Contribution of a Salt Bridge Triad to the Thermostability of a Highly Alkaline Protease from an Alkaliphilic Bacillus Strain*

Summary Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19–Glu271–Arg275 (in subtilisin BPN′ numbering), which is not found in less alkaline true subtilisins BPN′ and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN′, Gln residue was engineered into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal inactivation rate; the wild-type > Arg275 → Gln > Glu271 → Gln > Arg19 → Gln/Glu271 → Gln/Arg275 → Gln > Arg19 → Gln. This result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the highly alkaline M-protease.     

Genetic variations in humans associated with differences in the course of hepatitis C

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.     

Oxygen evolution in the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421

The oxygen-evolving reactions of the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 were compared with those of Synechocystis sp. PCC 6803. Four aspects were considered: sequence conservation in three extrinsic proteins for oxygen evolution, steady-state oxygen-evolving activity, charge recombination reactions, i.e., thermoluminescence and oscillation patterns of delayed luminescence on a second time scale and delayed fluorescence on the nanosecond time scale at − 196 °C. Even though there were significant differences between the amino acid sequences of extrinsic proteins in G. violaceus and Synechocystis sp. PCC 6803, the oxygen-evolving activities were similar. The delayed luminescence oscillation patterns and glow curves of thermoluminescence were essentially identical between the two species, and the nanosecond delayed fluorescence spectral profiles and lifetimes were also very similar. These results indicate clearly that even though the oxygen-evolving reactions are carried out in the periplasm by components with altered amino acid sequences, the essential reaction processes for water oxidation are highly conserved. In contrast, we observed significant changes on the reduction side of photosystem II. Based on these data, we discuss the oxygen-evolving activity of G. violaceus.     

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.     

Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1α and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1α protein, but the other was not. Administration of SB203580 (SB), an inhibitor of p38 MAPK, suppressed the increase in PGC-1α expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1α and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions.     

Bacterial cometabolic degradation of chlorinated paraffins

Summary Cometabolic dechlorination of chlorinated paraffins was demonstrated in the presence of n-hexadecane by bacterial strains (HK-3, HK-6, HK-8, and HK-10) isolated from soil samples.Eleven per cent of chlorine of chlorinated paraffin-150 (CP-150) was released by strain HK-3. The mixed culture of strain HK-3, catalyzing the dechlorination of terminal chlorine of chloroalkane, and strain H15-4, capable of releasing the chlorine from 2-chlorinated fatty acids, dechlorinated CP-150 up to 13%. The mixed culture of the four strains (HK-3, HK-6, HK-8, and HK-10) performed the dechlorination of CP-150 by cometabolism in a jar fermentor pH at 7.0. The amount of chloride released from the chlorinated paraffins tested was in the range of 15–57%.The activated sludge acclimatized to n-hexadecane for 60 days showed a little dechlorination activity to CP-150.     

Prolamellar bodies formed by cyanobacterial protochlorophyllide oxidoreductase in Arabidopsis

In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto‐plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.     

Acute exercise increases expression of extracellular superoxide dismutase in skeletal muscle and the aorta

Exercise dramatically increases oxygen consumption and causes oxidative stress. Superoxide dismutase (SOD) is important in the first-line defence mechanisms against oxidative stress. To investigate the effect of acute exercise on the expression of SOD, we examined the expression of mRNA for three SOD isozymes, in mice run on a treadmill to exhaustion. Six hours after exercise, the expression of extracellular SOD (EC-SOD) mRNA increased significantly in skeletal muscle and persisted for 24 h, whereas no change was observed for cytoplasmic and mitochondrial SOD mRNA. Moreover, acute exercise also induced EC-SOD mRNA in the aorta. These results suggest that a single bout of exercise is enough to augment the expression EC-SOD mRNA in skeletal muscle and the aorta, and may partly explain the beneficial effect of exercise.     

Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function.

Ultraviolet (UV) irradiation stimulates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) superfamily and implicated in stress-induced apoptosis. UV also induces the activation of another MAPK member, extracellular signal-regulated kinase (ERK), which is typically involved in a growth-signaling cascade. However, the UV-induced signaling pathway leading to ERK activation, together with the physiological role, has remained unknown. Here we examined the molecular mechanism and physiological function of UV-induced ERK activation in human epidermoid carcinoma A431 cells that retain a high number of epidermal growth factor (EGF) receptors. UV-induced ERK activation was accompanied with the Tyr phosphorylation of EGF receptors, and both responses were completely abolished in the presence of a selective EGF receptor inhibitor (AG1478) or the Src inhibitor PP2 and by the expression of a kinase-dead Src mutant. On the other hand, SAPK/JNK activation by UV was partially inhibited by these inhibitors. UV stimulated Src activity in a manner similar to the ERK activation, but the Src activation was insensitive to AG1478. UV-induced cell apoptosis measured by DNA fragmentation and caspase 3 activation was enhanced by AG1478 and an ERK kinase inhibitor (U0126) but inhibited by EGF receptor stimulation by the agonist. These results indicate that UV-induced ERK activation, which provides a survival signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGF receptors.     

Complex formation of amphotericin B in sterol-containing membranes as evidenced by surface plasmon resonance

Amphotericin B (AmB) is a membrane-active antibiotic that increases the permeability of fungal membranes. Thus, the dynamic process of its interaction with membranes poses intriguing questions, which prompted us to elaborate a quick and reliable method for real-time observation of the drug's binding to phospholipid liposomes. We focused on surface plasmon resonance (SPR) and devised a new modification method of sensor chips, which led to a significant reduction in the level of nonspecific binding of the drug in a control lane. With this method in hand, we examined the affinity of AmB for various membrane preparations. As expected, AmB exhibited much higher affinity for sterol-containing palmitoyloleoylphosphatidylcholine membranes than those without sterol. The sensorgrams recorded under various conditions partly fitted theoretical curves, which were based on three interaction models. Among those, a two-state reaction model reproduced well the sensorgram of AmB binding to an ergosterol-containing membrane; in this model, two states of membrane-bound complexes, AB and AB*, are assumed, which correspond to a simple binding to the surface of the membrane (AB) and formation of another assembly in the membrane (AB*) such as an ion channel complex. Kinetic analysis demonstrated that the association constant in ergosterol-containing POPC liposomes is larger by 1 order of magnitude than that in the cholesterol-containing counterpart. These findings support the previous notion that ergosterol stabilizes the membrane-bound assembly of AmB.