Prostaglandin E receptor type 4-associated protein interacts directly with NF-kappaB1 and attenuates macrophage activation

Macrophage activation participates pivotally in the pathophysiology of chronic inflammatory diseases, including atherosclerosis. Through the receptor EP4, prostaglandin E(2) (PGE(2)) exerts an anti-inflammatory action in macrophages, suppressing stimulus-induced expression of certain proinflammatory genes, including chemokines. We recently identified a novel EP4 receptor-associated protein (EPRAP), whose function in PGE(2)-mediated anti-inflammation remains undefined. Here we demonstrate that PGE(2) pretreatment selectively inhibits lipopolysaccharide (LPS)-induced nuclear factor kappaB1 (NF-kappaB1) p105 phosphorylation and degradation in mouse bone marrow-derived macrophages through EP4-dependent mechanisms. Similarly, directed EPRAP expression in RAW264.7 cells suppresses LPS-induced p105 phosphorylation and degradation, and subsequent activation of mitogen-activated protein kinase kinase 1/2. Forced expression of EPRAP also inhibits NF-kappaB activation induced by various proinflammatory stimuli in a concentration-dependent manner. In co-transfected cells, EPRAP, which contains multiple ankyrin repeat motifs, directly interacts with NF-kappaB1 p105/p50 and forms a complex with EP4. In EP4-overexpressing cells, PGE(2) enhances the protective action of EPRAP against stimulus-induced p105 phosphorylation, whereas EPRAP silencing in RAW264.7 cells impairs the inhibitory effect of PGE(2)-EP4 signaling on LPS-induced p105 phosphorylation. Additionally, EPRAP knockdown as well as deficiency of NF-kappaB1 in macrophages attenuates the inhibitory effect of PGE(2) on LPS-induced MIP-1beta production. Thus, PGE(2)-EP4 signaling augments NF-kappaB1 p105 protein stability through EPRAP after proinflammatory stimulation, limiting macrophage activation.     

Overproduction of highly active trypanosome alternative oxidase in Escherichia coli heme-deficient mutant

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in humans and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs, because it does not exist in the host. In this study, we established a system for overproduction of highly active TAO in Eschericia coli heme-deficient mutant. Kinetic analysis of recombinant enzyme and TAO in Trypanosoma brucei brucei mitochondria revealed that recombinant TAO retains the properties of native enzyme, indicating that recombinant TAO is quite valuable for further biochemical study of TAO.     

Selfing and inbreeding depression in seeds and seedlings of Neobalanocarpus heimii (Dipterocarpaceae)

We evaluated the degree of selfing and inbreeding depression at the seed and seedling stages of a threatened tropical canopy tree, Neobalanocarpus heimii, using microsatellite markers. Selection resulted in an overall decrease in the level of surviving selfed progeny from seeds to established seedlings, indicating inbreeding depression during seedling establishment. Mean seed mass of selfed progeny was lower than that of outcrossed progeny. Since the smaller seeds suffered a fitness disadvantage at germination in N. heimii, the reduced seed mass of selfed progeny would be one of the determinants of the observed inbreeding depression during seedling establishment. High selfing rates in some mother trees could be attributed to low local densities of reproductive individuals, thus maintenance of a sufficiently high density of mature N. heimii should facilitate regeneration and conservation of the species.     

A structural determinant for the control of PIP2 sensitivity in G protein-gated inward rectifier K+ channels

Inward rectifier K(+) (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), but G protein-gated Kir (K(G)) channels further require either G protein βγ subunits (Gβγ) or intracellular Na(+) for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of K(G) channel subunit Kir3.2 obtained in the presence and the absence of Na(+). The Na(+)-free Kir3.2, but not the Na(+)-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na(+)-dependent activation, lowered PIP(2) sensitivity. The conservation of these residues within the K(G) channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP(2) sensitivity.     

Proxy comparison in ancient peat sediments: pollen,macrofossil and plant DNA

We compared DNA, pollen and macrofossil data obtained from Weichselian interstadial (age more than 40 kyr) and Holocene (maximum age 8400 cal yr BP) peat sediments from northern Europe and used them to reconstruct contemporary floristic compositions at two sites. The majority of the samples provided plant DNA sequences of good quality with success amplification rates depending on age. DNA and sequencing analysis provided five plant taxa from the older site and nine taxa from the younger site, corresponding to 7% and 15% of the total number of taxa identified by the three proxies together. At both sites, pollen analysis detected the largest (54) and DNA the lowest (10) number of taxa, but five of the DNA taxa were not detected by pollen and macrofossils. The finding of a larger overlap between DNA and pollen than between DNA and macrofossils proxies seems to go against our previous suggestion based on lacustrine sediments that DNA originates principally from plant tissues and less from pollen. At both sites, we also detected Quercus spp. DNA, but few pollen grains were found in the record, and these are normally interpreted as long-distance dispersal. We confirm that in palaeoecological investigations, sedimentary DNA analysis is less comprehensive than classical morphological analysis, but is a complementary and important tool to obtain a more complete picture of past flora.     

Biodegradation of high molecular weight lignin under sulfate reducing conditions: lignin degradability and degradation by-products

This study is designed to investigate the biodegradation of high molecular weight (HMW) lignin under sulfate reducing conditions. With a continuously mesophilic operated reactor in the presence of co-substrates of cellulose, the changes in HMW lignin concentration and chemical structure were analyzed. The acid precipitable polymeric lignin (APPL) and lignin monomers, which are known as degradation by-products, were isolated and detected. The results showed that HMW lignin decreased and showed a maximum degradation capacity of 3.49 mg/l/day. APPL was confirmed as a polymeric degradation by-product and was accumulated in accordance with HMW lignin reduction. We also observed non-linear accumulation of aromatic lignin monomers such as hydrocinnamic acid. Through our experimental results, it was determined that HMW lignin, when provided with a co-substrate of cellulose, is biodegraded through production of APPL and aromatic monomers under anaerobic sulfate reducing conditions with a co-substrate of cellulose.     

Functional Characterization of the Vitamin K2 Biosynthetic Enzyme UBIAD1

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K₂ (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg²⁺/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.     

Association of Psychosocial Conditions,Oral Health,and Dietary Variety with Intellectual Activity in Older Community-Dwelling Japanese Adults

Background

This study examined the factors related to intellectual activity in community-dwelling elderly persons.

Methods

Self-administered questionnaires mailed to all people aged ≥65 years in a dormitory suburb in Japan (n = 15,210). The response rate was 72.2%. Analytical subjects (n = 8,910) were those who lived independently and completely answered questions about independent and dependent variables and covariates. Independent variables included psychosocial conditions (i.e., social activities, hobbies, and a sense that life is worth living (ikigai)), oral health (i.e., dental health behaviors and oral function evaluated by chewing difficulties, swallowing difficulties, and oral dryness), and dietary variety measured using the dietary variety score (DVS). A dependent variable was intellectual activity measured using the Tokyo Metropolitan Institute of Gerontology Index of Competence. Covariates included age, gender, family structure, pensions, body mass index, alcohol, smoking, medical history, self-rated health, medications, cognitive function, depression, and falling. Logistic regression was used to estimate the odds ratio (OR) for poor intellectual activity.

Results

Poor intellectual activity was reported by 28.9% of the study population. After adjustment for covariates and independent variables, poor intellectual activity was significantly associated with nonparticipation in social activities (OR = 1.90, 95%CI = 1.61–2.24), having neither hobbies nor ikigai (3.13, 2.55–3.84), having neither regular dental visits nor daily brushing (1.70, 1.35–2.14), the poorest oral function (1.61, 1.31–1.98), and the lowest DVS quartile (1.96, 1.70–2.26).

Conclusion

These results indicate that psychosocial conditions, oral health, and dietary variety are independently associated with intellectual activity in elderly persons. The factors identified in this study may be used in community health programs for maintaining the intellectual activity ability of the elderly.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.