Decellularization of camel corneas for tissue engineering applications.

Purification and amino acid compositions of the structural proteins of sindbis virus

To evaluate the potential use of decellularized porcine stromal matrix (PSM) for reconstruction of corneal stroma in a rabbit model. Ten chinchilla bastard rabbit corneas were exposed to a circular half-thickness keratotomy with a 3.0 mm diameter at the central cornea. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 K IU/ml) and 0.3% sodium dodecyl sulphate (SDS). The 3.0 mm in diameter decellularized corneal stromal xenograft was inserted into the pocket, and the incision was closed with four 10.0 nylon sutures. Clinical photographs were taken at day 1, day 7, day 30 and on a monthly basis for up to 6 months after transplantation. Six months after surgery, the rabbits were killed and eyes were enucleated. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the complete removal of the corneal cells after decellularization of porcine corneas and repopulation with rabbit cells. Alcian blue staining was performed for analysing the structure of the extracellular matrix (ECM). Efficient elimination of porcine cells was achieved by our decellularization protocol and confirmed via HE and DAPI stainings. Moreover, the major histoarchitectural ECM structure had been maintained as visualized by the alcian blue stain. Finally, the PSM was biocompatible with the host's epithelium evidenced as a regrowth covering the exposed xenograft. This novel technique of tissue engineering may provide one of many solutions to addressing anterior corneal pathological conditions in the face of a shortage of human corneal material.

Amino Acid Composition and Biological Value of Proteins In Some Aquatic Plant Species

The predictive limits of the amino acid composition for the secondary structural content (percentage of residues in the secondary structural states helix, sheet, and coil) in proteins are assessed quantitatively. For the first time, techniques for prediction of secondary structural content are presented which rely on the amino acid composition as the only information on the query protein. In our first method, the amino acid composition of an unknown protein is represented by the best (in a least square sense) linear combination of the characteristic amino acid compositions of the three secondary structural types computed from a learning set of tertiary structures. The second technique is a generalization of the first one and takes into account also possible compositional couplings between any two sorts of amino acids. Its mathematical formulation results in an eigenvalue/eigenvector problem of the second moment matrix describing the amino acid compositional fluctuations of secondary structural types in various proteins of a learning set. Possible correlations of the principal directions of the eigenspaces with physical properties of the amino acids were also checked. For example, the first two eigenvectors of the helical eigenspace correlate with the size and hydrophobicity of the residue types respectively. As learning and test sets of tertiary structures, we utilized representative, automatically generated subsets of Protein Data Bank (PDB) consisting of non-homologous protein structures at the resolution thresholds < or = 1.8A, < or = 2.0A, < or = 2.5A, and < or = 3.0 A. We show that the consideration of compositional couplings improves prediction accuracy, albeit not dramatically. Whereas in the self-consistency test (learning with the protein to be predicted), a clear decrease of prediction accuracy with worsening resolution is observed, the jackknife test (leave the predicted protein out) yielded best results for the largest dataset (< or = 3.0A, almost no difference to the self-consistency test!), i.e., only this set, with more than 400 proteins, is sufficient for stable computation of the parameters in the prediction function of the second method. The average absolute error in predicting the fraction of helix, sheet, and coil from amino acid composition of the query protein are 13.7, 12.6, and 11.4%, respectively with r.m.s. deviations in the range of 8.6 divided by 11.8% for the 3.0 A dataset in a jackknife test. The absolute precision of the average absolute errors is in the range of 1 divided by 3% as measured for other representative subsets of the PDB. Secondary structural content prediction methods found in the literature have been clustered in accordance with their prediction accuracies. To our surprise, much more complex secondary structure prediction methods utilized for the same purpose of secondary structural content prediction achieve prediction accuracies very similar to those of the present analytic techniques, implying that all the information beyond the amino acid composition is, in fact, mainly utilized for positioning the secondary structural state in the sequence but not for determination of the overall number of residues in a secondary structural type. This result implies that higher prediction accuracies cannot be achieved relying solely on the amino acid composition of an unknown query protein as prediction input. Our prediction program SSCP has been made available as a World Wide Web and E-mail service.

Data on the amino acid composition of proteins having various functions from organisms representing different evolutionary levels (83 superfamilies) are used in order to elucidate the trends in protein molecular evolution. The interconnections evolutionary rate (rate of mutation acceptance) — amino acid composition, and evolutionary level of the organism — amino acid composition (in case of proteins of the same or very similar function) are studied. The amino acid compositions of proteins performing jointly an evolutionarily old functions are also juxtaposed. The mean contemporary protein composition is used as a basis for comparison. The obtained results are evidence in favour of the existence of a trend for an increase of the special amino acids (Met, Ile, Gln, His, Lys, Asn, Phe, Tyr, Trp, Cys) at the expense of the usual ones (Thr, Pro, Ala, Ser, Arg, Gly, Leu, Val, Glu, Asp). The tests of statistical significance of the obtained results (comparison of the mean compositions of proteins from low evolutionary level organisms with that of all sequenced proteins; comparison of the mean contemporary protein composition with that obtained after simulation of the evolutionary process) confirm and universalize the observed trend. The above results direct the attention to the concept of a smaller number of amino acids in the ancient proteins and respectively simpler genetic code. A fluctuation around the initial primitive level is suggested to explain the conservatism of proteins of the same function in evolutionarily low level organisms. The observed trend could be applied for designing new proteins.

The expansion of the production of products with specified functional indicators (cereals, bakery products, pasta, desserts, muesli, yogurts) and feed with a certain amino acid composition is the main task of the food industry and the feed industry, and naked oats have firmly occupied their own niche in these areas of processing. The purpose of the research is to establish the amino acid composition of oat proteins of the hulled oat variety Konkur and naked varieties Bekas and Baget, to assess the balance of essential amino acids in oat proteins relative to the «reference protein» (amino acid scale according to FAO / WHO data) in order to use grain as an additive in food and feed to increase the proportion of essential amino acids in them. The research was carried out during 2018-2022 at the experimental base of the Samara Research Institute of Agriculture. Grain samples of the varieties of filmy and naked oats Konkur, Bekas and Baget were used as experimental material. It was found that the highest proportion of essential amino acids was observed in varieties of naked oats 39,9-41,0 g/kg. The sum of critical amino acids (lysine, methionine, tryptophan) in naked oat varieties was 9,4-9,6 g/kg, which is 1,1-1,3 g/kg higher than in the filmy variety Konkur. According to studies, 1 g of Konkur variety protein contains 249,7 mg of essential amino acids, proteins of naked oat variety Bekas - 268,4 mg and Baget variety - 262,9 mg, which corresponds to 69,4 %, 74,6 % and 73,0 % relative to FAO/WHO recommended standards. The excess of some essential amino acids in naked oat varieties (amino acid score over 100 %) contributed to the imbalance in the ratio of essential amino acids to the «reference» values, while increasing the coefficient of difference in amino acid scores (KRAS protein of Konkur variety was 24,2 %, Bekas variety 33,9 %, varieties Baget 35,5 %), thereby contributing to a decrease in the biological value of the proteins of the varieties Bekas and Baget in relation to the protein of the filmy variety Konkur. Grain of naked oats with a high content of essential amino acids should be used as an ingredient in the production of food and feed with high nutritional value.

Albumins, globulins, gliadins and glutenins presumably comprising 100 percent of the wheat seed proteins were sequentially extracted and electrophoresed on SDS-polyacrylamide gels. The SDS-electrophoretic patterns within each of the four fractions from T. boeotiaum, T. urartu, T. turgidum, T. timopheevii, T. aestivum, Ae. speltoides and Ae. squawosa were similar. They differed from one species to another only in a few minor components or density of certain components. Similarity in MW's of components, as indicated by the SDS-electrophoretic patterns, suggests that the wheats and Aegilops exhibit no variability for structural genes coding seed proteins. A minimum of 60 to 70 and a maximum of 360 to 420 structural genes with major or minor effects control the total seed protein in T. aestivum. Presumably, only one or the other homoeoallele was expressed in the polyploids. Different components of albumins and globulins presumably had distinct MW's and amino acid composition, while the components of gliadins and glutenins could be classified into a few groups each containing one or more components with the same MW and nearly identical amino acid composition. The genes for components with similar MW's and amino acid composition arose through multiplication of a single original gene and perhaps share the same regulatory mechanism. Seed protein content and quality in wheat might be improved through the incorporation of structural genes, coding for polypeptides with distinct MW's, from distantly related species, rather than by manipulation of the structural genes within the Triticum-Aegilops group. Regulatory mutants similar to opaque-2 of corn could be used to alter the proportion of gliadins in relation to albumins and globulins, to improve amino acid composition of wheat proteins.

Chapter 6 - Ultrastructure and Biomechanics of Skeletal Muscle ECM: Implications in Tissue Regeneration

d-Galactose-binding protein was purified from cell-free extracts of Escherichia coli B/r and crystallized using 2-methyl-2,4-pentanediol. This material appeared homogeneous following electrophoresis on polyacrylamide gels in either the presence or absence of sodium dodecyl sulfate. The amino acid composition of this protein was determined and found to be similar to the amino acid composition of d-galactose-binding protein isolated from E. coli K12 but different from the amino acid composition of l-arabinose binding protein from E. coli B/r. Ligand-binding specificities and affinities for the d-galactose-binding protein from E. coli B/r were determined and found to be distinctly different from the ligand-binding characteristics of the l-arabinose-binding protein isolated from the same organism. Peptide map patterns of peptides generated by tryptic digestions of both the d-galactose-binding protein and the l-arabinose-binding protein have demonstrated additional differences between these two proteins. Cross-reactivity was observed between antisera directed against either the d-galactose- or the l-arabinose-binding protein and the heterologous protein, suggesting that although these are two distinctly different proteins, they have some similar structural relationships.

The tobacco mosaic virus strains, C and W which were isolated from crucifer plants, were compared about their host ranges with those of the other TMV strains, HR, Lychnis strain Ly, two ordinary strains OM and O, cowpea strain BC-B, its tobacco form BC-T, and 06. Strains C and W infected all 18 kinds of crucifer plants systemically, and HR and Ly infected 11 and 15 kinds of them respectively. Sometimes OM infected 5 Brassica plants systemically, but their virus contents were very low. Strains 06, BC-B, and O were inoculated to 6 Brassica plants, but no plants were infected systemically. Strains HR, C, W, and Ly caused only local lesions on the leaves of petunia and most of Nicotiana plants including several tobacco varieties. Among cucurbitaceous plants tested, only M. Charantia showed local lesions by the inoculation of HR, C, W, Ly, and OM. Ph. vulgaris var. Kairyo-Otebo showed no local lesions by the inoculation of HR, C, W, and Ly. Strains HR, C, W, and Ly caused primary lesions on P. lanceolata, and HR and sometimes Ly caused systemic infection. From the similarity of the host ranges and the amino acid compositions of the coat proteins of HR, C, W, and Ly, the authors concluded that these strains should be classified as “HR group” and suggested that TMV strains having a similar native amino acid composition may have a similar host range.

A compact mitochondrial gene contains all essential information about the synthesis of mitochondrial proteins which play their roles in a small compartment of the mitochondrium. Almost no noncoding regions have been found through the gene, but a necessary set of tRNAs for the 20 amino acids is provided for biosynthesis, some of them coding different amino acids from those in a usual cell. Since the gene is so compact that the produced proteins would have some characteristic aspects for the mitochondrium, amino acid compositions of mitochondrial proteins (mt-proteins) were examined in the 20-dimensional composition space. The results show that compositions of proteins translated from the mitochondrial genes have a distinct character having more hydrophobic content than others, which is illustrated by a clustered distribution in the multidimensional composition space. The cluster is located at the tail edge of the global distribution pattern of a Gaussian shape for other various kinds of proteins in the space. The mt-proteins are rich in hydrophobic amino acids as is a membrane protein, but are different from other membrane proteins in a lesser content of Val. A good correlation found between the base and amino acid compositions for the mitochondria was examined in comparison to those of organisms such as thermophilic bacterium having an extreme G-C-rich base composition.

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

Modulation of antigen-specific T and B cell responses influence bacterial clearance of non-typeable Haemophilus influenzae from the lung in a rat model

In this study, cocoons and degummed silk samples of Bombyx mori and twenty Saturniidae species of the genera Actias, Attacus, Argema, Antheraea, Caligula, Callosamia, Cricula, Epiphora, Hyalophora, Loepa, Samia and Saturnia are studied to gain an insight into their morphology, chemical composition and physical structure. For this purpose, silk samples are characterized by optical microscopy and FTIR spectroscopy in attenuated total reflection mode (ATR-FTIR spectroscopy). Furthermore, degummed silk samples are analyzed for their amino acid (AA) composition by GC-FID. In the course of method development, various degumming methods are tested using alkalis, citric acid, enzymes and detergents. A mixture of 0.1% sodium carbonate and 2.5% ethylenediamine proves to be an effective agent for degumming Saturniidae and B. mori cocoons. After hydrolysis of the fibroin filaments with 6 N hydrochloric acid and derivatization with propyl chloroformate, fifteen AAs are identified and qualified. This method shows a satisfactory overall analytical performance with an average recovery rate of 95% at the medium concentration level. The chemical composition of the different silks was considered comparatively. Within a genus, the analyses usually show a high degree of similarity in AA composition and the resulting structural indices, whereas differences are found between genera.

The correlations between differentiation, the amino acid composition of total protein and of soluble protein, and the disc electrophoretic distribution of soluble protein of carrot tissue cultures and of carrot plants (leaves) were studied. In spite of pronounced and characteristic changes in the electrophoretic distribution of the components of soluble protein in various developmental stages of both bioassays, no significant differences in the amino acid composition of protein were observed. With progressive development of whole carrot plants and of carrot tissue cultures, the number of protein bands on disc electropherogramms increased.