The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

Transbilayer movement and metabolic fate of ether-linked phosphatidic acid (1-O-Octadecyl-2-acetyl-sn-glycerol 3-phosphate) in guinea pig peritoneal polymorphonuclear leukocytes.

1-O-Octadecyl-2-acetyl-sn-glycerol 3-phosphate (octadecylacetyl-GP) and its deacetylation product were used as a model of phosphatidic acid and its lyso derivatives, respectively. The binding, transbilayer movement, and intermembranous transport, which should be related to its metabolism in guinea pig peritoneal polymorphonuclear leukocytes, were studied. The albumin extraction procedure (Tokumura, A., Tsutsumi, T., Yoshida, J., and Tsukatani, H. (1990) Biochim. Biophys. Acta 1044, 91-100) was used for studying the transbilayer movement of [3H]octadecylacetyl-GP. The binding, translocation, and metabolism of octadecylacetylglycerol, a dephosphorylated product of octadecylacetyl-GP, in polymorphonuclear leukocytes were also investigated for comparison. The translocation of octadecylacetyl-GP was dependent on temperature, but not on its concentration (in the range of 1-100 nM). The rate of translocation of octadecylacetyl-GP was much slower than that of octadecylacetylglycerol. Treatment of polymorphonuclear leukocytes with N-ethylmaleimide did not affect the translocation of octadecylacetyl-GP. These results suggest that the transbilayer movement of octadecylacetyl-GP is driven by a diffusion process, not by a carrier protein. From these findings, the process of translocation of octadecylacetyl-GP is concluded to be a rate-limiting step in its metabolic conversion to triglyceride, phosphatidylethanolamine, and phosphatidylcholine.     

The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 Are Required for Autophagic Degradation in Arabidopsis

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit VACUOLAR PROTEIN SORTING2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.     

Characterization of Variant Creutzfeldt-Jakob Disease Prions in Prion Protein-humanized Mice Carrying Distinct Codon 129 Genotypes

To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.     

Isolation of Pyropheophorbide a as a Photodynamic Pigment from the Liver of Abalone,Haliotis discus hannai

A photodynamic agent was isolated from the liver of abalone, Haliotis discus hannai, and identified as pyropheophorbide a. This red fluorescent pigment was proved to induce photosensitization both in rats and cats by oral administration, and recognized as the sole photodynamic pigment in the liver.

The periodical examination on several kinds of herbivorous gastropods indicated that the liver becomes toxic only in spring.     

Substituted indanylacetic acids as PPAR-alpha-gamma activators

A series of oxazole-substituted indanylacetic acids were prepared which show a spectrum of activity as ligands for PPAR nuclear receptor subtypes.     

Hepatitis B core antigen-specific IFN-gamma production of peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the specificity of cellular immune response to hepatitis B virus (HBV) Ag in patients with chronic HBV infection, we have measured IFN-gamma production and proliferation of PBMC of 16 patients with chronic active hepatitis (CAH), 17 asymptomatic carriers of HBV (ASC), 6 anti-hepatitis B surface (HBs)-positive subjects, and 6 control individuals with ELISA procedure and [3H]thymidine incorporation. There was no significant increase in the mean proliferative response to recombinant HB surface and core Ag (rHBsAg and rHBcAg), nor was IFN-gamma production elicited with rHBsAg in any group. In contrast, PBMC of HBeAg-positive and anti-HBe-positive CAH patients, and anti-hepatitis B "e" Ag (HBe)-positive ASC showed significantly enhanced IFN-gamma production in response to HBcAg, whereas those of HBeAg-positive ASC and anti-HBs-positive subjects did not respond to HBcAg. The maximal response was observed in a 5-day culture with 500 ng/ml of rHBcAg when assessed by stimulation index value. Monocytes did not demonstrate an increased suppressor or helper activity for IFN-gamma production in these patients. T cell subset fractionation revealed that CD4+ cells were main population of IFN-gamma production specific for HBcAg and CD8+ cells did not suppress IFN-gamma production of CD4+ cells. Furthermore, CD4+ cells of HBeAg-positive ASC generated lesser amounts of IFN-gamma than HBeAg-positive CAH patients did. These results show that the measurement of IFN-gamma production is useful to determine cellular immune response to HBV Ag and suggest that IFN-gamma production depends on the helper activity of CD4+ T cells sensitized to HBcAg.     

A longitudinal large-scale objective sleep data analysis revealed a seasonal sleep variation in the Japanese population

In the contemporary era, when life habits are largely determined by social needs and individual preferences, sleep is nevertheless affected by seasonal environmental changes. Japan has large seasonal and geographical alterations of photoperiod and climate. Japan does not adopt the daylight saving time (DST) system, making it a suitable country for the study of seasonal variations in natural human sleep. The aim of this study was to analyze the seasonal changes in the sleep properties (timing and quality) and identify their relationship with environmental changes. Here, we report an analysis of objective sleep data of 691 161 nights collected from 1856 Japanese participants (age 20–79 years, male 91%, female 9%) for 3 years using contactless biomotion sensors. Sleep onset time did not show clear seasonal variation, but sleep offset time showed a seasonal change with a single latest peak in winter. Seasonal variation was larger during weekends than during weekdays. Sleep offset time well correlated with sunrise time but was different in spring and autumn even when the sunrise time was same, suggesting the role of temperature difference. Sleep quality, estimated by wake time after sleep onset and sleep efficiency, showed seasonal changes with the lowest trough around mid-summer. In conclusion, despite profound social influences, the timing and quality of sleep showed seasonal fluctuation indicating that they were influenced by climate factors even in the developed country.     

Proline- and alanine-rich Ste20-related kinase associates with F-actin and translocates from the cytosol to cytoskeleton upon cellular stresses

Proline- and alanine-rich Ste20-related kinase (PASK) is a Ste20-related protein kinase isolated from rat brain. Cell fractionation studies showed that PASK was present both in the cytosol and in Triton X-100-insoluble cytoskeletal fraction in rat tissues. In brain, PASK associated with protein complexes that contained actin and tubulin, confirming the association of PASK with the cytoskeleton in vivo. Glutathione S-transferase-PASK fusion protein cosedimented with F-actin, indicating that PASK binds to F-actin. In contrast to rat tissues, PASK was detected only in the Triton X-100-soluble cytosolic fraction in cultured PC12 and NIH 3T3 cells. Cytosolic PASK translocated to the cytoskeleton when these cells were stimulated with severe cellular stresses such as hypertonic sodium chloride, hydrogen peroxide, and heat shock at 45 degrees C. Our results suggest that PASK may be involved in the regulation of the cytoskeleton in response to cellular stresses such as hyperosmotic shock.