Moderately Different NADPH-Diaphorase Positivity in the Selected Peripheral Nerves after Ischemia/ Reperfusion Injury of the Spinal Cord in Rabbit

1. To vicariously investigate the nitric oxide synthase (NOS) production after spinal cord injury, NADPH-d histochemistry was performed on the selected peripheral nerves of adult rabbits 7 days after ischemia. The effect of transient spinal cord ischemia (15 min) on possible degenerative changes in the motor and mixed peripheral nerves of Chinchilla rabbits was evaluated.2. The NADPH-diaphorase histochemistry was used to determine NADPH-diaphorase activity after ischemia/reperfusion injury in radial nerve and mediane nerve isolated from the fore-limb and femoral nerve, saphenous nerve and sciatic nerve separated from the hind-limb of rabbits. The qualitative analysis of the optical density of NADPH-diaphorase in selected peripheral nerves demonstrated different frequency of staining intensity (attained by UTHSCSA Image Tool 2 analysis for each determined nerve).3. On the seventh postsurgery day, the ischemic spinal cord injury resulted in an extensive increase of NADPH-d positivity in isolated nerves. The transient ischemia caused neurological disorders related to the neurological injury—a partial paraplegia. The sciatic, femoral, and saphenous nerves of paraplegic animals presented the noticeable increase of NADPH-d activity. The mean of NADPH-diaphorase intensity staining per unit area ranged from 134.87 (±32.81) pixels to 141.65 (±35.06) pixels (using a 256-unit gray scale where 0 denotes black, 256 denotes white) depending on the determined nerve as the consequence of spinal cord ischemia. The obtained data were compared to the mean values of staining intensity in the same nerves in the limbs of control animals (163.69 (±25.66) pixels/unit area in the femoral nerve, 173.00 (±32.93) pixels/unit area in saphenous nerve, 186.01 (±29.65) pixels/unit area in sciatic nerve). Based on the statistical analysis of the data (two-way unpaired Mann–Whitney test), a significant increase (p≤0.05) of NADPH-d activity in femoral and saphenous nerve, and also in sciatic nerve (p≤0.001) has been found. On the other hand, there was no significant difference between the histochemically stained nerves of fore-limbs after ischemia/reperfusion injury and the same histochemically stained nerves of fore-limbs in control animals.4. The neurodegenerative changes of the hind-limbs, characterized by damage of their motor function exhibiting a partial paraplegia after 15 min spinal cord ischemia and subsequent 7 days of reperfusions resulted in the different sensitivity of peripheral nerves to transient ischemia. Finally, we suppose that activation of NOS indirectly demonstrable through the NADPH-d study may contribute to the explanation of neurodegenerative processes and the production of nitric oxide could be involved in the pathophysiology of spinal cord injury by transient ischemia.     

Evaluation of antioxidant properties of medical plants using microbial test systems

The antioxidant activities of extracts from leaves of the medicinal plants growing in Siberia were examined. Total antioxidant activity was determined using in vitro methods including DPPH (2,2-diphenyl-1-picrylhydrazyl radical) free radical scavenging assay, chelating capacity assay with ferrozine, evaluation of capacity to protect plasmid DNA against oxidative damage, measurement of H₂O₂ production, and measurement of total flavonoid and tannin content as well. Using in vivo experiments, we also evaluated capacities of the plant extracts to protect bacteria Escherichia coli against bacteriostatic and bactericidal effects of H₂O₂, and influence of the plant extracts on expression of antioxidant gene katG, encoding catalase. The extracts from Chamerion angustifolium, Filipendula vulgaris and Pyrola rotundifolia indicated the highest levels of antioxidant activity both in vivo and in vitro. Our data suggest that the extracts of the tested plants may provide antioxidant effects on bacteria simultaneously through different pathways, including direct radical scavenging, iron chelation and induction of genes encoding antioxidant enzymes.     

Influence of polyphenols on Escherichia coli resistance to oxidative stress

Among all polyphenols tested (tannic acid and flavonoids belonging to different subclasses) only tannin and quercetin significantly enhanced resistance of Escherichia coli to peroxide stress. Pretreatment of the cells with quercetin and tannin resulted in a decrease in the growth arrest duration under moderate H₂O₂ concentration (2 mM) and an increase in survival under high (10 mM) doses. The shorter growth recovery period in pretreated cells was connected with more rapid H₂O₂ elimination because of induced activity of scavenging enzymes. This effect was absent in the ΔoxyR mutant, which was unable to induce genes responding to peroxide stress. The data obtained suggest that the observed protection was a result of two overlapping effects: induction of OxyR regulon by low concentrations of H₂O₂, accumulated during extracellular autoxidation of quercetin and tannin, and protection of synthesis of OxyR-regulated antioxidant enzymes during H₂O₂ stress because of intracellular binding of iron by quercetin and tannin and suppressing Fenton chemistry.     

Identification of a mitochondrial-binding site on the N-terminal end of hexokinase II

Hexokinase II (HKII) is responsible for the first step in the glycolysis pathway by adding a phosphate on to the glucose molecule so it can proceed down the pathway to produce the energy for continuous cancer cell growth. Tumour cells overexpress the HKII enzyme. In fact, it is the overexpression of the HKII enzyme that makes the diagnosis of cancer possible when imaged by positron emission tomography (PET). HKII binds to the voltage-dependent anion channel (VDAC) located on the mitochondrial outer membrane (MOM). When bound to the MOM, HKII is blocking a major cell death pathway. Thus, HKII is responsible for two characteristics of cancer cells, rapid tumour growth and inability of cancer cells to undergo apoptosis. One method to identify novel compounds that may interfere with the HKII–VDAC-binding site is to create a molecular model using the crystal structure of HKII. However, the amino acid(s) responsible for HKII binding to VDAC are not known. Therefore, a series of truncations and point mutations were made to the N-terminal end of HKII to identify the binding site to VDAC. Deletions of the first 10 and 20 amino acids indicated that important amino acid(s) for binding were located within the first 10 amino acids. Next, a series of point mutations were made within the first 10 amino acids. It is clear from the immunofluorescence images and immunoblot results that mutating the fifth amino acid from histidine to proline completely abolished binding to the MOM.     

Modulating EGFR-MTORC1-autophagy as a potential therapy for persistent fetal vasculature (PFV) disease

ABSTRACT

Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway.     

Ribosomal acidic proteins of eucaryotic cells

Summary By the method of ethanol-salt extraction with ion-exchange chromatography on CM-cellulose an acidic protein of pea 80S ribosomes was isolated. This protein located in the large subunit, had a molecular weight of 14 000 and an IEP of 4.7. The protein is partially phosphorylated, alanine-rich and has methionine at the N-terminal position. Based on these characteristics and on the comparative study of tryptic hydrolyzates of the plant protein and E. coli L7/L12, the protein so obtained is found to be homologous to the L7/Ll2 of the procaryotic ribosomes.