Human Pepsin Research Articles

Portable and Rapid Fluorescence Turn-On Detection of Total Pepsin in Saliva Based on Strong Electrostatic Interactions.

Salivary pepsin has been proposed as a promising diagnostic marker for gastroesophageal reflux disease (GERD). However, the activity of human pepsin is strongly influenced by pH, and the acidic condition (pH ∼ 2) is optimal, which is a contradiction for the pepsin detection kit based on its catalytic activity. Thus, its accurate quantification in saliva (neutral pH) by readily rapid tools with simplicity and low cost is still challenging. Herein, a convenient fluorescence assay has been developed for the rapid detection of pepsin at neutral pH based on its electrostatic interaction with SYBR Green (SG) rather than the bioactivity. At neutral pH, the positively charged SG fluorophore can be effectively adsorbed by the negatively charged pepsin due to the low isoelectric point (pI) and large molecular size of pepsin. Thus, the molecular rotation of SG is limited, and its fluorescence intensity is significantly increased. The strategy was further confirmed to have the same fluorescence response as that of normally active and inactivated pepsin. Due to the unique pI of pepsin, the fluorescence strategy is highly selective for pepsin compared to other proteins. On the basis of this strategy, a smartphone-based fluorescence capture device integrated with a programmed Python program was fabricated for both color recognition and the accurate detection of pepsin within 3 min. Under the optimal conditions, this turn-on sensor allowed for the on-site analysis of pepsin with a detection limit of 0.2 μg/mL. Moreover, this strategy was successfully used to assess salivary pepsin to aid in the noninvasive diagnosis of GERD.

Effect of trans-nasal humidified rapid insufflation ventilatory exchange on reflux and microaspiration in patients undergoing laparoscopic cholecystectomy during induction of general anesthesia: a randomized controlled trial.

Reflux aspiration is a rare but serious complication during induction of anesthesia. The primary aim of this study is to compare the incidence of reflux and microaspiration in patients undergoing laparoscopic cholecystectomy during induction of general anesthesia using either a facemask or trans-nasal humidified rapid insufflation ventilatory exchange. We conducted a single-center, randomized, controlled trial. Thirty patients were allocated to either a facemask or a trans-nasal humidified rapid insufflation ventilatory exchange (THRIVE) group. Pre-oxygenation for 5 min with a facemask or THRIVE, positive pressure ventilation for 2 min or THRIVE for 2 min after anesthesia induction was followed. Before endotracheal intubation, the secretion above and below the glottis was collected to measure pepsin content and analyze blood gas. The ELISA assay for supra- and subglottic human pepsin content was used to detect the presence of reflux and microaspiration. The primary outcome was the incidence of reflux and microaspiration. Secondary outcomes were apnea time, PaO2 before tracheal intubation, and the end-expiratory carbon dioxide partial pressure. Patients in the THRIVE group had a significantly longer apnea time (379.55 ± 94.12 s) compared to patients in the facemask group (172.96 ± 58.87 s; p < 0.001). There were no differences observed in PaO2 between the groups. A significant difference in gastric insufflation, reflux, and microaspiration was observed between the groups. Gastric insufflation was 6.9% in the THRIVE group vs. 28.57% kPa in the facemask group (p = 0.041); reflux was 10.34% in the THRIVE group vs. 32.14% kPa in the facemask group (p = 0.044); and microaspiration was 0% in the THRIVE group vs. 17.86% kPa in the facemask group (p = 0.023). The application of THRIVE during induction of general anesthesia reduced the incidence of reflux and microaspiration while ensuring oxygenation and prolonged apnea time in laparoscopic cholecystectomy patients. THRIVE may be an optimal way to administer oxygen during the induction of general anesthesia in laparoscopic cholecystectomy patients. Chinese Clinical Trial Registry, No: ChiCTR2100054086, https://www.chictr.org.cn/indexEN.html.

THE PROGNOSTIC SIGNIFICANCE OF SALIVARY PEPSIN LEVEL IN THE DEVELOPMENT OF RECURRENT RESPIRATORY PATHOLOGY IN INFANTS WITH RUMINATION SYNDROME

Introduction. Infant rumination syndrome (IRS) is most common among infants with functional disorders of the gastrointestinal tract and can lead to the development of recurrent respiratory pathology. Pepsin is believed to play a key role in the upper airway mucosa impairment. The objective of the study was to determine the prognostic significance of salivary pepsin level in the development of recurrent respiratory pathology in infants with rumination syndrome. Materials and Methods. We analyzed the clinical and anamnestic data of 55 infants: the main group consisted of 38 children with apparent rumination syndrome, and the comparison group included 17 healthy children with no clinical manifestations of this syndrome. Salivary pepsin and IL-8 levels were measured three times a day by the enzyme-linked immunosorbent assay (Human Pepsin Elisa Kit and Human IL-8 ELISA Kit, USA). The study results were statistically processed using the Statistica v.6.1 software package. To assess the diagnostic significance of salivary pepsin level, ROC analysis was performed to determine the optimal cut-off point and calculate the area under the ROC curve (AUC) with 95% CI and operating characteristics of the criterion (sensitivity and specificity). Results and Discussion. Daily monitoring of salivary pepsin in infants showed that pepsin level was significantly higher in the main group vs. the comparison group, both for average daily values and intermediate indicators. No statistically significant difference was found between the three saliva samples and the daily average pepsin level in the main group of infants, which may be indicative of latent episodes of reflux during the day. A direct correlation was found between salivary levels of IL-8 and pepsin levels in the main group of children (r = 0.78, p &lt; 0.05). Among the potential predictors of recurrent respiratory pathology risk in infants with rumination syndrome, the following were established: fasting pepsin level &gt; 309.27 pg/ml, pepsin level in 1 hour after feeding &gt; 275.73 pg/ml, and pepsin level after regurgitation &gt; 532.31 pg/ml. Conclusions. Fasting pepsin level &gt; 309.27 pg/ml and pepsin level in 1 hour after feeding &gt; 275.73 pg/ml can be used in a multiple logistic regression model to predict the risk of recurrent respiratory pathology, taking into account other clinical, anamnestic, and laboratory data. A post-regurgitation pepsin level &gt; 532.31 pg/ml is an independent predictor and can be used alone.

Pepsin properties, structure, and its accurate measurement: a narrative review

: Pepsin is an aspartate protease that is generated from its proenzyme, pepsinogen by autocatalysis initiated by a fall in pH below 5. Human gastric juice contains eight isoenzymes of pepsin. The peptides released on conversion of pepsinogen to pepsin of which there are potentially five, have been shown to have antimicrobial activity against a wide range of bacteria including Escherichia coli, Pseudomonas and Staphylococcus which have also been shown to have biofilm formation inhibiting properties. The stability in response to changes in pH varies between pepsin and pepsinogen. Pepsinogen is stable up to pH 10, pepsin is only stable to pH just above 7.0 and is completely denatured at pH 8.0. Many diseases of the aerodigestive tract have been linked to reflux and the presence of pepsin. Therefore, the measurement of pepsin in tissue and lavages or in saliva or sputum, could be a good screening tool for the diagnosis of reflux related disease. However, there is no current consensus as to the best methods to measure it or the best time to sample it. For an effective pepsin enzyme-linked immunosorbent assay (ELISA), the following is required; a monoclonal/monospecific polyclonal antibody with a good lowest level of detection (LLOD) and sensitivity 1–25 ng/mL (depending on dilution) and an adequate supply of purified human pepsin as a standard for antibody-based assays. If possible, an activity assay for pepsin should also be used as the presence of pepsin protein does not indicate it is capable of damaging activity. Finally, if pepsin is associated with a disease large studies are required to confirm it with multiple samples. This review deals with several studies where pepsin quantitation is attempted, and their measurement techniques assessed.

Rapid and Simple Analysis of the Human Pepsin Secondary Structure Using a Portable Raman Spectrometer.

Human pepsin is a digestive protease that plays an important role in the human digestive system. The secondary structure of human pepsin determines its bioactivity. Therefore, an in-depth understanding of human pepsin secondary structure changes is particularly important for the further improvement of the efficiency of human pepsin biological function. However, the complexity and diversity of the human pepsin secondary structure make its analysis difficult. Herein, a convenient method has been developed to quickly detect the secondary structure of human pepsin using a portable Raman spectrometer. According to the change of surface-enhanced Raman spectroscopy (SERS) signal intensity and activity of human pepsin at different pH values, we analyze the change of the human pepsin secondary structure. The results show that the content of the β-sheet gradually increased with the increase in the pH in the active range, which is in good agreement with circular dichroism (CD) measurements. The change of the secondary structure improves the sensitivity of human pepsin SERS detection. Meanwhile, human pepsin is a commonly used disease marker for the noninvasive diagnosis of gastroesophageal reflux disease (GERD); the detection limit of human pepsin we obtained is 2 μg/mL by the abovementioned method. The real clinical detection scenario is also simulated by spiking pepsin solution in saliva, and the standard recovery rate is 80.7-92.3%. These results show the great prospect of our method in studying the protein secondary structure and furthermore promote the application of SERS in clinical diagnosis.

Pepsin-like aspartic proteases (PAPs) as model systems for combining biomolecular simulation with biophysical experiments.

Pepsin-like aspartic proteases (PAPs) are a class of aspartic proteases which shares tremendous structural similarity with human pepsin. One of the key structural features of PAPs is the presence of a β-hairpin motif otherwise known as flap. The biological function of the PAPs is highly dependent on the conformational dynamics of the flap region. In apo PAPs, the conformational dynamics of the flap is dominated by the rotational degrees of freedom associated with χ1 and χ2 angles of conserved Tyr (or Phe in some cases). However it is plausible that dihedral order parameters associated with several other residues might play crucial roles in the conformational dynamics of apo PAPs. Due to their size, complexities associated with conformational dynamics and clinical significance (drug targets for malaria, Alzheimer's disease etc.), PAPs provide a challenging testing ground for computational and experimental methods focusing on understanding conformational dynamics and molecular recognition in biomolecules. The opening of the flap region is necessary to accommodate substrate/ligand in the active site of the PAPs. The BIG challenge is to gain atomistic details into how reversible ligand binding/unbinding (molecular recognition) affects the conformational dynamics. Recent reports of kinetics (Ki, Kd) and thermodynamic parameters (ΔH, TΔS, and ΔG) associated with macro-cyclic ligands bound to BACE1 (belongs to PAP family) provide a perfect challenge (how to deal with big ligands with multiple torsional angles and select optimum order parameters to study reversible ligand binding/unbinding) for computational methods to predict binding free energies and kinetics beyond typical test systems e.g. benzamide–trypsin. In this work, i reviewed several order parameters which were proposed to capture the conformational dynamics and molecular recognition in PAPs. I further highlighted how machine learning methods can be used as order parameters in the context of PAPs. I then proposed some open ideas and challenges in the context of molecular simulation and put forward my case on how biophysical experiments e.g. NMR, time-resolved FRET etc. can be used in conjunction with biomolecular simulation to gain complete atomistic insights into the conformational dynamics of PAPs.

Influence of proton pump inhibitor therapy on occurrence of voice prosthesis complications.

It has been shown that the reflux of the gastric content to the proximal oesophagus influences incidence of voice prosthesis (VP) complications in laryngectomized patients. We conducted prospective randomised study to investigate the relationship between pepsin concentration in saliva and occurrence of VP complications before and after 3months of proton pump inhibitor (PPI) therapy. 60 laryngectomized patients with VP and 30 controls were included in the study. Saliva samples were collected in the morning and concentration of pepsin were measured by Human Pepsin (PG) ELISA kit. Thirty-Four (57%) patients reported one or more VP complication and were randomised in two groups, with and without PPI therapy, 40mg pantoprazole per day for 3months. Patients who had longer time since last VP change had higher incidence of periprosthetic and transprosthetic leakage and Candida colonisation. Pepsin was found in all saliva samples. Median saliva pepsin concentration level did not significantly differ between laryngectomized patients and control subjects, or between patients with and without VP complications, and there was no correlation between saliva pepsin concentration levels and type of VP complication. After 3months therapy, there was no difference in median saliva pepsin level or incidence of VP complication between patients with and without PPI therapy. Although reflux was proposed to be associated with VP complications and pepsin was proven as a most sensitive and specific marker of EER, we did not find any statistically significant correlation between pepsin levels and occurrence of VP complications. A 3months 40mg pantoprazole therapy was ineffective in reduction of VP complications in our study group.

Validation in China of a non‐invasive salivary pepsin biomarker containing two unique human pepsin monoclonal antibodies to diagnose gastroesophageal reflux disease

ObjectivesPeptest is a new non‐invasive reflux diagnostic test based on lateral flow technology that containing two highly specific human pepsin monoclonal antibodies for detecting pepsin, a biomarker for reflux disease. The primary aim of this multicenter clinical study was to validate the efficacy of Peptest in patients diagnosed with gastroesophageal reflux and healthy controls in China.MethodsPatients with suspected gastroesophageal reflux underwent an endoscopy and were classified into non‐erosive reflux disease and erosive esophagitis subgroups. A healthy control group was also recruited. All participants were given a reflux disease questionnaire—patients scoring greater than 12 and controls scoring zero. All participants provided a postprandial saliva sample and most patients gave an additional post‐symptom sample for pepsin analysis.ResultsAltogether 1032 participants aged between 19 and 78 years were recruited. They consisted of 488 patients with non‐erosive reflux disease, 221 with erosive esophagitis and 323 healthy controls. The number of postprandial and post‐symptom samples analyzed totaled 1031 and 692, respectively. The results across all centers showed an overall pepsin‐positive sensitivity of 85%, a specificity of 60%, a positive predictive value of 82%, a negative predictive value of 65% and a positive likelihood ratio of 2.12.ConclusionThe sensitivity of Peptest was high, but the specificity achieved in some centers was low, resulting overall in only a moderate specificity. Further diagnostic investigative studies are warranted.

Expression of milk-derived angiotensin I-converting enzyme-inhibitory peptides in Lactococcus lactis

ABSTRACTAngiotensin I-converting enzyme (ACE) plays a key role in the regulation of blood pressure. Currently, most single or tandem repeats of ACE-inhibitory (ACE-I) peptides have been expressed in Escherichia coli. However, in this study, a food-grade system was constructed using Lactococcus lactis (L. lactis) to simultaneously express four different milk-derived ACE-I peptides with antihypertensive activity. Mixed peptides (MPs) fused with green fluorescent protein (GFP) and eight histidines were synthesized. To ensure potent ACE inhibition by the MPs in human digestive juice, pepsin and trypsin cleavage sites were introduced among the four ACE-I peptides. The MP fusion gene was inserted into expression vector pSEC-E7 with nisin induction and expressed in L. lactis NZ9000, then purified by affinity chromatography. The transformants containing pSEC-MP:GFP were identified based on green fluorescence using Leica laser scanning confocal microscopy. The target proteins were detected by SDS-PAGE analysis and displayed obvious immunogenicity by western blot. After hydrolysis with digestive enzymes, the IC50 of the MPs was 118.63 μM. These results suggested that multiple milk-derived ACE-I peptides with antihypertensive properties could be produced using a food-grade lactococcal expression system.

Multiple target-based pharmacophore design from active site structures

Health care systems have benefitted from rational drug discovery processes like vHTS, virtual high throughput screening pharmacophores and quantitative structure–activity relationships, and many challenges have been explored using such techniques: decisions on specificity and selectivity are made after screening millions of molecules for multiple targets. Recent challenges in drug research emphasize the design of drugs that bind with more than one target of interest (multi-target) and do not bind with undesirable targets. This work attempts to use a three-dimensional interaction profile of the active site of a class of proteins, identify selective positions for the binding of functional groups, called features, and develop ensembles of multi-targeted pharmacophores that retain specificity and selectivity. The goal of this study is to develop multi-target pharmacophores by computational methods using protein structures alone to guide the discovery of novel inhibitors of plasmepsins, displaying selectivity over their human homologs, cathepsin D and pepsin. The development of such novel tools is attempted using a combination of different approaches such as the molecular interaction field, clique graph and inductive logic programming to identify and compare specific and selective complementary features. The identification of selective combinations of features has resulted in the design of multi-featured specific and selective pharmacophores that are validated using antimalarial compounds in ChEMBL that are known for their anti-plasmepsin II activity. This novel method is computationally less intensive and is applicable to any known class of target structures for finding specific and selective binders simultaneously.

Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.

Investigation of pepsin in tears of children with laryngopharyngeal reflux disease

ObjectivesNumerous investigations postulated that laryngopharyngeal reflux (LPR) is implicated in the pathogenesis of various upper airway inflammatory diseases as sinusitis or dacryostenosis.The presence of pepsin in tears might be confirmed the presuntive hypothesis of the arrival in the nasolacrimal ducts and precorneal tears film through the laryngopharyngeal reflux of either gastric acid or stomach secretions (pepsin) with inflammatory potentialities.The aim of this preliminary study was to identify the presence or absence of pepsin in the tears collected from children with a high suspicion of LPR who underwent 24-h pH (MII-pH) monitoring to confirm the disease. MethodsThis study enrolled 20 patients suffering from symptoms of laryngopharyngeal reflux that underwent 24-h multichannel intraluminal impedance (MII)-pH monitoring to confirm the disease. The findings of the study group were compared with those of a control group of patients with negative pH monitoring. The quantitative analysis of human pepsin concentration in the tear samples was performed by ELISA method in both groups. ResultsFour children (20%) of the study group showed pepsin in the tears. All of the subjects belonging to the control group were negative for its presence. No difference differences in the total number of reflux episodes and the number of weakly basic reflux in the pepsin positive patients vs. pepsin negative children were present. Conclusions20% of the children with diagnosed LPR showed pepsin in the tears. Our specific investigation might provide information regarding sinusitis or dacryostenosis.

S31 Pepsin detection despite the use of acid suppressant medication in patients with airway reflux related chronic cough

BackgroundChronic cough (CC) is an increasing problem that is not easy to treat with medication. Associated symptoms include hoarse voice, dysphonia, persistent tickling and irritation of the throat or chest....

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 103 and 6.52 × 103 M−1. The binding reactions were enthalpy driven with ΔHb values between −50.99 and −46.07 kJ mol−1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.

Potential Benefits of pH 8.8 Alkaline Drinking Water as an Adjunct in the Treatment of Reflux Disease

At the cellular level, tissue-bound pepsin is fundamental to the pathophysiologic mechanism of reflux disease, and although the thresholds for laryngeal damage in laryngopharyngeal reflux and for esophageal damage in gastroesophageal reflux disease differ, both forms of damage are due to pepsin, which requires acid for its activation. In addition, human pepsin remains stable at pH 7.4 and may be reactivated by hydrogen ions from any source. Thus, most tap and bottled waters (typically pH 6.7 to 7.4) would not be expected to affect pepsin stability. The purposes of these in vitro studies were to investigate whether artesian well water containing natural bicarbonate (pH 8.8) might irreversibly denature (inactivate) human pepsin, and to establish its potential acid-buffering capacity. Laboratory studies were performed to determine whether human pepsin was inactivated by pH 8.8 alkaline water. In addition, the buffering capacity of the alkaline water was measured and compared to that of the two most popular commercially available bottled waters. The pH 8.8 alkaline water irreversibly inactivated human pepsin (in vitro), and its hydrochloric acid-buffering capacity far exceeded that of the conventional-pH waters. Unlike conventional drinking water, pH 8.8 alkaline water instantly denatures pepsin, rendering it permanently inactive. In addition, it has good acid-buffering capacity. Thus, the consumption of alkaline water may have therapeutic benefits for patients with reflux disease.

Pepsin promotes proliferation of laryngeal and pharyngeal epithelial cells

Laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngeal squamous cell carcinoma (SCC), but causality has never been proven. It is accepted that chronic reflux into the esophagus can induce metaplastic changes in esophageal mucosa with subsequent increased risk of esophageal adenocarcinoma, but no similar associations have been established for LPR and laryngopharyngeal SCC. The objective of this study was to test the hypothesis that reflux of pepsin into the laryngopharynx can promote carcinogenesis. Translational research study. Normal human laryngeal primary epithelial cell cultures and hypopharyngeal FaDu SCC cells were exposed to human pepsin and analyzed by Human Cancer PathwayFinder and miRNA Superarrays, flow cytometry, and Western blot to determine the effect of pepsin on carcinogenesis. Laryngeal biopsy specimens taken from cancer patients and normal control subjects were analyzed for the presence of pepsin by Western blot. Microarray analysis demonstrated that pepsin significantly altered the expression of 27 genes implicated in carcinogenesis and also affected the expression of 22 microRNAs known to be altered in human head and neck cancers. Pepsin increased proliferation in both FaDu SCC cells and cultured normal laryngeal epithelial primary cells by increasing S phase distribution on flow cytometry analysis in a time- and dose-dependent manner. Furthermore, pepsin was detected in 60% (3/5) human laryngeal cancer biopsies, absent in all (0/5) normal control specimens. These data support a role for refluxed pepsin in the promotion of epithelial proliferation and carcinogenesis of the larynx and pharynx.

An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.

Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2

An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH₂ and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.

Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: A human Lymphatic Filarial parasite

Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.

Low-Acid Diet for Recalcitrant Laryngopharyngeal Reflux: Therapeutic Benefits and Their Implications

Laryngopharyngeal reflux (LPR) is an expensive, high-prevalence disease with a high rate of medical treatment failure. In the past, it was mistakenly believed that pepsin was inactive above pH 4; however, human pepsin has been reported to be active up to pH 6.5. In addition, it has been shown by Western blot analysis that laryngeal biopsy samples from patients with symptomatic LPR have tissue-bound pepsin. The clinical impact of a low-acid diet on the therapeutic outcome in LPR has not been previously reported. To provide data on the therapeutic benefit of a strict, virtually acid-free diet on patients with recalcitrant, proton pump inhibitor (PPI)-resistant LPR, I performed a prospective study of 20 patients who had persistent LPR symptoms despite use of twice-daily PPIs and an H2-receptor antagonist at bedtime. The reflux symptom index (RSI) score and the reflux finding score (RFS) were determined before and after implementation of the low-acid diet, in which all foods and beverages at less than pH 5 were eliminated for a minimum 2-week period. The subjects were individually counseled, and a printed list of acceptable foods and beverages was provided. There were 12 male and 8 female study subjects with a mean age of 54.3 years (range, 24 to 72 years). The symptoms in 19 of the 20 subjects (95%) improved, and 3 subjects became completely asymptomatic. The mean pre-diet RSI score was 14.9, and the mean post-diet RSI score was 8.6 (p = 0.020). The mean pre-diet RFS was 12.0, and the mean post-diet RFS was 8.3 (p < 0.001). A strict low-acid diet appears to have beneficial effects on the symptoms and findings of recalcitrant (PPI-resistant) LPR. Further study is needed to assess the optimal duration of dietary acid restriction and to assess the potential role of a low-acid diet as a primary treatment for LPR. This study has implications for understanding the pathogenesis, cell biology, and epidemiology of reflux disease.