Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.     

Effects of dietary soybean peptides on hepatic production of ketone bodies and secretion of triglyceride by perfused rat liver

Soybean protein isolate (SPI) was digested with protease to produce a peptides containing the low-molecular fraction (LD3) or a mixture of high- and low-molecular fractions (HD1). Rats were fed a diets containing SPI, LD3, or HD1 at a protein level equivalent to the 20% casein diet for 4 weeks. The serum triglyceride concentration was lower in rats fed SPI, LD3, and HD1 diets than in rats fed the casein diet, and the differences were significant for the cholesterol-enriched diet. The value for the LD3 group was the lowest among all groups for both the cholesterol-free and -enriched diets. The level of triglyceride in the post-perfused liver was significantly lower in the LD3 and HD1 groups and the SPI group than in the casein group irrespective of the presence of cholesterol in the diet. In the cholesterol-free diet, LD3 feeding as compared to casein feeding caused a reduction in triglyceride secretion from the liver to perfusate and an increment of hepatic ketone body production. The addition of cholesterol to the diets somewhat attenuated these effects of LD3. These results suggest that the low-molecular fraction in soybean peptides causes triglyceride-lowering activity through a reduction in triglyceride secretion from the liver to the blood circulation and the stimulation of fatty acid oxidation in the liver. There is a possibility that soybean peptides modulate triglyceride metabolism by changes in the hepatic contribution.     

Sensitivity analysis of ecosystem CO<Subscript>2</Subscript> exchange to climate change in High Arctic tundra using an ecological process-based model

Arctic terrestrial ecosystems are extremely vulnerable to climate change. A major concern is how the carbon balance of these ecosystems will respond to climate change. In this study, we constructed a simple ecological process-based model to assess how the carbon balance will be altered by ongoing climate change in High Arctic tundra ecosystems using in situ observations of carbon cycle processes. In particular, we simulated stand-level photosynthesis, root respiration, heterotrophic respiration, and hence net ecosystem production (NEP) of a plant community dominated by vascular plants and mosses. Analyses were carried out for current and future temperature and precipitation conditions. Our results showed that the tundra ecosystem was a CO₂ sink (NEP of 2.3–18.9 gC m^?2 growing season^?1) under present temperature conditions. Under rising temperature (2–6 °C), carbon gain is significantly reduced, but a few days’ extension of the foliage period caused by their higher temperatures compensated for the negative effect of temperature on NEP. Precipitation is the major environmental factor driving photosynthetic productivity of mosses, but it had a minor influence on community-level NEP. However, NEP decreased by a maximum 15.3 gC m^?2 growing season^?1 under a 30-day prolongation of the moss-growing season, suggesting that growing season extension had a negative effect on ecosystem carbon gain, because of poorer light conditions in autumn. Because the growing season creates a weak CO₂ sink at present, lengthening of the snow-free season coupled with rising temperature could seriously affect the future carbon balance of this Arctic tundra ecosystem.     

Identification, characterization, and site-directed mutagenesis of recombinant pentachlorophenol 4-monooxygenase

In a previous study, we constructed a three-dimensional (3D) structure of pentachlorophenol 4-monooxygenase (PcpB). In this study, further analyses are performed to examine the important amino acid residues in the catalytic reaction by identification of the proteins with mass spectrometry, circular dichroism (CD) and UV spectrometry, and determination of kinetic parameters. Recombinant histidine-tagged PcpB protein was produced and shown to have a similar activity to the native protein. Mutant proteins of PcpB were then produced (F85A, Y216A, Y216F, R235A, R235E, R235K, Y397A and Y397F) on the basis of the proposed 3D structure. The CD spectra of the proteins showed that there were no major changes in the structures of the mutant proteins, with the exception of R235E. Steady-state kinetics showed a 20-fold reduction in k(cat)/K(m) and a ninefold increase in K(m) for Y216F and a threefold reduction in k(cat)/K(m) and a sixfold increase in K(m) for Y397F compared to the wild type. On the other hand, the value of k(cat)/K(m) of R235K mutant was the same as that of wild type. As a result, it was confirmed that Y216 and Y397 play an important role with respect to the recognition of the substrate.     

Identification of a Novel Subgroup of Koala Retrovirus from Koalas in Japanese Zoos

We identified a new subgroup of koala retrovirus (KoRV), named KoRV-J, which utilizes thiamine transport protein 1 as a receptor instead of the Pit-1 receptor used by KoRV (KoRV-A). By subgroup-specific PCR, KoRV-J and KoRV-A were detected in 67.5 and 100% of koalas originating from koalas from northern Australia, respectively. Altogether, our results indicate that the invasion of the koala population by KoRV-J may have occurred more recently than invasion by KoRV-A.     

Chronic intracerebroventricular administration of relaxin-3 increases body weight in rats

Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.     

Selective proteolysis of apolipoprotein B-100 by Arg-gingipain mediates atherosclerosis progression accelerated by bacterial exposure

Epidemiological studies suggest the association of periodontal infections with atherosclerosis, however, the mechanism underlying this association remains poorly understood. Porphyromonas gingivalis is the primary etiologic agent of adult periodontitis and produces a unique class of cysteine proteinases consisting of Arg-gingipain (Rgp) and Lys-gingipain (Kgp). To elucidate key mechanisms for progression of atherosclerosis by P. gingivalis infection, we tested the effects of the disruption of genes encoding Rgp and/or Kgp and inhibitors specific for the respective enzymes on atherosclerosis progression in apolipoprotein E-knockout mice. Repeated intravenous injection of wild-type P. gingivalis resulted in an increase in atherosclerotic lesions as well as an increase in the serum LDL cholesterol and a decrease of HDL cholesterol in these animals. LDL particles in P. gingivalis-injected animals were modified as a result of selective proteolysis of apoB-100 in LDL particles. This modification of LDL by P. gingivalis resulted in an increase in LDL uptake by macrophages and consequent foam cell formation in vitro. The atherosclerotic changes induced by P. gingivalis infection were attenuated by disruption of Rgp-encoding genes or by an Rgp-specific inhibitor. Our results indicate that degradation of apoB-100 by Rgp plays a crucial role in the promotion of atherosclerosis by P. gingivalis infection.     

Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrP^Sc. Real-time quaking-induced conversion can amplify very small amounts of PrP^Sc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD₅₀) is reached approximately 10¹⁰/g brain (values varies 10^8.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrP^Sc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD₅₀ unit was equivalent to 0.06–0.27 fg of PrP^Sc. The SD₅₀ values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.     

Postmortem changes in uric acid and ascorbic acid in human cerebral cortex tissues excised after cardiac death

There has been no report on the determination of uric acid (UA) in human brain and heart tissues. UA and ascorbic acid (AA) in human cerebral cortex and heart tissues excised after cardiac death have been studied by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). It has been found that the levels of AA and UA in the human cerebral cortex tissues tend to decrease and increase, respectively, after cardiac death as a function of time between death and forensic operation. In addition, it has been found that there is no special relationship between UA levels in human heart tissues and time after cardiac death, also that the UA levels in the heart are high as compared with those in human cerebral cortex tissues. We have emphasized that the HPLC-ECD method is useful in determining UA and AA in mammalian tissues by one-time chromatography to gain a better understanding of the relationship between disease and serum urate level.