Effect Of Pronase Research Articles

The Effect of Pronase on Mollusk, Leech, and Frog Nerve Ganglia Causes the Formation of Neuron–Neuronal Gap Junctions

The Effect of Pronase on Mollusk, Leech, and Frog Nerve Ganglia Causes the Formation of Neuron–Neuronal Gap Junctions

Observation on the effect of body position intervention combined pronase in sifting gastroscopy

Objective To explore the application of body position intervention combined pronase in gastric mucosal cleaning in painless gastroscopy. Methods A total of 200 patients who underwent painless gastroscopy from July 2016 to July 2017 in the digestive endoscopy center were selected as the subjects. According to the random digital table method, the patients were randomly divided into the experimental group and the control group of 100 cases. In the experimental group, before the gastroscope was examined, pronase plus Dimethicone Powder and lidocaine mucilage was used, and then the body position intervention (right supine 5 min-supine 5 min-left lying position 5 min) was examined, and the control group was taken Dimethicone Powder and Lido before the intensive examination. The caking mortar was then placed on the left side of the examination bed 15 min for examination. The upper gastrointestinal tract visual field definition and endoscopic operation time were compared between the two groups under magnifying endoscopy under white light and narrowband imaging. Results In the experimental group, 72.0% (72/100), 20.0% (20/100), 6.0% (6/100) and 2.0% (2/100) of A, B, C, D grade of the visual field clarity of mucosa under white light were better than 32.0% (32/100), 30.0% (30/100), 13.0% (13/100) and 25.0% (25/100) of the control group, respectively. The difference was statistically significant (χ2= 39.54, P < 0.05). There were 0, 6, 29 and 65 cases of 1, 2, 3, 4 scores of microvascular visual field intelligibility scores under magnifying endoscopy combined with narrow band imaging in the experimental group, which were better than those in the control group (11, 31, 28 and 30 cases respectively). The difference between the two groups was statistically significant (Z=-6.07, P < 0.05). The examination time of the experimental group was (10.64 ± 3.83) minutes, which was lower than that of the control group (11.67 ± 4.89) minutes, and the difference was statistically significant (t=1.978, P < 0.05). Conclusions The effect of pronase as an anti mucilage agent combined with body position is obvious, and the effect of dispelling the mucus and removing the mucus is comprehensive, and it can effectively shorten the time of examination. It is worthy of clinical application. Key words: Posture intervention; Painless gastroscopy; Pronase; Visual clarity

P033 The effect of pronase on B-cell flow cytometry crossmatch results: A case study

Cross matching is crucial in pre-organ transplantation and FCXM is the most sensitive cell-based XM technique. Routinely in our laboratory, we pre-treat the cells with pronase to improve the sensitivity and specificity of the B-cell FCXM. Pronase treatment of B-cell is used to disrupt the Fc and CD20 to minimize nonspecific binding of immunoglobulins and interference with Rituximab respectively. Pronase-treated T cells are tested in a single tube T-cell/B-cell technique and it has been shown that pronase treatment is likely to give false-positive reactions in the T-FCXM test. T cells express fewer Fc receptors than B cells for that, pronase may expose cryptic epitopes. This phenomenon of falsely incompatible T-cell FCXM with pronase in the absence of DSA has been well documented and has been frequently seen in our laboratory. In this study, we are presenting a case of incompatible B-cell FCXM after pronase treatment. An 18-year-old Caucasian male with an end-stage renal disease secondary to Joubert syndrome was considered for renal transplantation. Prior antibody screening and single-antigen bead testing disclosed anti-HLA class I antibodies with 6% cPRA and the absence of anti-HLA class II antibodies. His mother was the potential living related donor and the recipient did not carry any DSA against his mother HLA antigen. Crossmatch was performed between the donor lymphocytes and the patient serum after incubating the isolated lymphocytes with pronase. An initial FCXM showed unexpected B-FCXM incompatibility with both current serum and a historic serum. The concurrent T-FCXM was compatible. One month later, we repeated the XM with and without pronase-treatment. Unexpectedly, pronase treatment caused incompatible B-FCXM, whereas non-treated cells were B-FCXM compatible. In all cases, the T-FCXM was compatible. The take-home message of that case that cautious should be taken in clinical labs using pronase treatment in single tube T/B FCXM, to avoid false-positive reporting of results and alternative methods for blocking Fc receptors in FCXM should be explored.

The effect of pronase on lymphocyte surface markers and implications for flow cytometric crossmatch

Aim Pronase treatment of lymphocytes has been used to improve the specificity and sensitivity of the flow cytometric crossmatch for almost 20 years, due to its ability to remove Fc receptors from B cells and reduce non-specific background. Due to controversy in the published data, and significant variations in laboratory practices about the use of pronase, our goal was to determine the concentration-dependent effect of pronase on lymphocyte surface molecules, such as CD32 (Fc γ RIIB), HLA class I and II, CD3, CD19, and CD20, and to provide a standardized procedure to improve the performance of flow cytometric crossmatch assays. Methods Lymphocytes from 8 donors were isolated from blood, lymph nodes (LN) or spleen, and were digested using different concentrations of pronase ranging from 0.3 through 2 mg/ml. Surface CD32, CD3, CD19, CD20, HLA class I and class II were monitored by antibody staining using multi-color flow cytometry. Results Digestion with pronase at 0.6 mg/ml concentration or higher decreased CD32 expression by 80% on peripheral blood B cells and 85–90% on LN and splenic B cells. Pronase treatment did not reduce expression of HLA class I, however, it increased W6/32 antibody staining by 20–40% on B cells, and 150–200% on T cells. Similarly, pronase did not decrease HLA class II expression, but increased Tu39 antibody staining by 10–30% on lymph node and blood B cells. Binding of the CD3 antibody on pronase-treated T cells was increased by 2–2.5 fold, compared to untreated cells. There was no significant effect on CD19 expression up to 1 mg/ml, but 45–55% decrease was observed at 2 mg/ml. Pronase reduced CD20 expression in a concentration-dependent manner by up to 98–99% at 2 mg/ml. Conclusions Pronase treatment increases specificity of the crossmatch assay since it reduces CD32 expression on B cells. Our data show that concentrations higher than 0.6 mg/ml do not decrease CD32 expression any further. Pronase treatment is safe since it does not decrease HLA class I or class II expression, but rather increases pan-HLA antibody binding, likely due to receptor “unmasking”, thus increasing the sensitivity of the crossmatch assay. Finally, increasing pronase concentration to 2 mg/ml eliminates B cell CD20 expression, a procedure that can help to eliminate interferences by rituximab-containing sera used for crossmatch.

Effect of pronase as mucolytic agent on imaging quality of magnifying endoscopy.

To investigate the efficacy of premedication with pronase, a proteolytic enzyme, in improving image quality during magnifying endoscopy. The study was of a blinded, randomized, prospective design. Patients were assigned to groups administered oral premedication of either pronase and simethicone (Group A) or simethicone alone (Group B). First, the gastric mucosal visibility grade (1-4) was determined during conventional endoscopy, and then a magnifying endoscopic examination was conducted. The quality of images obtained by magnifying endoscopy at the stomach and the esophagus was scored from 1 to 3, with a lower score indicating better visibility. The endoscopist used water flushes as needed to obtain satisfactory magnifying endoscopic views. The main study outcomes were the visibility scores during magnifying endoscopy and the number of water flushes. A total of 144 patients were enrolled, and data from 143 patients (M:F=90:53, mean age 57.5 years) were analyzed. The visibility score was significantly higher in the stomach following premedication with pronase (73% with a score of 1 in Group A vs 49% in Group B, P<0.05), but there was no difference in the esophagus visibility scores (67% with a score of 1 in Group A vs 58% in Group B). Fewer water flushes [mean 0.7±0.9 times (range: 0-3 times) in Group A vs 1.9±1.5 times (range: 0-6 times) in Group B, P<0.05] in the pronase premedication group did not affect the endoscopic procedure times [mean 766 s (range: 647-866 s) for Group A vs 760 s (range: 678-854 s) for Group B, P=0.88]. The total gastric mucosal visibility score was also lower in Group A (4.9±1.5 vs 8.3±1.8 in Group B, P<0.01). The addition of pronase to simethicone premedication resulted in clearer images during magnifying endoscopy and reduced the need for water flushes.

The effect of pronase to reduce false positive B cell flow crossmatches and on cell surface determinants

The effect of pronase to reduce false positive B cell flow crossmatches and on cell surface determinants

Pronase acutely modifies high voltage-activated calcium currents and cell properties of Lymnaea neurons.

Pronase E ('pronase') is one of the proteolytic enzymes that are used in preparative procedures such as cell isolation and to soften the sheath of invertebrate ganglia. Although several effects of proteolytic enzymes on the physiology of non-neuronal tissues have been described, the effects of these enzymes on central neurons have received little attention. We examined the effects of bath-applied pronase on neurons in the Lymnaea central nervous system and in vitro. Pronase caused action potential broadening in neurons that exhibit a shoulder on the repolarization phase of their action potentials. This effect of pronase was accompanied by, although unrelated to, a depolarization and decrease in action potential interval. Some, but not all, effects of pronase in the central nervous system were reversible. For example, the decreases in membrane potential and action potential interval were both reversed after approximately 1 h of washing with saline. However, the effect of pronase on the action potential duration was not reversed after a period of 90 min. The modulation of action potential width prompted us to examine Ca2+ currents. Exposure to pronase resulted in an increase in both peak and late high voltage-activated Ca2+ currents in isolated neurons. Pronase neither changed the inactivation rate nor caused a shift in the current-voltage relationship of the current. The changes in action potential duration could be prevented by application of 0.1 mM Cd2+, indicating that the action potential broadening caused by pronase depends on Ca2+ influx. This is the first systematic study of the acute and direct actions of pronase on Ca2+ currents and cell properties both in the CNS and in vitro.

Effect of pronase on high-incidence blood group antigens and the prevalence of antibodies to pronase-treated erythrocytes

Abstract Pronase is a useful and relatively nonspecific protease that cleaves many red blood cell (RBC) membrane proteins that carry blood group antigens. Unexpected findings in tests using pronase-treated RBCs during the investigation of a patient’s blood sample led us to test which high-incidence blood group antigens were sensitive and which were resistant to pronase treatment, and to determine the prevalence of antipronase in the serum of blood donors. Our results show that antigens in the Cromer and Lutheran blood group systems and the JMH antigen were sensitive to pronase treatment of RBCs. Antigens in the Dombrock blood group system and Sc1 were either sensitive to or markedly weakened by pronase treatment of RBCs. The following high-incidence antigens were resistant to treatment of RBCs with pronase: AnWj, Ata, Coa, Co3, Dib, EnaFR, Era, Fy3, Jk3, Jra, k, Kpb, Jsb, K14, Lan, Oka, Rh17, U, Vel, and Wrb. Over half of the serum samples from normal blood donors contained antibodies to pronase-treated RBCs. When testing human serum against pronase-treated RBCs, it is essential either to use an autocontrol or to perform the testing with an eluate.

The presence of glutamine synthetase in the thylakoids of nitrogen‐fixing cyanobacterium Nostoc muscorum

AbstractThe intracellular distribution of glutamine synthetase (GS) in cell‐free extracts of Nostoc muscorum has been investigated. 64–73% of the enzyme activity was associated with the thylakoid membranes. No enzyme activity was found in cytoplasmic membranes. 92% inhibition of the enzyme activity was obtained with an antiserum raised to the thylakoids using Triton‐treated membranes, in comparison to 7.95% inhibition with the intact membranes. This suggests that the majority of the enzyme is embedded inside these membranes. This was also supported by the effect of pronase on the exposed glutamine synthetase using intact membranes.

Chemical modification studies of Artocarpus lakoocha lectin artocarpin

The effect of chemical modification on an anti T-like lectin, artocarpin isolated from Artocarpus lakoocha seeds was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of carboxyl groups, arginine and lysine residues, did not affect the lectin activity. However, modification of tryptophan, tyrosine and histidine residues led to a complete loss of its activity, indicating the involvement of these amino acids in the saccharide-binding ability. A protection was observed in the presence of inhibitory sugar. A marked decrease in the fluorescence emission was found when the tryptophan residues of lectin were modified. The circular dichroism spectra showed the presence of an identical pattern of conformation in the native and modified lectin, indicating that the loss in activity was due to modification only. The effect of pronase on artocarpin showed loss of activity whereas papain and trypsin had no effect. The specific activity of artocarpin remained unaltered on treatment with glycosidases but remarkable increase in the activity (of the same) was observed with xylanase treatment. Immunodiffusion studies with chemically modified lectin showed no gross structural changes, indicating that the group specific modifying agents did not alter the antigenic sites of the modified lectin.

The intracellular localization of glycolate oxidoreductase in Escherichia coli

Distribution of glycolate oxidoreductase in cell-free extracts of E. coli grown in mineral medium containing sodium glycolate has been studied. The enzyme was found to be largely associated with the cytoplasmic membranes. Homologous antiserum to the membranes inhibited the enzyme activity completely after the solubilization of the intact membranes with Triton X-100. Results on the effect of pronase on glycolate oxidoreductase activity confirmed that part of the enzyme is exposed to the surface of the membrane.

Effect of a proteinase on the macromolecular distribution of Acacia senegal gum

Molecular size distribution of Acacia senegal gum on Sephacryl gel S-400 is compared before and after incubation with the proteolytic enzyme Pronase. The profile and elution volumes of chromatograms are greatly modified: a rather broad system of high molecular weight peaks near the exclusion volume is resolved into a single peak at lower molecular weight. Physicochemical data from isolated Pronase-treated samples tend to favour, for crude Acacia senegal gum, a structure where varying numbers of polysaccharide units of MW ca 2×10 5 are linked to a protein core. The effect of Pronase on Acacia mearnsii and Combretum nigricans gums was also investigated. Our results are discussed in relation to structural models proposed for these arabinogalactan-protein complexes.

Salt taste responses in the frog glossopharyngeal nerve: different receptor sites for Mg 2+ and Na +

The glossopharyngeal nerve of the frog responds to MgCl 2 and NaCl applied to the tongue. To learn whether or not Mg 2+ and Na + react with different receptor sites, a proteolytic enzyme was topically applied to the tongue dorsum. Responses were recorded from the frog glossopharyngeal nerve during stimulation of the tongue with 100 mM MgCl 2 and 500 mM NaCl. The magnitude of the MgCl 2 response was selectively reduced after application of 0.1% pronase E to the dorsal tongue surface. The selective suppression of the MgCl 2 response by the pronase treatment indicates that the receptor site responsible for the MgCl 2 response differs from that for the NaCl response. The effect of pronase on the MgCl 2 response was due to the proteolytic action. This suggests that the receptor site responsible for the MgCl 2 response is composed of a protein or interacts secondarily with a protein affected by pronase treatment. It is suggested that there are multiple receptor sites for cations in salt taste reception of the frog.

Different receptor sites for Ca 2+ and Na + in single water fibers of the frog glossopharyngeal nerve

Unitary discharges were recorded from single water-sensitive fibers (water fibers) of the frog glossopharyngeal nerve during stimulation of the tongue with chemical stimuli. Low CaCl 2 (1 mM CaCl 2) and relatively high NaCl (500 mM NaCl) are effective stimuli which excite water fibers. To learn whether or not Ca 2+ and Na + react with different receptors sites, a proteolytic enzyme was topically applied to the tongue dorsum. After treatment of the tongue with 0.05% pronase E, the magnitude of the NaCl response remained unchanged, but the magnitude of the CaCl 2 response markedly decreased. The selective elimination by the pronase E treatment indicates that there exist different receptor sites for Ca 2+ and Na + in single water fibers of the frog glossopharyngeal nerve. The effect of pronase E treatment was due to proteolytic action. The results suggest that the Ca 2+ receptor site may be composed of a protein.

Cross-reactivity of contact site A to antibody produced against a stage-specific antigen from a phenol/water extract of Dictyostelium discoideum.

The stage-specific antigen, gp68 (Hirano, T., Yamada, H., & Miyazaki, T. (1983) Biochim. Biophys. Acta 742, 224-234), was purified from a phenol/water extract of aggregation-competent cells of Dictyostelium discoideum by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Anti-gp68 was produced against purified gp68 which was determined to be homogeneous by silver staining on analysis by SDS-polyacrylamide gel electrophoresis. The cross-reactivity of anti-gp68 against cellular antigens was estimated by immuno-affinity chromatography on anti-gp68 immunoglobulin G (IgG)/Sepharose. When the whole cell lysate was applied to this affinity column, three major proteins, with molecular weights of 80,000, 68,000, and 56,000, were obtained in the absorbed fraction. When the butanol extract, which was enriched in contact site A, an adhesion mediating glycoprotein, was applied to the same column, two major proteins with molecular weights of 80,000 (corresponding to contact site A) and 56,000 were obtained in the absorbed fraction but, however, gp68 was negligible. Reversely, when the phenol/water extract was applied to anti-contact site A-IgG/Sepharose, only gp68 was obtained in the absobed fraction. Moreover, contact site A was seen to compete with [3H]mannose-labeled gp68 in a competition radioimmunoassay using anti-gp68 serum. The effect of Pronase or exo-alpha-mannosidase digestion on the antigenic activity of gp68 was examined by radioimmunoassaying. The results indicated that the alpha-mannosyl residue of the non-reducing terminal in the carbohydrate moiety of gp68 was a major immunodeterminant. However, the polypeptide chain did not participate in the antigenic reactivity against anti-gp68. Both anti-gp68 and anti-contact site A agglutinated heat killed-yeast cells. Also, both anti-sera inhibited EDTA-stable cell adhesion of aggregation-competent cells in the presence of Fab from goat anti-rabbit IgG. These results indicate that gp68 and contact site A have a common antigenic determinant against anti-gp68, and that the target antigen of anti-gp68 was somehow involved in cell adhesion.

The use of radioimmune assay in investigating reagents to be used in the immunocytochemical localization of hepatitis B surface antigen in immune complexes in the kidney of patients with membranous nephropathy and Australia antigenaemia.

Radioimmune assay (RIA) was used to investigate the effect of fixatives on antigenicity of the hepatitis B surface antigen (HBsAg) and the effect of pronase on the elution of antibody (Ab) from the HBsAG-Ab complex. The effect of pronase on Ab elution was also tested on sections of kidney from a patient with the immune complex disease systemic lupus erythematosus (SLE). Immunoglobulin G (IgG) was located in pronase treated and untreated sections using the indirect immunoperoxidase technique. Glutaraldehyde was shown to be the fixative of choice for studies involving HbsAG. All fixatives were shown to have less effect on antigenicity at 4 degrees C than at room temperature. Osmium tetroxide reduced antigenicity to one-third, even at 4 degrees C. RIA and SLE kidney section studies showed that Ab was eluted from immune complexes by pronase. Pre-fixation of the antigen (Ag) by glutaraldehyde appears to have no effect on the final elution, although fixation after pronase treatment seemed to enhance the elution effects. The availability of an RIA kit with HBsAg- and AB-coated beads was of great assistance in evaluating reagents to be used in immunoperoxidase studies of HBsAg in immune complexes of patients with membranous nephropathy and Australia antigenaemia.

Regulation of chitin synthase activity inSaccharomyces cerevisiae: Effect of the inhibition of cell division and of synthesis of RNA and protein

The effect of pronase and trypsin on the activation or deactivation (degradation?) of chitin synthase ofSaccharomyces cerevisiae occurs faster in membranous preparations than in toluene-treated cells. When the temperature is raised, the former preparation is deactivated earlier than the latter one. The activity found in growing cells is not modified after inhibition of protein synthesis by cycloheximide or amino acid starvation or by the inhibition of RNA synthesis. It was possible to activate the chitin synthase ofS. cerevisiae cdc 25 grown at 23°C by means of pronase, whereas trypsin had no effect. After the cells were grown at 37°C, chitin synthase could not be activated either with trypsin or with pronase. This effect occurred independently of protein synthesis but did not take place when the cells were toluenized prior to the transfer at 37°C. These results suggest that the low catalytic levels and stability of the chitin synthase found in actively growing cells ofS. cerevisiae may be due to the restrictions introduced in the system by its membrane location.

The effect of Pronase on choroid plexus transport

The effect of Pronase on choroid plexus transport

The pineal gland of the mole (talpa europaea L.). IV. Effect of pronase on material present in cisternae of the granular endoplasmic reticulum of pinealocytes.

The effect of a proteolytic enzyme, pronase, on material present in cisternae of the granular endoplasmic reticulum of mole pinealocytes demonstrates their proteinaceous nature.

The sheep egg: enzymatic removal of the zona pellucida and culture of eggs in vitro.

Summary. A total of 429 eggs was used to investigate procedures both for the removal of the zona pellucida and for the culture of sheep eggs in vitro. The effect of pronase, papain, bromelain, trypsin and α-chymotrypsin, on the zonae pellucidae of fertilized and unfertilized eggs was studied in the first series of experiments. All the enzymes were effective in removing the zonae pellucidae from unfertilized eggs. Pronase was the only enzyme that had a significant lytic effect on the zonae of fertilized eggs denuding 34% of eggs in less than 5 min and almost all the other eggs within 90 min. Investigation of some of the factors involved in the successful culture of normal sheep eggs in vitro constituted the second part of the study. Eggs of both the eight- to sixteen-cell and morula stages were cultured for 72 hr in either sheep serum, enriched tissue culture medium 199 or Brinster's medium. The development and viability of the eggs after culture was determined by transferring some eggs to recipient sheep and making accurate cell counts on the remainder. Eggs recovered at the eight-cell stage underwent only one or two cleavage divisions during the 72-hr culture period. These eggs did not develop into foetuses when transferred to the uteri of recipient sheep. On the other hand, the majority of morulae cultured for 72 hr in enriched M199 or sheep serum underwent cleavage and blastulation in vitro. That the cultured blastocysts were normal was demonstrated by their implantation and growth into foetuses when transferred to the uteri of suitable recipients.