Statistical optimization of solid state fermentation conditions for the enhanced production of thermoactive chitinases by mesophilic soil fungi using response surface methodology and their application in the reclamation of shrimp processing by-products

The mesophilic strains Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 are potent producers of extracellular thermoactive chitinases (endo-chitinase and β-N-acetylhexosaminidase). Chitinases have a wide range of applications in many areas including reclamation of seafood processing chitinous by-products. In the present study, the interactive effects of four fermentation conditions on thermoactive chitinase production by solid state fermentation (SSF) using commercial wheat bran (CWB) was investigated employing response surface methodology (RSM). Further, these chitinases were applied for the preparation of N-acetyl chitooligosaccharides from shrimp chitin. Statistical optimization resulted in the production (unit/g initial dry substrate, U/g IDS) of 19.8 endo-chitinase and 649.0 β-N-acetylhexosaminidase activity by A. flavus CFR 10, and 17.5 endo-chitinase and 319.9 β-N-acetylhexosaminidase activity by F. oxysporum CFR 8. Activity of crude endo-chitinase and β-N-acetylhexosaminidase were found to be optimum at 62?±?1 °C in a wide pH range. Hydrolysis of colloidal chitin with crude chitinases produced the maximum N-acetyl chitooligosaccharides yield (mmol/l) of 10.4?±?0.28 at 6 h and 10.2?±?0.01 at 30 h post-reaction initiation, respectively, by the enzymes of A. flavus CFR 10 and F. oxysporum CFR 8. HPLC analysis revealed the presence of N-acetyl chitooligosaccharides with N-acetyl chitotriose as the main end product of the colloidal chitin hydrolysis. These results indicate the potential of mesophilic A. flavus CFR 10 and F. oxysporum CFR 8 in the production of thermoactive chitinases employing the economical SSF process using CWB as an ideal substrate, as well as the potential of these chitinases for the reclamation of abundant shrimp processing by-products and production of defined N-acetyl chitooligosaccharides.     

Ethoxyzolamide Differentially Inhibits CO2 Uptake and Na+-Independent and Na+-Dependent HCO3- Uptake in the Cyanobacterium Synechococcus sp. UTEX 625

The effects of ethoxyzolamide (EZ), a carbonic anhydrase inhibitor, on the active CO2 and Na+-independent and Na+-dependent HCO3- transport systems of the unicellular cyanobacterium Synechococcus sp. UTEX 625 were examined. Measurements of transport and accumulation using radiochemical, fluorometric, and mass spectrometric assays indicated that active CO2 transport and active Na+-independent HCO3- transport were inhibited by EZ. However, Na+-independent HCO3- transport was about 1 order of magnitude more sensitive to EZ inhibition than was CO2 transport (50% inhibition = 12 [mu]M versus 80 [mu]M). The data suggest that both the active CO2 (G.D. Price, M.R. Badger [1989] Plant Physiol 89: 37-43) and the Na+ -independent HCO3 - transport systems possessed carbonic anhydrase-like activity as part of their mechanism of action. In contrast, Na+-dependent HCO3- transport was only partially (50% inhibition = 230 [mu]M) and noncompetitively inhibited by EZ. The collective evidence suggested that EZ inhibition of Na+ -dependent HCO3- transport was an indirect consequence of the action of EZ on the CO2 transport system, rather than a direct effect on HCO3- transport. A model is presented in which the core of the inorganic carbon translocating system is formed by Na+-dependent HCO3- transport and the CO2 transport system. It is argued that the Na+-independent HCO3 - utilizing system was not directly involved in translocation, but converted HCO3- to CO2 for use in CO2 transport.     

IDH1-Associated Primary Glioblastoma in Young Adults Displays Differential Patterns of Tumour and Vascular Morphology

Glioblastoma is a highly aggressive tumour with marked heterogeneity at the morphological level in both the tumour cells and the associated highly prominent vasculature. As we begin to develop an increased biological insight into the underlying processes driving the disease, fewer attempts have thus far been made to understand these phenotypic differences. We sought to address this by carefully assessing the morphological characteristics of both the tumour cells and the associated vasculature, relating these observations to the IDH1/MGMT status, with a particular focus on the early onset population of young adults who develop primary glioblastoma. 276 primary glioblastoma specimens were classified into their predominant cell morphological type (fibrillary, gemistocytic, giant cell, small cell, oligodendroglial, sarcomatous), and assessed for specific tumour (cellularity, necrosis, palisades) and vascular features (glomeruloid structures, arcades, pericyte proliferation). IDH1 positive glioblastomas were associated with a younger age at diagnosis, better clinical outcome, prominent oligodendroglial and small cell tumour cell morphology, pallisading necrosis and glomeruloid vascular proliferation in the absence of arcade-like structures. These features widen the phenotype of IDH1 mutation-positive primary glioblastoma in young adults and provide correlative evidence for a functional role of mutant IDH1 in the differential nature of neo-angiogenesis in different subtypes of glioblastoma.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.     

Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.

Biochemical and pharmacological data support the existence of multiple forms of the Na/H exchanger (NHE). Two isoforms, termed NHE-1 and NHE-2, have recently been isolated from rabbit ileal villus epithelial cells (Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. J. M., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouysségur, J., and Donowitz, M. (1991) EMBO J. 10, 1957-1967; Tse, C. M., Watson, A. J. M., Ma, A. I., Pouysségur, J., and Donowitz, M. (1991) Gastroenterology 100, A258). To identify additional molecular forms of the exchanger, rat brain, heart, kidney, stomach, and spleen cDNA libraries were screened for their presence using an NHE-1 cDNA probe under low stringency hybridization conditions. cDNAs encoding rat NHE-1 and two structurally related proteins, designated NHE-3 and NHE-4, have been isolated. Based on the deduced amino acid sequences, NHE-1, -3, and -4 are similar in size, having relative molecular masses of 91,506, 92,997, and 81,427, respectively. Overall, the proteins exhibit approximately 40% amino acid identity to each other and have similar hydropathy profiles, suggesting that they have the same transmembrane organization. The predicted N-terminal transmembrane regions of the three proteins, which span between 453 and 503 amino acids, exhibit the highest degree of identity (45-49%). In contrast, the C-terminal cytoplasmic regions, which span between 247 and 378 amino acids, exhibit very low amino acid identity (24-31%). Tissue distribution studies reveal that the NHE-1 mRNA is present at varying levels in all tissues examined, whereas NHE-3 and NHE-4 mRNAs exhibit a more limited distribution. NHE-3 mRNA is expressed at high levels in colon and small intestine, with significant levels also present in kidney and stomach. NHE-4 mRNA is most abundant in stomach, followed by intermediate levels in small intestine and colon and lesser amounts in kidney, brain, uterus, and skeletal muscle. These data suggest that the molecular basis for the functional diversity of the Na/H exchanger in mammals is based, at least in part, on expression of multiple members of a gene family.     

Copper oxide nanoparticles induce anticancer activity in A549 lung cancer cells by inhibition of histone deacetylase

Objectives

Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs).

Results

Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells.

Conclusion

Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.

     

Stimulation of acetoin production in metabolically engineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by increasing ATP demand

Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F₁-ATPase into a Lactococcus lactis strain engineered into producing acetoin, we show that production titer and yield both can be increased. At high F₁-ATPase expression level, the acetoin production yield could be increased by 10 %; however, because of the negative effect that the F₁-ATPase had on biomass yield and growth, this increase was at the cost of volumetric productivity. By lowering the expression level of the F₁-ATPase, both the volumetric productivity and the final yield could be increased by 5 % compared to the reference strain not overexpressing the F₁-ATPase, and in batch fermentation, it was possible to convert 176 mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream, deproteinized whey. Using this cheap and renewable feedstock, efficient acetoin production with a titer of 157 mM (14 g/L) acetoin was accomplished.     

High pressure freezing and freeze substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae ofBrassica

Summary High pressure freezing and freeze substitution methods significantly improve the antigenic preservation of S-locus specific glycoproteins (SLSG). The SLSG, which are implicated in the incompatibility response, are localized over the cell wall and cytoplasm. Labeling in the cytoplasm is mainly associated with dictyosomes and rough endoplasmic reticulum. Quantitative analysis show that in cryofixed papillae the labeling was enhanced by approximately 45% over the cell wall and approximately 90% over the dictyosomes compared to chemically fixed papillae.