Influence of the Carbohydrate Moieties on the Immunoreactivity and Digestibility of the Egg Allergen Ovomucoid

Background

Ovomucoid (OM) has two carbohydrate chains on each of the first and second domains and one in the third. The contribution of the covalently bound carbohydrate chains to the overall OM allergenicity is controversial. Another aspect directly related with the immunological properties of OM that has not been studied in depth is the importance of the carbohydrate chains on its digestibility.

Objective

The aim of the study was to assess the involvement of the carbohydrate moieties of OM in its digestibility and allergenic properties.

Methods

IgE-binding and basophil activation by glycosylated and enzymatically deglycosylated OM (dOM) were compared using blood from egg-allergic patients. The peptides obtained after digestion using a physiologically relevant model were identified by RP-HPLC-MS/MS and the IgE-binding of the resulting fragments was evaluated by DOT-Blot.

Results

No structural changes were observed after deglycosylation of OM. 80% of the patients showed lower IgE binding to dOM as compared with OM and, in some patients, IgE reactivity could not be inhibited by pre-incubation with dOM. A subtle reduction in the percentage of activated basophils was observed when incubated with dOM as compared to OM. Following simulated digestion, dOM was more extensively degraded than OM, particularly during the gastric phase and both, OM and dOM, yielded, after the duodenal phase, immunoreactive fragments that were totally or partially coincident with previously described epitopes.

Conclusion

& Clinical Relevance: this work demonstrated an enhanced IgE reactivity towards carbohydrate containing OM in some egg-allergic patients that could be attributed to cross-sensitization or sensitization to the glycosylated components. The carbohydrate chains contributed to an increased resistance to proteolysis, and thus, to its allergenic potency. Evaluation of the products of digestion of OM and dOM revealed the presence of high-frequency IgE-binding epitopes that could remain linked by disulphide bonds.     

Role of Catalase and Superoxide Dismutase Activities on Oxidative Stress in the Brain of a Phenylketonuria Animal Model and the Effect of Lipoic Acid

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase which leads to accumulation of phenylalanine and its metabolites in tissues of patients with severe neurological involvement. Recently, many studies in animal models or patients have reported the role of oxidative stress in PKU. In the present work we studied the effect of lipoic acid against oxidative stress in rat brain provoked by an animal model of hyperphenylalaninemia (HPA), induced by repetitive injections of phenylalanine and α-methylphenylalanine (a phenylalanine hydroxylase inhibitor) for 7 days, on some oxidative stress parameters. Lipoic acid prevented alterations on catalase (CAT) and superoxide dismutase (SOD), and the oxidative damage of lipids, proteins, and DNA observed in HPA rats. In addition, lipoic acid diminished reactive species generation compared to HPA group which was positively correlated to SOD/CAT ratio. We also observed that in vitro Phe inhibited CAT activity while phenyllactic and phenylacetic acids stimulated superoxide dismutase activity. These results demonstrate the efficacy of lipoic acid to prevent oxidative stress induced by HPA model in rats. The possible benefits of lipoic acid administration to PKU patients should be considered.     

Exploring Bacterioplankton Growth and Protein Synthesis to Determine Conversion Factors Across a Gradient of Dissolved Organic Matter

The effect of bacterial specific growth rates of abundance (micro) and protein synthesis (b) on conversion factor (CF) variability was explored in order to provide an alternative approach to the controversial application of just one universal CF to field data. Nine regrowth cultures (RCs) were set up from very diverse aquatic ecosystems, controlling temperature and adding N and P to avoid mineral limitation and force organic carbon limitation. The values of micro varied one order of magnitude from 0.26 to 3.34 d(-1), whereas b values varied two orders of magnitude from 0.28 to 34.87 d(-1). We found no relationships between micro or b values and the dissolved organic carbon (DOC) concentration or the dissolved organic matter (DOM) quality indexes assayed. Abundance and protein synthesis increased exponentially and synchronously in four RCs, leading to balanced growth (micro = b). In contrast, abundance and protein synthesis increased logistically in the other five RCs and b values were significantly higher than g values, leading to unbalanced growth (micro not equal b). CFs ranged from 0.0062 to 0.0576 x 10(18) cells mol leucine(-1) with an average of 0.0305 x 10(18) cells mol leucine(-1). CFs obtained in RCs with balanced growth were generally higher than CFs obtained in RCs with unbalanced growth and were not alike, impeding the establishment of an upper limit for CFs. A positive and significant relationship (n = 8, p < 0.0 1, r2 = 0.71) was found between CFs and DOC concentration (CF (x10(18) cells mol leucine(-1)) = 0.0104 + 0.0094 DOC (mM)) when the value for the most productive system was excluded. This function permits the estimation of site-specific CFs based on DOC concentration instead of the controversial use of a single CF for different systems.     

Quantification of the phosphorus released by zooplankton in an oligotrophic lake (La Caldera, Spain): regulating factors and adjustment to theoretical models

Using an in situ approach, we have evaluated the phosphorusinputs from zooplankton in a high-mountain oligotrophic lake.Values of the specific gross release rate (SGRR) fluctuatedbetween 0.2 and 2.9 µg P mg^–1 dry weight h^–1,and were higher when the nauplii of Mixodiaptomus laciniatusdominated the zooplankton community. The rate of P recyclingby the zooplankton was high, reaching 1.6 µg P l^–1day^–1, and showed a highly consistent seasonal patternfrom one year to the next, with maxima in midsummer. Zooplanktonsize accounted for as much as 85% of the variance obtained inthe measurements of the specific rate of P release, while otherfactors, such as the quality or quantity of food, did not significantlyinfluence the SGRR changes. Among the models tested, only theone proposed by Peters (Int, Ver. Theor. Angew. Limnol. Verh.,19, 273–279, 1975) was useful for predictions in thissystem. The stoichiometric model of Hessen and Andersen (Arch.Hydrobiol., 35, 111–120, 1992), applied in this oligotrophicsystem, adequately predicted the phyto- and zooplankton dynamics,whereas the values of P release estimated using this model werefar higher than the excretion rates obtained experimentally.These differences were related to the type of egestion (formationof faecal pellets) of metazooplankton and to the relative importanceof the food resistance to digestion. We believe that in communitieswhere copepods constitute a substantial percentage of the zooplankton,an evaluation of the P release which is readily available [solublereactive phosphate (SRP), total dissolved phosphate (TDP)] toalgae and bacteria would not fit the predictions of generalmodels of mass balance; under these circumstances, assimilationefficiency proves to be the key parameter for predicting thereadily available P (excreted P).     

Dual role of histamine in modulation of Leydig cell steroidogenesis via HRH1 and HRH2 receptor subtypes

Although several reports indicate effects of histamine (HA) on female reproductive functions, scant literature exists to suggest a physiological role of HA in the male gonad. In the present study, we report a dual concentration-dependent effect of HA on steroidogenesis in MA-10 murine Leydig cells and purified rat Leydig cells. Although 1 nM HA can stimulate steroid production and significantly increase the response to LH/hCG in these cells, 10 microM HA exerts an inhibitory effect. We also provide confirming evidence for the existence of functional HRH1 and HRH2 receptors in both experimental models. The use of HRH1 and HRH2 selective agonists and antagonists led us to suggest that HRH2 activation would be largely responsible for stimulation of steroidogenesis, while HRH1 activation is required for inhibition of steroid synthesis. Our results regarding signal transduction pathways associated with these receptors indicate the coupling of HRH2 to the adenylate cyclase system through direct interaction with a Gs protein. Moreover, we show HRH1 activation mediates increases in inositol phosphate production, possibly due to coupling of this receptor to Gq protein and phospholipase C activation. The data compiled in this report clearly indicate that HA can modulate Leydig cell steroidogenesis in the testis and suggest a possible new physiological site of action for HA. Given that many drugs binding to HRH1, HRH2, or both, are widely prescribed for the treatment of diverse HA-related pathologies, it seems necessary to increase the knowledge regarding histaminergic regulation of testicular functions, to avoid possible unexpected side effects of such substances in the testis.     

Prediction of methylated CpGs in DNA sequences using a support vector machine

DNA methylation plays a key role in the regulation of gene expression. The most common type of DNA modification consists of the methylation of cytosine in the CpG dinucleotide. At the present time, there is no method available for the prediction of DNA methylation sites. Therefore, in this study we have developed a support vector machine (SVM)-based method for the prediction of cytosine methylation in CpG dinucleotides. Initially a SVM module was developed from human data for the prediction of human-specific methylation sites. This module achieved a MCC and AUC of 0.501 and 0.814, respectively, when evaluated using a 5-fold cross-validation. The performance of this SVM-based module was better than the classifiers built using alternative machine learning and statistical algorithms including artificial neural networks, Bayesian statistics, and decision trees. Additional SVM modules were also developed based on mammalian- and vertebrate-specific methylation patterns. The SVM module based on human methylation patterns was used for genome-wide analysis of methylation sites. This analysis demonstrated that the percentage of methylated CpGs is higher in UTRs as compared to exonic and intronic regions of human genes. This method is available on line for public use under the name of Methylator at http://bio.dfci.harvard.edu/Methylator/.     

pH change induces shifts in the size and light absorption of dissolved organic matter

Dissolved organic matter (DOM) influences inland water ecosystems through its light absorbing qualities. We investigated how pH affects light absorption by DOM with pH manipulation experiments and with data from two lake surveys. We hypothesized that: (1) light absorption and photobleaching of DOM would increase with increasing pH, and (2) as a result of photobleaching, molar absorption (i.e. light absorbance at 440?nm/dissolved organic carbon concentration) would decrease among lakes with increasing pH. In experiments with filtered lake water both initial light absorption and photobleaching rates increased at higher (i.e. more basic) pH along with a concomitant shift in the size of DOM toward larger colloidal materials measured by dynamic light scattering (DLS). Both scanning electron microscopy (SEM) and atom force microscopy (AFM) revealed large colloidal to particulate-sized organic matter in alkaline relative to acidic treatments. In the lake surveys, molar absorption coefficients were negatively related to pH across gradients similar to the experiments. Our results are consistent with a conceptual model in which at low pH DOM polymers and colloids are condensed limiting exposure of chromophores to light; at higher pH, polymers and colloids are expanded exposing chromophores to light resulting in greater initial light absorption and faster photobleaching. Hence, water transparency, which is significantly controlled by DOM, is sensitive to environmental changes that influence the pH and chemical composition of inland waters.     

Study of absorption and retention of nitrogen in fast growing rats--II. Influence of IMAO (nialamide)

A study was made in fast growing rats (65 g) of the nutritive effects of protein and the effect on different organs when influenced by nialamide (IMAO), administered at a dosage of 10 mg/100 g diet, with food intake controlled (pair fed). The administration of nialamide reduces the protein absorbed and retained to a significant degree, both in males and females. No difference was observed between the sexes caused by the effect of the drug, due to the absence of the hormonal anabolizant situation caused by testosterone in adult rats. The administration of nialamide does not significantly modify nitrogen content per 100 g of body weight in any of the organs studied, with the exception of the longissimus dorsi.     

Influence of metazooplankton on interactions of bacteria and phytoplankton in an oligotrophic lake

Commensalism based on organic carbon supplied by phytoplanktonand competition for mineral nutrients are important interactionsbetween bacteria and phytoplankton in oligotrophic clear-watersystems. Both interactions are influenced by zooplankton activity.To examine the relation ship between algae and bacteria in LakeLa Caldera, we studied: the correlations among phyto plankton,bacteria and phosphorus (P) dynamics; the ratio of organic carbonsupplied by algae to organic carbon demand by bacteria; andthe importance of P remineralized by metazooplankton for bothcommunities. Phytoplankton and bacteria had a similar seasonaldynamics, and there was a sig nificant and positive relationshipbetween bacterial abundance and algal biomass (P<0.01). However,the release of organic carbon from phytoplankton was usuallyhigher than the bacterioplankton carbon requirement. P availablevia zooplankton remineralization satisfied between 74 and 316%of the minimum P demands of algae and bacteria. To elucidatewhether zooplankton operate similarly on algae and bacterialgrowth or indirectly influence bacterial growth through phytoplanktonmetab olism, we performed zooplankton manipulation experiments.High zooplankton biomass in these experiments stimulated bothprimary and bacterial production, but release of organic carbonfrom phytoplankton declined. These results suggest a directstimulus of bacterial growth, so algae and bac teria can balancegrazing losses by compensatory growth. Further, the algal decreaseof the organic carbon supply for bacteria could, over time,lead to a change in the algae-bacteria interaction from competitionto commensalism. This reduction in organic carbon excretioncould affect the balance of the competitive interaction.     

Integrating T-cell epitope annotations with sequence and structural information using DAS

Immunoinformatics is an emerging new field that benefits from computational analyses and tools that facilitate the understanding of the immune system. A large number of immunoinformatics resources such as immune-related databases and analysis software are available through the World Wide Web for the benefit of the research community. However, immunoinformatics developments have sometimes remained isolated from mainstream bioinformatics. Therefore, there is clearly a need for integration, which will empower the exchange of data and annotations within the scientific community in a quick and efficient fashion. Here, we have chosen the Distributed Annotation System (DAS), for integrating in house annotations on experimental and predicted HLA I-restriction elements of CD8 T-cell epitopes with sequence and structural information.