AREL1 resists the apoptosis induced by TGF-β by inhibiting SMAC in vascular endothelial cells

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-β (TGF-β), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-β treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-β. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-β treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-β induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.     

Chemistry and biology of enzymes in protein glutathionylation

Protein S-glutathionylation is emerging as a central oxidation that regulates redox signaling and biological processes linked to diseases. In recent years, the field of protein S-glutathionylation has expanded by developing biochemical tools for the identification and functional analyses of S-glutathionylation, investigating knockout mouse models, and developing and evaluating chemical inhibitors for enzymes involved in glutathionylation. This review will highlight recent studies of two enzymes, glutathione transferase omega 1 (GSTO1) and glutaredoxin 1 (Grx1), especially introducing their glutathionylation substrates associated with inflammation, cancer, and neurodegeneration and showcasing the advancement of their chemical inhibitors. Lastly, we will feature protein substrates and chemical inducers of LanC-like protein (LanCL), the first enzyme in protein C-glutathionylation.     

Common Epitopes In the Circumsporozoite Proteins of Plasmodium Berghei and Plasmodium Gallinaceum Identified By Monoclonal Antibodies to the P. Gallinaceum Circumsporozoite Protein

ABSTRACT. Monoclonal antibodies that react with the circumsporozoite protein of the avian malaria Plasmodium gallinaceum sporozoites also reacted with circumsporozoite protein of the rodent malaria Plasmodium berghei. Two types of reactivity were identified: 1) two monoclonal antibodies reacted with P. berghei sporozoite protein by enzyme-linked immunosorbent assay, Western blot and indirect immunofluorescence antibody, 2) six other monoclonal antibodies reacted with P. berghei sporozoites by ELISA and Western blot only. We studied whether these differences could be explained by reactivity in enzyme-linked immunosorbent assay with different P. berghei circumsporozoite peptides. Although all P. gallinaceum monoclonal antibodies reacted with the P. berghei repeats, the first group reacted with a conserved peptide sequence, N1, whereas the second group did not. These results suggest that circumsporozoite proteins from P. gallinaceum and P. berghei share common epitopes. the biological significance of our finding is not yet clear. Indeed, the cross-reactive monoclonal antibodies giving a positive indirect immunofluorescence antibody with the P. berghei sporozoites only caused a borderline effect on the living P. berghei parasites in vitro as measured by inhibition of sporozoite infectivity.     

Glycine: An important potential component of spinal shock

Amino acid neurotransmitters (AANTs) play a major role in maintenance of muscle tone. Abnormal AANT concentrations are associated with hyper- or hypotonic states. Flaccidity from spinal shock commonly occurs after spinal cord injury (SCI) and may be associated with changes in AANT concentrations. Ischemic SCIs created in the lumbar region of rabbits by intraaortic balloon occlusion produced spastic or flaccid injuries. Microdialysis sampling of AANTs from the injured segmental structures was done 3 days after SCI. Evoked potentials were used to monitor spinal cord stability. No significant changes in AANT levels occurred in the spastic or flaccid group after 4 hour sampling. However, flaccid animals had baseline glycine levels 2–3 times higher (p<0.001) than spastic animals or controls. High concentrations of the inhibitory AANT glycine is associated with flaccidity following SCI, or spinal shock, but not spasticity. Glycinergic compounds directed toward suppression of excess muscle tone deserve further study.     

Differential distribution of calpain in human lymphoid cells

Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both Calpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of Calpain (55–80%) whereas the monocytic line (U-937) showed prodominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases.     

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: Studies in the rat locus coeruleus

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi and Go, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.Special issue dedicated to Dr. Paul Greengard     

N-Terminal arginylation of proteins in explants of injured sciatic nerves and embryonic brains of rats

Posttranslational modification of proteins by arginine and lysine has been demonstrated in crude extracts of vertebrate nerves and brain but not in intact cells. In the present experiments we have exploited the fact that Arg is added posttranslationally only at the N-terminus of target proteins, to demonstrate these reactions in intact cells of sciatic nerves and embryonic brains of rats. Sciatic nerves were crushed in anaesthesized rats and 2 hrs later segments of nerve, including the site of the crush, were removed and incubated in media containing [³H]Arg. Incorporation of [³H]Arg into total proteins was analyzed by acid precipitation and the presence of label at the N-terminus was determined by a modification of the Edman degradation procedure. Approximately 25% of protein bound [³H]Arg was released from the N-terminus by the Edman reaction indicating that it was added posttranslationally rather than through protein synthesis. N-terminal labeling was not detectable in nerves not crushed prior to explant and incubation. Slices of embryonic day 20 visual cortex, when incubated under similar conditions as injured sciatic nerves, also showed approximately 25% of the protein incorporated [³H]Arg at the N-terminus, while arginylation was not detectable in adult rat brain slices. Since Lys is not added posttranslationally to the N-terminus, we have attempted to observe lysylation of proteins in intact cells by using cycloheximide (Cx) to block protein synthesis without interfering with protein modification. The posttranslational incorporation of Arg/Lys into proteins was found to be insensitive to up to 2.0 mM Cx in tissue extracts (in vitro). However, in intact cells, doses as low as 10 uM Cx completely inhibited the incorporation of [³H]Arg/Lys into proteins. One uM Cx allowed for some incorporation of [³H]Arg/Lys into protein and approximately 40% of the Cx insensitive Arg was incorporated into the N-terminal. These results show that in vivo but not in vitro, Cx can block protein modification, suggesting that either in intact cells protein modification requires protein synthesis, or that Cx has effects other than as an inhibitor of protein synthesis on cells in culture, effects that it does not have on the partially purified components of the reaction.     

Naphthalenesulfonamide derivatives ML9 and W7 inhibit catecholamine secretion in intact and permeabilized chromaffin cells

The role of protein phosphorylation in catecholamine secretion from bovine adrenomedullary chromaffin cells was studied using different protein kinase inhibitors. Naphthalenesulfonamide derivatives as ML9 and ML7, more specific for the myosin light chain kinase, and the calmodulin antagonist W7 inhibited catecholamine secretion 20 and 40% respectively in digitonin-permeabilized chromaffin cells. ML9 also decreased calcium evoked protein phosphorylation of different proteins including tyrosine hydroxylase in permeabilized cells. These naphthalenesulfonamide derivatives showed also an effect in intact cells, ML9 and W7 produced 50% inhibition in catecholamine secretion and⁴⁵Ca²⁺ uptake, however H8 had no effect. The partial [³H]nitrendipine binding displacement of these drugs to adrenomedullary membranes suggests that these sulfonamide derivatives could interact directly with L-type calcium channels in intact cells. The results obtained in permeabilized cells suggest a possible role of protein phosphorylation in the regulation of catecholamine secretion in chromaffin cells.The abbreviations used are ML9 1-(5-Chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine hydrochloride - ML7 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4 diazepine hydrochloride - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - H8 N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride - W7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride - PKI protein kinase A inhibitor - HEPES N-(2-hydroxyethylpiperazine-N-(2 ethanesulfonic acid) - PIPES piperazine-N, N-bis (2-ethanesulfonic acid) - EGTA [ethylene-bis (oxyethylenenitrilo)] tetraacetic acid - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DMEM Dulbecco's Modified Eagle's medium - MLC myosin light chain - MLCK myosin light chain kinase - TH tyrosine hydroxylase     

Purification and Characterization of a Protein Cryoprotective for Vibrio cholerae Extracted from the Prawn Shell Surface

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti-CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.     

Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.