Ontogenetic changes in digestive enzymatic capacities of the spider crab, Maja brachydactyla (Decapoda: Majidae)

Ontogenetic changes in digestive capabilities were analyzed in larvae and first juveniles of the spider crab Maja brachydactyla. Activities of five proteinases (total proteases, trypsin, chymotrypsin, pepsin-like and aminopeptidase), three carbohydrases (amylase, maltase and chitinase), an esterase and an alkaline phosphatase were studied to evaluate digestive enzyme profiles of the species. Both quantitative (spectrophotometry and fluorometry) and qualitative (SDS-PAGE) approaches were used. All assayed enzymes were active from hatching (zoea I-ZI) throughout larval development and in first juveniles. Significant variations during ontogeny were found only in total activities likely as a consequence of digestive system development. Specific activity varied little over ontogeny, being significant only for chitinase. Total proteases, trypsin and pepsin-like activities showed a similar pattern of increase as larval ontogeny advanced, decreasing significantly in juveniles. Chymotrypsin continued to increase, showing maximum activity after metamorphosis. Proteinase zymograms confirmed strong proteolytic activity in first zoeas, with increasing bands over the course of ontogeny, decreasing after metamorphosis. A group of bands with high molecular mass was specific to larval stages. Amylase and maltase showed a parallel pattern of continuous increase of total activity as development advanced. Gel-SDS-PAGE showed unchanged patterns of amylase activity in first zoeas of different ages and the most complex set of bands during larval ontogeny in second zoea. Esterase total activity increased significantly as ZI's aged likely reflecting introduction of a lipid-enriched diet. The importance of lipid accumulation at the beginning of ontogeny was also confirmed by the protease/esterase and amylase/esterase activity ratios, which decreased from hatch to late ZI and might be explained as an adaptation, ensuring the next molt. The results suggest that larvae of M. brachydactyla are capable of digesting a variety of dietary substrates as soon as they hatch.

When cholesterol is not cholesterol: a note on the enzymatic determination of its concentration in model systems containing vegetable extracts

Background: Experimental evidences demonstrate that vegetable derived extracts inhibit cholesterol absorption in the gastrointestinal tract. To further explore the mechanisms behind, we modeled duodenal contents with several vegetable extracts. Results: By employing a widely used cholesterol quantification method based on a cholesterol oxidase-peroxidase coupled reaction we analyzed the effects on cholesterol partition. Evidenced interferences were analyzed by studying specific and unspecific inhibitors of cholesterol oxidase-peroxidase coupled reaction. Cholesterol was also quantified by LC/MS. We found a significant interference of diverse (cocoa and tea-derived) extracts over this method. The interference was strongly dependent on model matrix: while as in phosphate buffered saline, the development of unspecific fluorescence was inhibitable by catalase (but not by heat denaturation), suggesting vegetable extract derived H2O2 production, in bile-containing model systems, this interference also comprised cholesterol-oxidase inhibition. Several strategies, such as cholesterol standard addition and use of suitable blanks containing vegetable extracts were tested. When those failed, the use of a mass-spectrometry based chromatographic assay allowed quantification of cholesterol in models of duodenal contents in the presence of vegetable extracts. Conclusions: We propose that the use of cholesterol-oxidase and/or peroxidase based systems for cholesterol analyses in foodstuffs should be accurately monitored, as important interferences in all the components of the enzymatic chain were evident. The use of adequate controls, standard addition and finally, chromatographic analyses solve these issues.

Identifying the preferred subset of enzymatic profiles in nonlinear kinetic metabolic models via multiobjective global optimization and pareto filters

Optimization models in metabolic engineering and systems biology focus typically on optimizing a unique criterion, usually the synthesis rate of a metabolite of interest or the rate of growth. Connectivity and non-linear regulatory effects, however, make it necessary to consider multiple objectives in order to identify useful strategies that balance out different metabolic issues. This is a fundamental aspect, as optimization of maximum yield in a given condition may involve unrealistic values in other key processes. Due to the difficulties associated with detailed non-linear models, analysis using stoichiometric descriptions and linear optimization methods have become rather popular in systems biology. However, despite being useful, these approaches fail in capturing the intrinsic nonlinear nature of the underlying metabolic systems and the regulatory signals involved. Targeting more complex biological systems requires the application of global optimization methods to non-linear representations. In this work we address the multi-objective global optimization of metabolic networks that are described by a special class of models based on the power-law formalism: the generalized mass action (GMA) representation. Our goal is to develop global optimization methods capable of efficiently dealing with several biological criteria simultaneously. In order to overcome the numerical difficulties of dealing with multiple criteria in the optimization, we propose a heuristic approach based on the epsilon constraint method that reduces the computational burden of generating a set of Pareto optimal alternatives, each achieving a unique combination of objectives values. To facilitate the post-optimal analysis of these solutions and narrow down their number prior to being tested in the laboratory, we explore the use of Pareto filters that identify the preferred subset of enzymatic profiles. We demonstrate the usefulness of our approach by means of a case study that optimizes the ethanol production in the fermentation of Saccharomyces cerevisiae.

Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap

This work describes the formation of transformation products (TPs) by the enzymatic degradation at laboratory scale of two highly consumed antibiotics: tetracycline (Tc) and erythromycin (ERY). The analysis of the samples was carried out by a fast and simple method based on the novel configuration of the on-line turbulent flow system coupled to a hybrid linear ion trap – high resolution mass spectrometer. The method was optimized and validated for the complete analysis of ERY, Tc and their transformation products within 10 min without any other sample manipulation. Furthermore, the applicability of the on-line procedure was evaluated for 25 additional antibiotics, covering a wide range of chemical classes in different environmental waters with satisfactory quality parameters. Degradation rates obtained for Tc by laccase enzyme and ERY by EreB esterase enzyme without the presence of mediators were ∼78% and ∼50%, respectively. Concerning the identification of TPs, three suspected compounds for Tc and five of ERY have been proposed. In the case of Tc, the tentative molecular formulas with errors mass within 2 ppm have been based on the hypothesis of dehydroxylation, (bi)demethylation and oxidation of the rings A and C as major reactions. In contrast, the major TP detected for ERY has been identified as the “dehydration ERY-A”, with the same molecular formula of its parent compound. In addition, the evaluation of the antibiotic activity of the samples along the enzymatic treatments showed a decrease around 100% in both cases

Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?

Aquest article descriu els sensors enzimàtics i immunosensors electroquímics que s’han desenvolupat als nostres grups per a la detecció de la biotoxina marina àcid okadaic (OA), i discuteix la possibilitat d’integrar-los en programes de seguiment. Els sensors enzimàtics per a OA que es presenten es basen en la inhibició de la proteïna fosfatasa (PP2A) per aquesta toxina i la mesura electroquímica de l’activitat enzimàtica mitjançant l’ús de substrats enzimàtics apropiats, electroquímicament actius després de la seva desfosforació per l’enzim. Els immunosensors electroquímics descrits en aquest article es basen en un enzimoimmunoassaig sobre fase sòlida competitiu indirecte (ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP) com a marcatges, i un sistema de reciclatge enzimàtic amb diaforasa (DI). Els biosensors presentats aquí s’han aplicat a l’anàlisi de dinoflagel·lats, musclos i ostres. Les validacions preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat la possibilitat d’utilitzar les bioeines desenvolupades per al cribratge preliminar de biotoxines marines en mostres de camp o de cultiu, que ofereixen informació complementària a la cromatografia. En conclusió, tot i que encara cal optimitzar alguns paràmetres experimentals, la integració dels biosensors a programes de seguiment és viable i podria proporcionar avantatges respecte a altres tècniques analítiques pel que fa al temps d’anàlisi, la simplicitat, la selectivitat, la sensibilitat, el fet de poder ser d’un sol ús i l’efectivitat de cost. This article describes the electrochemical enzyme sensors and immunosensors that have been developed by our groups for the detection of marine biotoxin okadaic acid (OA), and discusses the possibility of integrating them into monitoring programmes. The enzyme sensors for OA reported herein are based on the inhibition of immobilised protein phosphatase 2A (PP2A) by this toxin and the electrochemical measurement of the enzyme activity through the use of appropriate enzyme substrates, which are electrochemically active after dephosphorylation by the enzyme. The electrochemical immunosensors described in this article are based on a competitive indirect Enzyme- Linked ImmunoSorbent Assay (ciELISA), using alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as labels, and an enzymatic recycling system with diaphorase (DI). The biosensors presented herein have been applied to the analysis of dinoflagellates, mussels and oysters. Preliminary validations with colorimetric assays and LC-MS/MS have demonstrated the possibility of using the developed biotools for the preliminary screening of marine biotoxins in field or cultured samples, offering complementary information to chromatography. In conclusion, although optimisation of some experimental parameters is still required, the integration of biosensors into monitoring programmes is viable and may provide advantages over other analytical techniques in terms of analysis time, simplicity, selectivity, sensitivity, disposability of electrodes and cost effectiveness.

Anàlisi genètica i molecular de les malalties Niemann-Pick tipus A/B i tipus C. Estratègies terapèutiques.

Aquest treball es basa en l’estudi de dues malalties lisosòmiques: la malaltia de Niemann-Pick A/B (NPAB) i la malaltia de Niemann-Pick tipus C (NPC). En relació a la malaltia de NPAB, s’ha realitzat l’expressió in vitro d’algunes de les mutacions de canvi d’aminoàcid trobades en pacients espanyols per tal de detectar les activitats enzimàtiques residuals. Totes les mutacions presenten una activitat molt baixa, gairebé nul•la, excepte la p.L225P i la R608del que tenen un 11% i 20% d’activitat respectivament. Els resultats obtinguts són coherents amb la severitat del fenotip que presenten els pacients. D’altra banda, s’ha caracteritzat un al•lel amb una mutació que afecta a una posició poc conservada d’un donador de splicing i que produeix la generació de trànscrits aberrants corresponents a trànscrits minoritaris de SMPD1, prèviament descrits, que no codifiquen per proteïna funcional. Respecte a malaltia de NPC, s’ha realitzat una anàlisi molecular de pacients espanyols prèviament estudiats identificant, en la majoria dels casos, la segona mutació responsable de la patologia. S’ha descrit per primer cop per aquesta malaltia una gran deleció que inclou el gen NPC1 i altres gens flanquejants i s’ha estudiat l’efecte que tenen les mutacions de splicing trobades a nivell de RNA. Per una d’aquestes mutacions, c.1554-1009G&A, s’ha assajat amb èxit una estratègia terapèutica basada en la utilització d’oligonuclèotids antisentit. D’altra banda, s’està desenvolupant un model cel•lular neuronal de la malaltia de Niemann-Pick tipus C, basat en la utilització de RNAs d’interferència, sobre el qual es podran assajar possibles estratègies terapèutiques en un futur.

Modelització de la producció de melanina a partir de polifenol oxidasa d'Agaricus bisporus

Les melanines són un grup heterogeni de polímers producte de reaccions enzimàtiques en els teixits vegetals que contenen compostos fenòlics o polifenòlics. Estudis recents han descobert algunes propietats benèfiques de les melanines sobre la salut, tals com antioxidants, antiinflamatòries, immunològiques i propietats anti-tumorals. Així, no només la seva eliminació ha de ser examinada, sinó que també podria considerar-se la seva addició a aliments funcionals de nova creació. D’aquesta manera, es requereix conèixer el mecanisme cinètic de la lanogènesi abans de la seva possible utilització industrial. S’ha desenvolupat un model cinètic per explicar la formació de melanina a partir de L-tirosina utilitzant polifenol oxidasa d’Agaricus bisporus i monitoritzant l'absorbància de la solució. Aquesta expressió permet descriure la formació de melanina en funció del temps de reacció i obtenir alguns paràmetres importants que defineixen el producte, com el coeficient d'extinció. L’absorbància comença a créixer després d'un període de latència en què es produeixen productes intermedis incolors. El coeficient d'extinció dels productes resultants no és un valor constant, perquè depèn de les condicions de cada experiment. La tirosinasa tingué un menor efecte catalitzador sobre la L-tirosina (primera reacció que catalitza), que sobre L-DOPA (segona reacció).

CAV2 vector gene transfer into central nervious System

Estudi realitzat a partir d’una estada al Institut de Génétique Moléculaire de Montpellier, França, entre 2010 i 2012. En aquest projecte s’ha avaluat les avantatges dels vectors adenovirals canins tipus 2 (CAV2) com a vectors de transferència gènica al sistema nerviós central (SNC) en un model primat no-humà i en un model caní del síndrome de Sly (mucopolisacaridosis tipus 7, MPS VII), malaltia monogènica que cursa amb neurodegeneració. En una primera part del projecte s’ha avaluat la biodistribució, l’eficàcia i la durada de l’expressió del transgen en un model primat no humà, (Microcebus murinus). Com ha vector s’ha utilitzat un CAV2 de primera generació que expressa la proteïna verda fluorescent (CAVGFP). Els resultats aportats en aquesta memòria demostren que en primats no humans, com en d’altres espècies testades anteriorment per l’equip de l’EJ Kremer, la injecció intracerebral de CAV2 resulta en una extensa transducció del SNC, siguent les neurones i els precursors neuronals les cèl•lules preferencialment transduïdes. Els vectors canins, servint-se de vesícules intracel•lulars són transportats, majoritàriament, des de les sinapsis cap al soma neuronal, aquest transport intracel•lular permet una extensa transducció del SNC a partir d’una única injecció intracerebral dels vectors virals. En una segona part d’aquest projecte s’ha avaluat l’ús terapèutic dels CAV2. S’ha injectat un vector helper-dependent que expressa el gen la b-glucuronidasa i el gen de la proteïna verda fluorescent (HD-RIGIE), en el SNC del model caní del síndrome de Sly (MPS VII). La biodistribució i la eficàcia terapèutica han estat avaluades. Els nivells d’activitat enzimàtica en animals malalts injectats amb el vector terapèutic va arribar a valors similars als dels animals no afectes. A més a més s’ha observat una reducció en la quantitat dels GAGs acumulats en les cèl•lules dels animals malalts tractats amb el vector terapèutic, demostrant la potencialitat terapèutica dels CAV2 per a malalties que afecten al SNC. Els resultats aportats en aquest treball ens permeten dir que els CAV2 són unes bones eines terapèutiques per al tractament de malalties que afecten al SNC.

Analyzing peptides and proteins by mass spectrometry: principles and applications in proteomics

The study of proteins has been a key element in biomedicine and biotechnology because of their important role in cell functions or enzymatic activity. Cells are the basic unit of living organisms, which are governed by a vast range of chemical reactions. These chemical reactions must be highly regulatedin order to achieve homeostasis. Proteins are polymeric molecules that havetaken on the evolutionary process the role, along with other factors, of controlthese chemical reactions. Learning how proteins interact and control their up anddown regulations can teach us how living cells regulate their functions, as well asthe cause of certain anomalies that occur in different diseases where proteins areinvolved. Mass spectrometry (MS) is an analytical widely used technique to studythe protein content inside the cells as a biomarker point, which describesdysfunctions in diseases and increases knowledge of how proteins are working.All the methodologies involved in these descriptions are integrated in the fieldcalled Proteomics.

Selenium uptake by edible plants from enriched peat.

As a constituent of selenoproteins, selenium (Se) is considered an essential element for human health.The main way that Se enters the body is via the consumption of vegetables, whose concentration of thiselement depends on soil Se content. We grew cabbage, lettuce, chard and parsley, in peat enriched in Seby means of the additive Selcote Ultra®and Na2SeO3and Na2SeO4. Total Se in plants was determinedby acidic digestion and Se speciation by an enzymatic extraction. Both were measured by ICP/MS. Theconcentration ranges were between 0.1 mg Se kg−1and 30 mg Se kg−1for plants grown in Selcote Ultra®media, and between 0.4 mg Se kg−1and 1606 mg Se kg−1for those grown in peat enriched with Se sodiumsalts. We found Se (IV), Se (VI) and SeMet in all the extracts. Peat fortified with Selcote Ultra®gave slightlyhigher Se concentration than natural content values. For plants grown with selenium sodium salts, Secontent increases with the Se added and part of the inorganic Se was converted mainly to SeMet. A highSe fortification can damage or inhibit plant growth. Cabbage showed the greatest tolerance to Se.

Combining phospholipases and a liquid lipase for one-step biodiesel production using crude oils

Background Enzymatic biodiesel is becoming an increasingly popular topic in bioenergy literature because of its potential to overcome the problems posed by chemical processes. However, the high cost of the enzymatic process still remains the main drawback for its industrial application, mostly because of the high price of refined oils. Unfortunately, low cost substrates, such as crude soybean oil, often release a product that hardly accomplishes the final required biodiesel specifications and need an additional pretreatment for gums removal. In order to reduce costs and to make the enzymatic process more efficient, we developed an innovative system for enzymatic biodiesel production involving a combination of a lipase and two phospholipases. This allows performing the enzymatic degumming and transesterification in a single step, using crude soybean oil as feedstock, and converting part of the phospholipids into biodiesel. Since the two processes have never been studied together, an accurate analysis of the different reaction components and conditions was carried out. Results Crude soybean oil, used as low cost feedstock, is characterized by a high content of phospholipids (900 ppm of phosphorus). However, after the combined activity of different phospholipases and liquid lipase Callera Trans L, a complete transformation into fatty acid methyl esters (FAMEs >95%) and a good reduction of phosphorus (P <5 ppm) was achieved. The combination of enzymes allowed avoidance of the acid treatment required for gums removal, the consequent caustic neutralization, and the high temperature commonly used in degumming systems, making the overall process more eco-friendly and with higher yield. Once the conditions were established, the process was also tested with different vegetable oils with variable phosphorus contents. Conclusions Use of liquid lipase Callera Trans L in biodiesel production can provide numerous and sustainable benefits. Besides reducing the costs derived from enzyme immobilization, the lipase can be used in combination with other enzymes such as phospholipases for gums removal, thus allowing the use of much cheaper, non-refined oils. The possibility to perform degumming and transesterification in a single tank involves a great efficiency increase in the new era of enzymatic biodiesel production at industrial scale.

Gelatinase expression and proteolytic activity in giant-cell arteritis

Objectives: Gelatinases (MMP2 and MMP9) are expressed in giant-cell arteritis (GCA) and are thought to play a role in vessel disruption. However, their activation status and enzymatic activity have not been evaluated. Our aim was to investigate the distribution and proteolytic activity of gelatinases in GCA lesions at different stages. Methods: Expression of MMP2, MMP9, MMP2-activator MMP14 and their natural inhibitors TIMP1 and TIMP2 was determined by real-time PCR and immunohistochemistry in temporal artery sections from 46 patients and 12 controls. MMP activation status and enzymatic activity were assessed by gelatin and film in situ zymography. Results: Vascular smooth muscle cells from normal specimens constitutively expressed pro-MMP2 and its inhibitor TIMP2 with no resulting proteolytic activity. In GCA MMP2, MMP9 and MMP14 were strongly expressed in their active form by infiltrating leucocytes. Inflamed arteries also expressed TIMP1 and TIMP2. However, the MMP9/TIMP1 and MMP2/TIMP2 ratios were higher in patients compared with controls, indicating an increased proteolytic balance in GCA which was confirmed by in situ zymography. Maximal gelatinase expression and activity occurred at the granulomatous areas surrounding the internal elastic lamina (IEL). Myointimal cells also expressed MMPs and exhibited proteolytic activity, suggesting a role for gelatinases in vascular remodelling and repair. Conclusions: GCA lesions show intense expression of gelatinases. Activators and inhibitors are regulated to yield enhanced gelatinase activation and proteolytic activity. Distribution of expression and proteolytic activity suggests that gelatinases have a major role not only in the progression of inflammatory infiltrates and vessel destruction but also in vessel repair.

Determination of salivary glucose in healthy adults

Objectives: Our aim in this study was to determine the concentration of salivary glucose in healthy individuals and to compare it with the capillary glycemia. Study design: Samples of unstimulated whole saliva were collected from 63 non-diabetic patients. The concentration of salivary glucose and capillary blood was measured in all of the patients. The salivary glucose was determined by enzymatic method and spectrophotometry. The data was then analyzed using the Spearman correlation test, considering values of p<0.05 to be significant. Results: The whole sample consisted of 47.6% males and 52.4% women, with an average age of 37.5±15.7 years old. The average rates of unstimulated salivary flow were 0.41±0.21 ml/min among males and 0.31±0.15 ml/min among females. No significant difference was found based on these results (p=0.078). The average blood glucose among the males studied was 100.05±13.51 mg/dL, and among females, it was 99.5±13.9 mg/dL. The average salivary glucose for the whole sample was 5.97±1.87 mg/dL, with 5.91±2.19 mg/dL among males and 5.97±1.56 mg/dL among females, respectively, without presenting any significant differences (p=0.908). The concentration of salivary glucose did not present any statistically significant correlation with the capillary glycemia (p=0.732). Conclusions: The results suggest that the concentration of salivary glucose is not dependent on capillary glycemia and that the concentration of salivary glucose does not present significant differences between the measurements for males and females.

Preparation of (S)-1-halo-2-octanols using ionic liquids and biocatalysts

Preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol was carried out by the enzymatic hydrolysis of halohydrin palmitates using biocatalysts. Halohydrin palmitates were prepared by various methods from palmitic acid and 1,2-octanediol. A tandem hydrolysis was carried out using lipases from Candida antarctica (Novozym® 435), Rhizomucor miehei (Lipozyme IM), and “resting cells” from a Rhizopus oryzae strain that was not mycotoxigenic. The influence of the enzyme and the reaction medium on the selective hydrolysis of isomeric mixtures of halohydrin esters is described. Novozym® 435 allowed preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol after 1–3 h ofreaction at 40 °C in [BMIM][PF6].

Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation

Background: In the yeast biomass production process, protein carbonylation has severe adverse effects since it diminishes biomass yield and profitability of industrial production plants. However, this significant detriment of yeast performance can be alleviated by increasing thioredoxins levels. Thioredoxins are important antioxidant defenses implicated in many functions in cells, and their primordial functions include scavenging of reactive oxygen species that produce dramatic and irreversible alterations such as protein carbonylation. Results: In this work we have found several proteins specifically protected by yeast Thioredoxin 2 (Trx2p). Bidimensional electrophoresis and carbonylated protein identification from TRX-deficient and TRX-overexpressing cells revealed that glycolysis and fermentation-related proteins are specific targets of Trx2p protection. Indeed, the TRX2 overexpressing strain presented increased activity of the central carbon metabolism enzymes. Interestingly, Trx2p specifically preserved alcohol dehydrogenase I (Adh1p) from carbonylation, decreased oligomer aggregates and increased its enzymatic activity. Conclusions: The identified proteins suggest that the fermentative capacity detriment observed under industrial conditions in T73 wine commercial strain results from the oxidative carbonylation of specific glycolytic and fermentation enzymes. Indeed, increased thioredoxin levels enhance the performance of key fermentation enzymes such as Adh1p, which consequently increases fermentative capacity.