Immobilization of beta-galactosidase on fibrous matrix by polyethyleneimine for production of galacto-oligosaccharides from lactose

The production of galacto-oligosaccharides (GOS) from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth was studied. A novel method of enzyme immobilization involving PEI-enzyme aggregate formation and growth of aggregates on individual fibrils of cotton cloth leading to multilayer immobilization of the enzyme was developed. A large amount of enzyme was immobilized (250 mg/g support) with about 90-95% efficiency. A maximum GOS production of 25-26% (w/w) was achieved at near 50% lactose conversion from 400 g/L of lactose at pH 4.5 and 40 degrees C. Tri- and tetrasaccharides were the major types of GOS formed, accounting for about 70% and 25% of the total GOS produced in the reactions, respectively. Temperature and pH affected not only the reaction rate but also GOS yield to some extend. A reaction pH of 6.0 increased GOS yield by as much as 10% compared with that of pH 4.5 while decreased the reaction rate of immobilized enzyme. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme under the same reaction conditions, suggesting diffusion limitation was negligible in the packed bed reactor and the enzyme carrier. Increase in the thermal stability of PEI-immobilized enzyme was also observed. The half-life for the immobilized enzyme on cotton cloth was close to 1 year at 40 degrees C and 21 days at 50 degrees C. Stable, continuous operation in a plug-flow reactor was demonstrated for about 3 days without any apparent problem. A maximum GOS production of 26% (w/w) of total sugars was attained at 50% lactose conversion with a feed containing 400 g/L of lactose at pH 4.5 and 40 degrees C. The corresponding reactor productivity was 6 kg/L/h, which is several-hundred-fold higher than those previously reported.     

Characterization of humic materials extracted from hazelnut husk and hazelnut husk amended soils

This study was carried out to determine effects of composted hazelnut husk (CHH) on some chemical properties of soil and soil humic acid (HA). Compost application increases organic matter (OM) content of the soil considerably, OM value of 3.18% became 3.89% in 3 years time interval. Before application of compost, the soil pH was found to be 5.37, while after compost application it became 5.61. FTIR characteristics of humic acid/humic acid-like materials extracted from the original hazelnut husk, composted hazelnut husk and composted hazelnut husk amended soil have been investigated. C and O content of humic acid-like/humic acid materials were in the range of 41.4–50.8% and 37.8–50.5%, respectively. The N content of the humic acid/humic acid-like materials are in the expected range for humic materials which is 2–6%. Comparison of FTIR spectra of hazelnut husk and composted hazelnut husk humic acid-like fractions shows that both exhibit similar but not identical series of IR bands indicating the presence of the same functional groups in both samples. The only difference in the spectra seemed to be a decrement in the peak intensities of composted sample compared to uncomposted one. The similar differentiation of the intensities of IR bands of compost applied soil sample has also been observed. The FTIR spectral results show that the characteristics of composted material tend to become similar to that of soil humic acids characteristics in time.     

Cisplatin resistance induced by decreased apoptotic activity in non-small-cell lung cancer cell lines

We have investigated defective steps in apoptosis that might account for the development of resistance. For this purpose, A549 and Calu1 NSCLC (non-small-cell lung cancer) cell lines were treated with cisplatin to obtain resistant sub-lines. Gene expression profiles and the phosphorylation status of the BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) protein were determined for each cell line. Cell death and cytochrome c release were analysed after treating cell lines with their appropriate cisplatin doses. Gene expression of BAD, Bid, caspases 4 and 6 were clearly decreased in the resistant cell lines, and the differential phosphorylation status of BAD also seemed to play a role in the development of cisplatin resistance. Since this is a new cisplatin-resistant Calu1 cell line, it is noteworthy that DNA fragmentation, apoptotic cell ratio and cytochrome c levels were most decreased in the CR-Calu1 cell line.     

Establishment of higher passage PER.C6 cells for adenovirus manufacture

PER.C6 cells, an industrially relevant cell line for adenovirus manufacture, were extensively passaged in serum-free suspension cell culture to better adapt them to process conditions. The changes in cell physiology that occurred during this passaging were characterized by investigating cell growth, cell size, metabolism, and cultivation of replication-deficient adenovirus. The changes in cell physiology occurred gradually as the population doubling level, the number of times the cell population had doubled, increased. Higher passage PER.C6 (HP PER.C6) proliferated at a specific growth rate of 0.043 h(-1), 2-fold faster than lower passage PER.C6, and were capable of proliferation from lower inoculation cell densities. HP PER.C6 cell volume was 16% greater, and cellular yields on glucose, lactate, oxygen, and amino acids were greater as well. In batch cultures, HP PER.C6 cells volumetrically produced 3-fold more adenovirus, confirmed with three different constructs. The increase in productivity was also seen on a cell-specific basis. Although HP PER.C6 were more sensitive to the "cell density effect", requiring lower infection cell densities for optimal specific productivity, they proliferated more after infection than lower passage PER.C6, increasing the number of cells available for virus production. The extensive passaging established HP PER.C6 cells with several desirable attributes for adenovirus manufacture.     

Phytochemical Profiling and Biological Activity of Achillea sintenisii Hub.-Mor

Achillea (Asteraceae) species have been traditionally used for their different therapeutical properties. In this study, phytochemical composition of aerial parts of A. sintenisii which is endemic in Turkey was determined with Liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). To evaluate the wound healing potential, the cream formulation prepared from A. sintenisii was tested on the linear incision wound model in mice. In vitro enzyme inhibitory activity tests were performed on elastase, hyaluronidase, and collagenase. In the histopathological examination, angiogenesis and granulation tissue formation were significantly increased in A. sintenisii treatment groups compared to the negative control group. As a result of this study, it is thought that the enzyme inhibition and antioxidant activity of the plant may contribute to the wound healing process. According to LC/MS/MS analysis result, quinic acid (24.261 μg/mg extract) and chlorogenic acid (14.97 μg/mg extract) were identified as main constituents of the extract.     

Heligmosomoides polygyrus induces TLR4 on murine mucosal T cells that produce TGFbeta after lipopolysaccharide stimulation

Helminths are immune modulators that down-regulate colitis in inflammatory bowel disease. In animal models, intestinal bacteria drive colitis and in humans certain alleles of the LPS receptor protein TLR4 increase inflammatory bowel disease susceptibility. To understand helminthic immune modulation in the gut, we studied the influence of intestinal Heligmosomoides polygyrus colonization on LPS-induced lamina propria mononuclear cell (LPMC) cytokine responses in mice. LPS did not stimulate TGFbeta production from LPMC of uninfected mice. LPS strongly induced LPMC from worm-infected animals to secrete TGFbeta, but not TNF-alpha or IL-12. The TGFbeta derived from mucosal T cells. Helminth infection up-regulated TLR4 expression only in lamina propria T cells. LPMC from worm-infected TLR4 mutant animals did not respond to LPS, suggesting that LPS required TLR4 to stimulate TGFbeta secretion. Thus, during helminth infection, LPS challenge induces mucosal T cells to make TGFbeta through a TLR4-dependent process without promoting synthesis of proinflammatory cytokines.