Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH₄⁺-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate.

1. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue.

2. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibited by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate.

3. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitors of respiration or an uncoupler but not in their absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition.

On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibited by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Ultrasound effect on physical properties of corn starch

High power ultrasound (HPU) represents a non-thermal processing method that has been rapidly researched and used in the last 10 years. The application of power ultrasound offers the opportunity to modify and improve some technologically important compounds which are often used in food products. One of them is starch. The aim of this research was to examine the effect of the high power ultrasound of 24 kHz frequency on rheological and some physical properties of corn starch. Various ultrasound treatments were used; an ultrasound probe set with different intensities (34, 55, 73 W cm⁻²) and treatment times (15 and 30 min) and ultrasound bath of 2 W cm⁻² intensity and treatment times (15 and 30 min). Rheological parameters, turbidity and swelling power of corn starch suspensions were determined for native and ultrasonically treated corn starch suspensions. Differential scanning calorimetry was used in order to examine the pasting properties of corn starch. The results have shown that the ultrasound treatment of corn starch distorts the crystalline region in starch granules. The results of differential scanning calorimetry measurements have shown a decrease in enthalpy of gelatinization. A significant decrease in consistency coefficient (k) has also been observed. The consistency coefficient decreases stepwise jointly with the increasing ultrasound power. The increase in the swelling power is associated with water absorption capacity and corn starch granules solubility, respectively.     

Non-native and translocated fish species in Serbia and their impact on the native ichthyofauna

A total of 22 fish species have been introduced into the inland waters of Serbia, either intentionally or accidentally. This paper provides a summary of data concerning time and reason of introduction, mode of expansion, degree of acclimatization, impact on native fish and estimated area of recent distribution. Four of the non-native fish species currently occupy more than 51% of Serbian territory while 5 of them occupy between 21–50% of territory. This paper reviews impacts of introduced freshwater fish in Serbia based on collected data.     

Continuum simulations of acetylcholine diffusion with reaction-determined boundaries in neuromuscular junction models

The reaction-diffusion system of the neuromuscular junction has been modeled in 3D using the finite element package FEtk. The numerical solution of the dynamics of acetylcholine with the detailed reaction processes of acetylcholinesterases and nicotinic acetylcholine receptors has been discussed with the reaction-determined boundary conditions. The simulation results describe the detailed acetylcholine hydrolysis process, and reveal the time-dependent interconversion of the closed and open states of the acetylcholine receptors as well as the percentages of unliganded/monoliganded/diliganded states during the neuro-transmission. The finite element method has demonstrated its flexibility and robustness in modeling large biological systems.     

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates "suicide gene" expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.     

Antioxidant Properties of Selected <Emphasis Type="Italic">Boletus</Emphasis> Mushrooms

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O₂˙⁻ radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82 ± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical–scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016 ± 0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spin-trapping and spin-probing techniques, activity against relevant reactive species, ˙OH and O₂˙⁻ radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for ˙OH radical reduction, and both exhibited significant capacity for scavenging O₂˙⁻ radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource.     

Improving Education in Medical Statistics: Implementing a Blended Learning Model in the Existing Curriculum

Background

Although recent studies report on the benefits of blended learning in improving medical student education, there is still no empirical evidence on the relative effectiveness of blended over traditional learning approaches in medical statistics. We implemented blended along with on-site (i.e. face-to-face) learning to further assess the potential value of web-based learning in medical statistics.

Methods

This was a prospective study conducted with third year medical undergraduate students attending the Faculty of Medicine, University of Belgrade, who passed (440 of 545) the final exam of the obligatory introductory statistics course during 2013–14. Student statistics achievements were stratified based on the two methods of education delivery: blended learning and on-site learning. Blended learning included a combination of face-to-face and distance learning methodologies integrated into a single course.

Results

Mean exam scores for the blended learning student group were higher than for the on-site student group for both final statistics score (89.36±6.60 vs. 86.06±8.48; p = 0.001) and knowledge test score (7.88±1.30 vs. 7.51±1.36; p = 0.023) with a medium effect size. There were no differences in sex or study duration between the groups. Current grade point average (GPA) was higher in the blended group. In a multivariable regression model, current GPA and knowledge test scores were associated with the final statistics score after adjusting for study duration and learning modality (p<0.001).

Conclusion

This study provides empirical evidence to support educator decisions to implement different learning environments for teaching medical statistics to undergraduate medical students. Blended and on-site training formats led to similar knowledge acquisition; however, students with higher GPA preferred the technology assisted learning format. Implementation of blended learning approaches can be considered an attractive, cost-effective, and efficient alternative to traditional classroom training in medical statistics.     

Kinetic model of ethopropazine interaction with horse serum butyrylcholinesterase and its docking into the active site.

The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor.     

A high‐throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation

The serum half‐life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high‐throughput method for semi‐quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:235–241, 2016     

Inhibition of erythrocyte acetylcholinesterase by n-butanol at high concentrations

Erythrocyte acetylcholinesterase (AChE) is bound to the membrane by a complex glycosylphosphatidylinositol anchor, so the effect of alcohol on AChE activity may reflect direct and/or membrane-mediated effects. The indication of a direct interaction between n-butanol and AChE molecules is the activation/inhibition of AChE by occupation of the enzyme's active and/or regulatory sites by alcohol. The activation of AChE can occur only at low concentrations of alcohols, while at high concentrations AChE is inhibited. In this work the mechanism of inhibition of erythrocyte AChE by n-butanol at high concentrations was studied. The values of activity, calculated assuming parabolic competitive inhibition, which implies that one or two molecules of inhibitor bind to the enzyme, fit well to the experimental values. From the values of the inhibition constants it was concluded that at high n-butanol concentrations two alcohol molecules usually interact with AChE.