blocked by tetraethylammonium, the potassium currents were not decreased by 8-Br-cGMP. These experimental results suggest that 8-Br-cGMP changes the neuronal activity of vestibular nuclear cells by blocking the calcium-dependent potassium currents that underlie the afterhyperpolarization.

A new method for isolation and culture of tracheal epithelial cell of mice / 生理学报

Since most of the respiratory epithelial cell lines are descended from neoplastic tissues or have been fused with neoplastic cells when being selected to a cell line, their biological behaviors are far different from normal respiratory epithelial cells. Aiming at better reflecting the biological properties of epithelial cells under respiratory pathological conditions, our study probed into a new isolation technique and culture method of mice respiratory epithelial cell. Pronase was applied for cell isolation from mouse trachea, and a modified medium with collagen-coated plate for primary culture. The ciliary movement can be observed under microscope, which demonstrates that the original biological functions of the tracheal epithelial cell have been reserved with our method. The research presents the efficacy, convenience and reliability of the separation technique and culture method established by this study, and laying preferable condition for cell models of respiratory diseases. Meanwhile, this method may be used for other animal models.

Study on Effective Preservation of Bovine Pericardium Using Decellularization and alpha-galactosidase for Eliminating Xenoreactive Antigen / 대한흉부외과학회지

BACKGROUND: Effective decellularization and fixation process is critical, in order to use xenogenic valves clinically. In the present study, we decellularized bovine pericardium using sodium dodecyl sulfate (SDS) and N-lauroyl sarcosinate, treated with alpha-galactosidase, and then fixed in various manners, to find out the most effective tissue preservation & fixation procedure. MATERIAL AND METHOD: Bovine pericardium was decellularized with SDS and N-lauroyl sarcosinate, and treated with alpha-galactosidase. Both groups were fixed differently, by varying glutaraldehyde (GA) or EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide)/N-hydroxysuccinamide (NHS) treatment conditions. Thereafter, physical examination, tensile strength test, thermal stability test, cytotoxicity test, pronase test, pronase-ninhydrin test, purpald test, permeability test, compliance test, H&E staining, DNA quantification, and alpha-galactose staining were carried out to each groups. RESULT: GA fixed groups showed better physical properties and thermal stability than EDC/NHS fixed groups. EDC/NHS-GA dual fixed groups showed better physical properties and thermal stability than EDC/NHS fixed groups, and showed better thermal stability than GA fixed groups. In pronase test and pronase-ninhydrin test, GA fixed groups and EDC/NHS-GA dual fixed groups showed stronger crosslinks than EDC/NHS groups. Permeability and compliance tended to increase in EDC/NHS-GA dual fixed groups, compared to GA fixed groups. But, EDC/NHS-GA dual fixed groups had stronger tensile strength and lower cytotoxicity than GA fixed groups. CONCLUSION: We have verified that EDC/NHS-GA dual fixation can make effective crosslinks and lower the toxicity of GA fixation. Henceforth, we will verify if EDC/NHS-GA dual fixation can lower calcifications & tissue failure in vivo experiment.

The Relation Between Sox9, TGF-beta1, and Proteoglycan in Human Intervertebral Disc Cells

OBJECTIVE: The aim of this study is to elucidate the effects of transforming growth factor-beta (TGF-beta)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-beta1 in intervertebral disc cells. METHODS: Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in 37degrees C, 5% CO2 incubator. When intervertebral disc cells were cultured with TGF-beta1 or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-beta1 were detected by RT-PCR and Western blot analysis in each. RESULTS: The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-beta1 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-beta1 and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-beta1, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group. CONCLUSION: This study suggests that TGF-beta1 would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.

Adhesion of Weissella cibaria to the Epithelial Cells and Factors Affecting its Adhesion

We evaluated the ability of lactic acid bacteria, Weissella cibaria, isolated from the oral cavity to adhere to epithelial cells. W. cibaria efficiently adhered to KB cells and HeLa cells. In addition, W. cibaria efficiently adhered to Fusobacterium nucleatum. But the adhesiveness of W. cibaria disappeared upon exposure to LiCl or pronase, suggesting that the S-layer proteins of W. cibaria mediated the adhesiveness. The molecular mass of the S-layer proteins extracted from W. cibaria was approximately 50 kDa. When W. cibaria strains were washed with 0.45% saline, the bacteria were efficiently adhered to the epithelial cells. In conclusion, W. cibaria has the ability to adhere to epithelial cells through the S-layer proteins.

Ionic mechanisms underlying spontaneous firing in isolated type B medial vestibular nucleus neurons

Medial vestibular nucleus (MVN) neurons are found to have spontaneous electrical activity in the absence of any detectable synaptic input. To investigate the contributions of intrinsic mechanisms to the spontaneous activity of type B MVN neurons, we examined the effects of various channel blockers on spontaneous firing by means of patch clamp recordings. Coronal slice (400 micrometer) of the vestibular nucleus region was sequentially treated with pronase 0.2 mg/ml and thermolysin 0.2 mg/ml, then single neurons were mechanically dissociated. MVN neurons recorded in neonatal rat were shown to have either a single deep afterhyperpolarization (AHP; type A cells), or an early fast and a delayed slow AHP (type B cells). In 300 nM TTX, spontaneous firing was blocked in type B cells tested. In 8 of 11 cells, underlying fluctuation or oscillations in membrane potential was not remained, and hyperpolarization did not produce rebound low-threshold calcium spikes. Although type B MVN neurons possessed hyperpolarization activated cation current (Ih), cesium had no effect on firing rates. The spike AHP is calcium dependent. When Ca2+ influx was blocked in external Ca2+ free solution, repetitive firing was abolished and the cell rested at depolarized membrane potentials. Application of apamin (300 nM) caused a profound reduction in the amplitude of the AHP and produced rhythmic burst firing. These findings suggest that the spontaneous activity of type B MVN neurons is regulated by interactions between the membrane depolarization mainly due to a persistent sodium conductances and hyperpolarization due to the calcium-activated potassium conductances.

Voltage-Dependent Sodium And Potassium Currents In Acutely Isolated Rat Trigeminal Caudal Neurons

Comparative Study on Development of Mouse Embryos in Three Commercial Media and Hatching Rates of Mouse Embryos with/without Pronase / 대한불임학회지

OBJECTIVE: The purpose of this present study was to compare mouse embryo development in 3 commercial media and hatching competence of mouse embryo with or without enzymatic treatment. METHODS: Collected 375 mouse embryos were divided into three groups, and then cultured in IVF-20 (G2), Medicult IVF (M3), P-1 (blastocyst M), respectively. Three day mouse morulae were cultured in G2 media treated with pronase. The results were analyzed using Chi-square test, and considered statistically significant when p<0.01. RESULTS: The developmental rate of 2 cell mouse embryo after 72 hours was highest in IVF-20 (G2) among conventional 3 media. The hatching rate of mouse morulae was low when clultured in G2 media without pronase during 48 hours. However, it was higher when cultured in media treated with l mg/ml, 2.5 mg/ml, 5 mg/ml pronase, respectively. CONCLUSIONS: Using good media and digestion of zona pellucida with enzymatic treatment improve development and hatching rate of embryo. Therefore, implantation and pregnancy rate could be improved.

Voltage-Dependent Potassium Currents in Acutely Isolated Rat Medial Vestibular Nucleus Neurons / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Medial vestibular nucleus (MVN) neurons are second-order afferent neurons that are involved in the reflex control of the head and eyes. Results from several studies utilizing the intracellular microelectrode recording techniques suggest the presence of several ionic conductances contributes to the regulation of the MVN neuron excitability in rats. In this study, the types and characteristics of voltage-dependent potassium currents were investigated in acutely isolated MVN neurons of postnatal rats. Material and Methods: Electrophysiological recordings were performed by means of the whole cell patch clamp techniques. Coronal slice (400 nm) of the vestibular nucleus region was sequentially treated with pronase 0.2 mg/ml and thermolysin 0.2 mg/ml, then single neurons were mechanically dissociated RESULTS: In a Ca2+ -free solution, low-threshold transient (IA) and high-threshold sustained (IK) currents were recorded. IK was activated (gamma=4.0-12.4 ms at 10 mV) and inactivated (gamma=180-720 ms at 10 mV) more slowly than IA. The half-maximum activation and inactivation potential were -3.1+/-3.4 mV and -38.8+/-3.6 mV, respectively. IA was activated rapidly (gamma=1.0-2.3 ms at 10 mV) and inactivated in 10-60 ms. The half-maximum activation and inactivation potentials were -22.3+/-4.5 mV and -58.4+/-3.8 mV, respectively. When a 4-aminopyridine of 10 mM was applied, IA was almost totally blocked. In a solution with 2 mM Ca2+, calcium dependent potassium currents were identified by application of a Ca2+ free solution and consisted of a transient and a sustained components. Exposure to 0.3 nM apamin induced a reversible reduction of a sustained components. CONCLUSION: These results suggest that MVN neurons express a variety of voltage-dependent potassium currents which are responsible for proper membrane excitability and firing of MVN neurons.

Biochemical Assisted Hatching of Embryos in Human IVF-ET Program / 대한산부인과학회잡지

Human assisted reproductive technology programs have been being developed marvelously during this decade. However, implantation rates following embryo replacement remain low, regardless of increased fertilization rates by ICSI. One proposed possibility limiting the successful implantation is an impaired hatching caused by suboptimal culture conditions. As to improve the hatching potential of blastocysts, assisted hatching by an artificial alteration of zona pellucida(ZP) have been done in many laboratories using the various methods. We tried to investigate whether the supplementation of proteases into culture media has any effect on development, zona structure, and/or hatching of mouse embryos. Supplementation of either pronase E(PE) or proteinase K(PK) in culture media did not affect development up to blastocyst but significantly increased hatching rate. And we observed the alteration of ultrastructure and casein binding properties of ZP in mouse embryos. Also we investigated the effects of protease on development of human embryos and pregnancy rates in human ART program. From July 1994 to December 1996, 792 cycles(for study I) and 1095 cycles(for study II) undergoing the IVF-ET program in MizMedi Hospital were randomly selected for BAH. The concentrations of proteases used in this study were 1microgram/ml PE, 0.1microgram/ml PK and 1microgram/mlPE+0.1microgram/ml PK in HTF with 0.5% human serum albumin(HSA), and in vitro fertilized embryos were cultured for 24 hours. We analyzed the efficiency and stability of biochemical assisted hatching(BAH) according to the clinical profiles of patients and fertilization methods. After cultured in HTF with proteases for 24 hours of human embryos, the thinning in zona pellucida of embryos was observed but its development was not disturbed. Also, clinical pregnancy rates were higher in the PE, PK and PE+PK groups than the control group without proteases(36.0%(32/89), 35.3%(36/102), 35.1%(39/111) vs. 25.5%(125/490), p<0.05). The live birth rate in the PE, PK and PE+PK groups were increased than control, and the abortion rate were not different. They were showed a effect and safety of proteases treatment in human embryos. We selected PE as BAH for study II because of slightly better embryonic morphology and pregnancy rate. In patients over 35 years old, clinical pregnancy rates of the BAH group was higher than that of the control group(31.4%(58/185) vs. 22.2%(51/230); p<0.05). And in the cases with few oocytes retrieval, or less than 3 cycles of IVF-ET, clinical pregnancy rates of the BAH group was significantly higher than that of the control group(36.8%(86/234) vs. 27.2%(93/342), p<0.05; 36.8%(148/402) vs. 29.9%(168/562), p<0.05). In BAH groups, the clinical pregnancy rate was similar between conventional IVF and ICSI group. From above results, it is suggested that improved hatching by protease treatment is due to physiological alteration of ZP structure, giving rise to the similar hatching process to that in vivo. We suggest that BAH using protease is a simple, safe and economic technique compared to the other known assisted hatching techniques in human ART program.

Proteolytic and partial characterization of human sperm-surface glycopeptides

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatmentmainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.

Receptor presente en eritrocitos humanos para la variante RD del virus coxsackie B3 / Present receptor in human erythrocytes for the coxsackie virus B3 variant RD

Se efectuó un estudio comparativo de los receptores para la variante RD del virus coxsacki B3 presentes en la membrana plamática de eritrocitos humanos y de células de rhabdomiosarcoma (RD). Para ello se usó un anticuerpo monoclonal, obtenido a partir del receptor presente en la célula RD, el cual fue marcado con 125l. El ligando marcado se unió a los receptores de los eritrocitos humanos e impidió que el virus se uniera a ellos. El anticuerpo monoclonal saturó los receptores, lo que permitió calcular el número de sitios de unión, que fue de 1.95x10 3 por eritrocito, número aproximadamente 10 veces menor que los calculados para las células RD. Los receptores de ambas células fueron bloqueados en su unión al ligando, cuando dos proteasas usadas, la quimotripsina y la pronasa, ejercieron su acción proteolítica, modificando la estructura de los receptores. Los presentes en la membrana plasmática de las células RD fueron más sensibles a la acción proteolítica que los presentes en los eritrocitos. Otra proteasa usada, la tripsina, bloqueó los receptores sobre los eritrocitos en un porcentaje similar a como lo hicieron las otras dos enzimas. Por el contrario, los receptores presentes en las células RD fueron casi insensibles al tratamiento con la tripsina.Las glicoforinas A, tipos MN y MM, fueron capaces de competir por ambos receptores, no así la tipo NN. Se concluye de esto que los receptores presentes en la membrana plasmática de los eritrocitos humanos y de las células RD, son de naturaleza proteica y comparten epítopes. El receptor presente en los eritrocitos humanos para el virus coxsackie B3, variante RD, que es bloqueado por el anticuerpo monoclonal, podría ser la glicoforina A, tipo MN y/o MM