A functional model for pMMO (particulate methane monooxygenase): Hydroxylation of alkanes with H2O2 catalyzed by beta-diketiminatocopper(II) complexes

The reaction of copper(II) complexes supported by a series of beta-diketiminate ligands ((R1,R2)L, [(Dipp)N-C(R(2))-C(R(1))-C(R(2))-N(Dipp)](-), Dipp=2,6-diisopropylphenyl; see ) and H(2)O(2) has been examined spectroscopically at a low temperature. The beta-diketiminatocopper(II) complexes with R(2)=H (no substituent on the beta-carbon) provided a copper-oxygen intermediate that exhibited the same spectroscopic features as those of the bis(mu-oxo)dicopper(III) complex generated by the reaction of corresponding beta-diketiminatocopper(I) complex and O(2). On the other hand, the beta-diketiminatocopper(II) complexes with methyl substituent on the beta-carbon (R(2)=Me) did not produce such an intermediate in the same reaction. The beta-diketiminatocopper(II) complexes carrying an electron-withdrawing substituent on the alpha-carbon (R(1)=NO(2) or CN) but no beta-substituent (R(2)=H) exhibited a high catalytic activity in the oxygenation reaction of alkanes with H(2)O(2). Mechanism of the catalytic oxygenation reaction as well as the substituent effects of the ligands on the copper(II)-H(2)O(2) reactivity is discussed.     

HCV core protein localizes in the nuclei of nonparenchymal liver cells from chronically HCV-infected patients

Understanding the mechanism of hepatitis C virus (HCV) pathogenesis is an important part of HCV research. Recent experimental evidence suggests that the HCV core protein (HCcAg) has numerous functional activities. These properties suggest that HCcAg, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection. HCV is capable of infecting cells other than hepatocytes. Although the extrahepatic cellular tropism of HCV may play a role in the pathophysiology of this infection, the precise biological significance of the presence of HCV components in different liver cell types presently remains to be established. In this study, HCcAg was detected in nonparenchymal liver cells of six patients out of eight positive for serum HCV RNA. Immunostaining with anti-HCcAg mAbs revealed the presence of this protein in different liver cell types such as lymphocytes, Kupffer, polymorphonuclear, pit, endothelial, stellate, and fibroblast-like cells. Interestingly, HCcAg was immunolabeled not only in the cytoplasm but also in the nucleus of these cells. Remarkably, HCcAg co-localized with large lipid droplets present in stellate cells and with collagen fibers in the extracellular matrix. Moreover, HCcAg was immunolabeled in bile canaliculus suggesting the involvement of the biliary system in the pathobiology of HCV. Data suggest that nonparenchymal liver cells may constitute a reservoir for HCV replication. Besides, HCcAg may contribute to modulate immune function and fibrosis in the liver as well as steatosis.     

Role of Shiga toxin 2 (Stx2)-binding protein,human serum amyloid P component (HuSAP), in Shiga toxin-producing Escherichia coli infections: assumption from in vitro and in vivo study using HuSAP and anti-Stx2 humanized monoclonal antibody TMA-15

Shiga toxin 2 (Stx2) is a major pathogenic factor in Shiga toxin-producing Escherichia coli (STEC) infections. Some factor that neutralizes Stx2 in vitro had been shown to be specifically present in human serum and we recently identified it as human serum amyloid P component (HuSAP). Here, we report the role of HuSAP in STEC infections. HuSAP could not rescue Stx2-challenged mice from death, and it instead reduced the efficacy of the Stx2-neutralizing humanized monoclonal antibody TMA-15 when a lower dose of TMA-15 was injected to the mice. By contrast, the efficacy of TMA-15 at a higher dose was uninfluenced by the presence of HuSAP. These findings suggest that HuSAP acts as a carrier protein of Stx2 rather than as a Stx2-neutralizing factor in the human circulation and that passive immune therapy with Stx2-neutralizing antibodies such as TMA-15 is useful to prevent severe complications associated with STEC infections even in the presence of HuSAP.     

Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".     

A synaptic modification algorithm in consideration of the generation of rhythmic oscillation in a ring neural network

In consideration of the generation of bursts of nerve impulses (that is, rhythmic oscillation in impulse density) in the ring neural network, a synaptic modification algorithm is newly proposed. Rhythmic oscillation generally occurs in the regular ring network with feedback inhibition and in fact such signals can be observed in the real nervous system. Since, however, various additional connections can cause a disturbance which easily extinguishes the rhythmic oscillation in the network, some function for maintaining the rhythmic oscillation is to be expected to exist in the synapses if such signals play an important part in the nervous system. Our preliminary investigation into the rhythmic oscillation in the regular ring network has led to the selection of the parameters, that is, the average membrane potential (AMP) and the average impulse density (AID) in the synaptic modification algorithm, where the decrease of synaptic strength is supposed to be essential. This synaptic modification algorithm using AMP and AID enables both the rhythmic oscillation and the non-oscillatory state to be dealt with in the algorithm without distinction. Simulation demonstrates cases in which the algorithm catches and holds the rhythmic oscillation in the disturbed ring network where the rhythmic oscillation was previously extinguished.     

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

Differential response of type 2 deiodinase gene expression to photoperiod between photoperiodic Fischer 344 and nonphotoperiodic Wistar rats

The molecular basis of seasonal or nonseasonal breeding remains unknown. Although laboratory rats are generally regarded as photoperiod-insensitive species, the testicular weight of the Fischer 344 (F344) strain responds to photoperiod. Recently, it was clarified that photoperiodic regulation of type 2 iodothyronine deiodinase (Dio2) in the mediobasal hypothalamus (MBH) is critical in photoperiodic gonadal regulation. Strain-dependent differences in photoperiod sensitivity may now provide the opportunity to address the regulatory mechanism of seasonality by studying Dio2 expression. Therefore, in the present study, we examined the effect of photoperiod on Dio2 expression in photoperiod-sensitive F344 and photoperiod-insensitive Wistar rats. A statistically significant difference was observed between short and long days in terms of testicular weight and Dio2 expression in the F344 strain, while no difference was observed in the Wistar strain. These results suggest that differential responses of the Dio2 gene to photoperiod may determine the strain-dependent differences in photoperiod sensitivity in laboratory rats.     

Interaction Between Titanium and Cadmium in Various Guinea Pig Organs

Titanium (Ti) is used in many fields, while cadmium (Cd) is known to cause the itai-itai disease. In the present study, possible interactions between titanium and cadmium were investigated. Aorta, taenia coli, and liver were removed from male guinea pigs. Muscle tension was measured using intact aorta and taenia coli and using β-escin-permeabilized taenia coli in a physiological salt solution and a hyperpotassium solution containing Cd and/or Ti. Cellular Cd contents were determined using all tissues after washout with EDTA solution. Cadmium-induced relaxation in the hyperpotassium solution recovered significantly (P?<?0.01) following Ti treatment in taenia coli, but not in the aorta. In β-escin-permeabilized taenia coli, the percentage recoveries after Cd treatment and after Ti plus Cd treatment were 67.3?±?8.7 % (n?=?4) and 87.7?±?3.8 % (n?=?4), respectively, compared with Ca-induced control contraction. Cellular Cd contents in taenia coli decreased significantly following treatment with Ti 10^?4?M. Although similar results were obtained using the aorta and the liver, there were no significant differences between the control and Ti 10^?5?M. High concentrations of Ti may reduce cellular Cd content.     

Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.