pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.     

First detection of Rickettsia in soft-bodied ticks associated with seabirds, Japan

Rickettsia was first detected in seabird soft-bodied ticks, Carios capensis and C. sawaii in Japan. According to sequence analysis, Rickettsia in Japan was identical to Rickettsia scc31 in C. capensis in the U.S.A. This suggested that an environmental circulation had consisted among microorganisms, ticks and long distance migratory seabirds around the Pacific Ocean.     

Mangrove rehabilitation dynamics and soil organic carbon changes as a result of full hydraulic restoration and re-grading of a previously intensively managed shrimp pond

Hydraulic restoration by opening the shrimp pond banks facilitated the establishment of planted mangroves and colonisation by non-planted mangrove species and was shown to be an effective method of mangrove rehabilitation. Planted Rhizophora apiculata and Rhizophora mucronata had grown significantly in 6 years, to 300 and 350 cm, respectively. However, the growth rate of Bruguiera cylindrica was merely 150 cm in the same period despite vigorous growth in the initial stage. About 15 non-planted mangrove species had colonised within 6 years after reopening the banks, with the dominant species being Avicennia marina (46.9%) followed by B. cylindrica (27.0%) and Ceriops tagal (14.9%). After the enhancement, soil organic carbon increased considerably from 110 to 160 tonC ha⁻¹ in 2 years at the lower elevation, indicating that hydraulic restoration could stimulate carbon recovery through enhancement of mangrove growth. However, soil organic carbon decreased by almost half in the higher ground, suggesting that carbon decomposition was accelerated due to drying of soils.     

Volatile Anesthetic Sevoflurane Precursor 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) Exerts an Anti-Prion Activity in Prion-Infected Culture Cells

Prion disease is a neurodegenerative disorder with progressive neurologic symptoms and accelerated cognitive decline. The causative protein of prion disease is the prion protein (PrP), and structural transition of PrP from the normal helix rich form (PrP^C) to the abnormal β-sheet rich form (PrP^Sc) occurs in prion disease. While so far numerous therapeutic agents for prion diseases have been developed, none of them are still useful. A fluorinated alcohol, hexafluoro isopropanol (HFIP), is a precursor to the inhalational anesthetic sevoflurane and its metabolites. HFIP is also known as a robust α-helix inducer and is widely used as a solvent for highly aggregated peptides. Here we show that the α-helix-inducing activity of HFIP caused the conformational transformation of the fibrous structure of PrP into amorphous aggregates in vitro. HFIP added to the ScN2a cell medium, which continuously expresses PrP^Sc, reduced PrP^Sc protease resistance after 24-h incubation. It was also clarified that ScN2a cells are more susceptible to HFIP than any of the cells being compared. Based on these findings, HFIP is expected to develop as a therapeutic agent for prion disease.

     

Tetrathionate-Forming Thiosulfate Dehydrogenase from the Acidophilic,Chemolithoautotrophic Bacterium Acidithiobacillus ferrooxidans

Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg⁻¹) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg⁻¹) observed at pH 2.5 and 50°C.     

Two Alkaline Phosphatases from a Butirosin A Producer Bacillus vitellinus

In low-phosphate medium, a butirosin A producer B. vitellinus produced two alkaline phosphatases. These enzymes were fractionated by DEAE-cellulose column chromatography. One phosphatase (Pho I) was eluted with the lower concentration of NaCl compared with the other phosphatase (Pho II). In the wild type strain, Pho I was completely repressed in the high-phosphate medium, but 30% of the fully-derepressed level of Pho II was still produced.

The phosphatase-negative mutant, P-15, that was shown to accumulate butirosin A-6′-N-diphosphate in our previous study, produced only one phosphatase (Pho I) under the low-phosphate condition. Therefore, P-15 was characteristic of the deficiency in Pho II synthesis.

The partially purified preparations of Pho I and II were characterized. Although both enzymes had a similar molecular weight, they could be differentiated in control of synthesis, heat stability, substrate specificity and other properties. Kinetic properties showed that Pho-II was more specific than Pho I to aminoglycoside-phosphates; butirosin A-3′-phosphate, butirosin A-6′-N-diphosphate and 6′-deamino-6′-hydroxybutirosin A-6′-O-diphosphate. The roles of the two phosphatases in butirosin A biosynthesis were discussed.     

Wisp2/CCN5 up-regulated in the central nervous system of GM3-only mice facilitates neurite formation in Neuro2a cells via integrin-Akt signaling

Wisp2/CCN5 belongs to CCN family proteins which are involved in cell proliferation, angiogenesis, tumorigenesis and wound healing. Although a number of studies on the roles of Wisp2/CCN5 in cancers have been reported, no study on the expression and function of Wisp2/CCN5 in the central nervous system has been reported. In this study, we focused on Wisp2/CCN5 that was up-regulated in nervous tissues in GM3-only mice. Over-expression of Wisp2/CCN5 enhanced neurite outgrowth potently after serum withdrawal with increased phosphorylation levels of Akt and ERKs. When cells were cultured with recombinant Wisp2/CCN5 proteins, more and longer neurites were formed than in the controls. Thus, we demonstrated for the first time that Wisp2/CCN5 facilitates neurite formation in a mouse neuroblastoma cell line, Neuro2a. Akt phosphorylation induced by recombinant Wisp2/CCN5 was suppressed after knockdown of integrin β1. Moreover, Wisp2/CCN5-over-expressing cells were resistant to apoptosis induced by H₂O₂. These results suggested that secreted Wisp2/CCN5 induces Akt and ERK phosphorylation via integrins, and consequently facilitates neurite formation and conferred resistance to apoptosis. Up-regulation of Wisp2/CCN5 in GM3-only mice should be, therefore, a reaction to protect nervous tissues from neurodegeneration caused by ganglioside deficiency.     

Regulatory Mechanisms of Nervous Systems with Glycosphingolipids

A number of studies have suggested functions of sialic acid-containing glycosphingolipids (gangliosides) in the nervous system. However, results of analyses of the mutant mice lacking gangliosides suggested that they play crucial roles in the maintenance of integrity and repair of the nervous tissues. Furthermore, results of double knockout mice lacking all gangliosides except GM3 (GM3-only mice) suggested that deficiency of gangliosides induced complement activation and inflammation, leading to neurodegeneration. Generation of triple knockout mice by mating GM3-only mice and C3-deficient mice verified the involvement of complement systems in the inflammation and neurodegeneration. For the mechanisms of the complement activation, functional disorders of complement-regulatory proteins such as CD55 and CD59, which belong to GPI-anchored proteins, should be main factors. These results suggested that normal composition of gangliosides is essential for the maintenance of lipid rafts. Therefore, it was suggested that regulation of the complement systems and suppression of the inflammation should be important for the treatment of neurodegeneration, having common aspects with other neurodegenerative diseases such as Alzheimer disease.