Purification and properties of alpha-galactosidase from white-rot fungus Pleurotus florida

alpha-Galactosidase was strongly induced in the white-rot fungus Pleurotus florida by arabinose than its natural substrates and was purified to homogeneity by acetone precipitation, ultrafiltration and DEAE-Sepharose chromatography. The enzyme was a monomeric protein with a molecular mass of approximately equal to 99 kDa, as revealed by native-PAGE and SDS-PAGE. alpha-Galactosidase was optimally active at 55 degrees C for the hydrolysis of p-nitrophenyl-alpha-galactopyranoside (PNPalphaG) and lost its 20% and 50% of original activity in 30 min at 60 degres C and 70 degrees C, respectively. The pH optimum of the enzyme was between 4.6 and 5.0. It was stable in a wide pH range (pH 4.0 to 9.0) at 55 degrees C for 2 h. The Ag+ and Hg2+ strongly inhibited the enzyme activity. Galactose, glucose, maltose and lactose also inhibited the enzyme activity, whereas N-bromosuccinimide treatment resulted in near total loss of acitivity. The Km and Vmax values of the enzyme for PNPalphaG were found to be 1.1 mM, and 77 micromol min(-1) mg(-1), respectively. alpha-Galactosidase immobilized in agar was more effective for the degradation of raffinose than in the sodium alginate. TLC results indicated its potential for the removal of raffinose and stachyose in soymilk.     

Endothelial progenitor cells are integrated in newly formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a fibrin matrix

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue-engineered matrices. The aim of our study was to investigate the impact of a well-characterized murine embryonal EPC line (T17b-EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time-points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing β-galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell-free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices.     

From virgin education to real education

The now famous virgin headspace experiment from late Braja D. Mookherjee and Subha Patel, and its use in the creation of scent 'Virgin No 1' by Christophe Laudamiel and Christoph Hornetz for the novel by Patrick Süskind, and the movie by Tom Tykwer 'Perfume--The Story of a Murderer' (Constantin Film-Thierry Mugler Parfums-IFF, 2006) is discussed. Another fragrance from the luxury coffret, 'Salon Rouge', is described as well, illustrating how molecules and natural ingredients can be utilized, not only to create innovative or artistic fragrance compositions, but also to provide fundamental consumer education, improving the public image both of Chemistry and Perfumery.     

Identification and characterization of rabbit small intestinal villus cell brush border membrane Na-glutamine cotransporter

Glutamine, the primary metabolic fuel for the mammalian small intestinal enterocytes, is primarily assimilated by Na-amino acid cotransporters. Although Na-solute cotransport has been shown to exist in the brush border membrane (BBM) of the absorptive villus cells, the identity of Na-glutamine cotransport in rabbit small intestinal villus cells was unknown. Na-dependent glutamine uptake is present in villus BBM vesicles. An intravesicular proton gradient did not stimulate this Na-dependent glutamine uptake, whereas Li+ did not significantly suppress this uptake. These observations in concert with amino acid substitution studies suggested that Na-glutamine cotransporter in the villus cell BBM was the newly identified cotransporter B0AT1 (SLC6A19). Quantitative real-time PCR identified the message for this cotransporter in villus cells. Thus a full-length cDNA of B0AT1 was cloned and expressed in MDA-MB-231 cells. This expressed cotransporter exhibited characteristics similar to those observed in villus cells from the rabbit small intestine. Antibody was generated for B0AT1 that demonstrated the presence of this cotransporter protein in the villus cell BBM. Kinetic studies defined the kinetic parameters of this cotransporter. Thus this study describes the identification, cloning, and characterization of the Na-amino acid cotransporter responsible for the assimilation of a critical amino acid by the absorptive villus cells in the mammalian small intestine.     

A mercapto analogue of 5'-noraristeromycin.

5'-Noraristeromycin and its enantiomer have been found to possess a wide range of antiviral effects. In the search for analogues of and with improved activity, the synthesis of both enantiomers of 5'-mercapto-5'-deoxy-5'-noraristeromycin ( and ) has been accomplished. While (+)-7 was inactive, (-)- did show marginal activity against vaccinia virus, but not any other virus.     

Recurrence of Early Stage Colon Cancer Predicted by Expression Pattern of Circulating microRNAs

Systemic treatment of patients with early-stage cancers attempts to eradicate occult metastatic disease to prevent recurrence and increased morbidity. However, prediction of recurrence from an analysis of the primary tumor is limited because disseminated cancer cells only represent a small subset of the primary lesion. Here we analyze the expression of circulating microRNAs (miRs) in serum obtained pre-surgically from patients with early stage colorectal cancers. Groups of five patients with and without disease recurrence were used to identify an informative panel of circulating miRs using quantitative PCR of genome-wide miR expression as well as a set of published candidate miRs. A panel of six informative miRs (miR-15a, mir-103, miR-148a, miR-320a, miR-451, miR-596) was derived from this analysis and evaluated in a separate validation set of thirty patients. Hierarchical clustering of the expression levels of these six circulating miRs and Kaplan-Meier analysis showed that the risk of disease recurrence of early stage colon cancer can be predicted by this panel of miRs that are measurable in the circulation at the time of diagnosis (P = 0.0026; Hazard Ratio 5.4; 95% CI of 1.9 to 15).