Assessment of perfluorooctanoic acid toxicity in pancreatic cells.

Perfluorooctanoic acid (PFOA) was classified as a possible carcinogen for humans (Group 2B). The in vivo studies have reported that PFOA might lead to hepatic, testicular and pancreatic toxicities and cancers. However, its mechanisms in pancreatic tissue are still unclear and insufficiently discussed. Since inflammation is the most important mechanism leading to pancreatitis and ultimately cancer, we aimed to investigate the role of inflammation in PFOA-induced pancreatic toxicity. To this end, the effect of PFOA on cell viability, apoptosis, oxidative stress and inflammatory pathways, as well as levels of trypsin and chymotrypsin were assessed in the human pancreatic cell line (PANC-1). PFOA caused cell death in concentration dependent manner (IC50 195.6 µM), apoptosis appears to be the major cell death pathway. A significant increase in trypsin and chymotrypsin levels was detected in PANC-1 cells. Oxidative stress parameters and gene expression level-related inflammation were significantly altered with PFOA exposure. These results indicate oxidative stress plays a role in PFOA-induced pancreatic toxicity and highlight the incidence of inflammation with PFOA exposure. However, this data is preliminary. Advanced in vivo and in vitro mechanistic studies should be conducted in order to better understand the inflammation-induced oxidative stress role in the toxicity of PFOA.

Purification and identification of angiotensin I-converting enzyme-inhibitory peptides from apalbumin 2 during simulated gastrointestinal digestion.

BACKGROUND: Bee larvae are considered to be an important reservoir for proteins. However, little attention has been paid to the release of potential bioactive peptides from bee larva proteins. In this study the major protein in bee larvae was hydrolyzed in vitro by gastrointestinal enzymes. The peptide profile of the hydrolysis was characterized by gel filtration chromatography and tricine-SDS-PAGE. Furthermore, the bioactive peptide was isolated and identified by Q-TOF-MS/MS. RESULTS: The major bee larva protein was identified as apalbumin 2 and was more digestible into peptides with molecular weights lower than 3 kDa. The hydrolysate obtained after 3 h of digestion exhibited angiotensin I-converting enzyme (ACE)-inhibitory activity and was purified sequentially by gel filtration and RP-HPLC. The molecular weights of peptide fractions with ACE-inhibitory activity were distributed between 0.5 and 1.5 kDa. A novel peptide with highest ACE-inhibitory activity (IC50 54.9 µmol L(-1) ) was purified by further RP-HPLC. The amino acid sequence of this peptide was identified as LLKPY (632.40 Da). CONCLUSION: ACE-inhibitory peptides could be formed from bee larvae through gastrointestinal digestion. The most active peptide (LLKPY) is potentially useful as a therapeutic agent in treating hypertension.

Pancreatic function assessment.

Several non invasive tests are available to assess pancreatic function, but no one is routinely used in clinical practice to diagnose chronic pancreatitis, due to their poor sensitivity in diagnosing mild pancreatic insufficiency. (13)C breath tests share the same limits of the other non invasive functional tests, but the mixed triglyceride breath test seems to be useful in finding the correct dosage of enzyme substitutive therapy to prevent malnutrition in patients with known pancreatic insufficiency.

Simulated gastrointestinal digestion of Pru ar 3 apricot allergen: assessment of allergen resistance and characterization of the peptides by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry.

RATIONALE: Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. METHODS: Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4)HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. RESULTS: The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. CONCLUSIONS: An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology.

Assessment of proteasome impairment and accumulation/aggregation of ubiquitinated proteins in neuronal cultures.

The ubiquitin/proteasome pathway (UPP) is the major proteolytic quality control system in cells and involves tightly regulated removal of unwanted proteins and retention of those that are essential. In addition to its function in normal protein degradation, the UPP plays a critical role in the quality control process by degrading mutated or abnormally folded proteins. The proteolytic component of the UPP is a multiprotein complex known as the proteasome. Many factors, including the aging process, can cause proteasome impairment leading to formation of abnormal ubiquitin-protein aggregates that are found in most progressive neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. In this chapter, we describe protocols to measure proteasome activity, evaluate its state of assembly, and assess the accumulation and aggregation of ubiquitinated proteins in two types of neuronal cultures: human neuroblastoma cells and rat primary cortical cultures. These protocols can be used with different types of neuronal cultures to estimate proteasome activity and the levels and aggregation of ubiquitinated proteins. In addition, they can be used to identify compounds potentially capable of preventing a decline in proteasome activity and formation of ubiquitin-protein aggregates associated with neurodegeneration.

Quantitative assessment of multi-laboratory reproducibility of SDS-PAGE assays: Digestion pattern of beta-casein and beta-lactoglobulin under simulated conditions.

A digestion protocol was applied in triplicate by ten laboratories, simulating in vivo gastric and duodenal conditions. The intra- and inter-laboratory variability in the kinetics of protein degradation was quantified, focussing on the digestion of beta-casein under gastric conditions, and of beta-lactoglobulin (beta-Lg) under duodenal conditions. The addition of surfactants such as phosphatidylcholine (PC) in the digestion mix was also evaluated. Identification and quantification of peptide bands on SDS-PAGE gels formed the basis for analysis. An average intensity loss of 69% (SD=13.5) at 5 min (89% at 10 min, with SD=5.5) was observed for beta-casein, whereas the beta-Lg duodenal digestion showed an 82% loss at 30 min (SD=14.2). Constant rates of first-order reactions showed that for fast reactions, inaccuracies in the time of first sampling contributed to the variability, which were also affected by image quality, saturation, and the splitting of time courses across gels. Breakdown products for beta-casein included ten other polypeptides, with four detected in all and two in most gels, and for beta-Lg ten polypeptides, with five detected in most, and two in two-third of the cases. Addition of PC in the gastric phase led to beta-Lg intensity loss only a quarter as large as without PC and altered beta-Lg proteolysis in the duodenal compartment.

Assessment of enzymatic efficiency on protein digestion in the tilapia Oreochromis niloticus.

The present study develops an experimental procedure aimed to estimate the efficiency of protein digestion in fish by measuring both gut transit rate and total amount of the main intestinal proteases (trypsin and chymotrypsin). The selected species was the Nile tilapia (Oreochromis niloticus). Total time for digestion, calculated through the estimation of gut transit rate using differently colored feeds, was 7.15 h. Mean production of trypsin and chymotrypsin was 15.94 and 24.11 mU in the proximal intestine and much lower (2,39, 4.90 mU) in the distal intestine. The enzyme efficiency, calculated from the average enzyme activity and time of residence of the digesta in each intestinal section, points to the major role of proximal intestine in protein digestion for this species. Results are discussed in relation to the main features characterizing digestion in stomachless fish.

Assessment of the direct and indirect effects of MPP+ and dopamine on the human proteasome: implications for Parkinson's disease aetiology.

Mitochondrial impairment, glutathione depletion and oxidative stress have been implicated in the pathogenesis of Parkinson's disease (PD), linked recently to proteasomal dysfunction. Our study analysed how these factors influence the various activities of the proteasome in human SH-SY5Y neuroblastoma cells treated with the PD mimetics MPP+ (a complex 1 inhibitor) or dopamine. Treatment with these toxins led to dose- and time-dependent reductions in ATP and glutathione and also chymotrypsin-like and post-acidic like activities; trypsin-like activity was unaffected. Antioxidants blocked the effects of dopamine, but not MPP+, suggesting that oxidative stress was more important in the dopamine-mediated effects. With MPP+, ATP depletion was a prerequisite for loss of proteasomal activity. Thus in a dopaminergic neuron with complex 1 dysfunction both oxidative stress and ATP depletion will contribute independently to loss of proteasomal function. We show for the first time that addition of MPP+ or dopamine to purified samples of the human 20S proteasome also reduced proteasomal activities; with dopamine being most damaging. As with toxin-treated cells, chymotrypsin-like activity was most sensitive and trypsin-like activity the least sensitive. The observed differential sensitivity of the various proteasomal activities to PD mimetics is novel and its significance needs further study in human cells.

Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design.

The results of a protein design project are used to compare different predictive strategies with respect to protein-protein interactions. We have been able to generate variants of human pancreatic secretory trypsin inhibitor (hPSTI) optimized with respect to the affinity and specificity for human leukocyte elastase relative to trypsin and chymotrypsin, and in particular chymotrypsin. The extremely strong and specific human leukocyte elastase inhibitors were thus developed in three rounds of mutagenesis and two rounds of 3-D modelling; only 24 variants in total were synthesized, although variations at seven different amino acid positions were involved (i.e. from 20(7) possible variants). An excellent elastase inhibitor could be designed with the minimum of two amino acid exchanges. The value of structural modelling and actual structure determination is discussed in the light of the experimental results of the designed protein variants and the results of tertiary structure determinations of the free variant and the inhibitor-protease complex. Particular reference is given to the strategy to be followed in protein design projects in general and to the development of protease inhibitors in particular.

Solid-state NMR assessment of enzyme active center structure under nonaqueous conditions.

By using solid-state NMR spectroscopy, the integrity of the active center of alpha-chymotrypsin was investigated under a variety of nonaqueous conditions. Specifically, 13C cross-polarization/magic angle spinning NMR was used to analyze the ability of alpha-chymotrypsin to stabilize a transition state intermediate analog after freezing, drying, and addition of organic solvents (both anhydrous and hydrated) to the resultant powder. Lyophilization disrupted 42 +/- 5% of the active centers; it was determined that this occurred during drying, as opposed to freezing. Seven anhydrous solvents caused 0-50% additional disruption, which occurred immediately on addition of the solvent to the enzyme powder. The extent of structural integrity loss correlated with the solvent hydrophobicity, indicating that further dehydration, i.e. stripping of water retained by the enzyme during lyophilization, was the cause. Enzyme samples prepared with lyoprotecting additives, sucrose and ammonium sulfate, exhibited varying degrees of stabilization against the drying step of lyophilization. Moreover, when hydrophilic anhydrous solvents, which had the highest propensity to strip bound water, were added to the resultant enzyme powders, no additional damage occurred.

Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: assessment of binding specificity.

An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding of S. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4 +/- 3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bind S. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74 +/- 9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.

Automated docking of substrates to proteins by simulated annealing.

The Metropolis technique of conformation searching is combined with rapid energy evaluation using molecular affinity potentials to give an efficient procedure for docking substrates to macromolecules of known structure. The procedure works well on a number of crystallographic test systems, functionally reproducing the observed binding modes of several substrates.

Assessment of titrimetric and photometric methods for the determination of chymotrypsin catalytic activity in stool.

In this report a method for the spectrophotometric measurement of stool chymotrypsin (CT) is presented and compared to the more commonly used titrimetric method. Homogenisation of specimen and dissolution of CT is examined in detail. The advantages of the photometric method lie in the small size of sample required, and its easy adaptation to manual or automated analysis with a CT-specific amide substrate.

[Assessment of the in vitro digestibility of dietary proteins and of the degree of the breakdown of their antigenic structures by using polyenzyme systems]. / Otsenka perevarivaemosti in vitro pishchevykh belkov i stepeni rasshchepleniia ikh antigennykh struktur s ispol'zovaniem polifermentnykh sistem.

A comparative assessment was made of the digestion of bovine serum albumin (BSA), chicken ovalbumin (OVA), and casein by means of the gastric juice--duodenal contents floccular gel structures (FGS) system and a four-enzymic system including trypsin, chymotrypsin, peptidase, and bacterial protease preparations. Decomposition of the BSA and OVA antigenic structures with the use of the two systems was also studied. Significant differences in BSA and OVA digestion by the gastric juice--FGS system were detected both with respect to amino nitrogen content and to the degree of their antigenic structure decomposition, whereas no such differences were observed when the four-enzymic system was used. The systems most accurately simulating the 'proteolytic conveyer' conditions of the gastrointestinal tract are preferable for the studies. The developed method is recommended for use in comparative assessment of the nutrient protein sensitizing properties.