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.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

The protective effect of arbutin against potassium bromate-induced oxidative damage in the rat brain

This study aimed to investigate the protective effects of arbutin (ARB) against brain injury induced in rats with potassium bromate (KBrO₃). The rats were divided into four groups as Group 1: Control (0.9% NaCl ml/kg/day p.), Group 2: KBrO₃ (100 mg/kg (gavage), Group 3: ARB (50 mg/kg/day p.), and Group 4: KBrO₃ + ARB (100 mg/kg (gavage) + 50 mg/kg/day p.). At the end of the fifth day of the study, the rats in all groups were killed, and their brain tissues were collected. In the collected brain tissues, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels were measured, and routine histopathological examinations were made. The MDA levels in the group that was exposed to KBrO₃ were significantly higher than those in the control group (p ˂ 0.001). In comparison to the KBrO₃ group, the MDA levels in the KBrO₃ + ARB group were significantly lower (p ˂ 0.001). It was observed that SOD and CAT enzyme activity levels were significantly lower in the KBrO₃ group compared to the control group (p ˂ 0.001), while these levels were significantly higher in the KBrO₃ + ARB group than in the KBrO₃ group (p ˂ 0.001). Additionally, the group that was subjected to KBrO₃ toxicity, as well as ARB administration, had much lower levels of histopathologic signs than the group that was subjected to KBrO₃ toxicity only. Consequently, it was found that KBrO₃ exposure led to injury in the brain tissues of the rats, and using ARB was effective in preventing this injury.     

Oxidative brain and cerebellum injury in diabetes and prostate cancer model: Protective effect of metformin

The body can host the spread of prostate cancer cells. Metastases from prostate cancer are more frequently seen in the brain, liver, lungs, and lymph nodes. A well-known antidiabetic drug, metformin, is also known to have antitumor effects. Our study focuses on the evaluation of potential metformin protective effects on brain and cerebellum damage in streptozotocin (STZ)-induced diabetic and Dunning prostate cancer models. In this investigation, six groups of male Copenhagen rats were created: control, diabetic (D), cancer (C), diabetic + cancer (DC), cancer + metformin, and diabetic + cancer + metformin. The brain and cerebellum tissues of the rats were taken after sacrifice. Oxidative stress markers including reduced glutathione level, lipid peroxidation, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, catalase, superoxide dismutase activities, reactive oxygen species, total oxidant and total antioxidant status, lactate dehydrogenase, xanthine oxidase, acetylcholinesterase activities, protein carbonyl contents, nitric oxide and OH-proline levels, sodium potassium ATPase, carbonic anhydrase, and glucose-6-phosphate dehydrogenase activities; glycoprotein levels including hexose, hexosamine, fucose, and sialic acid levels; and histone deacetylase activity as a cancer marker were determined. Oxidative stress markers were impaired and glycoprotein levels and histone deacetylase activity were increased in the D, C, and DC groups. Metformin therapy reversed these effects. Metformin was found to protect the brain and cerebellum of STZ-induced diabetic rats with Dunning prostate cancer from harm caused by MAT-Lylu metastatic cells.     

Photoluminescence and thermoluminescence properties of pure and rare-earth-doped ZrO2 prepared by Pechini-type sol–gel

In this article, photoluminescence (PL) and thermoluminescence (TL) properties of ZrO₂, ZrO₂:Dy³⁺, ZrO₂:Dy³⁺–Gd³⁺, ZrO₂:Dy³⁺–Yb³⁺, ZrO₂:Dy³⁺–Er³⁺, and ZrO₂:Dy³⁺–Sm³⁺ phosphors synthesized by the Pechini method were investigated. The crystal structure, thermal properties, morphology, PL and TL properties were investigated using X-ray powder diffraction (XRD), differential thermal analysis/thermogravimetric analysis (DTA/TGA), scanning electron microscopy (SEM), PL and TL, respectively. The room temperature emission bands corresponding to ⁴F_9/2 → ⁶H_J (J = 9/2, 11/2, 13/2 and 15/2) transitions of Dy³⁺ ions were measured. The phosphors were analysed using T_m–T_STOP, variable dose, and computerized glow curve fitting methods. Reusability, dose–response, and fading characteristics were investigated. The phosphors have a natural TL emission that vanished by heating treatment. Moreover, new peaks with similar properties to the natural emissions were observed after high-dose irradiation and long-term fading experiments. The glow curves of the phosphors have 13 individual peaks and many low- and high-temperature satellite peaks. The origin of the peaks is ZrO₂ host material and doping with rare-earth ions (Gd³⁺, Dy³⁺, Yb³⁺, Er³⁺ and Sm³⁺) does not lead to a new glow peak. The dopants cause drastic changes in individual peak intensities of ZrO₂.The initial fading rates of all the phosphors are relatively fast, but they slow down as time goes on.     

Comparative bioinformatics analysis and abiotic stress responses of expansin proteins in Cucurbitaceae members: watermelon and melon

Protoplasma - Watermelon and melon are members of the Cucurbitaceae family including economically significant crops in the world. The expansin protein family, which is one of the members of the...     

Effect of Axial Ligand Length on Biological and Anticancer Properties of Axially Disubstituted Silicon Phthalocyanines

In this study, three new axially disubstituted silicon phthalocyanines ( SiPc1–3 ) and their quaternized phthalocyanine derivatives ( QSiPc1–3 ) were prepared and characterized. The biological properties (antioxidant, antimicrobial, antibiofilm, and microbial cell viability activities) of the water-soluble silicon phthalocyanines were examined, as well. A 1 % DMSO diluted with pure water was used as a solvent in biological activity studies. All the compounds exhibited high antioxidant activity. They displayed efficient antimicrobial and antimicrobial photodynamic therapeutic properties against various microorganisms, especially Gram (+) bacteria. Additionally, they demonstrated high antibiofilm activities against S. aureus and P. aeruginosa. In addition, 100 % bacterial reduction was obtained for all the studied phthalocyanines against E. coli viable cells. Besides, the DNA cleavage and binding features of compounds ( QSiPc1–3 ) were studied using pBR322 DNA and CT-DNA, respectively. Furthermore, the human topoisomerase I enzyme inhibition activities of compounds QSiPc1 – 3 were studied. Anticancer properties of the water-soluble compounds were investigated using cell proliferation MTT assay. They exhibited anticarcinogenic activity against the human colon cancer cell line (DLD-1). Compounds QSiPc1 and QSiPc3 displayed a high anticarcinogenic effect on the DLD-1 cell line. The obtained results indicated that all the studied compounds may be effective biological agents and anticancer drugs after further investigations.     

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.