Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221 --> Trp) and hydrogen peroxide

The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.     

Determination of the compositional distribution of copolymers by frontal analysis continuous capillary electrophoresis

Frontal analysis continuous capillary electrophoresis (FACCE) was carried out for a series of random copolymers of an ionic monomer, sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS), and a nonionic monomer, acrylamide (AAM). The electropherograms appeared in order of anionic content and were generally sigmoidal, in contrast to that of hyaluronic acid (HA), which was abrupt and discontinuous. This difference could be related to the compositional heterogeneity of the copolymers, completely absent in the biopolymer. Through the range of copolymer composition (10-100% AMPS) the relationship between average mobility and nominal AMPS content could be described by a calibration curve, making it possible to deduce the compositional distribution of copolymer samples.     

Hydrogen bond network in the distal site of peroxidases: spectroscopic properties of Asn70 --> Asp horseradish peroxidase mutant

The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, 1H NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the 1H NMR signal of heme peripheral 8-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.     

Decreasing resistances of membrane-electrode assemblies containing hydrocarbon-based ionomers for improving their anodic performances

To improve methanol-oxidation performances of membrane-electrode assemblies composed of a hydrocarbon-based ionomers, the resistances involved in the reaction were decreased. Electrochemical impedance spectroscopy revealed that the proton-conductive resistance (R_i) in the anode was decreased from 0.54 to 0.40 Ω cm² by increasing a loading ratio of platinum-ruthenium to carbon support of anode catalyst from 54 to 73 wt.%. In addition, R_i was decreased to be 0.25 Ω cm² by increasing ion-exchange capacity (IEC) of the ionomer from 1.4 to 2.9 mequiv. g⁻¹. Consequently, the polarization resistance of the anode was significantly decreased, in turn, increasing current density of methanol oxidation at the potential of 0.45 V from 0.110 to 0.244 A cm⁻².     

An application of the 2-nitrobenzenesulfenyl method to proteomic profiling of human colorectal carcinoma: A novel approach for biomarker discovery

In the development of novel biomarkers, the proteomic approach is advantageous because using it the cancer-associated proteins can be directly identified. We previously developed a 2-nitrobenzenesulfenyl (NBS) method to improve quantitative proteome analysis. Here, we applied this method to proteomic profiling of colorectal carcinoma (CRC) to identify novel proteins with altered expression in CRC. Each pair of tumor and normal tissue specimens from 12 CRC patients was analyzed, and approximately 5000 NBS-labeled paired peaks were quantified. Peaks with altered signal intensities (>1.5-fold) and occurring frequently in the samples (>70%) were selected, and 128 proteins were identified by MS/MS analyses as differentially expressed proteins in CRC tissues. Many proteins were newly revealed to be CRC related; 30 were reported in earlier studies of CRC. Six proteins that were up-regulated in CRC (ZYX, RAN, RCN1, AHCY, LGALS1, and VIM) were further characterized and validated by Western blot and immunohistochemistry. All six were found to be CRC-localized, either in cancer cells or in stroma cells near the cancer cells. These results indicate that the proteins identified in this study are novel candidates for CRC markers, and that the NBS method is useful in proteome mining to discover novel biomarkers.     

Outcomes of 50 leukemia patients who received bone marrow transplants from unrelated donors]

In this study, we examined the ability of tumour necrosis factor-alpha (TNF) to stimulate the mitogen-activated protein (MAP) kinase homologues p42/44 MAP kinase, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase and its effect upon DNA synthesis in primary cultures of bovine aortic endothelial cells (BAECs). TNF strongly stimulated p38 MAP kinase and JNK activity in both a time- and concentration-dependent manner. By contrast, TNF was a very poor activator of p42/44 MAP kinase relative to the known activator of p42/44 MAP kinase in endothelial cells, adenosine triphosphate (ATP). TNF-stimulated activation of p38 MAP kinase, and MAPKAP kinase-2, a known downstream target of p38 MAP kinase, was strongly inhibited by pre-incubation with the p38 MAP kinase inhibitor SB203580, whereas the minor activation of p42/44 MAP kinase was abolished by pre-incubation of the cell with the novel MAP kinase kinase 1 inhibitor PD098059. Addition of TNF resulted in a 50-60% decrease in DNA synthesis in BAECs. Pre-incubation with PD098059 or co-incubation with ATP failed to modify the inhibitory effect of TNF upon DNA synthesis. SB203580 reduced basal DNA synthesis by approximately 50%; however, if failed to modify the inhibition mediated by TNF. These results indicate that TNF strongly activates both p38 MAP kinase, JNK and, to a minor extent, p42/p44 MAP kinase. It is likely that only one of these kinases, JNK, plays a role in the regulation of DNA synthesis in these cells.     

Clinical survey of congenital cytomegalovirus infection in Japan

Contrast media-induced nephropathy is one of the leading causes of hospital-acquired renal failure, occurring most frequently in patients with pre-existing renal insufficiency. We prospectively studied 55 patients with chronic renal insufficiency (serum creatinine concentration 1.4 to 3.5 mg/dl) who underwent abdominal aortography and arteriography of the lower extremities. The patients were randomized into two groups. Group 1, 28 patients, received dopamine 2.5 mcg/kg beginning 1 hour before arteriography and continuing for 12 hours. Group 2 received an equal volume of saline for the same period of time. Serum creatinine and 12-hour creatinine clearance were measured before arteriography and for 4 consecutive days afterward. Acute contrast-induced decrease in renal function was defined as increase in the baseline serum creatinine concentration > or = 0.5 mg/dl. On day 1 postarteriography the serum creatinine increased from baseline .193 mg/dl for controls while the dopamine group decreased slightly from baseline .018 mg/dl (p = 0.002). Excepting day 1 postarteriography, there was no statistical difference between groups, and serum levels for both groups increased linearly from baseline across time (dopamine p = 0.028, control p = 0.025). In patients with pre-arteriography baseline serum levels greater than or equal to 2.0 mg/dl, however, the increase in serum creatinine from baseline levels was consistently and significantly greater in the control group through the fourth day (0.012 < or = p < or = 0.049). Creatinine clearance did not change significantly from baseline after arteriography in the dopamine group (baseline versus days 1 through 4, 0.238 < or = p < or = 0.968); however, the control group showed a significant linear decrease in creatinine clearance from baseline through the fourth day after arteriography (p = 0.016). Dopamine infusion prevented a rise in serum creatinine 24 hours after angiography in patients with pre-existing renal insufficiency, and protected against contrast-induced decrease in renal function in patients whose baseline serum creatinine was > or = 2.0 mg/dl.     

Portal-hepatic venous shunt through a portal aneurysm complicated by hepatic encephalopathy and pulmonary hypertension

We report a rare case of portal-hepatic venous shunt through an enormous portal aneurysm complicated by pulmonary hypertension. A 66-year-old woman was admitted to our hospital for hepatic encephalopathy. Chest roentgenography revealed pulmonary hypertension. Computed tomography and ultrasound examination demonstrated a shunt between the portal and hepatic veins through an enormous portal aneurysm. The diagnoses of portal-hepatic venous shunt and pulmonary hypertension were confirmed by hepatic venous catheterization and cardiac catheterization. Pulmonary hypertension might result from the effects of vasoconstrictive agents, which should be metabolized by the liver in normal subjects, passing through the intrahepatic shunt into the lung.     

New cleavable surfactants derived from glucono-1,5-lactone

New amido nonionic cleavable surfactants were synthesized in good yields by the acetalization of glucono-1,5-lactone with octanal, 2-octanone or 2-undecanone, followed by amidation with monoethanolamine, diethanolamine or morpholine. These compounds possessed good water solubilities. The compounds derived from 2-octanone showed higher critical micelle concentrations than the compounds from octanal. For the same hydrophobic chain, both the micelle-forming property and the ability to lower surface tension increased with the change in the terminal amide group in the order diethanolamide<morpholide<monoethanolamide. Interestingly, in spite of their relatively short hydrophobic chains, these compounds showed greater ability to lower surface tension than conventional nonionic surfactants, such as alcohol ethoxylates. Furthermore, their acid-decomposition properties were determined. Their decomposition rates were also compared with that of the corresponding carboxylate type of compound derived from glucono-1,5-lactone.     

A media conversion from speech to facial image for intelligentman-machine interface

An automatic field motion image synthesis scheme (driven by speech) and a real-time image synthesis design are presented. The purpose of this research is to realize an intelligent human-machine interface or intelligent communication system with talking head images. A human face is reconstructed on the display of a terminal using a 3-D surface model and texture mapping technique. Facial motion images are synthesized naturally by transformation of the lattice points on 3-D wire frames. Two driving motion methods, a text-to-image conversion scheme and a voice-to-image conversion scheme, are proposed. In the first method, the synthesized head image can appear to speak some given words and phrases naturally. In the second case, some mouth and jaw motions can be synthesized in synchronization with voice signals from a speaker. Facial expressions other than mouth shape and jaw position can be added at any moment, so it is easy to make the facial model appear angry, to smile, to appear sad, etc., by special modification rules. These schemes were implemented on a parallel image computer system. A real-time image synthesizer was able to generate facial motion images on the display at a TV image video rate