Comparison of ICP-MS and SIMS techniques for determining uranium isotope ratios in individual particles.

The determination of uranium isotope ratios in individual particles is of great importance for nuclear safeguards. In the present study, an analytical technique by inductively coupled plasma mass spectrometry (ICP-MS) with a desolvation sample introduction system was applied to isotope ratio analysis of individual uranium particles. In ICP-MS analysis of individual uranium particles with diameters ranging from 0.6 to 4.2 microm in a standard reference material (NBL CRM U050), the use of the desolvation system for sample introduction improved the precision of (234)U/(238)U and (236)U/(238)U isotope ratios. The performance of ICP-MS with desolvation was compared with that of a conventionally used method, i.e., secondary ion mass spectrometry (SIMS). The analysis of test swipe samples taken at nuclear facilities implied that the performance of ICP-MS with desolvation was superior to that of SIMS in a viewpoint of accuracy, because the problems of agglomeration of uranium particles and molecular ion interferences by other elements could be avoided. These results indicated that ICP-MS with desolvation has an enough ability to become an effective tool for nuclear safeguards.

Depositional records of plutonium and (137)Cs released from Nagasaki atomic bomb in sediment of Nishiyama reservoir at Nagasaki.

In a sediment core of Nishiyama reservoir at Nagasaki city, depth profiles of (240)Pu/(239)Pu isotopic ratio, (239+240)Pu and (137)Cs activities were determined. Sediments containing plutonium and (137)Cs, which were deposited immediately after a detonation of Nagasaki atomic bomb, were identified in the core. Observed below the sediments were macroscopic charcoals, providing evidence for initial deposit of the fallout of the Nagasaki atomic bomb. This is the first entire depositional records of plutonium and (137)Cs released from the Nagasaki atomic bomb together with those from atmospheric nuclear tests.

Plutonium isotopes derived from Nagasaki atomic bomb in the sediment of Nishiyama reservoir at Nagasaki, Japan.

The source of plutonium in sediments deposited at Nishiyama reservoir at Nagasaki was characterized by their (240)Pu/(239)Pu atom ratio. The average ratio was approximately 0.03, except in two layers. The main source of the plutonium was the Nagasaki atomic bomb. The plutonium continues to flow into the reservoir even now. The (240)Pu/(239)Pu atom ratios in two layers were higher than the average, which showed that plutonium in these layers were made of those of nuclear tests added to those of the atomic bomb.

Particle isolation for analysis of uranium minor isotopes in individual particles by secondary ion mass spectrometry.

A new technique to measure (234)U/(238)U and (236)U/(238)U isotope ratios for individual particles in environmental samples was developed, which was a combination of particle isolation under scanning electron microscope (SEM) and secondary ion mass spectrometry (SIMS). The technique was verified by measuring (234)U/(238)U and (236)U/(238)U isotope ratios in individual particles in a simulated environmental sample containing uranium standard (NBL CRM U010) and Pb metal particles. When the uranium particles were not isolated, the relative deviations of the measured isotope ratios from the reference values increased with increasing the signal intensity ratio of (208)Pb to (238)U, which was due to the molecular ion interferences by the Pb particles co-existing in the sputtered area. By the isolation of individual uranium particles, the interferences were eliminated and the measured isotope ratios were in good agreement with the reference values. The maximum relative deviations among 20 particles were 8.9% for (234)U/(238)U and 13.1% for (236)U/(238)U isotope ratios, respectively. The technique was also successfully applied to the analysis of a real swipe sample containing various kinds of elements.

Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts.

Indiscriminate invasion upon the endometrium by normal trophoblasts is strictly regulated unlike that by choriocarcinoma cells. In this study, we focused on the activity of matrix metalloproteinase (MMP)-2 and MMP-9 as parameters of invasion in normal human placenta. In situ hybridization (ISH), immunohistochemical staining (IH) and film in situ zymography (FIZ) were performed to identify cells having MMP-2 or MMP-9 expression and activity. Purified cytotrophoblasts (CTs) were used to examine the expression and activity of MMP-2 and MMP-9, and their invasive ability. In first trimester placental tissue, the MMP-2 expression was observed in extravillous trophoblasts (EVTs), and MMP-9 mainly in villous cytotrophoblasts (VCTs). FIZ revealed marked gelatinase activity in the EVTs which MMP-2 expression was observed in. In full-term placental tissue, the MMP-2 expressions was observed in the EVTs similar to that in first trimester, whereas the gelatinase activity in these cells was decreased or completely lost. Using purified CTs, the gelatinase activity was marked in early CTs, but not term CTs. Invasive ability of early CTs was inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 and MMP-2 antibody in a dose dependent manner. These suggests that the invasive ability of trophoblasts may be regulated by the enzyme activity of gelatinases, especially MMP-2.

Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers.

Hepatitis B virus (HBV) has been classified into six genotypes designated A-F by sequence divergence in the entire genome exceeding 8%. Very recently, the seventh genotype was reported and named genotype G. HBV genotype G is distinct from genomes of the other six genotypes in that it possesses an insertion of 36 nucleotides in the core gene, and has been found so far in France and the United States. A method for determining HBV genotype G was developed by polymerase chain reaction (PCR) with primers deduced from the 36-nucleotide (nt) insertion in five isolates of HBV genotype G the sequences of which have been deposited in DNA databases. The validity of this method, for specifically detecting HBV genotype G, was verified on a panel consisting of 142 HBV isolates of six major genotypes and four of genotype G. A total of 540 sera containing HBV in Japan covering symptom free carriers and patients with a spectrum of chronic liver disease were tested by this method, but not a single HBV genotype G sample was found. A possible method for serological determination of hepatitis B surface antigen of genotype G is suggested, without amplification or sequencing nucleotides, which would expand epidemiological and clinical researches on HBV genotype G.

Pharmacological effect of tamsulosin in relation to dog plasma and tissue concentrations: prostatic and urethral retention possibly contributes to uroselectivity of tamsulosin.

In the present study the pharmacokinetics and pharmacodynamics of tamsulosin were investigated in anesthetized male dogs. Hypogastric nerve stimulation elevated the intraurethral pressure (IUP), which was inhibited dose dependently by intraduodenal administration of tamsulosin (3-30 microg/kg). The inhibition peaked about 90 min after dosing and lasted up to 240 min. The basal mean blood pressure did not change significantly during the observation period. The plasma, prostatic, and urethral concentrations of tamsulosin were determined by the liquid chromatography-mass spectrometry/mass spectrometry method. The plasma concentration reached the maximal level within 30 min after dosing and gradually declined thereafter. The maximal total plasma concentration of tamsulosin (C(max, t)) and its unbound concentration (C(max, u)) correlated with the maximal effect on IUP response [r(2) = 0.81 (p<0.01, n = 15) and r(2) = 0.84 (p<0.01, n = 15), respectively]. Each individual unbound plasma concentration did not correlate, however, with its associated inhibition of IUP response (r(2) = 0.04, n = 126). Although the plasma concentration of tamsulosin decreased nearly to the lower limit of quantitation 240 min after dosing, the prostatic and urethral concentrations remained high, i.e., 13 to 44 times greater than the plasma concentration. Our data demonstrate that the maximal inhibition by tamsulosin of IUP response is well correlated with the maximal plasma concentration in the early phase. The sustained effect of tamsulosin on IUP response that follows may be related to prostatic and urethral retention of tamsulosin.

Clinical applications of serum placental protein 14 (PP14) measurement in the IVF-ET cycle.

OBJECTIVE: Placental protein 14 (PP14) is known to be one of the endometrial proteins that reflect endometrial functioning throughout the menstrual cycle. In this study, we examined PP14 as a marker for human endometrial receptivity in order to predict the outcome of in vitro fertilization and the embryo-transfer (IVF-ET) cycle. PATIENTS AND METHODS: The subjects were 72 women who had 96 IVF-ET cycles and who were examined at Tokyo Medical University Hospital during the period of January 1998 to June 1998 because of mechanical or unexplained infertility for a duration of at least 2 years. Serum samples were collected from all patients during treatment cycles, and serum PP14 concentrations were measured by a newly established enzyme-linked immunosorbent assay (ELISA). RESULTS: In the pregnant group, serum PP14 concentrations were markedly increased after ET, and a significant difference between the pregnant group and the nonpregnant group was observed 8 days following ET (p < 0.01). PP14 concentrations were higher in patients with endometria that exhibited homogenous patterns and that were more than 7 mm thicker than in other patients, as determined by ultrasound on the day of oocyte collection (p < 0.005). The pregnancy rates of patients with homogeneous patterns were lower than those of patients showing a trilaminar pattern. No pregnancies were observed when serum PP14 concentrations were greater than 6.85 U/l on the day of oocyte collection. CONCLUSION: PP14 might be a useful marker for human endometrial receptivity to predict the outcome of IVF-ET cycles.

Ventricular function and cardiac hypertrophy after coronary thrombolysis with tissue-type plasminogen activator (t-PA) in dogs with coronary artery thrombi.

Subacute prognosis of cardiac function after thrombolysis with a modified tissue-type plasminogen activator (t-PA) YM866 was determined in dogs with coronary artery thromboses induced by injection of a thrombin, fibrinogen and autogenous blood mixture. The left ventricular ejection fraction (LVEF) decreased 30 min after occlusion and had not improved 1 week later. Examination after sacrifice revealed myocardial infarction as well as increases in both the left ventricular myocardial area and heart mass. Occluded coronary arteries reperfused by YM866 (0.1 mg kg(-1) i.v.) treatment 30 min after occlusion, by contrast, had improved LVEF and inhibited myocardial infarction development. In addition, the left ventricular myocardial area and heart mass were significantly reduced compared with the vehicle control group 1 week after administration. Although occluded coronary arteries reperfused by YM866 (0.1 mg kg(-1) i.v.) treatment 3 h after occlusion did not show an improvement in the LVEF or inhibition of myocardial infarction development, the left ventricular myocardial area and heart mass decreased significantly compared with the vehicle control group 1 week after administration. In conclusion, early reperfusion by t-PA treatment 30 min after occlusion improved the ventricular function and cardiac hypertrophy, whereas late reperfusion by t-PA treatment 3 h after occlusion did not improve the ventricular function but did inhibit hypertrophy in dogs with coronary artery thrombi.

Development of analytical techniques for ultra trace amounts of nuclear materials in environmental samples using ICP-MS for safeguards

The authors have begun to develop analytical techniques for ultra trace amounts of nuclear materials and to prepare a clean chemistry laboratory for environmental sample analyses. The analytical techniques include bulk and particle analyses. For the bulk analysis, concentrations and isotopic ratios of U and/or Pu are determined by inductively-coupled plasma mass spectrometry (ICP-MS) and thermal ionization mass spectrometry (TIMS). In the particle analysis, isotopic ratios of U and/or Pu in each particle will be measured by secondary ion mass spectrometry (SIMS). This paper reports on the outline for the development of analytical techniques and the current situation of the development of the bulk analysis using ICP-MS is described.

Thrombopoietic activity of recombinant human interleukin-11 in nonhuman primates with ACNU-induced thrombocytopenia.

The effect of recombinant human interleukin-11 (rHuIL-11) on myelosuppressive nimustine (ACNU)-induced thrombocytopenia was assessed in nonhuman primates. A single intravenous (i.v.) injection of ACNU (15 mg/kg) was administered to cynomolgus monkeys on day 0. rHuIL-11 (100 microg/kg/day) or the vehicle was given subcutaneously (s.c.) from day 1 to day 21. In monkeys receiving ACNU, the circulating platelet count decreased to a low of 42 +/- 6 x 10(9)/L by day 21 but returned to pretreatment levels (375 +/- 48 x 10(9)/L) on day 30. Administration of rHuIL-11 prevented severe thrombocytopenia; the platelet count fell only to 138 +/- 23 x 10(9)/L on day 18, and platelet recovery was faster (458 +/- 91 x 10(9)/L by day 27) compared with that of the control animals. The size of bone marrow megakaryocytes from rHuIL-11-treated animals was larger than that of the controls, indicating that rHuIL-11 stimulated megakaryopoiesis in a myelosuppressive condition. Treatment with ACNU also caused leukopenia and moderate anemia. rHuIL-11 transiently and slightly decreased the white blood cell (WBC) and red blood cell (RBC) counts. Conversely, rHuIL-11 accelerated recovery of RBC count in the late administration period. These results support the assertion that rHuIL-11 may be an important therapeutic agent for reducing the severity and duration of thrombocytopenia following cancer chemotherapy.

Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product.

An enzyme-linked immunosorbent assay (ELISA) has been described for serological determination of hepatitis B virus genotypes, using monoclonal antibodies (mAb) against seven distinct epitopes (b, m, k, s, u, f and g) on the preS2-region products of hepatitis B surface antigen (HBsAg). The usefulness of this method for serological detection of genotype E, however, was theoretical, because no HBsAg samples of this genotype were included in the original test panel. Moreover, the predicted serotype of genotype E (bksufg) closely resembled that of genotype D (bksu, bksuf or bksug). Four HBsAg samples of genotype E were tested by the original described ELISA. The epitope g, predicted to be present in these samples by amino acid sequences, was not detected when HBsAg of genotype E was captured on a solid phase by mAb to the common determinant 'a' of HBsAg and then reacted with mAb to g (5156) labeled with horseradish peroxidase. However, the four examples of HBsAg of genotype E were captured by mAb 5156 to g on a solid phase; they were then detected by labeled mAb to the common determinant 'a'. Since epitopes f and g co-occurred on HBsAg of genotype E, HBsAg samples of this genotype were also detected, by 'sandwiching' them between immobilized mAb to g and labeled mAb to f. By contrast, HBsAg of genotype D in 90 sera was not reactive when sandwiched between mAb to f and g. Thus, this modified ELISA enables the serological determination of all six genotypes of HBsAg and, by inference, of hepatitis B virus.

Properties of a YAP powder scintillator as alpha-ray detector

A YAP scintillator (YAlO3: Ce crystal) for alpha counting has been produced in powder form and this paper describes its performance characteristics. By measuring pulse-height and rise-time distributions, it was found that the YAP powder, as a fast detector for alpha rays, could clearly distinguish alpha events from beta and gamma events. In addition, the YAP powder was used in a phoswich detector combined with a YAG (Y3Al5O12: Ce) crystal for beta and gamma detection.

Full-genome sequence analyses of hepatitis B virus (HBV) strains recovered from chimpanzees infected in the wild: implications for an origin of HBV.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus of the family Hepadnaviridae. Having been found in various animals (duck, heron, woodchuck, ground squirrel, and primates), hepadnaviruses must have undergone a long history of evolution and may comprise more members than currently recognized. Chimpanzees may also have their own hepadnavirus, even if it might be very close to HBV. We analyzed HBV-like sequences from three chimpanzees (Pan troglodytes) that were most likely infected during their life in Africa in the wild. Two chimpanzees (Ch256 and Ch258) possessed a viral genome of 3182 nt in length with a 33-nt deletion in the preS1 region, which could not be classified into any of the six genotypes (A-F) of human HBV but was very homologous to a previously reported isolate from a London Zoo chimpanzee. Phylogenetically distinct from the HBV-like sequences from gibbons, orangutans, and a gorilla so far reported, the Ch256 and Ch258 isolates would represent an indigenous chimpanzee HBV (tentatively ChHBV). A third chimpanzee (Ch195) had a 3212-nt genome, classifiable into the genotype E of HBV. Because HBV-E has been found mostly in Africans, Ch195 may have been infected from a human source in Africa. However, an inverse scenario is also possible: a spread of HBV-E might have occurred from chimpanzees to humans a long time ago in Africa. Analysis of the arginine-rich C-terminal region of the core protein, which is well conserved among mammalian hepadnaviruses, indicated that HBV-E/F and nonhuman primate hepadnaviruses are much closer than HBV-A/B/C/D to the hepadnaviruses of woodchuck and ground squirrel. Our results support an "ex-nonhuman primate" hypothesis for the origin of HBV.

Development and evaluation of a new canine myocardial infarction model using a closed-chest injection of thrombogenic material.

A new canine myocardial infarction model using thrombi induced by closed-chest injection of thrombin and autogenous blood with fibrinogen into coronary arteries was developed. Occlusive thrombi were formed in all treated animals. Occluded vessels did not spontaneously reperfuse 1 day after occlusion, but did so within 3 days. Infarction was confirmed by increased levels of creatine kinase-MB, glutamate-oxaloacetate transaminase and a-hydroxybutyrate dehydrogenase. Additionally, the left ventricular ejection fraction (LVEF) decreased within 0.5 h after occlusion and had not improved 4 weeks later. After 1 week, extensive transmural anteroinferior myocardial infarction was observed and heart mass had increased. By 4 weeks after occlusion, pulmonary capillary wedge pressure and central venous pressure were increased, and oxygen pressure was decreased. Dropout of nuclei in cardiomyocytes and increased amount of collagen fiber were observed in myocardial infarct regions of hearts excised 4 weeks after occlusion. This canine model may be useful and convenient in evaluating treatment efficacy and the long-term outcome of acute myocardial infarction.

Mutation and transcription analyses of the p63 gene in cervical carcinoma.

Genetic mutation of p53, which monitors DNA damage and operates cellular checkpoints, is a major factor in the development of human malignancies. A novel gene p63/p73L/p51, encoding a protein with significant homology to p53 and p73, was recently identified at 3q27-9. To investigate the penetration of p63 in cervical carcinogenesis, mutation and transcription analyses of p63 were performed in cervical carcinoma. A certain isotype of p63 called TAp63gamma encodes the acidic N-terminus and possesses a short C-terminus. Using reverse transcriptase-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) analysis for TAp63gamma, one mutation was found in the cervical carcinoma cell line SKG-I. However, no mutations causing amino acid substitutions or frameshifts were found in 54 cases examined for TAp63gamma, which is thought to be a tumor suppressor gene. While cervical carcinomas tended to yield a positive signal in the RT-PCR reaction designed to amplify transcripts encoding the acidic N-terminus, normal cervix and cervical intraepithelial neoplasia (CIN) did not express this transcript. These data suggest that the p63 gene does not play an essential role as a tumor suppressor gene, but expression of TAp63gamma may be speculatively associated with tumor growth in cervical carcinogenesis.

Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product.

An ELISA was developed for serological determination of the six genotypes of hepatitis B virus (HBV) designated A, B, C, D, E, and F. Monoclonal antibodies were raised against genotype-specific epitopes in the preS2-region product, and labeled with horseradish peroxidase. Hepatitis B surface antigen (HBsAg) in sera was captured by immobilized antibodies against the common determinant, and evaluated for reactivity with genotype-specific monoclonal antibodies labeled with the enzyme. Serological genotyping was in complete accord with genotypes determined by S-gene sequences in a panel of 68 sera containing HBV/HBsAg of different genotypes. Of 514 sera with HBsAg from Japan, 507 (98.6%) were genotyped serologically: genotype A was identified in 24 (4.7%); B in 196 (38.1%); C in 282 (54.9%); D in 2 (0.4%); and F in 3 (0.6%). There were no sera containing HBV of genotype E. Likewise, 425 of 446 (95.3%) sera with HBsAg from Brazil, China, India, Indonesia, Kenya, Korea, Nepal, Papua New Guinea, the Philippines, and Thailand were classified into A (25.6%), B (24.2%), C (33.9%), and D (11.7%) genotypes; there were no sera with HBsAg of genotype E or F among them. Some sera unclassifiable by ELISA revealed mixed infection with HBV of distinct genotypes, or contained HBsAg deprived of genotype-specific epitopes by point mutations. The ELISA would be useful for large-scale surveys, because it allows serological detection of HBV genotypes without sequencing nucleotides.

YM866, a novel modified tissue-type plasminogen activator, affects left ventricular function and myocardial infarct development in dogs with coronary artery thrombi.

YM866 is a novel modified tissue-type plasminogen activator (t-PA). Its effects on left ventricular function and myocardial infarct development in dogs with copper coil-induced coronary artery thrombosis were compared with those of a native t-PA, alteplase. YM866 (bolus injection) and alteplase (bolus plus infusion) were administered 15 min after coronary artery occlusion. YM866 and alteplase produced reperfusion in all animals, with a median time to reperfusion of 10 min. In contrast, no reperfusion occurred in the vehicle control group. Left ventricular ejection fraction (LVEF) significantly decreased 15 min after coronary occlusion. YM866 and alteplase improved LVEF 3 hr and 4 hr after administration, respectively, while LVEF did not improve in the vehicle control group. Only slight myocardial infarct areas were observed in both YM866- and alteplase-administered groups, while the area in the vehicle control group accounted for 18.2% of left ventricular myocardial area. In conclusion, although both YM866 and alteplase reperfused occluded coronary arteries, inhibited myocardial infarct development and improved LVEF in dogs with coronary artery thrombi, only a single bolus injection of YM866 was necessary to achieve these improvements. Therefore, YM866 shows promise as an improved clinical agent in treating acute myocardial infarction.

An enzyme-linked immunosorbent assay with monoclonal antibodies for the determination of phosphorylated hepatitis B core protein (p21c) in serum.

An enzyme-linked immunosorbent assay (ELISA) was developed for the determination of hepatitis B virus (HBV) core protein (p21c) using monoclonal antibody (mAb) directed to a phosphorylated C-terminal amino acid sequence that is not present in hepatitis B e antigen (HBeAg). HBV virions in the test serum were precipitated with horse polyclonal antibody to hepatitis B surface antigen (HBsAg), dissolved with Tween 20 and NaOH, and then neutralized. HBV core protein (p21c), released from HBV cores by this procedure, was sandwiched between immobilized mAb C33 directed to amino acids (aa) 133-140 of the core protein, fixed on a solid support and labeled mAb T2212 that recognizes aa 165-175, only when they are phosphorylated. The method was applied for the detection of phosphorylated p21c in sera from symptom-free carriers and patients with chronic hepatitis. The results indicated a higher extent of phosphorylation in p21c of HBV cores from symptom-free carriers than hepatitis patients.