In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 μM CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules.     

Identification of Novel Genetic Alterations in Samples of Malignant Glioma Patients

Glioblastoma is the most frequent and malignant human brain tumor. High level of genomic instability detected in glioma cells implies that numerous genetic alterations accumulate during glioma pathogenesis. We investigated alterations in AP-PCR DNA profiles of 30 glioma patients, and detected specific changes in 11 genes not previously associated with this disease: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2, KCNG2, PDE4D, KIR3DL3, and INPP5A. Further correlations revealed that 8 genes might play important role in pathogenesis of glial tumors, while changes in GP2, KCNG2 and KIR3DL3 should be considered as passenger mutations, consequence of high level of genomic instability. Identified genes have a significant role in signal transduction or cell adhesion, which are important processes for cancer development and progression. According to our results, LHFPL3 might be characteristic of primary glioblastoma, SGCG, HTR4, ITGB1, CPS1, PROS1 and INPP5A were detected predominantly in anaplastic astrocytoma, suggesting their role in progression of secondary glioblastoma, while alterations of PDE4D seem to have important role in development of both glioblastoma subtypes. Some of the identified genes showed significant association with p53, p16, and EGFR, but there was no significant correlation between loss of PTEN and any of identified genes. In conclusion our study revealed genetic alterations that were not previously associated with glioma pathogenesis and could be potentially used as molecular markers of different glioblastoma subtypes.     

Tel1 kinase and subtelomere-bound Tbf1 mediate preferential elongation of short telomeres by telomerase in yeast

Telomerase enables telomere length homeostasis, exhibiting increasing preference for telomeres as their lengths decline. This regulation involves telomere repeat-bound Rap1, which provides a length-dependent negative feedback mechanism, and the Tel1 and Mec1 kinases, which are positive regulators of telomere length. By analysing telomere elongation of wild-type chromosome ends at single-molecule resolution, we show that in tel1Delta cells the overall frequency of elongation decreases considerably, explaining their short telomere phenotype. At an artificial telomere lacking a subtelomeric region, telomere elongation no longer increases with telomere shortening in tel1Delta cells. By contrast, a natural telomere, containing subtelomeric sequence, retains a preference for the elongation of short telomeres. Tethering of the subtelomere binding protein Tbf1 to the artificial telomere in tel1Delta cells restored preferential telomerase action at short telomeres; thus, Tbf1 might function in parallel to Tel1, which has a crucial role in a TG-repeat-controlled pathway for the activation of telomerase at short telomeres.     

Coupling of permeabilized cells of Gluconobacter oxydans and Ralstonia eutropha for asymmetric ketone reduction using H2 as reductant

A combined two-cell reaction system containing Gluconobacter oxydans and Ralstonia eutropha was evaluated with regard to asymmetric ketone reduction using H₂ as the reductant. Whole cells permeabilized by EDTA/toluene were used, and synthesis was performed in a biphasic aqueous/organic reaction medium. The two-cell system was compared with a system in which G. oxydans alone was used for both ketone reduction and cofactor regeneration, using an alcohol as co-substrate. The two-cell system exhibited almost twice the initial reaction rate of the single-cell system, a higher yield (75% vs. 48%) but slightly lower enantiomeric purity (93% vs. 98%) of the product (S)-2-octanol. The permeabilized R. eutropha cells are worth evaluating for byproduct-free NADH regeneration in combination with other whole cell catalysts.     

A kinetic study of D-glucose oxidation by bromine in aqueous solutions

The kinetics of the oxidation of D-glucose to D-gluconic acid by bromine in aqueous solution were studied using potentiometric techniques and theoretical considerations of complex bromine-bromide-pH equilibria. The pH has a strong influence on reaction rate. At pH < 8 the reaction is very slow, while in the pH range pH 8-9.5 the reaction is sufficiently fast and seems optimal for the reaction. The proposed active species at that pH region is hypobromous acid. At pH > 9.5, the reaction is further accelerated due to the formation of hypobromite. The proposed kinetics expression for gluconic acid formation, based on the determined kinetic parameters at pH 9.24, is of the form dc(GA)/dt = 160c(2)(G)c(o)(HOBr)c(o)(H(+)c(o)(Br)     

EPR Spin-Trapping and Spin-Probing Spectroscopy in Assessing Antioxidant Properties: Example on Extracts of Catkin, Leaves, and Spiny Burs of Castanea sativa

Electron paramagnetic resonance (EPR) spin-trapping and spin-probing techniques were applied to determine antioxidant activity of extracts of catkin, leaves, and spiny burs of Castanea sativa against physiologically relevant reactive species—superoxide and hydroxyl radical generated in simple chemical systems and hydrogen peroxide applied on erythrocytes. Efflux of K⁺ was used as a marker of membrane integrity. Chemical composition of extracts was analyzed using HPLC/DAD and LC/MS. Extracts showed high antioxidative capacity against superoxide but lower activity against hydroxyl radical. They protected fluidity and integrity of membranes of erythrocytes exposed to hydrogen peroxide. Levels of derivatives of ellagitannins showed positive correlation with the antioxidative activity of extracts. Therefore, ellagitannins from chestnut extracts could represent easily accessible natural antioxidants and beneficial component of human diet in pathophysiological conditions related to oxidative stress. In conclusion, EPR spectroscopy represents a valuable tool for evaluation of antioxidant activity in both hydrophilic and lipophilic media. This work was supported by the Federal Ministry of Education and Science of Bosnia and Herzegovina Grant No. 614300 and the Ministry of Science, Technology, and Development of Republic of Serbia Grant No. 143016. We would like to thank Ms. Ana Martinović on technical support.     

Medium perfusion enables engineering of compact and contractile cardiac tissue

We hypothesized that functional constructs with physiological cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation and characterized in detail construct metabolism, structure, and function. Neonatal rat cardiomyocytes suspended in Matrigel were cultured on collagen sponges at a high initial density (1.35 x 10(8) cells/cm(3)) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity, cell cycle, presence of contractile proteins (sarcomeric alpha-actin, troponin I, and tropomyosin), and contractile function in response to electrical stimulation [excitation threshold (ET), maximum capture rate (MCR), response to a gap junctional blocker]. Interstitial flow of culture medium through the central 5-mm-diameter x 1.5-mm-thick region resulted in a physiological density of viable and differentiated, aerobically metabolizing cells, whereas dish culture resulted in constructs with only a 100- to 200-microm-thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR after treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.     

Artificial neural networks in the modeling and optimization of aspirin extended release tablets with eudragit L 100 as matrix substance

The purpose of the present study was to model the effects of the concentration of Eudragit L 100 and compression pressure as the most important process and formulation variables on the in vitro release profile of aspirin from matrix tables formulated with Eudragit L 100 as matrix substance and to optimize the formulation by artificial neural network. As model formulations, 10 kinds of aspirin matrix tablets were prepared. The amount of Eudragit L 100 and the compression pressure were selected as causal factors. In vitro dissolution time profiles at 4 different sampling times were chosen as responses. A set of release parameters and causal factors were used as tutorial data for the generalized regression neural, network (GRNN) and analyzed using a computer. Observed results of drug release studies indicate that drug release rates vary widely between investigated formulations, with a range of 5 hours to more than 10 hours to complete dissolution. The GRNN model was optimized. The root mean square value for the trained network was 1.12%, which indicated that the optimal GRNN model was reached. Applying the generalized distance function method, the optimal tablet formulation predicted by GRNN was with 5% of Eudragit L 100 and tablet hardness 60N. Calculated difference (f ₁ 2.465) and similarity (f ₂ 85.61) factors indicate that there is no difference between predicted and experimentally observed drug release profiles for the optimal formulation. This work illustrates the potential for an artificial neural network, GRNN, to assist in development of extended release dosage forms.     

Stereological analysis of thyroid mast cells in rats after exposure to extremely low frequency electromagnetic field and the following "off" field period

Influence of extremely low frequency electromagnetic field (ELF-EMF) on thyroid gland mast cells was investigated on male Mill Hill rats. Animals were exposed to EMF (50 Hz, 50 microT to 500 microT, 10 V/m) from 24 hours after birth, 7 hours/day, 5 days/week for three months when a part of animals (group I) was sacrificed, while the rest of them were subjected to recovery evaluation and sacrificed after one (group II), two (group II) and three (group IV) weeks following the exposure. Stereological analysis on toluidine blue-stained paraffin sections showed increased volume density of degranulated mast cells in all groups and, except in group III, and numerical density as well, implicating the sensitivity of thyroidal mast cells to power frequency EMFs. Since in our previous investigations, morphofunctional alterations of thyroid gland in rats exposed to ELF-EMF were found the contribution of released mast cell mediators to these changes could be presumed.