The adsorption of chymosin and lysozyme onto emulsion droplets and their association with casein

The proteolytic action of proteases present in cheese plays a major role in the ripening of cheese. These proteases originate from the rennet, the starter cultures and from the milk itself. The proteolysis in cheese results in the degradation of the casein proteins into smaller peptides and free amino acids, which act as flavour precursors. The ripening of cheese under conditioned storage is time consuming and costly. Addition of specific enzymes to the cheese milk is one of several options to accelerate ripening. A major problem then is that hardly any of these proteases end up in the cheese and most disappear with the whey stream. Entrapment of bacteria and milk fat globules into the casein matrix of the curd is due to their particle character. Immobilisation of proteases onto particles would thus result in retention of these proteins in the curd. Ideally, for reasons of acceptance, these particles should originate from the milk itself or at least be edible.In this study, in a more general approach, soya oil emulsion droplets and casein micelles, being protein aggregates, were tested as the carrier system. Chymosin and lysozyme were taken as the enzymes to be immobilized, because of their relevance to the dairy industry and because they are scientifically well-known. Moreover, their biochemical divergence make them suitable models for study.The literature provides several studies on adsorption of proteins onto interfaces. Few of these proteins are enzymes. In cases where lysozyme was studied, it was included because of its extraordinary properties as a protein and not because of its enzymatic activity. Apart from (phospho)lipases there is scarcely any literature that describes activity of enzymes adsorbed onto the oil/water or air/water interfaces. Proteins tend strongly to accumulate in interfaces and for that reason are said to be very surface active. This adsorption is accompanied by a conformational change of the three-dimensional structure of the protein that results in some unfolding, ranging from almost full stretching of the peptide chain to a more conserved conformation. Hydrophobic residues or patches of the protein, mostly buried inside the molecule, will tend to position themselves next to or even protrude partly into, the hydrophobic phase of an oil/water interface.The extent of conformational change depends on the conformational stability of the protein, which, in turn, depends on pH, temperature, ionic strength etc. Furthermore, the extent of unfolding will be dependent on the surface area available and on the time scale, and hence, on protein concentration. In a static condition, adsorption at the interface will be diffusion driven, whereas during emulsification the time of adsorption will be determined by convection and will be very much shorter. Consequently, conformational changes of proteins during emulsification should be smaller because full surface coverage may be reached before unfolding can occur. In the case of enzymes being adsorbed the emulsification process would therefore offer an opportunity to retain activity.The relation between the surface pressureΠi.e. the extent of surface tension decrease and the amount of protein adsorbed per unit surface area availableΓ, provides a possibility to relate the size of the adsorbed protein molecules, and thereby the extent of unfolding, to the surface load. As mentioned earlier the extent of unfolding should be less at a higherΓvalue. It has been calculated that lysozyme, an enzyme of high conformational stability, hardly unfolds at the air/water interface, even at low surface coverage. For the oil/water interface, however, a considerable increase in the radius of the protein molecules was observed at low surface load. At surface coverage of &gt; 1.5 mg.m -2, the radius remained more or less constant, indicating that substantial unfolding did not occur. Despite this rigidity, the enzyme had lost all of its enzymatic activity in situ and it even remained inactive after desorption.Apparently, conformational changes in the enzyme molecule do not necessarily become manifest in a larger size for the molecule. Chymosin, being an enzyme of smaller conformational stability, naturally lost all of its activity due to adsorption onto the oil/water interface. In experiments with the enzymes coadsorbed simultaneously with bovine serum albumin, or the one after the other, there was no retention of in situ activity. Chymosin also proved to be inactivated at the expanding air/water interface due to air incorporation, if this occurred e.g. during homogenization.During the cheese-making process enzymes like chymosin and lysozyme are retained in the curd. This retention must be due to association of the enzymes with the casein from milk. In order to adsorb the enzymes with retention of activity, the various casein fractions were used to make and stabilize a soya-oil emulsion, and the enzyme was subsequently allowed to associate with the casein. The extent of association of lysozyme with the casein fractions was in the orderα s -casein &gt;β-casein &gt;κ-casein. Only for theκ-casein stabilized emulsion, was lysozyme association dependent on pH within the range of pH 5.2 - 6.4 (greater for a lower pH). Furthermore, the association with the caseins was not dependent on temperature, indicating that hydrophobic interactions were not predominant. The same trends were found with the various caseins in solution, albeit that association withκ-casein hardly occurred. It should be kept in mind that casein adsorbed at an interface will expose other amino acid residues compared to its behaviour when free in solution. For that reason the association behaviour in the two systems may differ.Because the association varies between caseins the extent of association with lysozyme depended on the composition of the casein micelles (aggregates of many casein molecules and calcium phosphate, as occurring especially in milk). As expected, casein micelles containing a higher proportion ofκ-casein associated less with lysozyme. It was found that lysozyme did not lose activity due to association with casein adsorbed on soya oil droplets or free in solution. However, lysozyme activity was markedly reduced when the enzyme was associated with casein micelles. In this system lysozyme also associated with casein in the interior of the casein micelle. The apparent loss of activity was most probably due to internal diffusion limitation. The difference of association for the various systems was also reflected in the free equilibrium concentration at which the surface excess plateau value was reached. In the system of adsorbed caseins this value was reached at a free lysozyme concentration of about 3 _M, whereas for the micellar system this value was about 100 times higher.The association of chymosin with casein has been studied in the same three systems of casein adsorbed onto soya-oil emulsion droplets, caseins in solution and caseins aggregated in casein micelles. It appears that chymosin only associated with adsorbedκ-casein and not with adsorbedαs- orβ-casein. Preceding the association, the caseinomacropeptide part ofκ-casein is split off, followed immediately by the aggregation of the soya-oil emulsion droplets containing the remaining para-κ-casein. This coagulation behaviour is identical to the renneting of milk during the cheese-making process. The association characteristics for chymosin are also comparable. The association was strongly dependent on pH and ionic strength, and on chymosin and casein concentration. Theκ-casein stabilized emulsion has proven to be a good model system for studying chymosin retention in curd. The chymosin associated with para-κ-casein was shown to be still active on addedκ-casein or on a fluorescent small hexapeptide substrate. Consequently, the active centre of the enzyme is presumably not involved in the association with casein.The association of chymosin with caseins free in solution has also been studied. Only in a solution containingκ-casein will addition of chymosin result in protein flocculation and precipitation. This flocculation is due to splitting off the caseino-macropeptide part ofκ-casein and the consecutive aggregation of the fairly hydrophobic and almost electrically neutral para-κ-casein molecules. The precipitated protein fraction also contains associated chymosin, to an extent depending on conditions like pH, ionic strength and casein and chymosin concentrations. In this system time and temperature also affected the extent of chymosin association. The association decreased with increased contact time and was stronger at higher temperatures.The protein content in the supernatant after centrifugation increased not only due to dissociation of chymosin but also due to the presence of casein fragments. Apparently, the dissociation of chymosin was related to its proteolytic action. The dissociation rate increased with decreasing pH where chymosin becomes more active and less specific. The dissociation also increased with temperature for a given time of contact. However, when extrapolated to a contact time of t = 0 (i.e. when dissociation due to proteolysis has not occurred yet) the association was observed to be somewhat stronger for a higher temperature. The effect of temperature on the proteolysis-dependent dissociation, apparently was stronger than its effect on the increase of the association. Since chymosin association depends on mutual association of caseins (see below), it will also depend on the temperature dependence of the latter. Dissociation of chymosin was not found in the system of caseins adsorbed onto emulsion droplets.The addition of small amounts ofαs- orβ-casein strongly decreased the extent of association of chymosin with para-κ-casein. This effect was stronger forαs-casein than forβ-casein. It was also found that the extent of chymosin association (moles of chymosin per mole of para-κ-casein) was larger when the system was diluted or, in other words, when the casein concentration was reduced. Both phenomena can be explained by assuming that competitive association occurs between the caseins and chymosin for interaction with a para-κ-casein molecule. Chymosin is only able to associate with a para-κ-casein molecule when that is not associated with other casein molecules. Thermodynamically speaking, the extent of association of chymosin is determined by the association constants that exist between all caseins under conditions as in the system. These association constants vary with pH, ionic strength, casein concentration and temperature.The model of competitive association is further developed and applied to the association of chymosin with casein micelles of various composition. It follows that chymosin will associate less with casein micelles composed ofαs- andκ-casein than with micelles composed ofβ- andκ-caseins. Again, this behaviour can be explained by competitive association, since different association constants exist for the caseins and chymosin for association with para-κ-casein. The relations for association and dissociation found in this casein micelle system are comparable with those found with caseins in solution. The kinetic model for competitive association is only a crude approximation. It does not provide possibilities of calculating all the association constants occurring in milk from the relations found from retention of chymosin in curd.</p

Dysrhythmias in patients with a complete atrioventricular septal defect: From surgery to early adulthood

Contains fulltext : 203679.pdf (publisher's version ) (Open Access

Male–female differences in quality of life and coping style in patients with Marfan syndrome and hereditary thoracic aortic diseases

Hereditary thoracic aortic diseases (HTAD) such as Marfan syndrome (MFS) affect multiple organ systems and provide a risk of acute aortic dissection, which causes lifelong uncertainties. Although health-related quality of life (HRQOL) was found to be reduced in HTAD patients, no studies have evaluated male–female-specific aspects of HRQOL and coping in this population. This study aims to evaluate HRQOL in HTAD patients compared to the general population; assess male–female differences in HRQOL and factors associated with HRQOL; evaluate coping styles in male and female HTAD patients and identify factors associated with acceptance. All consecutive adult patients who visited the specialized HTAD outpatient clinic between 2013 and 2018 were asked to complete three HRQOL questionnaires: the Short Form 36 (SF-36), the Hospital Anxiety and Depression Scale (HADS), and the Nijmegen Clinical Screening Instrument (NCSI). In total, 142 patients were included (mean age 42.1 years, 65 females, 123 MFS). Compared to the general population, HTAD patients scored significantly lower on multiple SF-36 sub-domains (males: General Health 54.5 ± 18.8 vs. 71.6 ± 20.6, p <.001; Vitality 58.3 ± 20.4 vs. 71.9 ± 18.3, p <.001; females: Physical Functioning 67.5 ± 23.8 vs. 80.4 ± 24.2, p =.003; Role Physical 58.3 ± 45.1 vs. 73.8 ± 38.5, p =.047; General Health 49.4 ± 24.3 vs. 69.9 ± 20.6, p <.001; Social Functioning 73.5 ± 22.0 vs. 82.0 ± 23.5, p =.027). Females scored significantly lower than males on the SF-36 physical component score (41.6 [IQR 35.5–53.1] vs. 49.3 [IQR 42.3–54.6], p =.035). Males scored significantly higher on the coping style denial than females (2.75 [IQR 2.00–3.25] vs. 2.25 [IQR 1.75–3.25], p =.018). High scores on acceptance were found in 38 (26.8%) of HTAD patients, and these patients showed significantly better scores on the NCSI, SF-36, and HADS, except on NCSI Satisfaction Relationships and SF-36 Physical Functioning and Mental Health. Acceptance was associated with more medication use (beta blocker use, p =.008; angiotensin receptor blocker use, p =.003) and less hypertension (p =.001). In patients with MFS, employment was strongly associated with better scores on the NCSI. In conclusion, HTAD patients showed subnormal HRQOL, especially females. Interestingly, in both males and females factors such as employment, coping style, and disease acceptance seem more important for HRQOL than disease-related factors. This highlights the importance of genetic counseling and guidance for HTAD patients, and offers valuable leads for HRQOL improvement

Expert consensus recommendations on the cardiogenetic care for patients with thoracic aortic disease and their first-degree

Background: Thoracic aortic aneurysm (TAA) is a potentially life-threatening disorder with a strong genetic component. The number of genes implicated in TAA has increased exponentially over the last decade. Approximately 20% of patients with TAA have a positive family history. As most TAA remain asymptomatic for a long time, screening of at risk relatives is warranted to prevent complications. Existing international guidelines lack detailed instructions regarding genetic evaluation and family screening of TAA patients. We aimed to develop a consensus document to provide medical guidance for all health care professionals involved in the recognition, diagnosis and treatment of patients with thoracic aortic disease and their relatives. Methods: A multidisciplinary panel of experts including cardiologists, cardiothoracic surgeons, clinical geneticists and general practitioners, convened to review and discuss the current literature, guidelines and clinical practice on genetic testing and family screening in TAA. Results: There is a lack of high-quality evidence in the literature. This consensus statement, based on the available literature and expert opinions, summarizes our recommendations in order to standardize and optimize the cardiogenetic care for patients and families with thoracic aortic disease. In particular, we provide criteria to identify those patients most likely to have a genetic predisposition, and discuss the preferred modality and frequency of screening in their relatives. Conclusions: Age, family history, aortic size and syndromic features determine who is advised to have genetic testing as well as screening of first-degree relatives. There is a need for more prospective multicenter studies to optimize current recommendations

Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in √s = 7 TeV pp collisions with the ATLAS detector

A search for the direct production of charginos and neutralinos in final states with three electrons or muons and missing transverse momentum is presented. The analysis is based on 4.7 fb−1 of proton–proton collision data delivered by the Large Hadron Collider and recorded with the ATLAS detector. Observations are consistent with Standard Model expectations in three signal regions that are either depleted or enriched in Z-boson decays. Upper limits at 95% confidence level are set in R-parity conserving phenomenological minimal supersymmetric models and in simplified models, significantly extending previous results

Jet size dependence of single jet suppression in lead-lead collisions at sqrt(s(NN)) = 2.76 TeV with the ATLAS detector at the LHC

Measurements of inclusive jet suppression in heavy ion collisions at the LHC provide direct sensitivity to the physics of jet quenching. In a sample of lead-lead collisions at sqrt(s) = 2.76 TeV corresponding to an integrated luminosity of approximately 7 inverse microbarns, ATLAS has measured jets with a calorimeter over the pseudorapidity interval |eta| < 2.1 and over the transverse momentum range 38 < pT < 210 GeV. Jets were reconstructed using the anti-kt algorithm with values for the distance parameter that determines the nominal jet radius of R = 0.2, 0.3, 0.4 and 0.5. The centrality dependence of the jet yield is characterized by the jet "central-to-peripheral ratio," Rcp. Jet production is found to be suppressed by approximately a factor of two in the 10% most central collisions relative to peripheral collisions. Rcp varies smoothly with centrality as characterized by the number of participating nucleons. The observed suppression is only weakly dependent on jet radius and transverse momentum. These results provide the first direct measurement of inclusive jet suppression in heavy ion collisions and complement previous measurements of dijet transverse energy imbalance at the LHC.Comment: 15 pages plus author list (30 pages total), 8 figures, 2 tables, submitted to Physics Letters B. All figures including auxiliary figures are available at http://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/HION-2011-02

Measurement of the polarisation of W bosons produced with large transverse momentum in pp collisions at sqrt(s) = 7 TeV with the ATLAS experiment

This paper describes an analysis of the angular distribution of W->enu and W->munu decays, using data from pp collisions at sqrt(s) = 7 TeV recorded with the ATLAS detector at the LHC in 2010, corresponding to an integrated luminosity of about 35 pb^-1. Using the decay lepton transverse momentum and the missing transverse energy, the W decay angular distribution projected onto the transverse plane is obtained and analysed in terms of helicity fractions f0, fL and fR over two ranges of W transverse momentum (ptw): 35 < ptw < 50 GeV and ptw > 50 GeV. Good agreement is found with theoretical predictions. For ptw > 50 GeV, the values of f0 and fL-fR, averaged over charge and lepton flavour, are measured to be : f0 = 0.127 +/- 0.030 +/- 0.108 and fL-fR = 0.252 +/- 0.017 +/- 0.030, where the first uncertainties are statistical, and the second include all systematic effects.Comment: 19 pages plus author list (34 pages total), 9 figures, 11 tables, revised author list, matches European Journal of Physics C versio

Observation of a new chi_b state in radiative transitions to Upsilon(1S) and Upsilon(2S) at ATLAS

The chi_b(nP) quarkonium states are produced in proton-proton collisions at the Large Hadron Collider (LHC) at sqrt(s) = 7 TeV and recorded by the ATLAS detector. Using a data sample corresponding to an integrated luminosity of 4.4 fb^-1, these states are reconstructed through their radiative decays to Upsilon(1S,2S) with Upsilon->mu+mu-. In addition to the mass peaks corresponding to the decay modes chi_b(1P,2P)->Upsilon(1S)gamma, a new structure centered at a mass of 10.530+/-0.005 (stat.)+/-0.009 (syst.) GeV is also observed, in both the Upsilon(1S)gamma and Upsilon(2S)gamma decay modes. This is interpreted as the chi_b(3P) system.Comment: 5 pages plus author list (18 pages total), 2 figures, 1 table, corrected author list, matches final version in Physical Review Letter

Search for displaced vertices arising from decays of new heavy particles in 7 TeV pp collisions at ATLAS

We present the results of a search for new, heavy particles that decay at a significant distance from their production point into a final state containing charged hadrons in association with a high-momentum muon. The search is conducted in a pp-collision data sample with a center-of-mass energy of 7 TeV and an integrated luminosity of 33 pb^-1 collected in 2010 by the ATLAS detector operating at the Large Hadron Collider. Production of such particles is expected in various scenarios of physics beyond the standard model. We observe no signal and place limits on the production cross-section of supersymmetric particles in an R-parity-violating scenario as a function of the neutralino lifetime. Limits are presented for different squark and neutralino masses, enabling extension of the limits to a variety of other models.Comment: 8 pages plus author list (20 pages total), 8 figures, 1 table, final version to appear in Physics Letters

Measurement of the inclusive isolated prompt photon cross-section in pp collisions at sqrt(s)= 7 TeV using 35 pb-1 of ATLAS data

A measurement of the differential cross-section for the inclusive production of isolated prompt photons in pp collisions at a center-of-mass energy sqrt(s) = 7 TeV is presented. The measurement covers the pseudorapidity ranges |eta|<1.37 and 1.52<=|eta|<2.37 in the transverse energy range 45<=E_T<400GeV. The results are based on an integrated luminosity of 35 pb-1, collected with the ATLAS detector at the LHC. The yields of the signal photons are measured using a data-driven technique, based on the observed distribution of the hadronic energy in a narrow cone around the photon candidate and the photon selection criteria. The results are compared with next-to-leading order perturbative QCD calculations and found to be in good agreement over four orders of magnitude in cross-section.Comment: 7 pages plus author list (18 pages total), 2 figures, 4 tables, final version published in Physics Letters