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In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor., Graphical Abstract

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc1 complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.

Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo., Summary: Previous limitations are overcome in this first genetically tractable mouse model expressing invertebrate alternative oxidase, AOX, which can suppress pathological stresses in the mitochondrial respiratory chain.

A challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies.

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an "emergency shutdown" system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.

Made available in DSpace on 2018-11-26T16:18:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-17 Academy of Finland (CoE grant) European Research Council EU (Marie Curie International Incoming Fellowship) Tampere University Hospital Medical Research Fund Sigrid Juselius Foundation The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression. Tampere Univ, BioMediTech, FI-33014 Tampere, Finland Tampere Univ, Tampere Univ Hosp, FI-33014 Tampere, Finland Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, BR-14884900 Jaboticabal, SP, Brazil Univ Helsinki, Inst Biotechnol, FI-00014 Helsinki, Finland INSERM, UMR 1141, F-75019 Paris, France Univ Paris 07, Fac Med Denis Diderot, Hop Robert Debre, F-75019 Paris, France Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, BR-14884900 Jaboticabal, SP, Brazil Academy of Finland (CoE grant): 272376 European Research Council: 232738 EU (Marie Curie International Incoming Fellowship): 328988

The transcription factor hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the cellular response to low oxygen. HIF-1α protein accumulates in hypoxia due to inhibition of prolyl hydroxylase enzymes, which under normoxic conditions use molecular oxygen to hydroxylate HIF-1α on two conserved proline residues (Pro(402) and Pro(564)), thus targeting the protein for 26 S proteasome-dependent degradation. A functional mitochondrial electron transport chain is known to be necessary for HIF-1α stabilization in hypoxia. It has been reported that reactive oxygen species (ROS), produced under hypoxia by complex III of the mitochondrial electron transport chain, play a critical role in the stabilization of the HIF-1α protein, possibly by directly inhibiting prolyl hydroxylase enzymes. In contrast, we found that ROS production by complex III is not required for hypoxia-induced HIF-1α stabilization. Thus, reestablishing mitochondrial oxygen consumption in the presence of a complex III inhibitor by using an artificial electron donor to complex IV or by overexpressing Ciona intestinalis alternative oxidase results in HIF-1α protein stabilization in hypoxia. Furthermore, five inhibitors that target different sites of the mitochondrial electron transport chain have similar effects on the HIF-1α protein half-life in hypoxia but vary in their effects on mitochondrial ROS production. Finally, ROS do not regulate prolyl hydroxylase activity directly. We conclude that HIF-1α protein stabilization in hypoxia occurs independently of mitochondrial ROS production. However, mitochondria can modulate the cellular hypoxic response through altered respiratory activity, likely by regulating the cellular oxygen availability.

Twelve samples of cheeses (three types of Saint-Nectaire PDO cheeses and Savaron cheeses) differing by manufacturing and ripening conditions, from 12 different producers, were characterised by attenuated total reflectance mid-infrared (MIR) and front-face fluorescence spectroscopies, dynamic testing rheology and physico-chemical analysis. Fluorescence spectra (tryptophan residues, vitamin A and riboflavin) and MIR (3,000–2,800 (fat region), 1,700–1,500 (protein region) and 1,500–900 cm−1 (fingerprint region)) spectra were recorded on cheese samples. The potential of the data tables was investigated for discriminating the four different groups of cheeses. The results of factorial discriminant analysis (FDA) performed on the fluorescence and mid-infrared spectra showed a good discrimination of the four cheese groups. Considering cross-validation results, the best classifications (100%) were achieved from mid-infrared and fluorescence spectra, while only 91.7 and 72.2% of correct classification were obtained by applying FDA to rheology and physico-chemical data, respectively. Spectroscopic techniques could provide useful fingerprints and allow the identification of investigated cheeses according to manufacturing conditions. Simple and rapid spectroscopic methods offer a promising approach to the authentication of cheeses.

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1 -expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.

In plants as well as in a number of micro-organisms, and in several animal phyla, but not in mammals, the alternative oxidase (AOX) possibly by-passes the cytochrome segment of the respiratory chain. The AOX is located at the inner surface of the inner mitochondrial membrane, being activated by over-reduction of the quinone pool and accumulation of keto-acids such as pyruvate. Since these conditions are frequently encountered in patients with mitochondrial diseases, we hypothesized that the expression of the ascidian Ciona intestinalis AOX might alleviate the consequences of a blockade of the cytochrome segment of the respiratory chain in human cells. We previously expressed the C. intestinalis AOX in human embryonic kidney (HEK 293-T-derived) cells conferring cyanide-resistance to cell respiration without any detectable detrimental effect (Hakkaart et al. 2006). We have now expressed the AOX in human cultured fibroblasts either with a functional respiratory chain (foreskin immortalized fibroblasts, BJ1-htert) or presenting a cytochrome c oxidase deficiency resulting from an impaired heme aa3 biogenesis. We used immortalized COX15-deficient skin fibroblasts from a patient who died from an early fatal cardiomyopathy. We found that the expression of the AOX in these cells was well tolerated and corrected for the various consequences of the cytochrome c oxidase deficiency in COX15-mutant cells, i.e. decreased cell respiration, glucose and pyruvate dependency. We thus validated our hypothesis that AOX could compensate for cytochrome c oxidase deficiency in human cells.

Fluorescence spectroscopy is an emerging tool for the analysis of biomolecules from complex matrices. We explored the potentialities of the method for the pseudomonad taxonomic purpose at the genus and species level. Emission spectra of three intrinsic fluorophores (namely, NADH, tryptophan, and the complex of aromatic amino acids and nucleic acid) were collected from whole bacterial cells. Their comparisons were performed through principal component analysis and factorial discriminant analysis. Reference strains from the Xanthomonas, Stenotrophomonas, Burkholderia, and Pseudomonas genera were well separated, with sensitivity and selectivity higher than 90%. At the species level, P. lundensis, P. taetrolens, P. fragi, P. chlororaphis, and P. stutzeri were also well separated, in a distant group, from P. putida, P. pseudoalcaligenes, and P. fluorescens. These results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also provide additional information about strain relatedness. In the case of environmental isolates, the method allows good discrimination, even for strains for which ambiguity still remained after PCR and API 20NE identification. Rapid, easy to perform, and low cost, fluorescence spectroscopy provides substantial information on cell components. Statistical analysis of collected data allows in-depth comparison of strains. Our results strongly support the view that fluorescence spectroscopy fingerprinting can be used as a powerful tool in a polyphasic approach to pseudomonad taxonomy.

The objective of this preliminary study was to evaluate the usefulness of front face fluorescence spectroscopy to predict some chemical parameters [pH, fat, dry matter (DM), fat in DM, total nitrogen (TN) and water soluble nitrogen (WSN)] and cheese meltability of semi-hard and hard cheeses. Dynamic testing rheology was used to determine the melting point of cheeses corresponding to the temperature at which loss tangent (tan δ) = 1. Tryptophan and vitamin A fluorescence spectra were, also, recorded on cheese samples at 20 °C. The partial least squares (PLS) regression with the leave one-out cross-validation technique was used to build up calibration models. Excellent predictions were obtained from the tryptophan and vitamin A models for fat (R 2 = 0.99 and 0.97, respectively), DM (R 2 = 0.94 and 0.96, respectively), fat in DM (R 2 = 0.92 and 0.99, respectively), TN (R 2 = 0.91 and 0.91, respectively). Excellent predictions were also obtained for WSN (R 2 = 0.96) and melting point (R 2 = 0.97) from vitamin A spectra, while only good predictions for these two parameters (R 2 = 0.90 and R 2 = 0.87, respectively) were obtained from tryptophan spectra. The results for pH were good (R 2 = 0.82) and approximate (R 2 = 0.76) with tryptophan and vitamin A, respectively.

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by addingp-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

Ten traditional M1 (n = 5) and M2 (n = 5) soft cheeses produced from raw milk, and five other stabilised M3 (n = 5) cheeses manufactured from pasteurised milk, were studied using mid infrared (MIR) and front face fluorescence (FFFS) spectroscopies. MIR (3000–900 cm−1), tryptophan (excitation: 290 nm, emission: 305-450 nm), 400-640 emission spectra (excitation: 380 nm) and vitamin A (excitation: 280–350 nm, emission: 410 nm) spectra were recorded at two sampling zones (external (E) and central (C)) of the investigated cheeses. When the factorial discriminant analysis (FDA) was applied to the MIR spectra, the classification was not satisfactory. With tryptophan fluorescence spectra, correct classification of 94.4 and 69.4% was observed for the calibration and validation spectra, respectively. Better classification was obtained using vitamin A fluorescence spectra, since 91.8 and 80.6% of the calibration and validation spectra, respectively, were correctly classified. When the first five principal components (PCs) of the PCA extracted from each data set were pooled into a single matrix and analysed by FDA, the classification was considerably improved, obtaining a percentage of correct classification of 100 and 91.7% for the calibration and validation samples, respectively. It was concluded that concatenation of the physico-chemical and spectroscopic data sets is an efficient technique for the identification of soft cheese varieties.

Near infrared (NIR) spectroscopy was used to predict colour of European Emmental cheese samples. Colour values (L, a and b) were measured on 20 Emmental cheese samples using a Hunter-lab D25-D-2 optical head in the system according to Hunter to determine L (brightness), a (green-red component) and b (blue-yellow component). The diffuse reflectance of the investigated cheeses was also determined by a Buchi NIR Lab N-200 spectrometer using a rotating measuring cell in the range of 1000–2500 nm. The best results for L-value (squared correlation coefficient (R2) = 0.56, root mean square error of cross-validation (RMSECV) = 0.76, ratio of prediction deviation (RPD) = 1.89 and range error ratio (RER) = 7.91), a-value (R2 = 0.72, RMSECV = 0.15, RPD = 1.98 and RER = 7.6) and b-value (R2 = 0.82, RMSECV = 0.52, RPD = 2.56 and RER = 9.42) were obtained when the first 12 principal components (PCs) of the principal component analysis (PCA) applied on normalised NIR spectra were used. It can be concluded that NIR spectroscopy could be used to predict b-value. The a- and L-values can also be predicted from NIR technique with approximate quantitative prediction.

Twelve semi-hard (Raclette) cheeses, belonging to four brand products, namely A ( n = 3), B ( n = 3), C ( n = 3) and D ( n = 3), were produced during summer period and ripened at an industrial scale. Tryptophan, riboflavin and vitamin A fluorescence spectra were scanned on the 12 cheeses at 2, 30 and 60 days of ripening. The physico-chemical analyses were performed only at the end of the ripening stage (60 days). Common components and specific weights analysis (CCSWA) were applied on the four data tables. CCSWA showed that the common component 1 ( q 1 ), discriminating cheeses labelled A, B and C from those labelled D, expressed 94.4 and 59% of the inertia of vitamin A and tryptophan fluorescence spectra and a less amount for riboflavin fluorescence spectra and physico-chemical data (24.2 and 13.2%, respectively). Common component 3 ( q 3 ), differentiating between cheeses labelled B and those labelled A and C, explained 34.6 and 23.9% of the inertia of the physico-chemical data and tryptophan fluorescence spectra, respectively, and a tiny part of the inertia of riboflavin and vitamin A fluorescence spectra (3.2 and 0.7%, respectively). The CCSWA showed its ability to describe the overall information collected from fluorescence and physico-chemical data tables and to extract relevant information at the molecular level throughout ripening of the investigated semi-hard cheeses.

Front-face fluorescence spectroscopy was used for the first time to study the interactions between bovine beta-lactoglobulin variant A (beta-Lg A) and various beta-Lg-derived bioactive peptides. Fluorescence spectra were recorded for beta-Lg A-peptide mixtures at 25 degrees C and pH 6.8 with an excitation wavelength of 290 nm to characterize the molecular environment of tryptophan (Trp) residues present in the protein but absent in the peptides. Spectra remained unchanged following addition of peptides beta-Lg f92-100 and beta-Lg f125-135, while Phe-Phe interaction between beta-Lg f69-83 molecules interfered with analysis. Addition of beta-Lg f102-105 produced a blue shift (3 nm) and a significant increase in fluorescence intensity, while addition of beta-Lg f142-148 also caused a significant increase in fluorescence intensity but accompanied by a red shift (3 nm). These results indicate that the polarity of the Trp environment in the beta-Lg A structure may be modified differently depending on the peptide added.

The potential of front-face fluorescence spectroscopy combined with chemometric methods was investigated for discriminating different wines according to variety, typicality and vintage. A total of 120 wines produced in France and Germany were investigated. Emission (275–450 nm) and excitation (250–350 nm) fluorescence spectra of phenolic compounds were recorded directly on the wine samples using a front-face fluorescence accessory. The emission spectra were characterised by a maximum at 376 nm and a shoulder at 315 nm and the excitation spectra showed two peaks located at about 260 and 320 nm. The shape of the spectra changed with wine samples, varying mainly in the maximum/shoulder intensity. Wine samples were evaluated using principal component analysis (PCA) and classified by factorial discriminant analysis (FDA). PCA performed on the whole collection of excitation spectra allowed a good discrimination between French and German wines. Using FDA, correct classification of typical and non-typical Beaujolais amounting to 95% was observed for the emission fluorescence data set. These results showed that fluorescence spectroscopic technique may provide useful fingerprints and mainly allow the identification of wines according to variety and typicality.

There is a strong tendency towards exploring rapid and low cost methods for determining chemical parameters and degree of the ripening of cheeses. The visible-near infrared (VIS-NIR), mid infrared (MIR) and combination of VIS-NIR and MIR spectroscopic methods for measurements of some selected parameters of soft cheeses were compared. Fifteen traditional and stabilised retail soft cheeses, differing in manufacturing process were studied. Fat, dry matter (DM), pH, total nitrogen (TN) and water soluble nitrogen (WSN) contents were determined by reference methods and scanned with VIS-NIR and MIR spectrophotometers in reflectance mode. Three separate prediction models were developed from the VIS-NIR, MIR and the joint VIS-NIR-MIR spectra using the partial least square (PLS) regression and leave one-out cross-validation technique. Results showed that fat, DM, TN and WSN were the best predicted with the VIS-NIR models providing the lowest values of the root mean square error of prediction (RMSEP) of 1.32, 0.70, 0.11 and 0.10, respectively. The combination of the VIS-NIR and MIR spectral improved slightly the prediction of only the pH. This suggests using the VIS-NIR for the determination of fat, DM, TN and WSN. The pH can also be predicted from the two techniques with approximate quantitative prediction, while a difference between low and high levels of WSN/TN ratio could be determined by the VIS-NIR, MIR or joint use of VIS-NIR-MIR.

The determination of winter cheese chemical properties, namely, fat, sodium chloride (NaCl), pH, non protein nitrogen (NPN), total nitrogen (TN) and water soluble nitrogen (WSN) was done using spectroscopic technologies with different wavelength zones. The Emmental cheeses provided from different European countries were studied. A total of 91 cheeses produced during the winter time in Austria (n=4), Finland (n=6), Germany (n=13), France (n=30) and Switzerland (n=38) were analysed by near infrared (NIR) and mid infrared (MIR) spectroscopies. The combination of these two spectral regions (sum of their spectra) was also studied. The partial least square (PLS) regression with the leave one-out cross validation technique was used to build up calibration models using data set designated as calibration set. These models were validated with another data set designated as validation set. The obtained results suggest the use of the NIR for the determination of fat and TN contents, and the MIR for NaCl and NPN contents as well as for the pH. Similar results were obtained for WSN using the two techniques together. The combined spectra of both NIR and MIR did improve the results, while providing comparable results to those obtained from either the NIR or MIR spectroscopy.

The main objective of this study was to characterise the know-how diversity of small-scale facilities manufacturing traditional dry sausages and to investigate the relations between the know-how and the sensory diversity of the traditional dry sausages. First, a description of the know-how (typology) was observed on 108 out of 129 registered small-scale facilities in the Massif Central. The typology led to six groups with specific know-how differentiated mainly by the raw materials and drying. From these 6 groups, 12 small-scale facilities were selected (2 per group of know-how) in order to perform a Flash profile on the manufactured dry sausages with a panel of six experts. The Flash profile results showed discrimination between the dry sausages by the raw materials used. In addition, the canonical correlation analysis (CCA) showed a high correlation between the typology data and the sensory data. The similarity map 1–2 from the CCA illustrated the proximity between dry sausages from the same group of know-how and discriminated between dry sausages from different groups of know-how. Thus, it can be concluded that each group of know-how corresponded to traditional dry sausages with specific sensory characteristics.

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.

This study examines the feasibility of using the mid infrared (MIR) spectroscopy for the determination of some parameters in European Emmental cheeses produced in summer from different geographic origins. A total of 72 Emmental cheeses (4 samples from Finland, 6 samples from Germany, 8 samples from Austria, 27 samples from France and 27 samples from Switzerland) were investigated. Total nitrogen (TN), water soluble nitrogen (WSN), non protein nitrogen (NPN), sodium chloride (NaCl) and pH were analysed by the reference methods. The MIR-transmission of the investigated cheeses was measured by a Nicolet Magna 750 IR spectrophotometer in a measurement range between 4000 and 400 cm−1.

Three different fluorescence spectra were recorded following excitation at 250 nm (aromatic amino acids+nucleic acids, AAA+NA), 316 nm (NADH) and 380 nm (FAD) for 20 type strain collections of lactic acid bacteria (LAB). Evaluation of the data using principal component analysis and factorial discriminant analysis showed a good discrimination of considered LAB at the genus, species and genus-species level. AAA+NA fluorophores showed the highest percentage of good classification. From AAA+NA spectra recorded on LAB isolated from a small-scale facility producing traditional dry sausages, we succeeded to identify 28 of 29 wild strains. This method allowed us to discriminate between Lactobacillus sakei subsp. carnosus and Lactobacillus sakei subsp. sakei. Thus, intrinsic fluorescence is an economical and powerful tool for the identification of wild LAB isolated from meat and meat products.

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long-lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.

The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.

The tricarboxylic acid cycle enzyme fumarate hydratase (FH) has been identified as a tumor suppressor in a subset of human renal cell carcinomas. Human FH-deficient cancer cells display high fumarate concentration and ROS levels along with activation of HIF-1. The underlying mechanisms by which FH loss increases ROS and HIF-1 are not fully understood. Here, we report that glutamine-dependent oxidative citric acid cycle metabolism is required to generate fumarate and increase ROS and HIF-1 levels. Accumulated fumarate directly bonds the antioxidant glutathione in vitro and in vivo to produce the metabolite succinated glutathione (GSF). GSF acts as an alternative substrate to glutathione reductase to decrease NADPH levels and enhance mitochondrial ROS and HIF-1 activation. Increased ROS also correlates with hypermethylation of histones in these cells. Thus, fumarate serves as a proto-oncometabolite by binding to glutathione which results in the accumulation of ROS.

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects., Author Summary In mammalian mitochondria, the energy-producing machinery is powered by the electron transfer to molecular oxygen, a mechanism whose terminal step is mediated by the cyanide-sensitive cytochrome c oxidase (COX). In plants, fungi, microorganisms, and some lower animals (but not in mammals), in addition to the normal pathway, a cyanide-resistant alternative oxidase (AOX) exists. It maintains electron transfer to oxygen even when the normal pathway is blocked. This provides a bypath that releases constraints on the energy producing machinery and prevents the production of deleterious superoxide molecules. Thus, preventing the energy producing machinery blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that the AOX can be safely expressed in a mammal with major physiological and molecular parameters being unaffected. We also show that the AOX is active in vivo where it counteracts the energy producing machinery blockade and reduces in vitro the associated oxidative insult. Up to now, efficient therapies against mitochondrial-associated diseases are lacking dramatically. Therefore, in view of our results, the MitAOX mice represent a precious tool to assess the AOX therapeutic capacity against the panoply of inherited mitochondrial diseases.

Hydrolysis of β-lactoglobulin with thermolysin and pepsin at pressures ranging between 0.1 and 350 MPa showed a significant increase of cleavage rates. Pressure-induced changes of susceptibility to hydrolysis of β-lactoglobulin proteolytic sites were also observed. The pressure, raised to 200 MPa, accelerates the hydrolysis of β-lactoglobulin by thermolysin and changes obtained peptide profiles. Initially, higher pressure makes the N-terminal, and to a smaller extent, C-terminal peptide fragments of β-lactoglobulin molecule, more susceptible to removal by thermolysin. This indicates combined influence of pressure-induced thermolysin activation and partial unfolding of β-lactoglobulin by compression at neutral pHs. The rates of hydrolysis of β-lactoglobulin by pepsin (negligible at 0.1 MPa) are increased considerably with pressure up to 300 MPa. The Susceptibility of β-lactoglobulin proteolytic sites to peptic cleavage remains constant over all the studied pressure range. The lack of significant qualitative changes in the peptic peptide profiles produced at different pressures and at clearly pressure-dependent rates points to negative reaction volume changes as the major factor in peptic hydrolysis of β-lactoglobulin under high pressure. Thus the β-lactoglobulin molecule resists pressure-induced unfolding in acid pHs and yields to it in neutral pHs. © 1995 John Wiley & Sons, Inc.

Bovine β-lactoglobulin variant B was hydrolysed with thermolysin at various concentrations of calcium ions ranging between 0 and 50 m m . The changes in calcium concentration did not influence the overall rates of β-lactoglobulin proteolysis. However, HPLC analyses of the hydrolysates, obtained at nine different calcium concentrations, indicated that the rates of production and/or lysis of 3 of 17 identified peptides were calcium concentration-dependent. Proteolysis at low calcium concentrations yielded peptides V41-E45 and V43-L57. The peptide V43-L57, containing a cluster of dicarboxylic amino acids, was resistant to further hydrolysis by thermolysin even after 10 h incubation. An increase in calcium concentration triggered gradual hydrolysis of this peptide. Its hydrolytic product (peptide V43-D53) was obtained only by proteolysis of β-lactoglobulin with thermolysin at high calcium concentration.

International audience; The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10–7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10–7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.

The secondary structure transformation of beta-lactoglobulin from a predominantly beta-structure into a predominantly alpha-helical one, under the influence of solvent polarity changes is reversible. Independent of the alcohol used--methanol, ethanol, or 2-propanol--the midpoints of the observed structural transformation occur around dielectric constant epsilon approximately 60. The structural change destroying the hydrophobic core formed by the beta-barrel structure leads, at room temperature, to the dissociation of the retinol/beta-lactoglobulin complex in the neighborhood of dielectric constant epsilon approximately 50. However, when the dielectric constant of the medium is raised back to epsilon approximately 70 by the decrease of the temperature, both the refolding of BLG into a beta-structure and the reassociation of the retinol/beta-lactoglobulin complex are observed. The esterification of beta-lactoglobulin carboxyl groups has two effects: on the one hand it accelerates the beta-strand alpha-helix transition induced by alcohols. On the other hand, the esterification of beta-lactoglobulin strengthens its interaction with retinol as it may be deduced from the smaller apparent dissociation constant of retinol/methylated beta-lactoglobulin complex. The binding of retinol to modified or unmodified beta-lactoglobulin has no influence (stabilizing or destabilizing) on the folding changes induced by alcohol.

International audience; In this study, the potential of synchronous front-face fluorescence spectroscopy (SFS) coupled with chemometrics was investigated for the determination of heterocyclic aromatic amines (HAA) in cooked meat samples. Bovine meat samples (1-2 mm thick, 5 cm diameter) taken from Longissimus thoracis muscle were cooked at an average temperature of 237 A degrees C for 5, 7 and 10 min. Four HAA (4, 8-DiMeIQx, MeIQx, IQx and PhIP) were determined on the cooked meat samples using classical LC-APCI-MS/MS method. In parallel, SFS spectra were recorded using a spectrofluorimeter on the same cooked meat samples in an excitation wavelength range of 250-550 nm using offsets of 20, 30, 40, aEuro broken vertical bar, 160 nm between excitation and emission monochromators. The three-dimensional synchronous fluorescence data set was analyzed using PARAFAC (parallel factor) analysis and N-PLS (n-way partial least square) regression method. PARAFAC analysis allowed capturing the fluorescence changes occurring in meat during cooking: the best model was obtained with 2 components (core consistency of 100% and explained variance of 99.2%). Whereas the loading profile of component 1 showed a maximum excitation at about 495 nm and an optimal offset of 60 nm, the loading profile of component 2 was characterized by a maximum excitation at 367 nm and an optimal offset of 90 nm. The results obtained using N-PLS regression showed good correlation between the spectral and analytical data: average recovery of 104% for 4, 8-DiMelQx, 102% for both MelQx and IQx, and 103% for PhIP were obtained. In conclusion this study indicates that SFS along with chemometrics has the potential to be used as a rapid and non-destructive technique for the determination of HAA contents in meat.

The binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid to native, esterified, and alkylated beta-lactoglobulin was followed by enhancement of the ligand fluorescence. Three studied ligands bind to native or modified beta-lactoglobulin in apparent molar ratios varying between 1/8 and 2/1, with apparent dissociation constants in the range of 10(-8) M for ligand/beta-lactoglobulin complexes. The studied, chemically modified beta-lactoglobulin derivatives display higher binding affinities for all studied ligands, cis-parinaric acid excluded. The reductive alkylation of epsilon-NH2 lysyl residues of beta-lactoglobulin increases the apparent molar ratios of benzo(a)pyrene and cis-parinaric acid, and decreases it for ellipticine. The esterified and native beta-lactoglobulin complexed to the investigated ligands display similar stoichiometries. Dynamic light scattering study of ligand-beta-lactoglobulin complexes in solution shows the formation of aggregates: the apparent hydrodynamic radius value of beta-lactoglobulin dimer (3.4 nm) reaches 49, 46, and 74 nm upon addition and binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid, respectively.

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.

The attempt to use trypsin in order to monitor pH (7.5-9.0) induced beta-lactoglobulin conformation changes has revealed differences in the cleavage of specific sites. The tryptic cleavage of two dibasic X-Lys-Lys-Y sites (Lys 69, 70 and 100, 101) shows slighter predominance of symmetrical cut at pH 7.5 and 8.0. Mostly asymmetrical cleavage yielding two C-terminal lysines can be observed at pH 8.5 and 9.0. Atypical cleavage of the Tyr-20-Ser-21 site, which at pH 9.0 is relatively negligible, increases substantially in pH 7.5-8.5. This implies that Tyr-20 probably is the tyrosine reported to be exposed on the surface of the protein during transformation of beta-lactoglobulin molecule occurring in the studied pH range (Tanford et al. (1959) J. Am. Chem. Soc. 81, 4032-4036).

The present study was conducted to determine whether multispectral imagery combined with chemometrics could accurately distinguish and classify different blue cheeses. The images of the pre-packed PDO Bleu d'Auvergne (n = 12) and Fourme d'Ambert (n = 23) blue cheeses were acquired using a custom-design multispectral imager. The image acquisition was conducted in the ultraviolet (360 nm, 370 nm and 400 nm), visible (470 nm, 568 nm and 625 nm) and near- infrared (875 nm and 950 nm) spectral regions. The spectral functions of image texture based on the Fourier spectrum and image weights were extracted from the raw multivariate images using an image processing tool and a method of simultaneous decomposition of covariance matrices, respectively. Principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA) of the spectrum functions showed a reliable discrimination of the Bleu d'Auvergne and Fourme d'Ambert blue cheeses. Examination of the image weights using PLSDA allowed the pre- diction of the producers of the blue cheeses. Our data demonstrated the ability of the multispectral imagery combined with chemometrics to characterize the quality and identity of the blue cheeses in a rapid and inexpensive manner. blue cheese / characterization / multispectral imagery / image texture / chemometrics

International audience; The potential of front-face spectroscopy for grape ripening dates discrimination was investigated on Cabernet Franc grapes from three parcels located on the Loire Valley and for six ripening dates. The 18 batches were analysed by front-face fluorescence spectroscopy and visible spectroscopy. The excitation spectra (250–310 nm, emission wavelength = 350 nm) were characterised by a shoulder at 280 nm. Grapes spectra were classified by factorial discriminant analysis (FDA). Ripening dates were well predicted by fluorescence spectra: grapes before veraison were separated from grapes after veraison and almost every ripening date was identified. The common spectroscopic space obtained by CCSWA showed that wavelengths corresponding to anthocyanin absorption in the visible were correlated to fluorescence wavelengths around the starting and ending points of the shoulder (263 and at 292 nm). Then, regression models were investigated to predict total soluble solids (TSS), total acidity, malvidin-3G, total anthocyanins and total phenolics content from visible and fluorescence spectra. To predict technological indicators (TSS and total acidity), the PLS model with visible spectra (RMSECV = 0.82°Brix or 0.96 g L−1 H2SO4) was better than those with fluorescence one (RMSECV = 1.39°Brix or 2.06 g L−1 H2SO4). For malvidin-3G and total anthocyanins, all R c 2 and R cv 2 were superior to 0.90 and RMSECV were low. Visible and fluorescence spectroscopies succeeded in predicting anthocyanin content. Concerning total phenolic, the best prediction was provided by fluorescence spectroscopy.

Milk samples were collected in five dairy plants located in different regions of France (North, North-West, South-West and centre of France), during spring and autumn, at receipt (bulk-raw milk), and following pasteurisation and UHT sterilisation. Corresponding UHT milks were then stored at three temperatures (4, 20 and 40 °C) and analysed after different times (21, 42, 62, 90, 110 and 180 d). The physico-chemical characteristics of these different milks, including composition, micellar properties and stability as assessed by heat, ethanol and phosphate tests, were determined. The database was processed by principal component analysis and common components and specific weights analysis. The effects of season, milking zone, process and storage conditions were highlighted, and the involved physico-chemical characteristics were determined. For the region effect, numerous parameters related to the global composition and the casein micelles intervened. Some differences in milk stability as evaluated by the ethanol and phosphate tests were also observed. Considering the season, spring milks had higher values of pH, lactose, soluble phosphate and micellar hydration than milks collected in autumn. These spring milks also had lower values of fat and heat stability than autumn milks. The UHT process effect was observed through decreases in non-casein nitrogen content and in micellar hydration and by an increase in casein micelle size for UHT milks. The stability values derived from phosphate and ethanol tests were increased following the UHT process. Concerning storage conditions, the temperature of 40 °C led to a decrease in pH and increases in non-casein and non-protein nitrogen contents of milks. At 40 °C, low values of stability for the heat test and high values for the phosphate test were observed.

A technique that used multivariate data analysis to combine mid-infrared (MIR) spectroscopy with front-face fluorescence spectroscopy was used to discriminate between Emmental cheeses originating from different European countries: Austria (n=12), Finland (n=10), Germany (n=19), France (n=57), and Switzerland (n=65). In total, 163 Emmental cheeses produced in winter (n=91) and summer (n=72) periods were investigated. When Factorial Discriminant Analysis was applied to either the infrared or fluorescence spectral data the classifications were not satisfactory. Therefore, the first twenty principal components (PCs) of the PCA extracted from each data set (MIR and tryptophan fluorescence spectra) were pooled (concatenated) into a single matrix and analysed by Factorial Discriminant Analysis. Correct classifications were obtained for the samples for 89% of the calibration spectra and 76.7% of the validation spectra. The discrimination for cheeses from Finland was excellent, while Austrian, German, French and Swiss cheeses were also discriminated well although a few samples were misclassified. It was concluded that concatenation of the data from the two spectroscopic techniques is an efficient technique for authenticating Emmental cheeses independently of their manufacturing period.

Texture optimisation of dairy products is a major aim for manufacturers. A better knowledge of the structure and spatial organisation of, their main components would allow the opti- misation of their texture. In this study, using confocal scanning laser microscopy, a multiple fluorescent labelling of proteins, lipids and whey was developed to visualise these main components simultaneously in dairy products. Different extrinsic fluorescent probes were tested by confocal microscopy and fluorescence spectroscopy. Fuchsin acid, Bodipy" 665/676 and DM-NERF were selected to label pro- teins, lipids and whey, respectively. Methods for selecting stable and specifie fluorescent probes and for obtaining the multiple fluorescent labelling are presented. An application example on a dairy gel is also shown. © Inra/Elsevier, Paris. microscopy / fluorescence / protein /lipid / curd

The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/alpha-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion). Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thrl10Ala) expressed in Pichia pastoris, by small amounts of cathepsin L. The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC. The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide). The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with Ki values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The Ki for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 microM of the propeptide. The inhibitory activity was also found to be strongly pH-dependent. Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed Ki values of 11 nM and 2900 nM, respectively, against cathepsin L. The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity.

The effects of high hydrostatic pressures on the kinetics of hydrolysis of type III collagen from calf skin by collagenase and cathepsin B were studied. Collagen hydrolysates sampled at different time intervals (0-90 min) and at different pressures (0.1-300 MPa) were analysed by reverse-phase high pressure liquid chromatography. The rate of collagen hydrolysis decreased up to 300 MPa for both enzymes. The rate of collagen hydrolysis with cathepsin B was drastically reduced between 0.1 and 100 MPa. Significant differences in the populations of the peptides in cathepsin B hydrolysates were observed in chromatograms corresponding to different pressures. This indicated that some amino acid side-chains were less exposed to cathepsin B recognition on the surface of collagen molecules at high pressures. In contrast, the chromatograms of collagenase hydrolysates showed similar patterns, varying only by the peak heights in chromatograms corresponding to the 0.1-300 MPa pressure range. Pressureinduced decreases of the enzyme-apparent activities suggested that the activation volumes for the reaction of both enzymes were positive.

A peptide nitrile hydratase activity has been engineered into the cysteine protease papain by a single carefully selected mutation at the active site of the enzyme. The papain variant Gln19Glu hydrolyzes the substrate MeOCO-PheAla-CN to the corresponding amide with a kcat/KM value of 1.15 x 10(3) M-1 s-1. The reaction leads to an accumulation of the corresponding amide, which is then further hydrolyzed to the acid by the natural amidase activity of the enzyme. The pH-dependency of the nitrile hydratase activity of Gln19Glu supports the involvement of the acid form of the Glu19 residue in the reaction. The wild type enzyme displays very weak nitrile hydratase activity, and the introduction of a glutamic acid residue in the oxyanion hole of papain causes the kcat at pH 5 to increase by a factor of at least 4 x 10(5). Peptide nitriles react with cysteine proteases to form thioimidates, and the role of the glutamic acid residue introduced at position 19 in the Gln19Glu enzyme is to participate in the acid-catalyzed hydrolysis of the thiomidate to the amide by the provision of a proton to form the more reactive protonated thioimidate. This dramatically decreases the energy barrier for the hydrolysis of the thioimidate, as shown by the impressive increase in kcat. The success of the rational approach undertaken is a consequence of the level of understanding of the basic catalytic properties of cysteine proteases of the papain family.

The effects of pressure (0.1 MPa to 400 MPa) on intrinsic fluorescence of beta-lactoglobulin and on its binding of retinol and cis-parinaric acid have been studied at neutral and acid pHs. In neutral pH, fluorescence emission spectra of beta-lactoglobulin tryptophanes are characterized by an irreversible 14 nm red-shift indicating pressure-induced folding changes. The intensity of the fluorescence of retinol in beta-lactoglobulin-retinol complex is enhanced by a pressure increase up to 150 MPa. It decreases at higher pressures and disappears altogether at 300 MPa. beta-Lactoglobulin-retinol complex does not reassociate after decompression at neutral pH. At acid pH condition, the fluorescence quenching by pressure of beta-lactoglobulin tryptophans is coupled with a 2 nm spectral shift and is fully reversible demonstrating almost complete restoration of globulin folding. The evolution of retinol fluorescence in beta-lactoglobulin-retinol complex is also entirely reversible between 0.1 MPa and 400 MPa and the complex never dissociates in the studied pressure range. beta-lactoglobulin-cis-parinaric acid complexes at neutral and acid pH values dissociate irreversibly at 200 MPa and 350 MPa, respectively.

Fourier transform infrared spectroscopy has been applied to investigate the secondary structural changes of beta-lactoglobulin in water/ethanol mixtures. The studies were carried out at two different pHs and at high protein concentrations. The spectra were recorded using an attenuated total reflection cell. The amide I band of beta-lactoglobulin in water reveals large amounts of intra extended beta-sheet structure. About 20% ethanol, beta-lactoglobulin unfolds and beta-strand formation is observed. alpha-Helices are built up by increasing the ethanol concentration up to 30%. In 50% ethanol, beta-lactoglobulin gels providing the apparent pH are neutral. The secondary structural changes of beta-lactoglobulin were observed on the similarity maps obtained by Principal Component Analysis.

It has been demonstrated using circular dichroism that methanol induces important structural changes of beta-lactoglobulin (BLG). The secondary structure of BLG dissolved in 45% methanol (v/v) at 30 degrees C (dielectric constant epsilon approximately 50) is predominantly alpha-helical and unable to complex retinol any more. However, when the dielectric constant of such a medium is raised back to epsilon approximately 70 by decreasing the temperature, both the refolding of BLG into a beta-structure and the formation of the retinol/BLG complex are observed. On the other hand, in the case of BLG solution in 30% methanol, the decrease of the dielectric constant from epsilon approximately 69 to epsilon approximately 53 by heating from 20 degrees C to 60 degrees C leads to the transition from a predominantly beta-structure into a predominantly random one and the dissociation of the retinol/BLG complex. The reversibility of the conformation changes by cooling was demonstrated by circular dichroism curves and tryptophan fluorescence. The retinol fluorescence intensity could not be completely recovered.