Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.
The effects of fullerene bisadduct regioisomers on solar cell performance have been examined for the first time and the two substituent positions on C(60) have been found to have a large impact on the solar cell performance.
The effects of recombinant human hepatocyte growth factor (HGF) on liver growth and function of normal and partially hepatectomized rats have been examined. HGF was continuously administered into the jugular vein because it was rapidly eliminated from the plasma (t1/2 alpha; approximately 4.5 min) and degraded. In normal rats, the labeling index of hepatocytes was increased about 6 times by the administration of HGF. HGF also decreased the prothrombin time and increased the hepaplastin and serum albumin content. In 70%-hepatectomized rats, HGF stimulated liver regeneration and increased the level of blood proteins such as hepaplastin in a dose-dependent manner. The stimulation of serum protein level seemed to result from not only the increase of hepatic cell number but also the direct effect of HGF on the protein production in hepatocytes, because HGF rapidly enhanced the protein synthesis prior to the increase of cell number and increased the mRNA content of albumin in the liver in vivo. In addition, a combination of heparin with HGF further accelerated the effects of HGF described above, possibly due to the decrease of HGF clearance. These findings suggest that HGF accelerates both the hepatic regeneration and function in vivo, and that rhHGF is clinically expected to be a potent therapeutic agent in hepatectomy and liver injury.
Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.
Substantial increase in amantadine-resistant influenza A (H3N2) was reported in Asia and North America in 2005. In this study the frequency and genetic characteristics of amantadine-resistant influenza A, circulated in Japan in 2005-2006 season, were investigated. Isolates were tested by amantadine susceptibility test (TCID(50)/0.2 ml method), and sequencing of the M2 gene to identify mutations that confer resistance. Additionally, the hemagglutinin (HA) and neuraminidase (NA) genes of the viruses were examined. In total, 415 influenza A isolates from six prefectures were screened, and 231 (65.3%) of 354 influenza A (H3N2) were amantadine-resistant, with a serine to asparagine (S31N) change in the M2 gene. However, none of 61 A (H1N1) isolates were resistant. In addition, genetic analyses of the HA gene showed all amantadine-resistant viruses clustered in one (named clade N), possessing specific double mutations at 193, serine to phenylalanine (S193F), and at 225, asparatic acid to asparagine (D225N), and sensitive viruses belonged to another group (clade S). The clinical presentations at the clinical visit did not differ between patients shedding clade N virus and those shedding clade S virus. None of the patients had received previous treatment with amantadine. The results indicate an unusually high prevalence and wide circulation of the amantadine-resistance influenza A (H3N2) in Japan in the 2005-2006 season. These strains had the characteristic double mutations in the HA, in addition to the M2 mutation responsive for resistance. Antiviral resistance monitoring should be intensified and maintained for rapid feedback into treatment strategies, and selection of alternative therapeutic agents.
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