Promoting effect of amino acids added to a chemically defined medium on blastocyst formation and blastomere proliferation of bovine embryos cultured in vitro

This study was conducted to elucidate the role of amino acids added singly or in groups to a chemically defined culture medium in blastocyst formation and blastomere proliferation of bovine embryos. Embryos were generated by in vitro fertilization, and blastocyst formation and hatching, and blastomere number of blastocysts were subsequently monitored after the culture of embryos in synthetic oviduct fluid medium (SOFM). First, one of four non-essential amino acids (asparagine, aspartate, glutamate or serine) was added to SOFM and, compared with no addition, a significant (P <0.05) increase in blastocyst formation was found after the addition of asparagine, aspartate, or glutamate (35-42% versus 22%). Second, one of four essential amino acids (arginine, cystine, isoleucine or leucine) was added and arginine or isoleucine greatly improved blastocyst formation (30-36% versus 16%). Third, the addition of five stimulatory amino acids (aspartate, asparagine, glutamate, arginine and isoleucine) to SOFM significantly improved blastocyst formation compared with no addition (12% versus 21%) and such value was similar to that obtained after the addition of 19 amino acids consisting of MEM amino acid solutions (21-27%). However, five amino acids yielded fewer hatched blastocysts than 19 amino acids. Finally, although five amino acids yielded more cell number of blastocysts than no addition (93 versus 74 cells per blastocyst), it was lower than that from 19 amino acids (131 cells per blastocyst). In conclusion, either single or combined addition of asparagine, aspartate, glutamate, arginine and isoleucine stimulated blastocyst formation, while other amino acids might be necessary for further stimulating blastomere proliferation and blastocyst hatching.     

Taxonomic discrimination of higher plants by pyrolysis mass spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum and has been widely applied to the discrimination of closely related microbial strains. Leaf samples of six species and one variety of higher plants (Rosa multiflora, R. multiflora var. platyphylla, Sedum kamtschaticum, S. takesimense, S. sarmentosum, Hepatica insularis, and H. asiatica) were subjected to PyMS for spectral fingerprinting. Principal component analysis of PyMS data was not able to discriminate these plants in discrete clusters. However, canonical variate analysis of PyMS data separated these plants from one another. A hierarchical dendrogram based on canonical variate analysis was in agreement with the known taxonomy of the plants at the variety level. These results indicate that PyMS is able to discriminate higher plants based on taxonomic classification at the family, genus, species, and variety level.Abbreviations ANNs Artificial neural networks - CVA Canonical variate analysis - GC/MS Gas chromatography/mass spectrometry - PCA Principal component analysis - PyMS Pyrolysis mass spectrometry - UPGMA Unweighted pair group method with arithmetic mean Communicated by I.S. Chung     

Metabolomic analysis of Strychnos nux-vomica, Strychnos icaja and Strychnos ignatii extracts by 1H nuclear magnetic resonance spectrometry and multivariate analysis techniques

1H Nuclear magnetic resonance spectrometry and multivariate analysis techniques were applied for the metabolic profiling of three Strychnos species: Strychnos nux-vomica (seeds, stem bark, root bark), Strychnos ignatii (seeds), and Strychnos icaja (leaves, stem bark, root bark, collar bark). The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between all samples, using the three first components. The key compounds responsible for the discrimination were brucine, loganin, fatty acids, and Strychnos icaja alkaloids such as icajine and sungucine. The method was then applied to the classification of several "false angostura" samples. These samples were, as expected, identified as S. nux-vomica by PCA, but could not be clearly discriminated as root bark or stem bark samples after further statistical analysis.     

Clinical application of fresh fibroblast allografts for the treatment of diabetic foot ulcers: a pilot study

Diabetic foot ulcers often pose a difficult problem for health care professionals because of the defects associated with fibroblast functioning. Although there has been much interest recently in the use of topical growth factors for the treatment of diabetic foot ulcers, the effects are generally not very dramatic. Cryopreserved fibroblast implants, which are able to adjust to a wound's environment and provide the desired growth factors and other substances that may be lacking in a chronic wound, represent an exciting development and a major advance. These products may well provide growth factors in the right concentration and in the right sequence, something that has proved difficult to achieve with the topical application of recombinant growth factors. However, cell activities are impaired by cryopreservation. The purpose of this study was to assess the effects of fresh human allogeneic fibroblast grafting for the treatment of diabetic foot ulcers. Eight patients with diabetic foot ulcers ranging from 6 to 17 weeks in duration were treated. The size of the wounds ranged from 2.0 to 6.0 cm2, with three patients exhibiting exposed bones. A history of diabetic foot ulcers was present in five patients. Human dermal fibroblasts from healthy teenagers were cultured in Dulbecco's modified Eagle medium/Ham's F-12 supplemented with 10% autologous serum. The cultured cells were applied over the wounds immediately after debridement; fibrin was used as a cell carrier. A dressing was then applied with Tegaderm and kept moist until healing was complete. The progress and time for complete wound closure and patient satisfaction were assessed, with follow-up time ranging from 6 to 18 months. Complete wound healing occurred in all patients. Eleven to 21 days were needed for complete reepithelization of the wound, and no clinical or laboratory abnormalities were noted. Patient satisfaction was also very positive. In this study, the use of fresh human fibroblast allografts was found to be a safe and effective treatment for diabetic foot ulcers.     

Hyaluronic acid inhibits adhesion of hepatic stellate cells in spite of its stimulation of DNA synthesis

Unbalanced accumulation of fibers in extracellular matrix (ECM) results from attachment and activation of hepatic stellate cells (HSCs) during chronic liver diseases, in which the content of hyaluronic acid (HA), a glycosaminoglycan, in ECM changes. No information is available on the effect of HA on adhesion and activation of HSCs although that of collagen (Col) on HSCs was extensively studied. This study investigated the effects of HA with or without Col on adhesion of HSCs or the rate of DNA synthesis. Attachment of primary cultured HSCs was microscopically monitored in the plate simultaneously coated with HA or other ECM components. HA inhibited adhesion of quiescent HSCs at least up to 7 days after seeding, whereas HSCs were adherent to plastic or type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV) or fibronectin. Both microscopy and alpha-smooth muscle actin immunocytochemistry revealed that the number of HSCs, which had been re-seeded after 15 days of culture, attached to HA-coated area was remarkably lower compared to that of HSCs on Col-I or plastic. Incorporation of HA into Col-I prevented adhesion of activated HSCs to matrix film. The number of HSCs adherent to HA at early times after seeding was minimal and significantly lower than that of the cells adherent to plastic. In contrast, either Col-I or Col-IV increased the number of adherent cells. Attachment of HSCs to plastic was inhibited by soluble HA in culture medium. CD44, the cell surface receptor to which HA binds, was immunochemically detected in HSCs. Adhesion of HSCs to plastic, HA or Col-I was not changed by anti-CD44 antibody. Either HA or Col increased the basal or platelet-derived growth factor-inducible rate of thymidine incorporation into DNA in HSCs. In conclusion, HA inhibits adhesion of quiescent or activated HSCs in spite of its stimulation of DNA synthesis, whereas Col increases HSC attachment and DNA synthesis, and inhibition of HSC adhesion by HA does not involve CD44.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.     

Lead biosorption by waste biomass of Corynebacterium glutamicum generated from lysine fermentation process

Biomass waste, mainly Corynebacterium glutamicum, is generated from large-scale lysine fermentation process. In this study, protonated C. glutamicum biomass was evaluated as a biosorbent for the removal of lead from synthetic wastewater. As Pb2+ were bound to the biomass, the solution pH deceased, indicating that protons in the biomass were exchanged with lead ions. The Corynebacterium biomass bound Pb2+ at up to 2.74 mmol g(-1) at pH 5, where lead does not precipitate. Compared with other biosorbents and conventional sorbents, such as natural zeolite, activated carbon and synthetic ion exchange resin, the protonated C. glutamicum biomass was considered to be a useful biomaterial for lead biosorption.