Hemoglobin recognition by imprinting in semi-interpenetrating polymer network hydrogel based on polyacrylamide and chitosan

Semi-interpenetrating polymer network (semi-IPN) hydrogel was prepared to recognize hemoglobin, by molecularly imprinted method, in the mild aqueous media of chitosan and acrylamide in the presence of N,N'-methylenebisacrylamide as the cross-linking agent. The hydrogel obtained has been investigated by using thermal analysis, X-ray diffraction, differential scanning calorimetry (DSC), and environmental scanning electron microscope (ESEM). Langmuir analysis showed that an equal class of adsorption was formed in the hydrogel, and the adsorption equilibrium constant and the maximum adsorption capacity were evaluated to be 4.27 g/mL and 36.53 mg/g wet hydrogel, respectively. The imprinted semi-IPN hydrogel has a much higher adsorption capacity for hemoglobin than the nonimprinted hydrogel with the same chemical composition and also has a higher selectivity for the imprinted molecule.     

Chemoenzymatic synthesis and hydrogelation of amylose-grafted xanthan gums

This paper reports the chemoenzymatic synthesis of an amylose-grafted xanthan gum. An amine-functionalized maltooligosaccharide was chemically introduced to xanthan gum by condensation with its carboxylates using a condensing agent to produce a maltooligosaccharide-grafted xanthan gum. Then, a phosphorylase-catalyzed enzymatic polymerization of glucose 1-phosphate from the graft chain ends on the xanthan gum derivative was performed, giving an amylose-grafted xanthan gum. Furthermore, the product formed a gel with an ionic liquid, which was converted into a hydrogel with high water content by replacement of the ionic liquid with water. The ionically cross-linked hydrogel was also provided by soaking the primary formed hydrogel in FeCl₃ aqueous solution. The mechanical properties of the resulting hydrogels were evaluated by compressive testing.     

Structural studies on periodate-oxidized chicken ovomucoid. Spectral behaviour of the tyrosine residues.

About 50% of the carbohydrate moiety of ovomucoid was destroyed by periodate oxidation. The oxidation was carried out for 6 h or 24 h. The data obtained showed that in the carbohydrate chain 2-5 glucosamines and 1-2 neutral sugar residues were decomposed with the consumption of 16 mol and 29 mol of periodate respectively. Periodic oxidation slightly changed the inhibitory activity of the ovomucoid, but altered its spectral properties. An increase of the absorption maximum at 278 nm was noted, as well as a tendency for normalization of phenolic ionization and an increase of the relative fluorescence. The reactivity of tyrosine residues towards tetranitromethane is also changed. It was suggested that even in native ovomucoid the tyrosines could be regarded as 'dissolved' in the 'carbohydrate solvent'. This contact could be achieved by the hydrogen bonds in the formation of which the NHCOCH3 groups of the glucosamine residues play an essential role. Peroxidate oxidation seems to lead to an alteration of the nature of the 'sugar solvent' and disturbs the conformation of the sugar chain.     

Ion-pairing separation of bioactive peptides using an aqueous/octan-1-ol micro-extraction system from bovine haemoglobin complex hydrolysates

The ion-pair concept was applied on complex haemoglobin hydrolysates to extract two opioid peptides, LVV-haemorphin-7 and VV-haemorphin-7, in an aqueous/octan-1-ol micro-extraction system in the presence of alkyl-sulfonic acid as a surfactant agent and in relation to the haemorphin physico-chemical properties (charge, hydrophobicity). The effect of combined alkyl chain length/aqueous phase pH and the haem behaviour during the extraction, on the haemorphin recovery yield and enrichment has been determined. It has proved that transport over the organic phase is mediated by the alkyl-sulfonic acids, whatever be the aqueous phase pH. However, increasing both the alkyl chain length and the pH in the aqueous phase shows an haemorphin enrichment ratio increase but a recovery decrease of the extracted opioid peptides in the organic phase. Therefore, the best conditions to extract LVVh-7 and VVh-7 are the use of the octane-sulfonic acid at aqueous phase pH of 5 or 7 and the octane or the heptane-sulfonic acid with an aqueous phase pH of 5 or 7 respectively. In these conditions, a partition coefficient of 1.64 and 1.60 respectively for LVVh-7 and VVh-7 are obtained and represent about 40 times that acquired without agent.     

Crystallization,crystal structure analysis and molecular model of the third domain of Japanese quail ovomucoid,a Kazal type inhibitor

The third domain of Japanese quail ovomucoid, a Kazal type inhibitor, has been crystallized and its crystal structure determined at 2.5 Å resolution using multiple isomorphous replacement techniques. The asymmetric unit contains four molecules. In the crystal the molecules are arranged in two slightly different octamers with approximate D4 symmetry. The molecules are held together mainly by interactions of the N-terminal residues, which form a novel secondary structural element, a β-channel.The molecule is globular with approximate dimensions 35 Å × 27 Å × 19 Å. The secondary structural elements are a double-stranded anti-parallel β-sheet of residues Pro22 to Gly32 and an α-helix from Asn33 to Ser44. The reactive site Lys18-Asp19 is located in an exposed loop. It is close to Asn33 at the N terminus of the helical segment. The polypeptide chain folding of ovomucoid bears some resemblance to other inhibitors in the existence of an anti-parallel double strand following the reactive site loop.     

Matrix metalloprotease triggered delivery of cancer chemotherapeutics from hydrogel matrixes

Glioblastoma multiforme (GBM) is a highly advanced and invasive brain tumor due to which current treatments cannot completely treat GBM or prevent recurrence. Therefore, adjunctive treatments are required. As part of the invasive and angiogenic nature of GBM, it has been well established that matrix metalloprotease-2 (MMP-2) and MMP-9 are overactive. To better treat GBM using chemotherapy, we have designed a hydrogel-based delivery system that can control the release of drugs based on the activity of MMPs. A model chemotherapeutic agent, cisplatin (CDDP), complexed to an MMP substrate (peptide-linker) was incorporated into poly(ethylene glycol) diacrylate hydrogel wafers having different poly(ethylene glycol) chain lengths (M(n) approximately 574 and 4000). Hydrogel wafers were studied for physical characteristics and drug release in the presence and absence of MMPs. There was a substantial increase in CDDP release for the poly(ethylene glycol) 4000 hydrogel indicating that this chain length provides a mesh size that is sufficient to permit MMP activity within the hydrogel. CDDP bioactivity increased when the cell media was spiked with MMPs (0% cell survival) in case of the longer chain length as compared to in the absence of MMPs (approximately 50% cell survival). The results suggest that this system can be used for selective, local delivery of drugs where higher amounts of the drug are released in response to metastasis, angiogenesis, and invasion-promoting proteases. This strategy may prove to be a novel and effective method to overcome inadequacies in current controlled drug release systems.     

Human IgE antibody to the carbohydrate-containing third domain of chicken ovomucoid

Two kinds of the third domain, either with or without a carbohydrate chain, were prepared from chicken ovomucoid. The immunoreactivity of the domain preparations to human IgE antibody directed against ovomucoid was examined by using the sera from patients of egg allergy. About 30% of the serum antibody to ovomucoid reacted with the carbohydrate-containing domain, whereas little or no antibody with reactivity to the carbohydrate-free domain was detected, suggesting that the carbohydrate chain attached to the third domain played an important role in antigenic determinants of the carbohydrate-containing third domain against the human IgE antibody.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

Release of hydrophobic anticancer drug from a newly designed self-assembling peptide

A newly designed self-assembling peptide, P4 (Ac-NH-LDLKLELKLDLKLELK-CONH(2)), capable of stabilizing hydrophobic compounds in aqueous solution has been discovered. The ionic self-complementary peptide P4 has 16 amino acids, ～5 nm in size, with an alternating polar and non-polar pattern. Circular dichroism analysis demonstrated that P4 forms stable β-sheet structures, and self-assembles into nanofibers, which was demonstrated by atomic force microscopy. These nanofibers can form a scaffold hydrogel consisting of >99% water. It showed that the P4 hydrogel had stable hydrogel rheological properties. The ability of the peptide to stabilize the hydrophobic anticancer agent ellipticine was tested in this research. The results showed that the state of ellipticine in the complexes was dependent on the concentration of the peptide, which also affected the size and morphology of the complex. The anticancer activity of the complexes was studied by testing the viability with a MTT assay and a LIVE/DEAD Viability/Cytotoxicity kit in two cancer cell lines including SMMC7721 and EC9706. The viability results showed that complexes of protonated ellipticine could significantly reduce the viability of the two cell lines. The results described herein provide further incentives for basic studies on self-assembling peptide-based delivery of hydrophobic anticancer drugs.     

Ovomucoid intervening sequences specify functional domains and generate protein polymorphism

Two thirds of the natural chicken ovomucoid gene has been sequenced, including all exons and the intron sequences surrounding all fourteen intron/ exon junctions. The junction sequences surrounding four of the introns are redundant; however, the sequences surrounding the other three introns contain no redundancies and thus the splicing sites at either end of these three introns are unambiguous. The splicing in all cases conforms to the GT-AG rule. The ovomucoid gene sequence around intron F can be used to predict the cause of an internal deletion polymorphism in the ovomucoid protein, which is an apparent error in the processing of the ovomucoid pre-mRNA. We also compare the structural organization of the ovomucoid gene with the ovomucoid protein sequence to examine theories of the evolution of ovomucoids as well as the origin of intervening sequences. This analysis suggests that the present ovomucoid gene evolved from a primordial ovomucoid gene by two separate intragenic duplications. Furthermore, sequence analyses suggest that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Finally, the positions of the introns within the ovomucoid gene support the theory that introns separate gene segments that code for functional domains of proteins and provide insight on the manner by which eucaryotic genes were constructed during the process of evolution.     

Structure and sorption properties of regenerated hemosorbent SCN

Stepwise desorption of protein components from the hemosorbent SCN after hemoperfusion was studied, and their molecular weights were determined. It was found that hard alkaline regeneration and oxidation with nitric acid do not affect the hemosorbent structure, composition and potentiometric characteristics. The regenerated hemosorbent can be successfully used for adsorbtion of extraneous low- and medium-molecular weight components to clarify microbiological liquids.     

Isolation and characterization of the chicken ovomucoid gene.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.     

Photo-cross-linked hydrogels with polysaccharide-poly(amino acid) structure: new biomaterials for pharmaceutical applications

The aim of this work has been the preparation and characterization of novel hydrogels with polysaccharide-poly(amino acid) structure having suitable physicochemical properties for pharmaceutical applications. In the first step, hyaluronic acid (HA) and alpha,beta-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) have been derivatized with methacrylic anhydride (AMA), thus obtaining HA-AMA and PHM derivatives, respectively. In the second step, aqueous solutions of both these derivatives have been irradiated at 313 nm to obtain chemical hydrogels. The hydrogel obtained by irradiating for 15 min an aqueous solution containing 4% w/v of HA-AMA and 4% w/v of PHM resulted in the highest yield. Its swelling ability was dependent on the pH and nature of the external medium. Besides, this hydrogel undergoes a partial hydrolysis, especially in the presence of enzymes, such as esterase or hyaluronidase, but the entity of this degradation is lower than that observed for a hydrogel based on HA-AMA alone. The ability of this hydrogel to entrap drug molecules has been evaluated by using thrombin as a model drug. In vitro release studies and a platelet aggregation test demonstrated that the HA-AMA/PHM hydrogel is able to release thrombin in the active form, thus suggesting its suitability for the treatment of hemorrhages.     

Lipid transfer between phosphatidylcholine vesicles and human erythrocytes: exponential decrease in rate with increasing acyl chain length

The rate of phospholipid transfer from sonicated phospholipid vesicles to human erythrocytes has been studied as a function of membrane concentration and lipid acyl chain composition. Phospholipid transfer exhibits saturable first-order kinetics with respect to both cell and vesicle membrane concentrations. This kinetic behavior is consistent either with transfer during transient contact between cell and vesicle surfaces (but only if the fraction of the cell surface susceptible to such interaction is small) or with transfer of monomers through the aqueous phase. The acyl chain composition of the transferred phospholipid affects the transfer kinetics profoundly; for homologous saturated phosphatidylcholines, the rate of transfer decreases exponentially with increasing acyl chain length. This behavior is consistent with passage of phospholipid monomers through a polar phase, which might be the bulk aqueous phase( as in the monomer transfer model) or the hydrated head-group regions of a cell-vesicle complex (transient collision model). Collisional transfer also predicts that intercell transfer of phospholipids should be slow compared to cell-vesicle transfer, as surface charge and steric effects should prevent close apposition of donor and acceptor membranes. This is not found; dilauroylphosphatidylcholine transfers rapidly between red cells. Thus, the observed relationship between acyl chain length and intermembrane phospholipid transfer rates likely reflects the energetics of monomer transfer through the aqueous phase.     

Turtle-dove ovomucoid, a glycoprotein proteinase inhibitor with P1-blood-group antigen activity.

Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.     

Superabsorbent conducting hydrogel from poly(acrylamide-aniline) with thermo-sensitivity and release properties

Interpenetrating networks (IPN) poly(acrylamide-aniline) polymer was synthesized by a two-steps aqueous polymerization method, which aniline monomer was absorbed in the network of polyacrylamide and followed by a polymerization reaction between aniline monomers. The poly(acrylamide-aniline) hydrogel possessed a conductivity of 25.28 mS cm⁻¹. An interpenetrating network structure model with a three-dimensional network of polyacrylamide and a one-dimensional chain of polyaniline for poly(acrylamide-aniline) conducting hydrogel was proposed, and a conduction mechanism with charge carriers (protons) hopping along the polyaniline chain was suggested. The poly(acrylamide-aniline) hydrogels have predominant thermo-sensitivity. Poly(acrylamide-aniline) hydrogels possess loading and releasing properties, an anomalous release mechanism is found.     

Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli

The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens. Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children. A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end. Upon induction, the E. coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni²⁺ nitrilotriacetic acid agarose affinity chromatography. Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue. The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum. CD spectra indicated that that the recombinant ovomucoid has more -helix and less -structure than native form. These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid.     

Will heat-stable proteins nonspecifically protect cells from thermal stress?

In cell-free systems, stress-resistant proteins nonspecifically stabilize stress-susceptible proteins. This mechanism has been suggested to contribute to thermotolerance in cells (Minton et al.: Proc. Natl. Acad. Sci. USA, 79: 7107-7117, 1982). To test this hypothesis, red-blood-cell-mediated microinjection was used to transfer macromolecules into monolayers of CHO cells. We introduced the heat-stable proteins fetuin and ovomucoid into RBCs during hypotonic hemolysis and then fused the RBCs to CHO cells with polyethylene glycol as fusogen. Fetuin and ovomucoid were successfully transferred into 36-55% of the CHO cells as demonstrated by fluorescence of FITC-conjugated proteins. The plating efficiency of these CHO cells after fusion ranged from 35% to 60%. Three hours after fusion, CHO cells microinjected with fetuin or ovomucoid were exposed to 43 degrees C for 0-180 min or 45 degrees C for 0-40 min, and thermal survival was determined. There was no difference in cell survival between control untreated cells, control cells fused with nonloaded RBCs, and cells fused with RBCs loaded with fetuin or ovomucoid. While our results do not support the hypothesis that heat-stable proteins nonspecifically protect cells from thermal stress, several possible explanations are provided for this observation.     

Structural characterization of nanoscale meshworks within a nucleoporin FG hydrogel

The permeability barrier of nuclear pore complexes (NPCs) controls all exchange of macromolecules between the cytoplasm and the cell nucleus. It consists of phenylalanine-glycine (FG) repeat domains apparently organized as an FG hydrogel. It has previously been demonstrated that an FG hydrogel derived from the yeast nucleoporin Nsp1p reproduces the selectivity of authentic NPCs. Here we combined time-resolved optical spectroscopy and X-ray scattering techniques to characterize such a gel. The data suggest a hierarchy of structures that form during gelation at the expense of unstructured elements. On the largest scale, protein-rich domains with a correlation length of ~16.5 nm are evident. On a smaller length scale, aqueous channels with an average diameter of ~3 nm have been found, which possibly represent the physical structures accounting for the passive sieving effect of nuclear pores. The protein-rich domains contain characteristic β-structures with typical inter-β-strand and inter-β-sheet distances of 1.3 and 0.47 nm, respectively. During gelation, the formation of oligomeric associates is accompanied by the transfer of phenylalanines into a hydrophobic microenvironment, supporting the view that this process is driven by a hydrophobic collapse.     

Galactosyltransferase activities in mitochondria outer membrane: biosynthesis of galactosylated proteins

1. Mitochondria outer membranes prepared from mouse livers were purified on a discontinuous sucrose gradient. Control in electron microscopy and marker enzymes assays confirmed purity and homogeneity of this fraction. 2. Purified mitochondria outer membranes exhibited significant UDP-galactose: glycoprotein galactosyltransferase activities when incubated with endogenous or exogenous glycoprotein acceptors in presence of detergent (Nonidet P40). 3. Some properties of two distinct mitochondrial galactosyltransferases, acting respectively on ovomucoid and ovine asialo-mucin were investigated. 4. Transfer of galactose on ovomucoid was maximal for a pH of 7.6 at 33 degrees C whereas asialo-mucin galactosyltransferase exhibited an optimum pH of 5.6 for an optimal temperature of 46 degrees C. 5. These two distinct membrane-bound enzymes were both inhibited by diacylglycerophospholipids whereas lysophospholipids modulated both enzymes in a different way: at 5 mM lysophosphatidylcholine, asialo-mucin galactosyltransferase was slightly stimulated while ovomucoid galactosyltransferase was markedly activated. 6. The most important activating effect on ovomucoid galactosyltransferase was obtained with a phospholipid containing a long aliphatic side chain linked by an ester bond in sn-1 of glycerol, an hydroxyl group or hydrogen atoms in sn-2 and a phosphorylcholine head group in sn-3.