Should Carbohydrate Intake Be More Liberal during Oral and Enteral Nutrition in Type 2 Diabetic Patients?

Carbohydrate (CHO) intake in oral and enteral nutrition is regularly reduced in nutritional support of older patients due to the high prevalence of diabetes (usually type 2-T2DM) in this age group. However, CHO shortage can lead to the lack of building blocks necessary for tissue regeneration and other anabolic processes. Moreover, low CHO intake decreases CHO oxidation and can increase insulin resistance. The aim of our current study was to determine the extent to which an increased intake of a rapidly digestible carbohydrate-maltodextrin-affects blood glucose levels monitored continuously for one week in patients with and without T2DM. Twenty-one patients (14 T2DM and seven without diabetes) were studied for two weeks. During the first week, patients with T2DM received standard diabetic nutrition (250 g CHO per day) and patients without diabetes received a standard diet (350 g of CHO per day). During the second week, the daily CHO intake was increased to 400 in T2DM and 500 g in nondiabetic patients by addition of 150 g maltodextrin divided into three equal doses of 50 g and given immediately after the main meal. Plasma glucose level was monitored continually with the help of a subcutaneous sensor during both weeks. The increased CHO intake led to transient postprandial increase of glucose levels in T2DM patients. This rise was more manifest during the first three days of CHO intake, and then the postprandial peak hyperglycemia was blunted. During the night's fasting period, the glucose levels were not influenced by maltodextrin. Supplementation of additional CHO did not influence the percentual range of high glucose level and decreased a risk of hypoglycaemia. No change in T2DM treatment was indicated. The results confirm our assumption that increased CHO intake as an alternative to CHO restriction in type 2 diabetic patients during oral and enteral nutritional support is safe.

Glycosidases in porcine follicular fluid and their effect on zona pellucida-AWN 1 spermadhesin interaction.

Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, ß-d-galactosidase, ß-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and ß-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and ß-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both ß-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.

Life cycle assessment comparison of photocatalytic coating and air purifier.

This article presents a comparison of 2 very different options for removal of undesirable microorganisms and airborne pollutants from the indoor environment of hospitals, schools, homes, and other enclosed spaces using air purifiers and photocatalytic coatings based on nano titanium dioxide (TiO2 ). Both products were assessed by life cycle assessment (LCA) methodology from cradle-to-grave. The assessment also includes comparison of 2 different nano TiO2 production technologies, one by continuous hydrothermal synthesis and the other by a sulfate process. Results of the study showed a relatively large contribution of photocatalytic coatings to reducing the effects of selected indices in comparison with an air purifier, regardless of which nano TiO2 production method is used. Although the impacts of the sulfate process are significantly lower compared to those of hydrothermal synthesis when viewed in terms of production alone, taken in the context of the entire product life cycle, the net difference becomes less significant. The study has been elaborated within the Sustainable Hydrothermal Manufacturing of Nanomaterials (SHYMAN) project, which aims to develop competitive and sustainable continuous nanoparticle (NP) production technology based on supercritical hydrothermal synthesis. Integr Environ Assess Manag 2016;12:478-485. © 2016 SETAC.

Chicken and rabbit antibodies against porcine pepsinogen A.

Isolated porcine pepsinogen A was used for the preparation of polyclonal rabbit and polyclonal chicken anti-pepsinogen A antibodies. Immunochemical properties of both immunoglobulin fractions were compared. The rabbit anti-serum was further purified using immobilized porcine pepsinogen A on magnetic cellulose beads and the resulting anti-pepsinogen A fraction proved to be applicable for the separation and the determination of porcine pepsinogen A. In contrary, antibodies prepared from chicken eggs by the same way have been found not suitable for the evaluation of the pepsinogen A level. Unexpectedly, the pre-immune fraction of chicken antibodies showed reactivity against porcine pepsinogen A and the affinity separation of specific polyclonal chicken anti-pepsinogen A antibodies on immobilized porcine pepsinogen A did not result in an enrichment of anti-pepsinogen A antibodies.

The antimicrobial action of histones in the reproductive tract of cow.

An infection of any part of female reproductive tract can severely interfere with fertility and reproduction. The fluids and epithelium from the lumen of the female reproductive tract (uterus, oviduct and ovarian follicle) are a known source of antimicrobial action in several species. In this study, we compared the antimicrobial properties of fluids from the reproductive tract of a cow. After removal of small molecules, we demonstrated that there is an antimicrobial activity connected with a fraction of compounds with a molecular mass range between 3500 and 30,000. The most probable candidates responsible for the observed antimicrobial effect were subsequently identified by mass spectroscopy as histones H2A type 2-C, H2B type 1-K, H3.3, and H4. The antimicrobial role of histone H2B was further confirmed by using an antibody against this histone.

Immobilized endoproteinase Glu-C to magnetic bead cellulose as a tool in proteomic analysis.

Magnetic bead cellulose activated with divinyl sulfone was used for the immobilization of Staphylococcus aureus endoproteinase Glu-C (EC 3.4.21.19). The immobilized proteinase was characterized by increased thermostability, by decreased self-cleavage activity, and a possibility of repeated use. The prepared immobilized enzyme was applied for the proteolytic cleavage of α-casein and BSA under different conditions (different composition of buffers, different pH, and different time of digestion). The possibilities of the direct use of enzyme reaction products for MALDI TOF MS analysis were shown.

Application of heptapeptides containing D-amino acid residues immobilized to magnetic particles and Sepharose for the study of binding properties of gastric aspartic proteases.

Synthetic heptapeptides containing D-amino acid residues and differing in the content of L-phenylalanine and L-tyrosine residues and their position (Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu, Val-D-Leu-Pro-Phe-Tyr-Val-D-Leu) were immobilized to two types of carriers: glyoxal-activated magnetic agarose particles and CNBr-activated Sepharose. In both cases, peptides were immobilized via their terminal amino group. Immobilized peptides were used for the study of binding properties of two gastric aspartic proteases (porcine pepsin A and rat pepsin C). Porcine pepsin A was adsorbed to all studied peptide-modified magnetic carriers, while rat pepsin C interacted with immobilized ligands only slightly. Similar results were obtained in affinity chromatographic experiments using heptapeptides immobilized to Sepharose.

Magnetic bead cellulose as a suitable support for immobilization of α-chymotrypsin.

Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol-gel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of α-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. α-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.

Native red electrophoresis--a new method suitable for separation of native proteins.

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state).

Phosphoprotein electrophoresis in the presence of Fe(III) ions.

Preparation of affinity polyacrylamide gels containing immobilized Fe(III) ions for the separation of proteins exhibiting metal ion binding properties is described. The presented method enables uniform distribution of immobilized metal ions in the affinity part of the polyacrylamide separating gel. Affinity gels prepared by this way are suitable to follow the effect of different concentrations of metal ions immobilized in polyacrylamide gel on a protein electrophoretic behavior. Polyacrylamide gels containing immobilized Fe(III) ions were used to study the electrophoretic behavior of two model proteins differing in their phosphate group content: chicken ovalbumin and bovine α-casein. For the electrophoretic separation, both the native and the denaturating conditions were used.

Native polyacrylamide electrophoresis in the presence of Ponceau Red to study oligomeric states of protein complexes.

Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.

Iminodiacetic acid-modified magnetic poly(2-hydroxyethyl methacrylate)-based microspheres for phosphopeptide enrichment.

Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) were employed for the IMAC separation of phosphopeptides. Fe(3+) and Ga(3+) ions immobilized on IDA-modified magnetic microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of the prepared Fe(3+)- and Ga(3+)-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.

Peptide inhibitor modified magnetic particles for pepsin separation.

Synthetic heptapeptide containing D-amino acid residues (Val-D-Leu-Pro-Phe-Phe-Val-D-Leu) was coupled to glyoxal-activated magnetic agarose particles via the free peptide amino group. The peptide-modified magnetic particles were used for the separation of pepsins. Porcine pepsin A and human pepsin A were adsorbed to the magnetic peptide-modified affinity carrier, while the rat pepsin C and human pepsin C did not interact with the immobilized ligand. Conditions of pepsin adsorption to peptide-modified magnetic particles, as well as elution buffers were optimized. Porcine pepsin A did not interact with the immobilized peptide in the presence of pepsin inhibitor pepstatin A, indicating that the enzyme binding site is involved in the studied interaction. The elaborated method represents a rapid and simple technique not only for the separation of pepsins but also, in combination with MS, for the enzyme detection and determination.

Interaction of pepsin with aromatic amino acids and their derivatives immobilized to Sepharose.

The interaction of porcine pepsin A with immobilized derivatives of aromatic amino acids was investigated. Divinyl sulfone-activated Sepharose was used to immobilize N-acetyl-l-phenylalanine and 3,5-diiodo-l-tyrosine via their free carboxyl groups and l-tyrosine via its amino group. Immobilized l-tyrosine was iodinated after coupling. The optimum conditions for the separation of porcine pepsin A using the prepared affinity carriers were studied and the following parameters were established: enzyme recovery, reproducibility of analyses, capacity and dependence of the elution peak area on the concentration of the loaded enzyme. The ability of the prepared affinity carriers to retain various types of proteins was compared under optimum conditions for porcine pepsin A separation. While immobilized 3,5-diiodo-l-tyrosine and iodinated l-tyrosine-Sepharose adsorbed relatively high amounts of bovine serum albumin and ovalbumin, only negligible amounts of these proteins were adsorbed to immobilized N-acetyl-l-phenylalanine. The behavior of porcine pepsin A was the same as its complex with pepstatin A on the prepared affinity carriers, indicating that the enzyme active site is not involved in the studied interaction.

Magnetic IDA-modified hydrophilic methacrylate-based polymer microspheres for IMAC protein separation.

Preparation of a new type of magnetic non-porous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres with hydrophilic properties containing coupled iminodiacetic acid (IDA) is described. The prepared microspheres were used for the immobilization of Ni(II) or Fe(III) ions to show their application in protein binding studies. Human IgG was bound to magnetic Ni(II)-IDA-modified microspheres and conditions of its adsorption and elution were optimized. Non-specific binding of the protein to magnetic microspheres in the absence of Ni(II) ions was low. Fe(III) ions immobilized on magnetic IDA-modified microspheres were used for the specific binding of porcine pepsin, as a model phosphoprotein. The ability of phosphate buffer to release the adsorbed enzyme from the microspheres and a low adsorption of the dephosphorylated protein indicate the participation of phosphate groups in the pepsin interaction. The elaborated method represents a rapid technique that can be used not only for the separation of proteins but also for analytical purposes.

Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins.

Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma.

Saccharide-mediated interactions of boar sperm surface proteins with components of the porcine oviduct.

The role of boar seminal plasma proteins attached to the sperm plasma membrane during ejaculation has been studied in saccharide-mediated events in the female reproductive tract. Heparin-binding (Hep(+)) proteins (DQH sperm surface protein, and AQN and AWN spermadhesins) and their aggregated forms (fractions II and III) interacted more strongly with both oviductal epithelium cells and fluid than non-heparin-binding (Hep(-)) proteins (PSP I and PSP II spermadhesins) and their heterodimer (fraction IV), and interactions correlate with affinity of these proteins to yeast mannan. Indirect immunofluorescence (IMF) showed that the AQN 1 spermadhesin and fraction II bind to the apical glycocalyx of the ampulla, as well as the isthmic and uterine tubal junction regions of the oviductal sections. IMF demonstrated the recognition of AQN 1 and fraction II and mannosyl components of oviductal epithelium. We suggest that Hep(+) proteins (especially AQN 1, fraction II) on sperm could enable sperm binding to oviductal epithelium and thus participate in formation of the sperm oviductal reservoir. Interactions of Hep(+) proteins to oviductal epithelium were inhibited by mannan, hyaluronic acid and sialylated O-glycoproteins. No or slight inhibition was observed with sulphated polysaccharides (heparin, chondroitin sulphate) and simple monosaccharides. Besides that, attachment of boar seminal plasma proteins to oviductal epithelium cells was affected by oviductal fluid, the natural environment in the oviduct. Moreover, the ability of hyaluronic acid to inhibit the interaction of sperm surface proteins to the oviduct might play a role in sperm release from the oviductal reservoir and in the capacitation process.

Preparation and testing of stationary phases and modified capillaries for affinity chromatography and affinity capillary electrophoresis of pepsin.

Three stationary phases have been prepared for affinity liquid chromatography isolation and separation of porcine and human pepsin. The phases contain 3,5-diiodo-L-tyrosine (DIT) bound to the supports HEMA BIO VS, HEMA BIO E and EPOXY TOYOPEARL. These phases have been tested on a model sample of porcine pepsin A and applied to human pepsin. Fractions have been collected and the chymase activity determined in selected analyses. For affinity CE, capillaries have been prepared by modifying the wall with 3-aminopropyltriethoxysilane, followed either by direct binding of DIT, or by binding L-tyrosine that was subsequently iodated. The dissociation constant K(d) has been determined for the pepsin-DIT complex from the changes in the electrophoretic mobilities.

Characterization of human seminal plasma proteins homologous to boar AQN spermadhesins.

Spermadhesins, proteins secreted by the boar sexual accessory glands, are believed to play an important role in sperm capacitation and primary contact of sperm and egg. We have previously found human seminal plasma proteins immunobiochemically related to boar AQN and AWN spermadhesins. In this study, we characterized further the AQN spermadhesin-related proteins, here designated as hSA (human spermadhesin-like) proteins. On Western blot, we immunodetected 14, 16 and 18 kDa forms of hSA proteins (hSA-14, hSA-16 and hSA-18, respectively) cross-reacting with rabbit antibody against AQN spermadhesins. Each relative molecular-mass form of hSA comprised three isoelectric isoforms (6.0, 6.8 and 8.4) as shown by 2D-PAGE. Glycoprotein analysis revealed that all hSA-16 and hSA-18 isoforms were N-glycosylated, and those of hSA-14 were non-glycosylated. Two isoforms of hSA-14 (pI 6.0 and 8.4) had affinity to heparin. Size-exclusion chromatography of human seminal plasma indicated that hSA proteins formed high molecular-mass complexes either with other hSA proteins or with seminal plasma lactoferrin and/or its fragments. Similarity of biochemical properties (relative molecular masses, isoelectric points and existence of non- and N-glycosylated forms) of hSA proteins and those of boar AQN spermadhesins, together with a previously described N-terminal amino acid sequence of one hSA protein identical to AQN spermadhesins, imply that hSA proteins are structurally related to boar AQN spermadhesins. However, localization of hSA proteins on the sperm tail and neck suggests that their biological role differs from that of boar AQN spermadhesins located on the sperm head.

Mannan-binding proteins from boar seminal plasma.

The interaction of boar seminal plasma proteins and sperm with yeast mannan was investigated by the enzyme-linked binding assay (ELBA) and specific detection of proteins after SDS electrophoresis and blotting using biotinylated derivative of the polysaccharide. Heparin-binding proteins (especially AQN 1 and DQH proteins) and their aggregated forms showed affinity to yeast mannan. Besides that, these proteins were shown to bind to oviductal epithelium. The mannan-binding activity of boar proteins and sperm was inhibited most efficiently by ovomucoid, ovalbumin and N-glycans released from ovalbumin, but not with d-glucose, d-mannose and their phosphates. On the other hand, yeast mannan inhibited both the interaction of boar seminal plasma and sperm with heparin and the binding of these proteins to porcine oviductal epithelium. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. Proteins adsorbed to the immobilized polysaccharide were analyzed by RP-HPLC, SDS electrophoresis and N-terminal amino acid sequencing. AQN and AWN spermadhesins and DQH protein (names are derived from the N-terminal amino acid sequence) were identified as components of the isolated fraction. The results suggest an involvement of mannan-binding proteins in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both the complex d-mannose-containing saccharide structures and the heparin may also play an important role in sperm release from the oviductal reservoir or the capacitation process.