Percepcija teksture namirnica u ovisnosti o sadržaju ulja, te debljini lipidnog sloja na površini usne šupljine

Lipid content in food strongly influences food perception on the level of textural properties. Lipids in contact with the tongue and palate are substantially responsible for the sensory impact of a product. The aim of this study is to investigate the impact of oil content on the thickness of lipid deposition on oral surface as well as on the mouthfeel perception. The fluorescent probe method was used to study the thickness of lipid deposition on oral surface. We observed an increase in the thickness of lipid deposition depending on the increase of oil content in oil/water dispersions. Clear correlation was shown between the thickness of lipid deposition on oral surfaces and the perception of mouthfeel. A direct measure of undisrupted deposition of food components on oral surface contributes to the understanding of the behaviour of food components in the mouth and their influence on mouthfeel perception.Sadržaj lipida u hrani snažno utječe na percepciju teksture namirnica. Lipidi u kontaktu s jezikom i nepcem u najvećoj su mjeri odgovorni za senzorska svojstva hrane. Svrha je ovoga rada bila istražiti utjecaj udjela ulja u hrani na debljinu lipidnog sloja na površini usne šupljine, kao i na percepciju teksture namirnica. Za mjerenje debljine lipidnog sloja na površini usne šupljine upotrijebljena je fluorescentna proba. Uočili smo da je debljina lipidnog sloja na površini usne šupljine proporcionalna udjelu ulja u disperzijama ulja i vode. Dokazana je izrazita korelacija između debljine lipidnog sloja na površini usne šupljine i njegove percepcije u ustima. Direktno mjerenje debljine lipidnog sloja na površini usne šupljine pridonosi razumijevanju promjene sastojaka hrane u ustima i njihovog utjecaja na percepciju teksture namirnice

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivative

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives

The humoral response to Plasmodium falciparum VarO rosetting variant and its association with protection against malaria in Beninese children

<p>Abstract</p> <p>Background</p> <p>The capacity of <it>Plasmodium falciparum</it>-infected erythrocytes to bind uninfected erythrocytes (rosetting) is associated with severe malaria in African children. Rosetting is mediated by a subset of the variant surface antigens PfEMP1 targeted by protective antibody responses. Analysis of the response to rosette-forming parasites and their PfEMP1 adhesive domains is essential for understanding the acquisition of protection against severe malaria. To this end, the antibody response to a rosetting variant was analysed in children recruited with severe or uncomplicated malaria or asymptomatic <it>P. falciparum </it>infection.</p> <p>Methods</p> <p>Serum was collected from Beninese children with severe malaria, uncomplicated malaria or <it>P. falciparum </it>asymptomatic infection (N = 65, 37 and 52, respectively) and from immune adults (N = 30) living in the area. Infected erythrocyte surface-reactive IgG, rosette disrupting antibodies and IgG to the parasite crude extract were analysed using the single variant Palo Alto VarO-infected line. IgG, IgG1 and IgG3 to PfEMP1-varO-derived NTS-DBL1α₁, CIDRγ and DBL2βC2 recombinant domains were analysed by ELISA. Antibody responses were compared in the clinical groups. Stability of the response was studied using a blood sampling collected 14 months later from asymptomatic children.</p> <p>Results</p> <p>Seroprevalence of erythrocyte surface-reactive IgG was high in adults (100%) and asymptomatic children (92.3%) but low in children with severe or uncomplicated malaria (26.1% and 37.8%, respectively). The IgG, IgG1 and IgG3 antibody responses to the varO-derived PfEMP1 domains were significantly higher in asymptomatic children than in children with clinical malaria in a multivariate analysis correcting for age and parasite density at enrolment. They were essentially stable, although levels tended to decrease with time. VarO-surface reactivity correlated positively with IgG reactivity to the rosetting domain varO-NTS-DBL1α₁. None of the children sera, including those with surface-reactive antibodies possessed anti-VarO-rosetting activity, and few adults had rosette-disrupting antibodies.</p> <p>Conclusions</p> <p>Children with severe and uncomplicated malaria had similar responses. The higher prevalence and level of VarO-reactive antibodies in asymptomatic children compared to children with malaria is consistent with a protective role for anti-VarO antibodies against clinical falciparum malaria. The mechanism of such protection seems independent of rosette-disruption, suggesting that the cytophilic properties of antibodies come into play.</p

Structured fiber supports for gas phase biocatalysis

Pseudomonas cepaciae lipase adsorbed onto non-porous structured fiber supports in the form of woven fabrics, was used to catalyze hydrolysis and transesterification reactions in the gas phase. The enzyme adsorbed onto carbon fiber support exhibited much higher catalytic activity compared to the enzyme immobilized onto glass fiber carrier. The effect of temp. and relative humidity on reactions catalyzed by P. cepaciae lipase adsorbed onto structured fiber carbon support was studied in the gas system. Under the conditions investigated (up to 60 DegC and 80% relative humidity), the immobilized enzyme showed a high thermostability and could be efficiently used to catalyze hydrolytic and transesterification reactions in continuous mode. Structured fiber supports, with a high sp. surface area and a high mech. resistance, showed a low-pressure drop during the passage of reactants through a reactor. The approach proposed in this study could be suitable for immobilization of a wide variety of enzymes. [on SciFinder (R)

Allelic Diversity of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Entails Variant-Specific Red Cell Surface Epitopes

The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α1 PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α1 domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquistion of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity

Engineering of the human O6-alkylguanine DNA alkyltransferase

Recently, a new method for the specific covalent labeling of fusion proteins in vitro and in living cells has been developed. This method is based on the unusual mechanism of a DNA repair protein: the O6-alkylguanine DNA alkyltransferase (AGT). In addition to its natural substrate, O6-alkylated guanine incorporated in DNA, the AGT protein can transfer the benzyl group of O6-benzylguanine to a reactive cysteine residue, leading to its irreversible alkylation. The labeling system relies on the fact that affinity or fluorescent tags can be attached to the para position of the benzyl ring and thus be transferred to the AGT protein, leading to its covalent labeling. Although this system has already been successfully applied, it still suffers from two major drawbacks: the low reactivity of the protein toward O6-benzylguanine derivatives and the limitation to cells deficient in endogenous AGT. The work reported here describes directed evolution strategies that were developed to overcome these drawbacks. The first goal was to increase the labeling efficiency in living cells by increasing the reactivity of AGT toward O6-benzylguanine derivatives. Structural data available on AGT and the results of docking experiments pointed out four residues, 140, 157, 159 and 160, which are close enough to the O6-benzylguanine to allow interactions with this substrate. Therefore, these four positions were fully randomized and selections were performed using a phage display system based on the phagemid technology. Two mutants, PGEAhAGT and PGEGhAGT, showed a 15- and 20-fold increase in the reaction rate in vitro. It has subsequently been shown that this increase in reaction rate in vitro is also reflected in living cells by a more efficient labeling of AGT fusion proteins. In conclusion, the selected mutant proteins allow for a highly efficient covalent labeling of AGT fusion proteins in vitro as well as in living cells and therefore should become an important tool for studying protein function. The second aim was to enable the selective labeling of AGT fusion proteins in living cells in the presence of endogenous AGT. For this purpose, four positions, 131, 132, 134 and 135, which are believed to interfere with N9-substituted O6-benzylguanines were simultaneously completely randomized. Phage display selections allowed the identification of a clone, AGT54, with increased reactivity toward O6-benzylguanine and complete resistance toward an N9-substituted O6-benzylguanine. This mutant was further engineered to render the protein more stable, non-DNA binding and to decrease the overall protein size, leading to the so-called MAGT. This new mutant therefore allowed the specific labeling of fusion proteins in living cells in presence of endogenous AGT by simply pre-incubation with the N9-substituted O6-benzylguanine. In conclusion, the possibility to specifically label AGT fusion proteins in the presence of endogenous protein should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells. Finally, a transcription-based yeast three-hybrid system was adapted and successfully employed to identify clones with a 2-fold increased activity toward O6-benzylguanine out of an error prone library based on MAGT