Preparation of polymer gel dosimeters based on less toxic monomers and gellan gum.

New polymer gel dosimeters consisting of 2-hydroxyethyl methacrylate (HEMA), triethylene glycol monoethyl ether monomethacrylate (TGMEMA), polyethylene glycol 400 dimethacrylate (9G), tetrakis (hydroxymethyl) phosphonium chloride as an antioxidant, and gellan gum as a gel matrix were prepared. They were optically analyzed by measuring absorbance to evaluate a dose response. The absorbance of the polymer gel dosimeters that were exposed to (60)Co γ-rays increased with increasing dose. The dosimeters comprising HEMA and 9G showed a linear increase in absorbance in the dose range from 0 to 10 Gy. The dose response depended on the 9G concentration. For others comprising HEMA, 9G and TGMEMA, the absorbance of the polymer gel dosimeters drastically increased above a certain dose, and then leveled off up to 10 Gy. The optical variations in these polymer gel dosimeters were also induced by x-irradiation from Cyberknife radiotherapy equipment. Furthermore, the exposed region of the latter polymer gel dosimeter exhibited a thermo-responsive behavior.

Prevalence of hepatitis C virus and its genotypes among a cohort of drug users in Kenya.

BACKGROUND: Prevalence of hepatitis C virus and that of its main genotypes varies between the worlds geographic regions. The risk factors for infection with HCV include blood transfusion, tattoing and injecting drug use. OBJECTIVES: To examine the prevalence of HCV and determine its main genotypes among a cohort of drug users in Kenya. DESIGN: A laboratory based study. SETTING: Hepatitis research laboratory in the Centre for Virus Research at the Kenya Medical Research Institute, Nairobi. SUBJECTS: Three hundred and fourteen male and 19 female intravenous and non-intravenous drug users aged between 15-55 years. RESULTS: Seventy four (22.2%) out of 333 samples tested positive for anti-HCV. Sixty nine out of the 74 serum samples were assayed for HCV RNA and 38 (55.5%) were positive. The RNA positive samples were further subjected to sequencing and 19 (73%) of the samples were classified as genotype 1a, while seven (27%) samples were classified as genotype 4. Genotypes 2, 3, 5 and 6 were not identified in this study. CONCLUSIONS: These results demonstrate a high HCV infection prevalence among this cohort of drug users (22.2%) as compared to that of the general population, which is estimated to be 0.2-0.9%. The study also confirms the presence of at least two major genotypes among Kenyan drug users (genotypes 1 and 4).

Observation of phonons in multiferroic BiFeO(3) single crystals by Raman scattering.

We have grown BiFeO(3) bulk single crystals by a flux method and characterized the phonon spectra in detail by Raman scattering in the temperature range 4-1100 K. All the 13 Raman-active phonon modes predicted by group theory, 4A(1)+9E, were observed at low temperature and successfully assigned by a polarized Raman measurement. Moreover, drastic spectral changes in the Raman spectra were observed at temperatures 600-700 K and 1000-1100 K. These features are discussed from the viewpoint of phonon coupling with the magnetic ordering and the structural phase transition, respectively.

Concentration of 17beta-estradiol using an immunoaffinity porous hollow-fiber membrane.

We describe attempts to achieve high throughput of 17beta-estradiol (E2) analysis, including the development of an immunocleanup membrane using polyclonal antibodies and an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. An epoxy-group-containing monomer, glycidyl methacrylate (GMA), was graft-polymerized onto a porous hollow-fiber membrane. Subsequently, anti-estrogen (ES) antibody, as a ligand, was coupled with the epoxy group. The ligand density ranged from 3.1 to 5.8 mg/g of the GMA-grafted porous hollow-fiber membrane. A 1.0 microg/L E2 solution was forced to permeate through pores rimmed by the anti-ES-antibody-immobilized polymer chains, at a constant permeation rate. A breakthrough curve, that is, the change in the E2 concentration of the effluent penetrating the outside of the hollow fiber with a change of the effluent volume, was determined. Bound E2 in amounts ranging from 0.42 to 0.80 microg was quantitatively eluted with 3-5 mL of methanol in the permeation mode. The higher permeation rate of the E2 solution resulted in the higher overall binding rate of E2 to the anti-ES-antibody-immobilized porous hollow-fiber membrane because of the negligible diffusional mass-transfer resistance of E2 to the antibody.

Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation.

Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.

Selective inhibition of Ras interaction with its particular effector by synthetic peptides corresponding to the Ras effector region.

Ras proteins possess multiple downstream effectors of distinct structures. We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms. Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2. The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity. The peptide corresponding to the residues 17-44 competitively inhibited Ras association with all the three effectors at the Ki values of 1 approximately 10 microM, and the inhibition was considerably attenuated by the D38A mutation. The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase. Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide. These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function.

Aminoguanidine attenuates endotoxin-induced acute lung injury in rabbits.

OBJECTIVE: To assess the effect of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, on endotoxin-induced acute lung injury in rabbits. DESIGN: Prospective, blinded, controlled laboratory study. SETTING: University research laboratory. SUBJECTS: Twenty-eight male rabbits. INTERVENTIONS: The animals were randomly assigned to receive one of four treatments (n = 7 for each group): infusion of saline only (S-S group), infusion of saline and aminoguanidine (S-AG group), infusion of Escherichia coli endotoxin (5 mg/kg over 60 mins) (E-S group), and infusion of endotoxin and aminoguanidine (E-AG group). Fifteen minutes before infusion of endotoxin (E-S and E-AG groups) or saline (S-S and S-AG groups), the animals received an intravenous injection of 1 mg/kg of aminoguanidine (S-AG and E-AG groups) or saline (S-S and E-S groups). The same dose of aminoguanidine or saline was given 1 hr after the end of endotoxin or saline infusion. The lungs of the rabbits were ventilated with 40% oxygen. MEASUREMENTS AND MAIN RESULTS: Hemodynamics, peripheral leukocyte counts, and PaO2 were recorded during the ventilation period (6 hrs). After these observations were made, lung mechanics, cell fraction of bronchoalveolar lavage fluid, and concentrations of thromboxane A2 and prostacyclin metabolites in bronchoalveolar lavage fluid were determined. The wet weight/dry weight ratio of the lung and albumin concentrations in bronchoalveolar lavage fluid were analyzed as indices of pulmonary edema. Endotoxin decreased the lung compliance and PaO2 and increased the wet weight/dry weight ratio, neutrophil counts, and albumin concentrations in bronchoalveolar lavage fluid. The bronchoalveolar lavage fluid concentrations of thromboxane B2 in bronchoalveolar lavage fluid were increased by infusion of endotoxin. Aminoguanidine attenuated these changes. Endotoxin caused extensive morphologic lung damage, which was lessened by aminoguanidine. CONCLUSIONS: Aminoguanidine given intravenously before and after endotoxin attenuated endotoxin-induced lung injury in rabbits. These findings suggest that inducible nitric oxide synthase inhibition may be useful in the treatment of endotoxin-induced lung injury. However, further studies are required to determine the optimal dosage of aminoguanidine, when the inhibitor is given alone as therapy after lung injury.

Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector.

Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, we carried out a yeast two-hybrid screening for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibited a strong structural homology with human AF-6, rat Afadin and Drosophila melanogaster Canoe and possessed both the Ras-associating (RA) domain and the PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associated with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras.

Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

Synergistic activation of c-fos promoter activity by Raf and Ral GDP dissociation stimulator.

Ral, a member of small GTP-binding protein (G protein) superfamily, has been suggested to act downstream of Ras, since Ral GDP dissociation stimulator (RalGDS) has been found to be an effector protein of Ras. In this study, we examined the effects of RalGDS and Ral on gene expression using c-fos promoter linked to the luciferase reporter gene (c-fos-luciferase). RalGDS interacted with RasG12V/E37G (in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf in COS cells. RafCAAX is an active Raf kinase targeted to the plasma membranes by virtue of the addition of a C-terminal localization signal from K-Ras. Transfection of either RalGDS or RafCAAX into NIH3T3 cells slightly stimulated c-fos-luciferase expression and cotransfection of both proteins greatly enhanced the expression. RalGDS and an activated Rac (RacG12V) did not act synergistically to stimulate c-fos-luciferase expression. Transfection of an activated Ral (RalG23V) stimulated c-fos-luciferase expression. Furthermore, cotransfection of RalG23V and an activated Ras (RasG12V) enhanced RasG12V-dependent c-fos-luciferase expression. However, RalG23V did not synergize with RafCAAX, RacG12V or RalGDS to stimulate the expression. These results show that RalGDS and Ral regulate c-fos promoter activity and suggest that RalGDS may activate c-fos promoter synergistically with the signal from Raf by transmitting the signal to a target other than Ral.

Characterization of a potent uveitopathogenic site derived from rat phosducin.

Phosducin is a retinal and pineal phosphoprotein assumed to play an important role in visual phototransduction. Phosducin is also a uveitopathogenic retinal antigen, but its potency has been reported to be mild. During the course of studies aimed at identifying uveitopathogenic sites in phosducin, we found that rat phosducin possessed a potent uveitopathogenic site. In this study, we characterize the potent uveitopathogenic site by using synthetic peptides. Several synthetic peptides from this region plus adjuvants were injected into Lewis rats, and the uveitopathogenic core sequence was defined. We also determined the pivotal amino acid residues by using synthetic peptides with single residue substitution. Immunization with PDC(R)65-96 (amino acid residues 65 through 96 derived from rat phosducin) at doses of 0.83 nmol or more induced severe experimental autoimmune uveitis (EAU) in all rats within 12 days. Experimental autoimmune pinealitis (EAP) was also observed in all rats after immunization with 0.83 nmol or higher doses of the peptide. The lowest dose of the peptide to induce EAU and EAP was 0.24 nmol. The smallest peptide that induced EAU as severe as PDC(R)65-96 was PDC(R)77-87, which consisted of 11 amino acid residues (YELIHQDKEDE). The core sequence within the uveitopathogenic site was a pentapeptide (LIHQD), amino acid residues from 79 to 83. To determine the role of individual residues within PDC(R)77-87, we tested the uveitopathogenicity of analogues of PDC(R)75-85 and PDC(R)77-89, respectively, in which each of the residues from 77 to 87 was replaced by alanine (A). Analogous peptides bearing a single residue substitution at 80 (I-->A) and 82 (Q-->A), respectively, were not uveitopathogenic. Our findings demonstrated the presence of a potent uveitopathogenic site in PDC(R)65-96 whose potency in Lewis rats was comparable to that of S-antigen. The pivotal amino acid residues for uveitopathogenicity were the residues at 80 (I) and 82 (Q). The clinical and histological features of this EAU closely resembled those of the EAU induced by S-antigen and recoverin.

Membrane recruitment of Raf-1 is not the only function of Ras in Raf-1 activation.

Ras interacts with Raf-1 and stimulates its kinase activity, which results in activation of the mitogen-activated protein (MAP) kinase cascade. It has been proposed that the main function of Ras in Raf-1 activation is to recruit Raf-1 to the plasma membrane, where a separate activation event such as phosphorylation takes place. Here, we examined the activities of various mutants of human Ha-Ras to induce membrane translocation of Raf-1 and to activate Raf-1 in vivo. Overexpression of an activator region mutant Ha-Ras(V45E) in COS7 cells induced membrane translocation of Raf-1 as effectively as wild-type Ha-Ras. However, the activity of this mutant to activate Raf-1 and extracellular signal-regulated kinase-2 (ERK2) was attenuated by approximately 70% compared to that of wild-type Ha-Ras. The decrease in the specific activity was further demonstrated by measuring the activity of the Ha-Ras(V45E)-associated Raf-1 purified from the membrane fraction. These results imply that the association of Ras with Raf-1 has another important consequence, presumably dependent on the interaction between its activator region and Raf-1, than the simple recruitment of Raf-1 to the plasma membrane.

Difference in the mechanism of interaction of Raf-1 and B-Raf with H-Ras.

Ras is known to possess multiple cellular targets including Raf-1. Here, we measured both direct binding of various H-Ras mutants to two representative mammalian Ras targets, Raf-1 and B-Raf, and the activity of the mutants to stimulate Raf-1 and B-Raf, and analysed the difference in their Ras-interaction mechanisms. B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-1 by examining binding of the H-Ras mutants to Raf-1 and B-Raf in the yeast two-hybrid and in vitro binding assays. Mutants, Y32F, A59E, and V45E bound to Raf-1 in Sf9 cells coexpressing them, but failed to activate Raf-1. On the other hand, Y32F activated B-Raf in a cell-free system which consisted of rat brain cytosol and recombinant MEK. These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-1 and B-Raf.

Differential structural requirements for interaction of Ras protein with its distinct downstream effectors.

Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2- phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.

Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras.

The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.

Omeprazole reduces preoperative gastric fluid acidity and volume in children.

To explore the effects of oral omeprazole on preoperative gastric fluid pH and volume in children, 104 healthy in-patients aged 4-9 yr were randomly allocated to four groups (n = 26). Subjects in the Omeprazole-Omeprazole Group received two doses of omeprazole (20 mg per dose), those in the Placebo-Placebo Group, two doses of placebo, those in the Placebo-Omeprazole and Omeprazole-Placebo Groups, one dose each of the two preparations by mouth. For each treatment regimen, the first medication was administered at 9:00 p.m. on the night before surgery and the second at 5:30 a.m. on the morning of the day of surgery (three hours preoperatively). Children undergoing elective surgery were offered 10 ml.kg-1 of apple juice three hours before induction of anaesthesia. After induction of anaesthesia and tracheal intubation, gastric fluid was aspirated through a large-bore, multiorifice orogastric tube and analyzed for pH and total fluid volume. The administration of omeprazole at bedtime before surgery increased gastric pH (3.3 +/- 1.3 vs 2.0 +/- 0.6, P < 0.05) in comparison with placebo, as did two doses of omeprazole (pH = 4.8 +/- 1.6, P < 0.05). A single dose of omeprazole administration on the morning of the day of surgery failed to increase gastric pH. There was a reduction in the number of children with a pH < 2.5 and a volume > 0.4 ml.kg-1 in the Omeprazole-Omeprazole and Omeprazole-Placebo Groups, compared with the Placebo-Placebo or Placebo-Omeprazole Groups.(ABSTRACT TRUNCATED AT 250 WORDS)

[Coronary artery spasm during coronary artery bypass surgery].

We report two cases of coronary artery spasm during coronary artery bypass surgery. As one of the complications during cardiac surgeries, coronary vasoconstriction occurs mainly after coming off cardiopulmonary bypass. The factors responsible for the spasm include high endogenous catecholamine levels due to inadequate anesthesia and hypothermia, exogenous catecholamines for circulatory support, various chemical mediators and combination of these factors. Coronary artery spasm was suspected strongly because of sudden ischemic change in electrocardiography and simultaneous aggravation of circulatory parameters, which improved immediately after direct injection of coronary vasodilators into vein graft. This method, popular in coronary angiography and catheterization, is effective for release of coronary-artery spasm observed particularly after cardiopulmonary bypass. Then mechanical circulatory assist is readily available to treat possible systemic side effect of the vasodilators.

Pancreatic excretion of dimethadione and trimethadione by repeated oral administration of trimethadione in dogs.

To examine pancreatic excretion of dimethadione (DMO), a weak organic acid, as well as of its precursor trimethadione (TMO), TMO was given orally to dogs with pancreatic fistulae at a dose of 10-160 mg/kg/day over a period of 14 days. Blood samples were taken once a day during the administration of TMO and for 7 days after discontinuation of the drug. On the 15th day, pancreatic juice was collected under stimulation by secretin (2 Crick-Haper-Raper units/kg/h). DMO concentration in plasma reached a maximal plateau around the 10th day after starting TMO administration, and depended directly on the dose of TMO. Pancreatic excretion of DMO at a steady state closely depended on both the dose of TMO and the DMO concentration in plasma. The pancreatic juice/plasma concentration ratio for DMO exceeded 1.0 at a steady rate and decreased with the increased flow rate. Pancreatic DMO clearance (DMO output/DMO concentration in plasma) increased, depending on the flow rate, the bicarbonate concentration, and pH of pancreatic juice. Pancreatic excretion of TMO was zero or extremely low.