Meteorological conditions and sports deaths at school in Japan, 1993–1998

We evaluated the association between meteorological conditions and sports deaths at elementary, junior high and senior high schools. Data were collected from attached documents such as accident reports and death certificate records in the National Agency for the Advancement of Sports and Health in Japan. Evaluation of seasonal variation showed a significant concentration of deaths from heat disorders and drowning in July and August. When heart disease was evaluated according to the sports situation, significant seasonal variation with a high number of deaths in September–December was observed in sports events. Concerning circadian variation, deaths from heart disease showed a high peak at 10:00–11:00 a.m. in physical education classes and sports events, and at 2:00–5:00 p.m. in sports club activities. Analysis using a multiple logistic model showed a significantly lower odds ratio from heart disease and a significantly higher odds ratio from heat disorders at a wet bulb globe temperature of 21.0°C than at <21.0°C. According to the sports situation in heart disease, the odds ratio in sports club activities was significantly lower on days with rainfall than on days without rainfall. According to the school categories in heart diseases, the odds ratio in girls in elementary school was significantly higher than that in boys, but the odds ratio in girls in senior high school was significantly lower than that in boys.     

A test for ectopic exchange catalyzed by Cre recombinase in maize

A maize line expressing Cre recombinase as well as the recipient line without the transgene were assayed for evidence of ectopic recombination within the maize genome. Such a test is valuable for understanding the action of Cre as well as for its use to recombine two target lox sites present in the chromosomes. Pollen examination and seed set tests of material expressing Cre provided no evidence of ectopic recombination, which would be manifested in the production of translocations or inversions and result in pollen abortion and reduced seed set. Root-tip chromosome karyotype analysis was also performed on material with and without Cre expression. Chromosomal aberrations in Cre+ material were not observed above the background level.     

Rational design of a mononuclear metal site into the archaeal Rieske-type protein scaffold

Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.     

Aggrecanase-1 (ADAMTS-4) interacts with alpha1-antitrypsin

In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1 (ADAMTS--4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY 48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified alpha1-antitrypsin, a member of the family of plasma serine proteinase inhibitors, as a prey. Recombinant aggrecanase-1 and alpha1-antitrypsin were expressed in mammalian cells and used in co-immunoprecipitation experiments, which showed that full-length aggrecanase-1 and alpha1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of alpha1-antitrypsin against elastase, and alpha1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and alpha1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and alpha1-antitrypsin remains unclear.     

Relocation of water molecules between the Schiff base and the Thr46-Asp96 region during light-driven unidirectional proton transport by bacteriorhodopsin: an FTIR study of the N intermediate

A key event in light-driven proton pumping by bacteriorhodopsin is the formation of the L intermediate, whose transition to M is accompanied by the first proton transfer step, from the Schiff base to Asp85 on the extracellular side. Subsequent reprotonation of the Schiff base from the other side of the membrane to form the N intermediate is crucial for unidirectional proton transport. Previous FTIR studies have suggested that the intense water O-D stretching vibration bands which appear in L at 2589, 2605, and 2621 cm(-)(1) are due to a cluster of polarized water molecules connecting the Schiff base to the Thr46-Asp96 region closer to the cytoplasmic surface. In the present study the difference spectrum was obtained of the N intermediate with its photoproduct N', formed after irradiating N at 80 K. The water O-D stretching vibrations of N appear as a broad feature in a similar frequency region with a similar intensity to those of L. This feature is also affected by T46V like in L. However, the intensities of these water vibrations of N nearly returned to the initial unphotolyzed state upon formation of N', unlike those of L which are preserved in L'. An exception was V49A, which preserved the intense water vibrations of N in N'. The results suggest that both L and N have a water cluster extending from the Schiff base to Thr46. The surrounding protein moiety stabilizes the water cluster in L, but in N it is stabilized mostly by interaction with the Schiff base.     

Factor H is a dermatan sulfate-binding protein: identification of a dermatan sulfate-mediated protease that cleaves factor H

Dermatan sulfate mediates the blood coagulation cascade by binding to heparin cofactor II and potentiating the antithrombin activity. In order to explore another function of dermatan sulfate, a dermatan sulfate affinity column was prepared from biotinylated dermatan sulfate and Streptavidin Sepharose. When human plasma was applied on the dermatan sulfate column, factor H was bound and cleaved. The cleavage products, a 30-kDa N-terminal fragment and a 120-kDa fragment, were eluted from the column with 500 mM NaCl and detected after Western blotting with anti-factor H. The bond between the tandem arginine residues in the sixth domain of factor H was cleaved. When purified factor H was applied on the column, the factor H was not cleaved and was recovered from the column as an intact 150-kDa fraction. The finding that dermatan sulfate-mediated cleavage of factor H was inhibited by (p-amidinophenyl) methanesulfonyl fluoride, but not N-ethylmaleimide or EDTA, indicates that a serine protease in the plasma was activated on the dermatan sulfate column and factor H was cleaved without intervention of the plasma protease inhibitors. Amidase activity was detected in the effluent from the dermatan sulfate column but was abolished by pretreatment of the plasma with dermatan sulfate. Therefore, dermatan sulfate participates in the activation of a protease as well as having the protease inhibitory action.     

Recognition and fluorescence sensing of specific amino acid residue on protein surface using designed molecules

Many biological processes are mediated by surface recognition between proteins. Small molecules that recognize and bind a specific region of a protein surface may be promising agents for disrupting certain protein-protein surface interactions, which consequently leads to regulation of cellar functions. This article describes our recent efforts toward the development of the designed small molecules, which can recognize histidine or phosphorylated amino acid residues on peptide surfaces in a sequence-selective manner. These results demonstrate that cooperative metal-ligand interaction is powerful for tight and selective binding to the specific amino acid residues of proteins in aqueous medium.     

Detection of the novel autoantibody (anti-UACA antibody) in patients with Graves' disease

Uveal autoantigen with coiled coil domains and ankyrin repeats (UACA) is an autoantigen in patients with panuveitis such as Vogt-Koyanagi-Harada disease. The prevalence of IgG anti-UACA antibodies in patients with uveitis is significantly higher than healthy controls, suggesting its potential role as an autoantigen. Originally, UACA was cloned from dog thyroid tissue following TSH stimulation. So, we presumed UACA could be a novel autoantigen in autoimmune thyroid diseases. We measured serum anti-UACA antibody titer using ELISA in patients with autoimmune thyroid diseases (Graves' disease, Hashimoto's thyroiditis, subacute thyroiditis, and silent thyroiditis). The prevalence of anti-UACA antibodies in Graves' disease group was significantly higher than that in healthy group (15% vs. 0%). Moreover, the prevalence of anti-UACA antibodies in Graves' ophthalmopathy was significantly higher than that in Graves' patients without ophthalmopathy (29% vs. 11%). Especially, 75% of severe ocular myopathy cases showed high UACA titer. Immunohistochemical analysis revealed that UACA protein is expressed in eye muscles as well as human thyroid follicular cells. Taken together, UACA is a novel candidate for eye muscle autoantigens in thyroid-associated ophthalmopathy.     

Temperature-dependent modulation of farnesyl diphosphate/geranylgeranyl diphosphate synthase from hyperthermophilic archaea

Enzyme characteristics of trans-prenyl diphosphate synthase (Tk-IdsA) from Thermococcus kodakaraensis, which catalyzes the consecutive trans-condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate, were examined. Product analysis revealed that Tk-IdsA is a bifunctional enzyme, farnesyl diphosphate (FPP, C(15))/geranylgeranyl diphosphate (GGPP, C(20)) synthase, and mainly yields both C(15) and C(20). The FPP/GGPP product ratio increases with the rise of the reaction temperature. The kinetic parameters obtained at 70 and 90 degrees C demonstrated that the rise of the temperature elevates the k(0) value for the C(10) allylic substrate to more than those for the C(5) and C(15) allylic substrates. These data suggest that Tk-IdsA contributes to adjust the membrane composition to the cell growth temperature by modulating its substrate and product specificities. Mutation study indicated that the aromatic side chain of Tyr-81 acts as a steric hindrance to terminate the chain elongation and defines the final product length.