Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins

The function of bcl-2 in preventing cell death is well known, but the mechanisms whereby bcl-2 functions are not well characterized. One mechanism whereby bcl-2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl-2 can protect cells against oxidative injury resulting from post-hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat-1 and their bcl-2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl-2 transfectants exhibited the cell viability superior to that of their parent non-transfectants upon treatment with reoxygenation after 24-, 48-, or 72-h hypoxia, but not upon normoxic serum-deprivation or upon serum-supplied hypoxic treatment alone. Thus bcl-2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time-sequential events of H/R-induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1-12 h after H/R, (2) activation of caspases-1 and -3, at 1-3 h and 3-6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (DeltaPsi) at 3-12 h after H/R. These cell death-associated events were prevented entirely except caspase-1 activation by bcl-2 transfection, and were preceded by Bcl-2 upregulation which was executed as early as at 0-1 h after H/R for the bcl-2 transfectants but not their non-transfected counterpart cells. Thus upregulation of Bcl-2 proteins may play a role in prevention of H/R-induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoprotection through blockage of either ROS generation, caspase-3 activation, or DeltaPsi decline.     

Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.     

Down-regulation of endodermal Shh is required for gland formation in chicken stomach

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Here we present evidence that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. We also show that gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required.     

In vitro cytotoxicity of non-cyt inclusion proteins of a Bacillus thuringiensis isolate against human cells, including cancer cells

A soil isolate designated 90-F-45-14, belonging to Bacillus thuringiensis serovar dakota (H15), was examined for characterization of in vitro cytotoxicity, associated with parasporal inclusion proteins, against human cells. When activated with proteolytic processing, inclusion proteins of the isolate 90-F-45-14 exhibited a moderate cytotoxicity against the human uterus cervix cancer cells (HeLa) with an EC(50) value of 60.8 microg ml(-1), while showing extremely high activities on the human leukaemic T cells (MOLT-4) and the normal T cells with EC(50) values of 0.27 and 0.20 microg ml(-1), respectively. Anti-leukaemic cell activity of the 90-F-45-14 proteins was eight to nine times greater than that of the B. thuringiensis serovar israelensis proteins containing the Cyt1 protein, a broad-spectrum cytolysin. The cytopathy by the 90-F-45-14 proteins was characterized by marked cell-ballooning, while the israelensis proteins induced early breakdown of the cells due to cytolysis. Inclusions of the isolate consisted of five major polypeptides of 170, 103, 73, 40 and 32 kDa. A 100% homology was observed in the sequence of 15 N-terminal amino acids between the proteins of 170 and 103 kDa. There was no N-terminal sequence homology between 90-F-45-14 proteins and the existing Cry/Cyt proteins of B. thuringiensis. Proteolytic processing by proteinase K yielded several proteins with molecular masses ranging from 40 to 28 kDa.     

Dynamics of a hybrid system of a brain neural network and an artificial nonlinear oscillator

In the brain, many functional modules interact with each other to execute complex information processing. Understanding the nature of these interactions is necessary for understanding how the brain functions. In this study, to mimic interacting modules in the brain, we constructed a hybrid system mutually coupling a hippocampal CA3 network as an actual brain module and a radial isochron clock (RIC) simulated by a personal computer as an artificial module. Return map analysis of the CA3-RIC system's dynamics showed the mutual entrainment and complex dynamics dependent on the coupling modes. The phase response curve of CA3 was modeled regarding the CA3 as a nonlinear oscillator. Using the phase response curves of CA3 and RIC, we reconstructed return maps of the hybrid system's dynamics. Although the reconstructed return maps almost agreed with the experimental data, there were deviations dependent on the coupling mode. In particular, we noted that the deviation was smaller under the bidirectional coupling conditions than during the one-way coupling from RIC to CA3. These results suggest that brain modules may flexibly change their dynamical properties through interaction with other modules.     

Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA

Adenine paired with 8-hydroxyguanine (oh⁸G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells. Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His₆-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh⁸G and A:G mismatches by DNA cleavage assay and gel mobility shift assay. We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q³²⁴ and 2-Q³¹⁰ proteins with type 1-H³²⁴ and 2-H³¹⁰ proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain. In a reaction buffer with a low salt (0–50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh⁸G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh⁸G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh⁸G, was proportionally reduced. The glycosylase activity on A:oh⁸G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins. These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh⁸G under physiological salt concentrations.     

Quantitative structure-intestinal permeability relationship of benzamidine analogue thrombin inhibitor

The intestinal permeability of benzamidine analogue thrombin inhibitor is correlated with molecular volume, lipophilicity (calculated log P and IAM column capacity factor), hydrogen bond acidity/basicity and dipolarity.