Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates

In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (q _Ab, SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816

The patients suffering from acidosis usually sign psychological deficits. The cerebral dysfunction is reportedly caused by an acid-induced functional impairment of GABAergic neurons; however, the role of pyramidal neurons in this process remains unclear. By using electrophysiological method and changing extracellular pH, we investigated the influence of acidic environment on pyramidal neurons in the cortical slices, such as their ability of firing spikes and response to synaptic inputs. A low pH of artificial cerebral spinal fluid elevates the responses of pyramidal neurons to excitatory synaptic inputs and their ability of encoding digital spikes, as well as reduces the signal transmission at GABAergic synapses. The elevated ability of neuronal spiking is associated with the decreases of refractory periods and threshold potentials. Therefore, acidosis deteriorates brain functions through making the activities between cortical pyramidal neurons and GABAergic neurons imbalanced toward the overexcitation of neural networks, a process similar to neural excitotoxicity.     

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh⁸dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh⁸dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh⁸dG-treated cells were also cell growth inhibited and arrested at G₁ in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh⁸dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh⁸dG treatment of KG-1 cells induces cellular senescence.     

Enrichment of electrochemically active bacteria using a three-electrode electrochemical cell

Electrochemically active bacteria were successfully enriched in an electrochemical cell using a positively poised working electrode. The positively poised working electrode (+0.7 V vs. Ag/AgCl) was used as an electron acceptor for enrichment and growth of electrochemically active bacteria. When activated sludge and synthetic wastewater were fed to the electrochemical cell, a gradual increase in amperometric current was observed. After a period of time in which the amperometric current was stabilized (generally 8 days), linear correlations between the amperometric signals from the electrochemical cell and added BOD (biochemical oxygen demand) concentrations were established. Cyclic voltammetry of the enriched electrode also showed prominent electrochemical activity. When the enriched electrodes were examined with electron microscopy and confocal scanning laser microscopy, a biofilm on the enriched electrode surface and bacterium-like particles were observed. These experimental results indicate that the electrochemical system in this study is a useful tool for the enrichment of an electrochemically active bacterial consortium and could be used as a novel microbial biosensor.     

Antioxidant and anticancer activity of extract from Betula platyphylla var. japonica

The antioxidant and anticancer properties of a medicinal plant, Betula platyphylla var. japonica were investigated. The total methanol extract of B. platyphylla var. japonica had protective effects against hydrogen peroxide (H2O2) in the Chinese hamster lung fibroblast (V79-4) cell line and induced apoptotic cell death in human promyelocytic leukemia (HL-60) cells, a cancer cell line. B. platyphylla var. japonica extract significantly increased cell viability against H2O2. The extract also showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 2.4 microg/ml) and lipid peroxidation inhibitory activity (IC50 below 4.0 microg/ml). Furthermore, B. platyphylla var. japonica extract reduced the number of V79-4 cells arrested in G2/M in response to H2O2 treatment and increased the activities of several cellular antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. Treatment with B. platyphylla var. japonica extract induced cytotoxicity and apoptosis in HL-60 cells, as shown by nucleosomal DNA fragmentation, increases in the subdiploid cell population, and fluorescence microscopy. B. platyphylla var. japonica extract gradually increased the expression of pro-apoptotic Bax and led to the activation of caspase-3 and cleavage of PARP. These findings suggest that B. platyphylla var. japonica exhibits potential antioxidant and anticancer properties.     

Induction of endothelial apoptosis by 4-hydroxyhexenal.

Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.     

Molecular Cloning and Characterization of Transferrin from Wax Moth, Galleria mellonella

Complete mRNA sequence of transferrin from Galleria mellonella was obtained, and compared with those of other species. Until now, two types of insect transferrin were reported. Transferrins in cockroach and termite have two iron binding sites while those in most other insect groups, studied for the protein, have only one. It was suggested that the presence of two types of transferrin was related with transferrin evolution, because vertebrate transferrins have two iron binding sites, called N and C terminal lobe. It was shown that G. mellonella transferrin also has only one iron binding site (N terminal lobe), and the deduced amino acid sequence was most similar to those of Manduca sexta and Bombyx mori.     

Facile synthesis of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N 6-ethenoadenosine nucleosides

Facile synthetic methods of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N ⁶-ethenoadenosine nucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction were proposed. The synthetic products were isolated and purified by preparative HPLC and their structures were confirmed by¹H NMR (500 MHz) and FAB-MS including high resolution mass measurement. These modified nucleoside analogs have not been reported yet. Therefore, these modified nucleoside analogs are of potential value to be studied further for biological activity such as anticancer or antiviral.