Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at ?0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Proteomic insight into phenolic adaptation of a moderately halophilic Halomonas sp. strain AAD12

A gram-negative, moderately halophilic bacterium was isolated from ?amalt? Saltern area, located in the Aegean Region of Turkey. Analysis of its 16S rRNA gene sequence and physiological characteristics showed that this strain belonged to the genus Halomonas ; hence, it was designated as Halomonas sp. strain AAD12. The isolate tolerated up to 800 mg?L(-1) phenol; however, at elevated concentrations, phenol severely retarded cell growth. The increase in lag phase with increasing phenol concentrations indicated that the microorganism was undergoing serious adaptative changes. To understand the physiological responses of Halomonas sp. strain AAD12 to phenol, a 2-dimensional electrophoresis approach combined with mass spectrometric analysis was used. This approach showed that the expression of 14 protein spots were altered as phenol concentration increased from 200 to 800 mg?L(-1). Among the identified proteins were those involved in protein biosynthesis, energy, transport, and stress metabolism. So far, this is the first study on phenolic adaptation of a gram-negative, moderately halophilic bacteria using proteomic tools. The results provided new insights for understanding the general mechanism used by moderately halophilic bacteria to tolerate phenol and suggested the potential for using these microorganisms in bioremediation.     

BCL::Fold—Protein topology determination from limited NMR restraints

When experimental protein NMR data are too sparse to apply traditional structure determination techniques, de novo protein structure prediction methods can be leveraged. Here, we describe the incorporation of NMR restraints into the protein structure prediction algorithm BCL::Fold. The method assembles discreet secondary structure elements using a Monte Carlo sampling algorithm with a consensus knowledge‐based energy function. New components were introduced into the energy function to accommodate chemical shift, nuclear Overhauser effect, and residual dipolar coupling data. In particular, since side chains are not explicitly modeled during the minimization process, a knowledge based potential was created to relate experimental side chain proton–proton distances to C_β–C_β distances. In a benchmark test of 67 proteins of known structure with the incorporation of sparse NMR restraints, the correct topology was sampled in 65 cases, with an average best model RMSD100 of 3.4 ± 1.3 Å versus 6.0 ± 2.0 Å produced with the de novo method. Additionally, the correct topology is present in the best scoring 1% of models in 61 cases. The benchmark set includes both soluble and membrane proteins with up to 565 residues, indicating the method is robust and applicable to large and membrane proteins that are less likely to produce rich NMR datasets. Proteins 2014; 82:587–595. © 2013 Wiley Periodicals, Inc.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

The expression of HER-2/neu (c-erbB2), survivin and cycline D1 in serous ovarian neoplasms: their correlation with clinicopathological variables

Ovarian cancer is the most common cause of death among all gynecologic malignancies and a result of complex interaction of multiple oncogenes and tumor suppressor genes. The aim of this study was to evaluate expression of HER-2/neu (c-erbB2), survivin and cycline D1 biomarkers in serous ovarian neoplasms and their correlations with clinicopathological variables in serous ovarian cancers. We analyzed pathological specimens of 62 patients with benign (n = 25), borderline (n = 14) and malignant (n = 23) serous ovarian neoplasms. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded specimens. Significantly more immunoreactivity with HER-2/neu was detected in malignant tumors (100 %) compared to borderline (78.6 %) and benign tumors (48 %) (P < 0.01). Survivin expression was significantly higher in malignant tumors (91.3 %) than those found in borderline (71.4 %) and benign tumors (24 %) (P < 0.001). Similarly, higher cyclin D1 expression was observed in malignant tumors (95.6 %) compared to borderline (85.7 %) and benign tumors (48 %) (P < 0.001). Expression of all biomarkers analyzed significantly and gradually increased from benign to borderline and borderline to malignant serous tumors. In terms of clinicopathological variables, only tumor grade was associated with the expression of all biomarkers others exhibited different correlations in serous ovarian cancers. The expressions of HER-2/neu (c-erbB2), survivin and cycline D1 are positively correlated with the malignant potential of serous ovarian neoplasms.     

Phenolic constituents in flax bast tissue and inhibition of cellulase and pectinase

Flax bast tissue was sequentially extracted using hexane, propanol, methanol and water as solvents and extracts were analyzed using reverse phase HPLC and ¹³C NMR. Results indicated a large variety of aromatic constituents including flavonoids and hydroxy-methoxy cinnamic acids linked to oligosaccharides and hydroxy acids through glycosidic linkages. The extracts inhibited cellulase and pectinase activities and can thus influence retting.     

Rumen fungi: morphological types from Georgia cattle and the attack on forage cell walls

Fungal colonies developing in anaerobic media from zoospores in rumen fluid from cows eating Cynodon dactylon or Medicago sativa included types showing monocentric and polycentric growth. High energy supplements added to diets of Sorghum bicolor silage increased fungal numbers in the rumen, but increases were also affected by the history and predisposition of the animal. Mixed fungal types in rumen fluid and pure cultures of isolates showing monocentric and polycentric growth degraded and weakened lignocellulosic tissues and penetrated the cuticle of C. dactylon leaf blades. By weakening or degrading recalcitrant structures in forages, rumen fungi may alter physical parameters of plants that influence utilization of fibre by ruminants.     

Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis

BACKGROUND: The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm. METHODS: The mechanism of PEI-mediated DNA transfer was investigated using quantitative methods to study individual steps in the overall transfection process. In addition to transfection efficiency, the cellular uptake, local pH environment, and stability of vectors were analyzed. N-Quaternized (and therefore non-proton sponge) versions of PEI and specific cell function inhibitors were used to further probe the proton sponge hypothesis. RESULTS: Both N-quaternization and the use of bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately two orders of magnitude. Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N-quaternized PEIs and polylysine by 2-3-fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Significantly lower average pH environments were observed for permethyl-PEI (pH 5.4), perethyl-PEI (pH 5.1), and polylysine (pH 4.6) vectors. Cellular uptake levels of permethyl-PEI and perethyl-PEI vectors were found to be 20 and 90% higher, respectively, than that of parent PEI vectors, indicating that the reduction in transfection activity of the N-quaternized PEIs is due to a barrier downstream of cellular uptake. A polycation/DNA-binding affinity assessment showed that the more charge dense N-quaternized PEIs bind DNA less tightly than PEI, demonstrating that poor vector unpackaging was not responsible for the reduced transfection activity of the N-quaternized PEIs. CONCLUSIONS: The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes.     

Degradation of polysaccharides and lignin by ruminal bacteria and fungi.

Bermudagrass (Cynodon dactylon) leaf blades and whole cordgrass (Spartina alterniflora) fiber were evaluated for degradation of cell walls by microbial groups in ruminal fluid. The groups were selected by the addition of antibiotics to the inoculum as follows: (i) whole ruminal fluid (WRF), no antibiotics; (ii) cycloheximide (C) to inhibit fungi, thus showing potential bacterial activity; (iii) streptomycin and penicillin (S,P) to inhibit fiber-degrading bacteria, showing potential fungal activity; (iv) streptomycin, penicillin, and chloramphenicol (S,P,CAM) to inhibit all bacteria including methanogens; (v) streptomycin, penicillin, and cycloheximide (S,P,C) to inhibit all microbial activity as a control; and (vi) autoclaved ruminal fluid (ARF) to inhibit all biological activity as a second control. Scanning electron microscopy of tissue degradation indicated that tissues not giving a positive histological reaction for lignin were more readily degraded. Cordgrass was more highly lignified, with more tissues resisting degradation than in bermudagrass. Patterns of degradation due to treatment resulted in three distinct groups of data based on the extent of fiber or component losses: WRF and C greater than S,P and S,P,CAM greater than S,P,C and ARF. Therefore, bacterial activity was responsible for most of the fiber loss. Fiber degradation by anaerobic fungi was significantly less (P = 0.05). Cupric oxide oxidation of undigested and digested bermudagrass fiber indicated that phenolic constituents differed in their order of resistance to removal or solubilization. Vanillyl and syringyl components of lignin were the most resistant to decomposition, whereas ferulic acid was readily solubilized from fiber in the absence of microbial activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological degradation of tannins in sericea lespedeza (Lespedeza cuneata) by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus analyzed by solid-state 13C nuclear magnetic resonance spectroscopy.

Leaves of sericea lespedeza exhibit a high proportion of condensed tannin, resulting in poor forage quality. The white rot fungi Ceriporiopsis subvermispora and Cyathus sterocoreus are known to preferentially degrade lignin in a variety of plants and were evaluated for their ability to degrade condensed tannin from sericea leaves with the aim of improving digestibility. Relative levels of condensed tannin, cutin, pectin, and cellulose were monitored as a function of fungal treatment by solid-state cross-polarization and magic angle spinning 13C nuclear magnetic resonance spectroscopy. Total soluble phenolics, soluble tannins, and soluble and insoluble proanthocyanidin levels in fungus-treated and control samples were measured by established chemical techniques. Results indicate that both species of fungus preferentially degrade condensed tannin and that C. subvermispora is markedly superior to C. stercoreus in this capacity.