[Current situations and problems of quality control for medical imaging display systems].

Diagnostic imaging has been shifted rapidly from film to monitor diagnostic. Consequently, Japan medical imaging and radiological systems industries association (JIRA) have recommended methods of quality control (QC) for medical imaging display systems. However, in spite of its need by majority of people, executing rate is low. The purpose of this study was to validate the problem including check items about QC for medical imaging display systems. We performed acceptance test of medical imaging display monitors based on Japanese engineering standards of radiological apparatus (JESRA) X-0093*A-2005 to 2009, and performed constancy test based on JESRA X-0093*A-2010 from 2010 to 2012. Furthermore, we investigated the cause of trouble and repaired number. Medical imaging display monitors had 23 inappropriate monitors about visual estimation, and all these monitors were not criteria of JESRA about luminance uniformity. Max luminance was significantly lower year-by-year about measurement estimation, and the 29 monitors did not meet the criteria of JESRA about luminance deviation. Repaired number of medical imaging display monitors had 25, and the cause was failure liquid crystal panel. We suggested the problems about medical imaging display systems.

An alkaline serine-proteinase from a bacterium isolated from bat feces: purification and characterization.

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 degrees C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H(2)O(2), 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P(1) position, in descending order. The k(cat), K(m) and k(cat)/K(m) values for Z-Val-Lys-Met-MCA were 16.8+/-0.14 min(-1), 5.1+/-0.28 microM, and 3.3+/-0.28 microM(-1) min(-1) respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.

Microcirculatory responses to acupuncture stimulation and phototherapy.

BACKGROUND: Acupuncture stimulation and phototherapy have been reported to have analgesic effects and improve the microcirculation. However, few studies have directly examined changes in peripheral blood vessels, either quantitatively or objectively. We assessed the responses of arteriolar blood flow to acupuncture stimulation and phototherapy under direct vision to examine the effects of these treatments. METHODS: We used 40 rabbits with a rabbit ear chamber attached to the auricle. The rabbit ear chamber was fixed to the auricle under a dissecting microscope. Arterioles were selected and observed with the use of a microscope video camera. Pentobarbital was injected IV. The trachea was intubated and spontaneous respiration was maintained. Rabbits were randomly assigned to receive acupuncture stimulation (acupuncture group, n = 10), near-infrared lamp irradiation (lamp group, n = 10), near-infrared low-powered laser irradiation (laser group, n = 10), or no irradiation (control group, n = 10). In the acupuncture group, an acupuncture needle was placed in the auricle for 20 min. The lamp group repeatedly received 1 s of near infrared irradiation (1540 mW) followed by 4 s of treatment cessation. The laser group continuously received 60 mW of laser irradiation. In the lamp and laser groups, the auricle (same site as that of the acupuncture needles in the acupuncture group) was irradiated for 10 min with a contact probe. Arteriolar diameter and blood flow velocity were measured at baseline and for 60 min after acupuncture or irradiation treatment. Blood flow rate was calculated by multiplying the blood flow velocity by the cross-sectional area of the vessels. RESULTS: Arteriolar diameter significantly increased to 131% +/- 14% in the acupuncture group (P < 0.005), 129% +/- 19% in the lamp group (P < 0.005), and 128% +/- 11% in the laser group (P < 0.005) when compared with the pretreatment value (100%). Maximum values were reached 20 min after the end of the acupuncture stimulation, and 10 min after the end of lamp and laser irradiation. The three groups showed significant increases in arteriolar diameter when compared with the control group (P < 0.005). Blood flow velocity and blood flow rate showed similar trends to arteriolar diameter. Treatment effect persisted for 40-50 min after the end of stimulation and irradiation. CONCLUSIONS: Acupuncture stimulation and phototherapy were directly confirmed to increase the diameter and blood flow velocity of the peripheral arterioles. Acupuncture stimulation and phototherapy, associated with minimal systemic and local side effects, can enhance the microcirculation and may be a useful supportive treatment for diseases caused by poor peripheral blood flow.

Crystal Structural and Functional Analysis of the Putative Dipeptidase from Pyrococcus horikoshii OT3.

The crystal structure of a putative dipeptidase (Phdpd) from Pyrococcus horikoshii OT3 was solved using X-ray data at 2.4 A resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of the alpha/beta fold, and the C-terminal domain consists of five long mixed strands, four helices, and two 3(10) helices. The structure of Phdpd is quite similar to reported structures of prolidases from P. furiosus (Zn-Pfprol) and P. horikoshii (Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However, Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) of Phdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those of Phdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein with Zn(2+) ions is inactive.

Permissive range of hypercapnia for improved peripheral microcirculation and cardiac output in rabbits.

OBJECTIVES: Permissive hypercapnia improves outcomes in patients with respiratory failure, most likely because of a reduction in ventilator-induced lung injury. Because hypercapnia is a potent vasoactive stimulus, adequate tissue perfusion and oxygen delivery to dilated microvessels may be restored. We examined how Paco2 affects microvascular changes, hemodynamics, and cardiac output in rabbits. We evaluated the permissive range of Paco2 required for maintenance of the peripheral circulation. DESIGN: Prospective experimental animal study. SETTING: Animal research laboratory. SUBJECTS: A total of 31 Japanese domestic white rabbits. INTERVENTIONS: The animals were anesthetized with pentobarbital. An ear chamber was prepared to examine blood vessels by intravital microscopy. The rabbits were mechanically ventilated with air, oxygen, and CO2. The values of Paco2 were adjusted to about 20 (hypocapnia), 40 (normocapnia), 60, 80, 100, 125, 150, and >250 mm Hg (hypercapnia). After stabilization at each Paco2 level, microvascular changes were recorded with a microscope-closed video camera to permit analysis of arteriolar diameter and blood flow. MEASUREMENTS AND MAIN RESULTS: The pH and heart rate decreased and mean blood pressure increased progressively as the Paco2 was increased. When Paco2 was increased from 20 to 80 mm Hg, vessel diameter, blood-flow velocity, and blood-flow rate increased markedly. Cardiac output increased slightly. When Paco2 exceeded 100 mm Hg, all of these variables decreased. When Paco2 exceeded 150 mm Hg, all variables were significantly lower than the control values (p < .01). CONCLUSION: Intravital microscopic visualization of the rabbit ear microcirculation showed that 150 mm Hg is the permissive upper limit of acute hypercapnia with respect to maintenance of the peripheral microcirculation.

Crystal structure of scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis.

Scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum is the founding member of the newly discovered\ family of peptidases, G1, so far found exclusively in fungi. The crystal structure of SGP revealed a previously undescribed fold for peptidases and a unique catalytic dyad of residues Gln53 and Glu136. Surprisingly, the beta-sandwich structure of SGP is strikingly similar to members of the carbohydrate-binding concanavalin A-like lectins/glucanases superfamily. By analogy with the active sites of aspartic peptidases, a mechanism employing nucleophillic attack by a water molecule activated by the general base functionality of Glu136 has been proposed. Here, we report the first crystal structures of SGP in complex with two transition state peptide analogs designed to mimic the tetrahedral intermediate of the proteolytic reaction. Of these two analogs, the one containing a central S-hydroxyl group is a potent sub-nanomolar inhibitor of SGP. The inhibitor binds non-covalently to the concave surface of the upper beta-sheet and enables delineation of the S4 to S3' substrate specificity pockets of the enzyme. Structural differences in these pockets account for the unique substrate preferences of SGP among peptidases having an acidic pH optimum. Inhibitor binding is accompanied by a structuring of the region comprising residues Tyr71-Gly80 from being mostly disordered in the apoenzyme and leading to positioning of crucial active site residues for establishing enzyme-inhibitor contacts. In addition, conformational rearrangements are seen in a disulfide bridged surface loop (Cys141-Cys148), which moves inwards, partially closing the open substrate binding cleft of the native enzyme. The non-hydrolysable scissile bond analog of the inhibitor is located in the active site forming close contacts with Gln53 and Glu136. The nucleophilic water molecule is displaced and a unique mode of binding is observed with the S-OH of the inhibitor occupying the oxyanion binding site of the proposed tetrahedral intermediate. Details of the enzyme-inhibitor interactions and mechanistic interpretations are discussed.

A halophilic serine proteinase from Halobacillus sp. SR5-3 isolated from fish sauce: purification and characterization.

A halophilic bacterium was isolated from fish sauce, classified, and named Halobacillus sp. SR5-3. A purified 43-kDa proteinase produced by this bacterium showed optimal activity at 50 degrees C and pH 9-10 in 20% NaCl. The activity of the enzyme was enhanced about 2.5-fold by the addition of 20-35% NaCl, and the enzyme was highly stabilized by NaCl. It was found to be a serine proteinase related to either chymotrypsin or subtilisin. It absolutely preferred Ile at the P(2) position of substrates. Thus, the enzyme was found to be a halophilic serine proteinase with unique substrate specificity.

Exploring the subsite-structure of vimelysin and thermolysin using FRETS-libraries.

Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS-libraries, revealing a significant difference at the P3' position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3' subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases.

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.

Effects of colloid resuscitation on peripheral microcirculation, hemodynamics, and colloidal osmotic pressure during acute severe hemorrhage in rabbits.

We examined the effects of hydroxyethyl starch (HES) on the microcirculation, hemodynamics, and colloidal osmotic pressure in a rabbit model of hemorrhagic shock. A total of 40 rabbits was anesthetized with pentobarbital and isoflurane, and they were mechanically ventilated. An ear chamber was prepared to examine blood vessels by intravital microscopy. Shock was induced by removing nearly half of the circulating blood volume. Twenty rabbits received 20 mL of HES by intravenous infusion immediately after blood letting. Additional HES was then administered intravenously to a total volume of 100 mL. The other 20 rabbits (control) were intravenously given 40 mL of lactated Ringer's solution (LR), followed by additional LR to a total volume of 200 mL, administered under the same conditions as HES. After blood letting, arteriolar diameter decreased similarly in the the HES and LR groups (HES, 40.5% +/- 14.8% of the baseline value versus LR, 43.3% +/- 13.1%). After the completion of infusion, arteriolar diameter significantly recovered to 90.8% +/- 10.2% of the baseline value in the HES group as compared with only 62.6% +/- 10.7% in the LR group (P < 0.005). Recovery of arterial blood flow velocity and blood flow rate was also significantly better in the HES group than in the LR group (P < 0.005). Mean arteriolar pressure, central venous pressure, and plasma colloid osmotic pressure after the completion of infusion were significantly greater in the HES group than in the LR group (P < 0.005). We conclude that intravenous infusion of HES effectively maintains the microcirculation, hemodynamics, and colloidal osmotic pressure in a rabbit model of acute severe hemorrhage.

A single local application of recombinant human basic fibroblast growth factor accelerates initial angiogenesis during wound healing in rabbit ear chamber.

Local angiogenic therapy with recombinant human basic fibroblast growth factor (rhbFGF) has been used to promote wound healing. To obtain useful information for the development of optimal angiogenic therapy, we chronologically evaluated the effects of a single local application of rhbFGF on angiogenesis in a rabbit ear chamber model of wound healing by observing the subcutaneous vessel bed intravitally. New vessel formation during wound healing was macroscopically and microscopically evaluated for 5 wk. Each rabbit ear chamber received a single dose of 6 microg rhbFGF (treatment B1: n = 13), 18 microg rhbFGF (treatment B2: n = 16), or physiological saline as control (n = 13). At 1 wk the newly vascularized area was significantly larger in groups B1 and B2 than in control. At 2 wk, the vascularized areas in groups B1, B2, and control were similar. At 5 wk, the percentage of rabbits with complete vascularization was significantly larger in group B1 than in control. Capillary density at 5 wk was similar among the three groups. These results suggest that locally applied rhbFGF accelerated angiogenesis during early wound healing in rabbits; however, this effect was transient and no increase in capillary density occurred at the completion of vascularization.

Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5.

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10-11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and alpha-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15-25% NaCl respectively using Suc-Ala-Ala-Phe-pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.

Mammalian D-aspartyl endopeptidase: a scavenger for noxious racemized proteins in aging.

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease, cataracts, and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600kDa) and is localized in the inner mitochondrial membrane of mouse and rabbit, but DAEP activity was not detected in Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans. A specific inhibitor for DAEP was newly synthesized, and inhibited DAEP activity (IC(50), 3microM), a factor of 10 greater than lactacystin on DAEP. On the other hand, the inhibitor did not inhibit either the 20S or 26S proteasome.

Substrate specificity of alkaline serine proteinase isolated from photosynthetic bacterium, Rubrivivax gelatinosus KDDS1.

A novel type of fluorescence resonance energy transfer (FRET) combinatorial libraries were used for the characterization of alkaline serine proteinase produced from Rubrivivax gelatinosus KDDS1. This enzyme was the first serine proteinase characterized from photosynthetic bacteria. The proteinase was found to prefer Met and Phe at the P1 position, Ile and Lys at the P2 position, and Arg and Phe at the P3 position. To date, no serine proteinase has exhibited a preference for Met at the P1 position. Thus, the alkaline serine proteinase from R. gelatinosus KDDS1 is very unique in terms of substrate specificity. A highly sensitive substrate, Boc-Arg-Ile-Met-MCA, was synthesized for kinetic study based on the results reported here. The optimum pH of the enzyme for this substrate was pH 10.7, and the values of kcat, Km, and kcat/Km were 23.7 s(-1), 15.4 microM, and 1.54 microM(-1) s(-1), respectively.

Urinary trypsin inhibitor improves peripheral microcirculation and bronchospasm associated with systemic anaphylaxis in rabbits in vivo.

Using intravital microscopy of the rabbit ear for quantitative studies of microvascular dynamics, we examined the impact of urinary trypsin inhibitor (UTI), a proteolytic enzyme inhibitor, on microvascular changes during immune complex-mediated anaphylaxis. A total of 50 rabbits, previously sensitized with horse serum, were anesthetized and mechanically ventilated with pentobarbital and isoflurane for the intravital microscopy. Rabbits were then challenged with intravenous injection of horse serum to induce systemic anaphylaxis. One minute after the challenge, each rabbit was randomly assigned to receive saline (group C), 50,000 units x kg(-1) of UTI (group U1), or 150,000 units x kg(-1) (group U2). There were no statistical differences between hemodynamic variables, including heart rate (HR), mean arterial pressure (MAP), and central venous pressure (CVP), among the survivors in each treatment group. Peak inspiratory pressure rose in all three groups but at a much higher rate in group C (P < 0.05). In contrast with the moderate effects of UTI on the above parameters, microscopic evaluation revealed a substantial difference among treatment groups: upon the initiation of anaphylaxis, the arteriole started to reduce in diameter, but UTI prevented vasoconstriction in the arteriole in a dose-dependent manner. Similar results were observed with blood flow velocity. Because flow rate was calculated as the product of blood flow velocity and vascular cross-sectional area proportional to the square of the vessel diameter, these results indicate that UTI preserves microvascular flow rate during anaphylaxis. Rabbit ear microcirculation is highly preserved in the UTI-treated groups during systemic anaphylactic shock.

Effect of ulinastatin on microcirculation during excessive hemorrhage using fluid therapy.

Microcirculation kinetics during excessive hemorrhage and fluid therapy under anesthesia, with or without treatment with ulinastatin (urinary trypsin inhibitor), was studied by using the rabbit ear chamber. Sixteen rabbits were divided into 2 groups: 8 rabbits without ulinastatin treatment (group C) and 8 rabbits treated with ulinastatin (group U). The vascular diameter, blood-flow velocity, blood-flow rate and urinary output were maintained well in the ulinastatin group as compared to those in the control group. It is concluded that ulinastatin is effective in maintaining microcirculation during excessive hemorrhage and fluid therapy at the same volume.

Macroscopic sequential pictures of angiogenesis in a rabbit ear chamber.

In angiogenic research, quantitative measurement of newly formed vessels is essential for precise evaluation of angiogenesis. Although the rabbit ear chamber (REC) model provides a system for in vivo viewing of capillaries, macroscopic photographs of capillaries are unsatisfactory due to the transparent reflecting material used in the windows/chambers. We designed a new filming system to obtain clear macroscopic pictures of REC with good contrast and sharp edges without flare. A Nikon F4s with a Medical Nikkor on a Nikon Multi photo stand were used. A strobe, Nikon macro speed light SB-21, and a modeling lamp were placed in a black light box. A 1-cm-diameter hole was made on top of the light box at the edge. Filming was performed by placing the REC on top of the light box. Filming was done without difficulty. Without anesthetizing or stressing the animal, photographs were taken for 1 or 2 min. The picture quality was satisfactory, with good contrast and sharp edges without flare. Without treatment, angiogenesis was observed at day 7 and vascularization was complete at 4 weeks. The photographs of the REC field were in good quality up to 6 months. Our results suggest that this system is excellent to obtain direct evidence of angiogenesis in vivo, and maintains a good condition for weekly monitoring over a long period from immediately after set up.