Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A.

Protein aggregation is related to numerous pathological conditions like Alzheimer's and Parkinson's disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg2Cl2 (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen ß-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native ß-barrel conformation to ß-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg2Cl2 treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the ß-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.

Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens.

Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies.

In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress.

Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice-liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as -15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near -10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (TCD ∼ -17 °C; calculated via isothermal chemical denaturation and Gibbs-Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near -5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

Trypsinogen and chymotrypsinogen: potent anti-tumor agents.

Introduction: Trypsinogen and chymotrypsinogen have been used clinically in tissue repair due to their ability to resolve inflammatory symptoms. Recently, novel evidence has supported the anti-tumourigenic potential of a mixture of trypsinogen and chymotrypsinogen.Areas covered: First, we analyze the structure of these proteases and the effects of pancreatic proteinases on tissue repair, inflammation and the immune system. Second, we summarize studies that provided evidence of the effects of pancreatic (pro)enzymes on tumor cells both in vitro and in vivo and some successful clinical applications of pancreatic (pro)enzymes. Finally, we study pancreatic (pro)enzymes potential molecular targets, such as the proteinase-activated receptors (PARs).Expert opinion: This novel therapy has been shown to have effective antitumor effects. Treatment with these (pro) enzymes sensitizes Cancer Stem Cells (CSCs) which may allow chemotherapy and radiotherapy to be more effective, which could positively affect the recovery of cancer patients.

Trypsinogen and chymotrypsinogen: the mysterious hyper-reactivity of selected cysteines is still present after their divergent evolution.

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (KD  = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times.

Orders-of-Magnitude Larger Force Demonstrated for Dielectrophoresis of Proteins Enabling High-Resolution Separations Based on New Mechanisms.

Proteins are perhaps the most important yet frustratingly complicated and difficult class of compounds to analyze, manipulate, and use. One very attractive option to characterize and differentially concentrate proteins is dielectrophoresis, but according to accepted theory, the force on smaller particles the size of proteins is too low to overcome diffusive action. Here, three model proteins, immunoglobulin G, α-chymotrypsinogen A, and lysozyme, are shown to generate forces much larger than predicted by established theory are more consistent with new theoretical constructs, which include the dipole moment and interfacial polarizability. The forces exerted on the proteins are quantitatively measured against well-established electrophoretic and diffusive processes and differ for each. These forces are orders of magnitude larger than previously predicted and enable the selective isolation and concentration of proteins consistent with an extremely high-resolution separation and concentration system based on the higher-order electric properties. The separations occur over a small footprint, happen quickly, and can be made in series or parallel (and in any order) on simple devices.

Macromolecular crowding stabilises native structure of α-chymotrypsinogen-A against hexafluoropropanol-induced aggregates.

Cell interior is extremely congested with tightly packed biological macromolecules that exerts macromolecular crowding effect, influencing biophysical properties of proteins. To have a deeper insight into it we studied consequences of crowding on aggregation susceptibility and structural stability of α-chymotrypsinogen-A, pro-enzyme of serine protease family, upon addition of co-solvent reported to exert stress on polypeptides crafting favourable conditions for aggregation. Hexafluoropropan-2-ol (HFIP), a fluorinated alcohol caused structural disruption at 5% v/v unveiled by reduced intrinsic intensity and blue shifted ANS spectra. Significantly enhanced, red-shifted ThT and Congo red spectra sustained conformational changes concomitant with aggregation. FTIR and CD results confirmed transition of native structure to non-native extended, cross-linked beta-sheets. Transmission electron micrographs visibly exhibited incidence of amorphous aggregates. Macromolecular crowding, typically mimicked by concentrated solutions of dextran 70, was noticeably witnessed to defend conformational stability under denaturing condition. The native structure was retained maximally in presence of 100 mg/ml followed by 200 and 300 mg/ml dextran indicating concentration dependent deceleration of aggregate formation. It can be established that explicit consideration of crowding effects using relevant range of inert crowding agents must be a requisite for presumptions on intracellular conformational behaviour of proteins deduced from in vitro experiments.

Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins.

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.

Delayed Desorption Improves Protein Analysis by Desorption Electrospray Ionization Mass Spectrometry.

Protein analysis by desorption electrospray ionization mass spectrometry (DESI-MS) is limited and often accompanied by a mass-dependent loss in sensitivity as protein molecular weight increases. Previously, incomplete dissolution was identified as a potential contributing factor to this limitation for larger proteins. Here, we developed a unique two-step configuration in which a prewetting solvent is applied to the sample surface proximal to DESI analysis by a wetting quill to increase dissolution time and the detection of larger proteins. After optimizing the system with a mixture of proteins containing cytochrome c, myoglobin, and chymotripsinogen, we demonstrate the ability of delayed desorption to improve the analysis of larger proteins such as bovine serum albumin. Albumin and other serum proteins, including even larger ones, were also detected directly from diluted goat serum. An additional feature of this technique is the ability to deliver multiple solvents with potential synergistic or cooperative effects. For example, when using acetonitrile solutions of formic acid and ammonium bicarbonate as the prewetting and DESI spray solvent, respectively, the intensity of chymotrypsinogen improved dramatically compared to controls but less so for smaller proteins such as myoglobin and cytochrome c. Adduct removal was also observed for all proteins. These early results demonstrate the ability of this two-step technique for the use of multiple additives and increased dissolution times compared to standard DESI-MS experiments.

Quantifying protein aggregation kinetics using electrospray differential mobility analysis.

Protein aggregation is a critical concern in bioprocessing, where its presence can result in serious adverse interactions in clinical end-use applications. In this study, an aerosol-based technique, electrospray differential mobility analysis (ES-DMA), was used to quantify thermally-induced protein aggregation kinetics for bovine serum albumin (BSA) and α-chymotrypsinogen A (α-chymo), employing a new methodology to modify the solution for compatibility with the electrospray process. Results are compared orthogonally with asymmetrical-flow field-flow fractionation (AF4), a hydrodynamic separation technique with UV detection. Measurements were conducted over a range of protein concentrations and temperatures. Both techniques successfully resolved the protein monomer and dimer populations, allowing quantification of monomer loss. BSA and α-chymo exhibited second and first order kinetics, respectively, confirming different limiting steps for the two species. The Arrhenius equation yielded activation energies for BSA of (240 ±â€¯20) kJ mol-1 and (190 ±â€¯10) kJ mol-1 by ES-DMA and AF4, respectively. The rates determined by ES-DMA were equal to or slightly faster than those measured by AF4, so instrumental differences were analyzed to identify potential sources of bias. An important factor may be the applicable concentration range for each method; notably, AF4 operates at the mg mL-1 level, while ES-DMA is sensitive at µg mL-1 and therefore requires much smaller samples for analysis (typically several µL are injected). The limitations of each method are detailed in the discussion and demonstrate the importance of orthogonal measurement strategies for the analysis of protein kinetics. ES-DMA provides a potentially useful alternative to size exclusion chromatography to screen the stability of formulation conditions for protein therapeutics; neither ES-DMA nor AF4 rely on column interactions for separation.

Pancreatic (pro)enzymes treatment suppresses BXPC-3 pancreatic Cancer Stem Cell subpopulation and impairs tumour engrafting.

Cancer stem cells (CSCs) subpopulation within the tumour is responsible for metastasis and cancer relapse. Here we investigate in vitro and in vivo the effects of a pancreatic (pro)enzyme mixture composed of Chymotrypsinogen and Trypsinogen (PRP) on CSCs derived from a human pancreatic cell line, BxPC3. Exposure of pancreatic CSCs spheres to PRP resulted in a significant decrease of ALDEFLUOR and specific pancreatic CSC markers (CD 326, CD 44 and CxCR4) signal tested by flow cytometry, further CSCs markers expression was also analyzed by western and immunofluorescence assays. PRP also inhibits primary and secondary sphere formation. Three RT2 Profiler PCR Arrays were used to study gene expression regulation after PRP treatment and resulted in, (i) epithelial-mesenchymal transition (EMT) inhibition; (ii) CSCs related genes suppression; (iii) enhanced expression of tumour suppressor genes; (iv) downregulation of migration and metastasis genes and (v) regulation of MAP Kinase Signalling Pathway. Finally, in vivo anti-tumor xenograft studies demonstrated high anti-tumour efficacy of PRP against tumours induced by BxPC3 human pancreatic CSCs. PRP impaired engrafting of pancreatic CSC's tumours in nude mice and displayed an antigrowth effect toward initiated xenografts. We concluded that (pro)enzymes treatment is a valuable strategy to suppress the CSC population in solid pancreatic tumours.

Solution thermodynamic approach to analyze protein stability in aqueous solutions.

Protein thermal stability was analyzed by a solution thermodynamic approach. The small energetic differences in hydrogen-bonds (HB) among amino acid resdues and water molecules were proved to be amplified by the large number of HB involved to bring about the equilibrium shift from folding to unfolding of proteins. In aqueous solutions, water activity (Aw) plays a key role in protein stability. Therefore, Aw was precisely determined for various solutions and its relationship with solution structure was discussed. Wyman-Tanford analysis based on Aw showed linear regressions, without exception, between protein unfolding-ratio and Aw for lysozyme, ribonuclease A, and α-chymotrypsinogen A in various solutions with sugars, osmolytes, alcohols, and protein denaturant. From this linear regression, the free energy difference, ΔΔG, for a protein in a solution and in pure water, was easily obtained. Protein stability in a solution was proved to be determined by a balance between hydration and solute-binding effects to the protein and also by solution structure, which indirectly affects the hydrophobic interaction in a protein molecule. Temperature dependence of HB on protein stability suggested its interrelationship with hydrophobic interaction.

Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization.

Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO2 to conventional liquid mobile phases. The addition of liquefied CO2 increases diffusivity and decreases viscosity, which inherently leads to a more efficient separation. Herein, EFLC is applied to hydrophobic interaction chromatography (HIC) stationary phases for the first time to study the impact of liquefied CO2 to the chromatographic behavior of proteins. The effects of liquefied CO2 on chromatographic properties, charge state distributions (CSDs), and ionization efficiencies were evaluated. EFLC offered improved chromatographic performance compared to conventional liquid chromatography (LC) methods including a shorter analysis time, better peak shapes, and higher plate numbers. The addition of liquefied CO2 to the mobile phase provided an electrospray ionization (ESI)-friendly and "supercharging" reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in large-scale application.

Kirkwood-Buff-Derived Alcohol Parameters for Aqueous Carbohydrates and Their Application to Preferential Interaction Coefficient Calculations of Proteins.

The CHARMM36 carbohydrate parameter set did not adequately reproduce experimental thermodynamic data of carbohydrate interactions with water or proteins or carbohydrate self-association; thus, a new nonbonded parameter set for carbohydrates was developed. The parameters were developed to reproduce experimental Kirkwood-Buff integral values, defined by the Kirkwood-Buff theory of solutions, and applied to simulations of glycerol, sorbitol, glucose, sucrose, and trehalose. Compared to the CHARMM36 carbohydrate parameters, these new Kirkwood-Buff-based parameters reproduced accurately carbohydrate self-association and the trend of activity coefficient derivative changes with concentration. When using these parameters, preferential interaction coefficients calculated from simulations of these carbohydrates and the proteins lysozyme, bovine serum albumin, α-chymotrypsinogen A, and RNase A agreed well with the experimental data, whereas use of the CHARMM36 parameters indicated preferential inclusion of carbohydrates, in disagreement with the experiment. Thus, calculating preferential interaction coefficients from simulations requires using a force field that accurately reproduces trends in the thermodynamic properties of binary excipient-water solutions, and in particular the trend in the activity coefficient derivative. Finally, the carbohydrate-protein simulations using the new parameters indicated that the carbohydrate size was a major factor in the distribution of different carbohydrates around a protein surface.

Green synthesis of silver nanoparticles, its characterization, and chaperone-like activity in the aggregation inhibition of α-chymotrypsinogen A.

Consumption of silver nanoparticles (AgNPs) has been increased many folds due to its antimicrobial actions resulting in its widespread incorporation into a wide range of biomedical and consumer products. Still, enough research is needed to clearly understand the effect of these nanoparticles on the conformations of important macromolecules like proteins under different pathophysiological conditions. Pointing towards the situation, we carefully designed an in vitro study to elucidate the effect of green AgNPs on the aggregation pattern of α-chymotrypsinogen A at a human pathological body temperature. We observed that the B-AgNPs inhibited the aggregation in αCgn-A in a concentration-dependent manner showing maximum inhibition at 30 µg/ml above which the effect of aggregation inhibition was reduced as evident at 40 and 50 µg/ml concentrations of B-AgNPs. Further, in our in vitro analysis, we found that the B-AgNPs of lower sizes has potential chaperone-like activity at pathological body temperature, which can be used as a component of the drug to prevent protein aggregation after further verification in animal models.

Parallel chromatography and in situ scattering to interrogate competing protein aggregation pathways.

Protein aggregation can follow different pathways, and these can result in different net aggregation rates and kinetic profiles. α-chymotypsinogen A (aCgn) was used as a model system to quantitatively and qualitatively assess an approach that combines ex situ size-exclusion chromatography (SEC) with in situ laser scattering (LS) to monitor aggregation vs. time. Aggregation was monitored for a series of temperatures and initial dimer (ID) levels for starting conditions that were primarily (> 97%) monomer, and under initial-rate conditions (limited to low monomer conversion-less than 20% monomer mass loss), as these conditions are of most to interest to many pharmaceutical and biotechnology applications. SEC results show that modest decreases of ID levels can greatly reduce monomer loss rates, but do not affect the effective activation energy for aggregation. The normalized aggregation rates determined from LS were typically ∼ 1 order of magnitude higher than the corresponding rates from SEC. Furthermore, LS signals vs. time became variable and highly nonlinear with decreasing ID level, temperature, and/or total protein concentration. Temperature-cycling LS experiments showed this corresponded to conditions where dimer/oligomer "seeding" was suppressed, and high levels of reversible oligomers ("prenuclei") were formed prior to "nucleation" and growth of stable aggregates. In those conditions, aggregation rates inferred from LS and SEC are greatly different, as the techniques monitor different stages of the aggregation process. Overall, the results illustrate an approach for interrogating non-native protein aggregation pathways, and potential pitfalls if one relies on a single method to monitor aggregation-this holds more generally than the particular methods here.

Control of the adsorption properties of alginate - guar gum matrix functionalized with epichlorohydrin through the addition of different flexible chain polymers as toll for the chymotrypsinogen isolation.

This work addresses the obtaining and characterization of alginate-guar gum matrix, cross-linked with epichlorohydrin in the presence of different flexible chain polymers: polyvinyl alcohol, polyvinyl pyrrolidine and Pluronic® F68. These matrixes were used for the adsorption of chymotrypsinogen and showed an increasing uptake in presence of the flexible chain polymer in the sense: none < Pluronic 68 < polyvinyl pyrrolidine < polyvinyl alcohol. The adsorption process was found to follow a first order kinetics model and was not influenced by the polymer type. It was found that Freundlich model was more suitable for our data. Polyvinyl alcohol and polyvinyl pyrrolidine addition increase the adsorption capacity of the original bed due to an increment in the rigidity of the gel caused by the formation of hydrogen bound between the polysaccharides and synthetics polymers.

Adsorption characteristics of various proteins on a metal surface in the presence of an external electric potential.

The effect of the properties of a protein on its adsorption to a metal surface in the presence of external electric potential was investigated. Protein adsorption processes at different surface potentials were measured for fifteen types of proteins using an in-situ ellipsometry. The tested proteins were classified into three groups, based on the amount of protein that was adsorbed as a function of the surface potential: In First group of proteins, an increasing trend for the amount adsorbed with a more positive surface potential was found; The amount adsorbed of α-chymotrypsinogen A and ribonuclease A (Second group) were roughly constant and independent of the applied surface electric potentials; In Third group, the amount adsorbed decreased with increasing surface potential. This protein classification was correlated with the isoelectric points of the proteins (First group: ≤9.3; Second group: 9.3-10; Third group: >10). Increasing the pH positively and negatively shifted the surface potentials, allowing ß-lactoglobulin (First group) and lysozyme (Third) to become adsorbed, respectively. The surface potential range for protein adsorption was also markedly shifted depending on the metal substrate type. These findings were interpreted based on the electrostatic interactions among the protein, surface hydroxyl groups, and the applied external electric field.

Effect of different salinities on gene expression and activity of digestive enzymes in the thick-lipped grey mullet (Chelon labrosus).

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture.

Chymotrypsinogen C Genetic Variants, Including c.180TT, Are Strongly Associated With Chronic Pancreatitis in Pediatric Patients.

OBJECTIVES: Genetic studies in adults/adolescent patients with chronic pancreatitis (CP) identified chymotrypsinogen C (CTRC) genetic variants but their association with CP risk has been difficult to replicate. To evaluate the risk of CP associated with CTRC variants in CP pediatric patients-control study. METHODS: The distribution of CTRC variants in CP pediatric cohort (n = 136, median age at CP onset 8 years) with no history of alcohol/smoking abuse was compared with controls (n = 401, median age 45). RESULTS: We showed that p.Arg254Trp (4.6%) and p.Lys247_Arg254del (5.3%) heterozygous mutations are frequent and significantly associated with CP risk in pediatric patients (odds ratio [OR] = 19.1; 95% CI 2.8-160; P = 0.001 and OR = 5.5; 95% CI 1.6-19.4; P = 0.001, respectively). For the first time, we demonstrated that the c.180TT genotype of common p.Gly60Gly variant is strong, an independent CP risk factor (OR = 23; 95% CI 7.7-70; P < 0.001) with effect size comparable to p.Arg254Trp mutation. The other novel observation is that common c.493+51C>A variant, both CA and AA genotype, is significantly underrepresented in CP compared with controls (15% vs 35%; OR = 0.33; 95% CI 0.19-0.59; P < 0.001 and 2.8% vs 11%; OR = 0.24; 95% CI 0.06-0.85; P = 0.027, respectively). CONCLUSIONS: Our study provides evidence that CTRC variants, including c.180TT (p.Gly60Gly) are strong CP risk factors. The c.493+51C>A variant may play a protective role against CP development.