Effect of Digestion with Phospholipase A2 on Endogenous Protein Phosphorylation in Particulate Fractions from Rat Brain Synaptosomes

The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Acceptors for cyclic AMP-dependent and calcium ion-dependent protein kinases in rat brain cytosol fractions: A comparison of occluded (synaptosomal) cytosol with non-occluded cytosol

Endogenous protein phosphorylation patterns were compared in occluded and non-occluded cytosol fractions prepared from rat forebrain. The occluded fraction was taken as representative of synaptosomal cytosol. One- and two-dimensional autoradiographs revealed the presence in non-occluded cytosol of a substrate for cAMP- and Ca²⁺/calmodulin-dependent protein kinase activities of M_r 300kD, corresponding to phosphorylated microtubule-associated protein-2 (MAP-2); this protein was absent in occluded cytosol. In contrast, a major substrate for protein kinase C was observed exclusively in occluded cytosol after phosphorylation under basal conditions. However, after phosphorylation in the presence of exogenous lipids, approximately equal amounts of the 82kD substrate were detected in both fractions, suggesting that protein kinase C in the occluded fraction was present in a partially activated state. Other minor differences in phosphorylation patterns between the two fractions were observed.Special Issue dedicated to Prof. Eduardo De Robertis.     

Structure-Activity Relationship of Methyl 4-Aminobenzoate Derivatives as Being Drug Candidate Targeting Glutathione Related Enzymes: in Vitro and in Silico Approaches

A thiol compound, glutathione, is essential for healthy cell defence against xenobiotics and oxidative stress. Glutathione reductase (GR) and glutathione S-transferase (GST) are two glutathione-related enzymes that function in the antioxidant and the detoxification systems. In this study, potential inhibitory effects of methyl 4-aminobenzoate derivatives on GR and GST were examined in vitro. GR and GST were isolated from human erythrocytes with 7.63 EU/mg protein and 5.66 EU/mg protein specific activity, respectively. It was found that compound 1 (methyl 4-amino-3-bromo-5-fluorobenzoate with K_i value of 0.325±0.012 μM) and compound 5 (methyl 4-amino-2-nitrobenzoate with K_i value of 92.41±22.26 μM) inhibited GR and GST stronger than other derivatives. Furthermore, a computer-aided method was used to predict the binding affinities of derivatives, ADME characteristics, and toxicities. Derivatives 4 (methyl 4-amino-2-bromobenzoate) and 6 (methyl 4-amino-2-chlorobenzoate) were estimated to have the lowest binding energies into GR and GST receptors, respectively according to results of in silico studies.     

Facile Synthesis of Hybrid Nanoflowers Using Glycine and Phenylalanine and Investigation of Their Catalytic Activity

In the context of the proposed work, two different amino acids (Glycine, Phenylalanine) have interacted with copper ions in a phosphate buffer (PBS) in place of enzymes. This interaction resulted in the nucleation of copper phosphate crystals and the formation of flower-shaped amino acid-copper hybrid nanostructures (AA-hNFs), which grew through self-assembly. While Cu (II) ions in the structure of AA-hNFs were used as Fenton's agent for the catalytic activity. SEM, energy dispersive X-ray spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy measurements were used to define the AA-hNFs′ characterisation. The peroxidase-like activities of AA-hNFs were investigated by UV/VIS spectrophotometer. Metal nanoparticles have peroxidase-like activity. A class of enzymes known as peroxidases is able to catalyze the conversion of hydrogen peroxide into hydroxyl radicals. These radicals also take part in electron transfers with substrates, which results in color during oxidation. When cupric oxide nanoparticles are added to the peroxidase substrate while H₂O₂ is present, a blue color product with a maximum absorbance at=652 nm can result, demonstrating the catalytic activity of a peroxidase. The morphology and composition of AA-hNFs were carefully characterized and the synthesized parameters were optimized systematically. Results showed that the nanoparticles were dispersed with an average diameter of 7–9 μm and indicated a uniform flower shape. The results of the investigation are anticipated to significantly advance a number of technical and scientific sectors.     

Suppression of TLR4/MyD88/TAK1/NF-κB/COX-2 Signaling Pathway in the Central Nervous System by Bexarotene,a Selective RXR Agonist,Prevents Hyperalgesia in the Lipopolysaccharide-Induced Pain Mouse Model

Neurochemical Research - A selective RXR agonist, bexarotene, has been shown to have anti-inflammatory, anti-nociceptive, and neuroprotective effects in several models of numerous neurological...     

Molecular characterization of acutely and gradually heavy metal acclimated aquatic bacteria by FTIR spectroscopy

In the environment, bacteria can be exposed to the concentration gradient of toxic heavy metals (gradual) or sudden high concentration of them (acute). In both situations, bacteria get acclimated to toxic heavy metal concentrations. Acclimation causes metabolic and molecular changes in bacteria. In this study, we aimed to understand whether there are differences between molecular profiles of the bacteria (Brevundimonas, Gordonia and Microbacterium) which are under acute or gradual exposure to cadmium or lead by using ATR‐FTIR spectroscopy. Our results revealed the differences between the acclimation groups in membrane dynamics including changes in the structure and composition of the membrane lipids and proteins. Furthermore, protein concentrations decreased in acclimated bacterial groups. Also, a remarkable increase in exopolymer production occurred in acclimated groups. Interestingly, bacteria under acute cadmium exposure produced the significantly higher amount of exopolymer than they did under gradual exposure. On the contrary, under lead exposure gradually acclimate strains produced significantly higher amounts of exopolymer than those of acutely acclimated ones. This information can be used in bioremediation studies to obtain bacterial strains producing a higher amount of exopolymer.