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.     

Overexpression of the Tn5 transposase in Escherichia coli results in filamentation, aberrant nucleoid segregation, and cell death: analysis of E. coli and transposase suppressor mutations.

Overexpression of the Tn5 transposase (Tnp) was found to be lethal to Escherichia coli. This killing was not caused by transposition or dependent on the transpositional or DNA binding competence of Tnp. Instead, it was strictly correlated with the presence of a wild-type N terminus. Deletions removing just two N-terminal amino acids of Tnp resulted in partial suppression of this effect, and deletions of Tnp removing 3 or 11 N-terminal amino acids abolished the killing effect. This cytotoxic effect of Tnp overexpression is accompanied by extensive filament formation (i.e., a defect in cell division) and aberrant nucleoid segregation. Four E. coli mutants were isolated which allow survival upon Tnp overexpression, and the mutations are located at four discrete loci. These suppressor mutations map near essential genes involved in cell division and DNA segregation. One of these mutations maps to a 4.5-kb HindIII region containing the ftsYEX (cell division) locus at 76 min. A simple proposition which accounts for all of these observations is that Tnp interacts with an essential E. coli factor affecting cell division and/or chromosome segregation and that overexpression of Tnp titrates this factor below a level required for viability of the cell. Furthermore, the N terminus of Tnp is necessary for this interaction. The possible significance of this phenomenon for the transposition process is discussed.     

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.     

Melatonin ameliorates cardiac remodelling in fructose-induced metabolic syndrome rat model by using genes encoding cardiac potassium ion channels

Molecular Biology Reports - Metabolic syndrome comprises a group of disorders, including cardiac abnormalities. Ventricular arrhythmias observed in metabolic syndrome are due to the impaired...     

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 Chitinase Genes from a Local Isolate of Serratia marcescens and Their Contribution to the Insecticidal Activity of Bacillus thuringiensis Strains

The chitinase B (chiB) and C (chiC) genes and flanking regions from a local isolate of Serratia marcescens were cloned individually and sequenced. Results showed that these chiB and chiC genes have a 96 % maximum similarity with chiB and chiC from different S. marcescens species (GenBank numbers Z36295.1 and AJ630582.1, respectively). The amplified chiB fragment, including some upstream and downstream regions, is 1,689-bp long with an open reading frame of 1,500 bp. The amplified fragment of chiC is 1,844 bp with an open reading frame of 1,443 bp. These sequences were submitted to the GenBank with accession numbers JX847796 (chiB) and JX847797 (chiC). Putative promoter regions and Shine–Dalgarno sequences were identified in both genes. The genes were cloned into a shuttle vector and the constructs were designated as pHYSB and pHYSC, respectively. Both plasmids were introduced separately into kurstaki and israelensis strains of Bacillus thuringiensis and the insecticidal activities of the engineered B. thuringiensis strains were assayed in larvae of Galleria mellonella and adult of Drosophila melanogaster. Engineered B. thuringiensis strains showed higher insecticidal activity than parental strain and the parental S. marcescens. In addition, pHYSB and pHYSC were stable over 16 daily passages under non-selective conditions in transformed B. t. israelensis 5724 strain.