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The circadian clock, an internal time-keeping system with a period of about 24 h, coordinates many physiological processes with the day–night cycle. We previously demonstrated that BML-259 [N-(5-isopropyl-2-thiazolyl) phenylacetamide], a small molecule with mammal CYCLIN DEPENDENT KINASE 5 (CDK5)/CDK2 inhibition activity, lengthens Arabidopsis thaliana (Arabidopsis) circadian clock periods. BML-259 inhibits Arabidopsis CDKC kinase, which phosphorylates RNA polymerase II in the general transcriptional machinery. To accelerate our understanding of the inhibitory mechanism of BML-259 on CDKC, we performed structure–function studies of BML-259 using circadian period-lengthening activity as an estimation of CDKC inhibitor activity in vivo. The presence of a thiazole ring is essential for period-lengthening activity, whereas acetamide, isopropyl and phenyl groups can be modified without effect. BML-259 analog TT-539, a known mammal CDK5 inhibitor, did not lengthen the period nor did it inhibit Pol II phosphorylation. TT-361, an analog having a thiophenyl ring instead of a phenyl ring, possesses stronger period-lengthening activity and CDKC;2 inhibitory activity than BML-259. In silico ensemble docking calculations using Arabidopsis CDKC;2 obtained by a homology modeling indicated that the different binding conformations between these molecules and CDKC;2 explain the divergent activities of TT539 and TT361.

During and after the domestication of crops from ancestral wild plants, humans selected cultivars that could change their flowering time in response to seasonal daylength. Continuous selection of this trait eventually allowed the introduction of crops into higher or lower latitudes and different climates from the original regions where domestication initiated. In the past two decades, numerous studies have found the causal genes or alleles that change flowering time and have assisted in adapting crop species such as barley (Hordeum vulgare), wheat (Triticum aestivum L.), rice (Oryza sativa L.), pea (Pisum sativum L.), maize (Zea mays spp. mays), and soybean (Glycine max (L.) Merr.) to new environments. This updated review summarizes the genes or alleles that contributed to crop adaptation in different climatic areas. Many of these genes are putative orthologs of Arabidopsis (Arabidopsis thaliana) core clock genes. We also discuss how knowledge of the clock’s molecular functioning can facilitate molecular breeding in the future.

Study question What was the impact on treatment age in Japan after a subsidy policy change that set age limits for assisted reproductive technology (ART) treatment? Summary answer The national policy introducing age limits in the subsidy may have promoted ART treatment among younger women. What is known already Japan has provided partial subsidies for ART treatment since 2004. To promote treatment at a younger age, the government introduced a subsidy policy in 2016 that set age limits: up to six treatment cycles for women younger than 40 years of age; and up to three cycles for women between 40 and 42 years old. However, two out of 47 prefectures continued to provide subsidies to women aged 43 and older. Study design, size, duration We conducted a time series analysis of the utilisation of ART before and after the introduction of age limits, using data from the Japanese national ART registry from 2012 to 2016. Participants/materials, setting, methods We described the number of fresh and frozen treatment cycles, comparing the number between 45 prefectures that followed the national policy change (hereafter, prefectures with age limits) and two prefectures that did not (hereafter, prefectures without age limits). Ordinary least squares regression models were used to assess the impact of the policy change by prefecture on the number of ART cycles by women of different ages. Main results and the role of chance The overall number of fresh and frozen ART cycles continuously increased in all age groups from 2012 to 2016. Meanwhile, the number of fresh ART cycles among women aged ≤ 36 and 37-39 years in 2016 increased from the previous year by + 4.0% and +1.8% in prefectures with age limits, whereas it decreased in prefectures without age limits: -3.1% and -2.3%, respectively. The number of fresh ART cycles among women aged 40-42 and 43-45 years in prefectures with age limits in 2016 changed by + 1.5% and -0.1%, respectively, whereas it increased considerably in prefectures without age limits by + 9.6% and +65.4%, respectively. Similar changes were shown for the frozen cycles. After controlling for underlying time trends and prefectural characteristics, the policy change significantly increased the number of fresh and frozen ART treatment cycles among women aged ≤ 36 years and decreased the treatment cycles of women aged 40-42 years. Limitations, reasons for caution We evaluated the change observed in the year of the policy change and could not assess longer-term trends. Additionally, unobserved factors might have contributed to the change in treatment numbers. Wider implications of the findings The introduction of a policy to set an age limit for the partial ART subsidy resulted in a significant increase in treatment even among age groups younger than the boundary groups. The policy change might have conveyed educational messages regarding the benefits of early treatment. Trial registration number not applicable

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional–translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

Further improvement of high-temperature strength is required for the martensitic oxide-dispersion-strengthened (ODS) steels that are the promising structural materials for the advanced fission and fusion reactors, since they have better formability and isotropic mechanical property emphasized as the engineering structural materials. We conducted severe hot rolling at the elevated temperature and subsequent air-cooling for the martensitic 9CrODS steels, and their tensile and creep strength recorded the highest worldwide for the engineering class of ferritic-martensitic steels. Significant strength improvement is originated by replacing the martensitic structure with a transformed coarse ferrite during hot rolling and subsequent air-cooling. The formation of the transformed coarse ferrite doesn’t follow the widely accepted ultrafine grain formation by the similar processing in the ferrite-martensite steels. New structural control and improving high-temperature strength for the ODS steels are greatly important and opened the window for realizing high-performance fission and fusion reactor materials.

UNLABELLED Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified in clades by single nucleotide polymorphisms (SNPs), but this analysis requires significant laboratory effort. As the distribution of insertion sequence (IS) 629 insertions has been reported to be biased among different clades, O157 isolates can be putatively classified in clades by comparison with an IS629 distribution database. A database of the IS629 distribution in O157 strains isolated in Chiba Prefecture and their classification in clades was determined by SNP analysis and IS-printing, an easy and quick analytical tool for IS629 in the O157 genome. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing. These strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. Concordance Ratios were calculated, which compared the number of strains putatively classified in a clade by Relative Likelihood to the number of strains classified in that clade by SNP analysis. For the Fukuoka and Yamagata strains, the Concordance Ratios for clades 3, 6 and 8 were 97-100%, for clade 7 about 88%, and for clades 2 and 12 over 90%. In conclusion, O157 clade 2, 3, 6, 7, 8 and 12 strains could be putatively classified by IS-printing. SIGNIFICANCE AND IMPACT OF THE STUDY This study demonstrated that enterohemorrhagic E. coli O157 (O157) strains could be putatively classified in clades using an IS-printing system. IS-printing was previously developed as a relatively quick and easy tool for analysis of insertion sequence 629 in the O157 genome. Since most local government public health institutes in Japan carry out IS-printing for early detection of O157 outbreaks, these data should be useful for putative classification of O157 strains in each area.

Large and reliable components by wrought γ′ strengthened Ni and Ni-Fe based superalloy are strongly demanded for advanced ultra-supercritical (A-USC) power generation, which is one of the most promising and efficient technologies with steam temperature of above 700 °C. In this work, the effect of phosphorus on the creep properties and microstructure change of wrought γ′ strengthened Ni-based superalloy (alloy 282), was investigated, focused on the effects of carbides precipitation. In an alloy with phosphorus content of 8 ppm, precipitation of M23C6 carbides was observed in both grain boundaries and grain interior prior to the creep tests. Grain boundary coverage by carbide increased until the phosphorus content up to about 30 ppm. On the other hand, the amount of M23C6 in grain interior decreased with increasing phosphorus content. Results of the creep tests revealed the relationship between the time to rupture and the grain boundary coverage by carbide. Microstructure of the crept specimen showed the existence of misorientation at the vicinity of grain boundary without carbides by means of an electron backscattered diffraction (EBSD) analysis. These results suggest that the improvement of the time to rupture is due to the grain boundary precipitation strengthening mechanism by grain boundary carbides and phosphorus content affects precipitation behavior of M23C6 carbides in grain interior and grain boundaries.

Production cross sections of astatine isotopes 207−211At in the 29–57 MeV 7Li + natPb reaction have been measured by α- and γ-ray spectrometry. Excitation functions of production cross sections have been compared with a statistical calculation to study the reaction mechanism of the 7Li + natPb reaction. Production reactions of 207−211At in the 7Li + natPb have been derived. Considerably small experimental cross sections of 210At and 209At compared with the statistical calculation were clearly observed at incident energies higher than 44 MeV, indicating that the effects of breakup reaction play a role. A chemical separation of astatine from an irradiated lead target has been studied with a dry-distillation method. A complementary way to produce astatine isotopes has been developed.

Palpation has been considered as one of the important diagnostic skills for doctors, which is conducted on a patient to identify the size, the consistency, and the location of a subcutaneous tumor. Accurate palpation skill often detects the cause of disease early. Even in the field of dentistry, palpation has been considered as the important diagnostic skill for dentists. By the way, any practical training of palpation against a patient did not implemented in the dental education because the patient safety has a priority more than its educational effect. Then we propose a training system for maxillofacial palpation by using virtual reality and haptic interface. The details of modeling a virtual patient model for the training system are shown in this paper, and the experimental results are also described.

In order to utilize a 211At isotope, a promising α-emitter for radionuclide therapy, the chemical properties of astatine isotopes are studied. We have examined wet chemistry methods through the distribution ratios of astatine in liquid–liquid extraction. The astatine isotopes have been found to be well extracted into DIPE and MIBK. We observed that the distribution ratio of astatine isotopes increases with concentrations of HCl greater than 3 M, while it decreases with the HCl concentration less than 2 M. The results will be useful for development of the 211Rn–211At generator., In Press

Aims/hypothesis. The cumulative effects of several thrifty factors could contribute to the pathogenesis of Type II (non-insulin-dependent) diabetes mellitus. We screened the human UCP1 gene (UCP1) for polymorphisms associated with susceptibility to Type II diabetes. Methods. By using PCR and single-strand conformation polymorphism analysis, UCP1 were screened for mutations in 25 Type II diabetic subjects and 25 healthy control subjects. The allele frequencies of the detected polymorphisms were determined by PCR and restriction fragment length polymorphism analysis in 320 diabetic subjects and 250 control subjects. Results. An A→C transition in the 5' untranslated region (UTR) of exon 1 (112 bp upstream of the translation initiation codon) and a Met229→Leu variant were detected. The allele frequencies for the C variant and for the Leu229 variant were higher in the Type II diabetic group than in the control group (p = 0.017 and p = 0.038, respectively). These polymorphisms were in linkage disequilibrium (p < 0.00001). Luciferase assay showed that the former variant in the 5' UTR may affect the promoter activity of UCP1. Conclusion/interpretation. Both the A→C polymorphism and the Met229→Leu polymorphism of UCP1 are in linkage disequilibrium and could be one of the diabetes associated single nucleotide polymorphisms (SNPs). [Diabetologia (2001) 44: 373–376]

Aims The genetic differences of enterohaemorrhagic Escherichia coli O157 (O157) strains isolated from humans in three widely-separated areas in Japan were analysed to provide information on possible geographic aspects of O157 pathogenicity. Methods and Results Epidemiologically unlinked O157 strains were isolated in Chiba (300 strains), Fukuoka (260 strains) and Yamagata (81 strains) prefectures. These strains were classified in clades by single nucleotide polymorphism in seven loci and lineage-specific polymorphism assay-6, and differences between the strains in each clade were compared by population genetic analyses using the IS-printing system. Analysis of the clades from the three areas showed linkage disequilibrium of the strains in each clade. Comparison of the genetic differences of strains from the three areas in each clade, from calculated ΦPT values, indicated that the strains in each clade were the same population in all three areas, except possibly the clade 12 strains. Conclusions Population genetics analyses confirmed that the distribution of O157 strains in the clades isolated in three areas in Japan were similar and stable. Significance and Impact of the Study The pathogenicity of O157 strains infecting humans was comparable due to the similar, stable geographic distribution of O157 clades.

Prevalences of Cryptosporidium spp. oocysts in cattle (n = 486) on five selected farms in Morogoro municipality and three species of herbivorous wildlife (n = 87) from Mikumi National Park, Morogoro, Tanzania, were determined using the modified Ziehl-Neelsen staining technique. Of 486 bovine faecal samples, 5.3% were positive for Cryptosporidium spp. The prevalence of Cryptosporidium was higher in calves less than 3 months of age compared to weaned calves and adults. Cryptosporidium spp. oocysts were detected in both diarrhoeic and non-diarrhoeic animals, but there was a significantly higher prevalence (P < 0.001) of oocyst shedding in diarrhoeic than in non-diarrhoeic animals. Of the 87 faecal specimens from the wildlife species, 36 were from the African buffaloes (Syncerus caffer), 25 from zebra (Equus zebra) and 26 from the wildebeest (Connochaetes gnou). Cryptosporidium spp. oocysts were detected in eight (22%) buffaloes, seven (28%) zebras and seven (27%) wildebeests. Confirmation of the diagnosis was performed using anti-Cryptosporidium monoclonal antibody specific for Cryptosporidium muris. Cryptosporidium parvum and Cryptosporidium baileyi (Pathasure Cryptosporidium test kit).

Makene, V.M., Muhairwa, A.P., Mtambo, M.M.A., Kusiluka, L.J.M., Kambarage, D.M. and Maeda, G.E. 1996. Prevalence of canine gastrointestinal parasites in Morogoro, Tanzania. J. Appl. Anim. Res., 10: 149–153. Out of 235 domestic dogs from Morogoro municipality and Mgeta area in Morogoro region coproscopically screened for gastrointestinal parasitic infections, 174 (74%) were found positive for one or the other parasite. Ancylostoma caninum was the most common parasite (72%). Protozoan parasites and other helminths were less prevalent (

The distribution of Taenia saginata cysts among organs and muscle groups was determined by slicing the heart, masseters, liver and muscles of the half carcass of 21 naturally infected Zebu cattle from Morogoro abattoir, Tanzania. The carcasses harboured from 1 to 310 cysts. In the muscle groups, normally considered predilection sites, the heart and Musculus triceps brachii harboured the highest proportions of cysts (mean 17% and 12%, respectively, of the total cysts in the carcass) while lower proportions were found in the tongue, masseters and psoas muscles (3%, 2% and 5%, respectively). A relatively high proportion was found in the liver (10%). Relative cyst density was calculated for the different muscle groups by dividing the mean proportion of total weight of the muscle group into the mean proportion of cysts located in that site. The cysticerci in the examined tissues were found in the following order of relative density: heart (10.4), tongue (2.1), masseters (1.9), M. triceps brachii (1.7), liver (1.4), M. psoas (1.3), hindlimb muscles (0.71), trunk muscles (0.67), and other forelimb muscles (0.32). The proportion of cysts expected to be found at the surfaces exposed by visual examination or incision at meat inspection was calculated using an indirect method, which incorporated the area revealed by incision and visual inspection of an organ and the proportion of cysts located in the particular organ. It was estimated that 17% of the cysts would be located at an inspected site if the regulations were followed carefully. Based on cyst density, we recommend that the heart should be paid particular attention during meat inspection.

The present study was conducted to see the effect of probucol on streptozotocin diabetes in rats. After 2 weeks of a 1% probucol diet, 35 or 50 mg/kg of streptozotocin were intravenously injected into male Wistar rats. All the rats became diabetic 2 days after treatment. Thereafter, in order to see the effect of probucol on spontaneous recovery from streptozotocin diabetes, 25 mg/kg of streptozotocin was injected into rats after two weeks of probucol diet and the diet was continued for additional two weeks. All the rats with a standard diet (group CS, n = 13) and 12 of 13 rats with probucol diet (group PS) became diabetic 2 days after streptozotocin injection. One rate from group PS did not develop diabetes. Two weeks after injection, only 4 of 13 rats in groups CS showed recovery, while 11 of 12 rats in group PS showed recovery from streptozotocin diabetes (p less than 0.05). The average blood glucose levels in group PS were significantly lower than group CS (10.5 +/- 4.6 vs 18.5 +/- 0.6 mM, p less than 0.05). In addition, the pancreatic insulin content of group PS was 8 times greater than that of group CS (0.75 +/- 0.24 vs 0.09 +/- 0.03 mmol/pancreas, p less than 0.01). Thus, the in vivo diabetogenic action of streptozotocin could not be reduced by pretreatment with probucol. However, recovery from streptozotocin diabetes was induced by subsequent treatment with probucol. The precise mechanisms for this phenomenon were not known; but the present findings suggest the protective effect of probucol on beta-cell damage induced by small dose of streptozotocin.

A computer-aided approach to the planning of SS/TDMA network operation is proposed. SS/TDMA is a sophisticated satellite communication network based on an onboard switch matrix, whose operation planning is formulated as a large-scale scheduling problem. An overall planning and scheduling model is presented to deal with practical SS/TDMA systems, such as a two-frequency channel network and nondisjoint beam coverage. For systematic planning suitable for a computer-aided approach, the entire scheduling process is divided into several steps, for each of which an efficient mathematical model is proposed. A linear programming model is used for distribution of traffic on a beam-to-beam basis and the generated switching sequence is further refined by a model using the traveling salesman problem. Burst scheduling is carried out by the Bin packing algorithm and scheduling models for practical networks allowing transponder hopping and multidestination bursts. An algorithm called enhanced rescheduling is presented to further improve the scheduling capability of the existing methods. The performance of the proposed methods is demonstrated by simulation results. >

An efficient discrete cosine transform (DCT) coding of chest X-ray images is presented. This coding method has a great ability to reduce the redundancy in the coding of chest X-ray images. Excellent performance is demonstrated in terms of signal to noise ratio (SNR), and in also by direct comparison of original and reconstructed images. >

A new general purpose method for object extraction and detection, RONPaC (Robust Object Extraction (Detection) using NPaC Features) method is presented. RONPaC employs normalized principal component (NPaC) features as a measure of similarity between corresponding regions of a target image and a background image. No a priori knowledge of objects and no assumptions about the environment are required. The object extraction problem is dealt with as a discriminant problem of two classes, 'object' and 'background', in the feature space. The performance of the method is quantitatively evaluated using various real images and compared with conventional methods using two criteria, separability between two classes in feature space and ease of binarizing expressed by the maximum discriminant criterion. Experimental results confirm the extraction accuracy and applicability of the proposed method. >

Spontaneous activity in cultured networks of cortical neurons was investigated by extracellular recordings of their spatiotemporal firing patterns, using planar electrode arrays which comprised 64 microelectrodes. In the cultured cortical networks synchronous bursts states, asynchronous bursts states and non-burst firings states were observed by changing the Ca/sup 2+/, Mg/sup 2+/ and K/sup +/ concentration of the extracellular solution and by adding NMDA and non-NMDA receptor antagonists. Observed phenomena, such as burst generation and asynchronous firing activity as a function of the activation of random synaptic inputs, could be simulated by using a simplified single neuron model.

In this paper, we describe an advanced wiring RC timing design techniques for the gigahertz era. Our new technique of wiring capacitance extraction makes it possible to calculate more than 1 M nets within 3 hours as accurately as carrying out net-by-net 3-D simulations. Furthermore, we introduced the timing window for estimating crosstalk effects on delay time so as to distinguish harmful nets from harmless nets and reduce surplus design guard-bands.

The allele frequencies for a Pro12--Ala substitution in peroxisome proliferator-activated receptor-gamma differ among ethnic groups, and its relationship with diabetes and associated diseases is controversial. The prevalence of this polymorphism and its effects on clinical characteristics have now been evaluated with a large number of Japanese individuals with type 2 diabetes (n = 2,201) and normal control subjects (n = 1,212) recruited by 10 institutions located in seven different cities in Japan. The allele frequency for the Ala12 variant was significantly lower in the type 2 diabetic group than in the control group (2.39 vs. 4.13%, P = 0.000054). However, compared with subjects without the Ala12 variant, the diabetic subjects with this variant exhibited a significantly higher serum concentration of total cholesterol (P = 0.001), manifested a reduced capacity for insulin secretion as evaluated by homeostasis model assessment (P = 0.007), and tended to possess a higher level of HbA1c. These data suggest that the Ala12 variant is associated with a reduced risk for the development of diabetes in the general population, but that it may be also a risk factor for insulin deficiency and disease severity in individuals with type 2 diabetes.

Primary hyperoxaluria Type 1 (PH1) is caused by a functional deficiency of a liver enzyme, serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), which catalyzes transamination between L-serine or l-alanine as an amino acid substrate and glyoxylate or pyruvate as an alpha-keto acid substrate. A high affinity for glyoxylate is a notable feature of this enzyme, suggesting a role in glyoxylate metabolism in vivo. Another conspicuous feature of SPT/AGT is its species-specific and food habit-dependent subcellular distribution. Thus, the enzyme is located in peroxisomes in herbivores and man, largely in mitochondria in carnivores, and in both the organelles in rodents. The mechanism of the species-specific dual organelle localization of SPT/AGT is either transcription of the gene from two different start sites or loss of the upstream translation initiation ATG codon by mutations. It appears that the mitochondrial versus peroxisomal distribution of SPT/AGT in different animal species is indispensable in meeting the metabolic needs caused by their respective food habits. As for the peroxisomal localization, glycolate is contained in plants much more than in animal tissues, and when ingested, it is converted to glyoxylate, an immediate precursor of oxalate, in liver peroxisomes. Therefore, peroxisomal localization of SPT/AGT may be indispensable for herbivores to convert the glyoxylate formed in peroxisomes into glycine in situ rather than forming oxalate. On the other hand, our recent studies showed that SPT/AGT contributed substantially to serine metabolism in rabbit, human, and dog livers; i.e., irrespective of its mitochondrial or peroxisomal localization. Thus, the mitochondrial localization of SPT/AGT was not a prerequisite for the metabolism of L-serine. Another source of glyoxylate is the metabolism of L-hydroxyproline, and in this case, the enzyme responsible for the glyoxylate formation has been reported to be a mitochondrial matrix enzyme. Collagen accounts for about 30% of total animal proteins and contains about 13% (w/w) hydroxyproline. It is therefore possible that both mitochondrial and peroxisomal SPT/AGT contribute to the metabolism of glyoxylate and serine, but the subcellular site for glyoxylate metabolism is different in herbivores and carnivores.

Effect of blanching time, salt concentration and fermentation on texture of green beans (var. Tuf) was determined. Beans were blanched at 90 degrees C for 1, 10 or 20 minutes and fermented with brine containing 3.08 and 4.6% salt (corresponding to 1.2 and 1.8% salt in the finished product). The beans were assessed for texture mechanically using Universal Testing Machine (Model 1011, Instron). Sensory evaluation was carried out to assess the taste, texture and overall acceptability of the products. Pectolytic enzyme (pectinestarase and exopolygalacturonase) activities were determined in fresh and fermented beans. Increase in blanching time improved the texture significantly. Significant (p0.05) decreases in shearing energy of the beans were observed in the first two days of fermentation and, thereafter, there were no significant (p0.05) changes except for samples blanched for one minute. Salt concentration showed a small but significant effect on texture only in samples blanched for one minute and this was probably due to activation by salt of residual pectolytic enzymes in the beans. Most acceptable beans were those blanched for 20 minutes and fermented with 1.2% salt brine.

A full-term baby was born to an epileptic mother treated with two anti-epileptic drugs, sodium valproate and phenobarbital, throughout the pregnancy. She was given no information about the risk of teratogenesis of these drugs. At birth the patient was hypotonic and had clinical features specific for the fetal valproate syndrome. After a viral infection at three months of age, he had intractable and persistent wheezing. Suspecting the presence of congenital respiratory tract abnormality, we performed tracheobronchoscopy, which revealed a relatively big submucosal tumor on the left trachial wall below the vocal cord. Dyspnea and wheezing were remarkably improved by tracheolaryngotomy, but he died suddenly and unexplainedly at seven months of age.

The first victim of four serial murders occurring in Hiroshima in 1996 was an unidentified young female when she was found. In the lower jaw, the left and right deciduous second molars were identified, and the left and right permanent second premolars were revealed to be congenitally defective by X-ray examination. Comparison of the dental findings and study model after death with those before death identified the victim as a 16-year old female.

The polishing agent, mainly composed of cerium oxide (CeO2), is used for fine polishing of products such as liquid crystal panels. The impurities of iron oxide, silica, and alumina get mixed in the cerium oxide-based polishing waste water. In order to reuse the polishing agent, it is necessary to remove these impurities. In this study, the impurities were removed utilizing the difference of magnetic susceptibility in order to recover the polishing agent. In our previous study, we proposed a technique to recover cerium oxide in high purity. In the first stage, iron oxide was removed from the polishing waste water by high-gradient magnetic separation. In the second stage, silica and alumina were removed by magneto-Archimedes method utilizing the magnetic force in vertical direction. In this study, we proposed a magnetic separation method utilizing the magnetic force in horizontal direction, and examined the application possibility of the method for continuous processing in practical use.

The recently cloned factor thrombopoietin (TPO) has been shown to exhibit megakaryocyte colony-stimulating activity in vitro. In this investigation, to further evaluate the action of TPO on megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), GpIIb/IIIa+ and GpIIb/IIIa- populations of CFU-MK were prepared from rat bone marrow cells based on their reactivity with P55 antibody, a monoclonal antibody against rat GpIIb/IIIa, and their responsiveness to recombinant human TPO (rhTPO) and recombinant rat interleukin-3 (rrIL-3) was examined using a megakaryocyte colony-forming assay (Meg-CSA). rhTPO supported only megakaryocyte colony growth from both fractions in a dose-dependent fashion. The mean colony size observed with the GpIIb/IIIa+ population was smaller than that seen with the GpIIb/IIIa- population. With the optimal concentration of either rhTPO or rrIL-3, similar numbers of megakaryocyte colonies were formed from the GpIIb/IIIa+ population previously shown to be highly enriched for CFU-MK. In contrast, the maximum number of megakaryocyte colonies from the GpIIb/IIIa- population stimulated by rhTPO was only 24.2% of that achieved with rrIL-3. Morphologic analysis of rhTPO-promoted megakaryocyte colonies from the GpIIb/IIIa+ population showed that the average colony size was smaller but that the mean diameter of individual megakaryocytes was larger than in megakaryocyte colonies promoted with rrIL-3. rhTPO plus rrIL-3, each at suboptimal concentrations, had an additive effect on proliferation of CFU-MK in the GpIIb/IIIa+ fraction, whereas rhTPO plus murine IL-6 or murine granulocyte-macrophage colony-stimulating factor (mG-M-CSF) modestly but significantly reduced megakaryocyte colony growth. These results indicate that TPO preferentially acts on GpIIb/IIIa+ late CFU-MK with lower proliferative capacity and interacts with some other cytokines in CFU-MK development.

Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.

We investigated the genetic defects in two patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Their clinical manifestations including corneal opacities, anemia, proteinuria, and hypoalphalipoproteinemia were identical for familial LCAT deficiency. Their LCAT activities and the cholesterol esterification rate (CER) were nearly zero, and their LCAT masses were below 10% of normal control values. Sequence analysis of the amplified DNA of case 1 revealed one base deletion of G at base 873 (first position of Val264) in exon 6, leading to a premature termination by frameshift. Sequence analysis of amplified DNA of case 2 revealed a single G to A converting Gly (GGT) to Ser (AGT) substitution at residue 344. When COS-1 cells were transfected with these mutants, LCAT activity in the medium was nearly zero, and the LCAT mass was undetectable (0.01 microgram/ml). In contrast, LCAT activity in the medium of COS-1 cells, transfected with wild-type LCAT, was 1.7 nmol/h per ml and the LCAT mass was 0.09 micrograms/ml. The LCAT mass in the cell lysates of the mutants was less than 12% of control for case 1 and 18% of control for case 2. Northern blot analysis of the mRNA of COS-1 cells transfected with the mutants showed the same amounts of LCAT mRNA as compared with wild-type LCAT. Biosynthesis of mutant LCATs was analyzed by pulse-chase and immunocytochemistry in transfected baby hamster kidney cells. SDS-PAGE/fluorography demonstrated that wild-type LCAT was synthesized as a high-mannose type of 56 kDa, which was very slowly converted to a mature form of 67 kDa and was secreted into the media. In contrast to the wild-type LCAT, the mutant precursors were not processed into the mature form but slowly degraded along with chase times. On steady and continuous labeling in the case of wild-type LCAT, the mature 67 kDa form was observed in both the cell lysate and media, whereas no mature form was detected in the cell lysates and media which were transfected mutant LCATs. These data suggest that the mutant LCATs are actually synthesized in an amount comparable to that of wild-type, but they are slowly degraded without being processed into the mature form. The immunocytochemistry revealed that mutant LCATs were mainly retained in the endoplasmic reticulum. These data suggest that these two mutations may disrupt the mutant LCATs' transport from the endoplasmic reticulum into Golgi apparatus, resulting in LCAT deficiency.

We measured the lactate concentration in whole blood of healthy infant with use of the recently developed assay system consisting of an enzyme-coated electrode and a small meter. The mean values of blood lactate concentration at each age in days were not showed the significant difference between male and female infants. The trace of lactate concentration change in days showed the peak at the age in 1 days and then, gradually decreased to the almost constant level at the age in 6 days after. At the age in 6-7 days, the concentration mean values of infants, born in 38-40 gestational weeks were lower than that of 37 and 41 weeks one. It was assumed by mathematically that these equilibrium constant level of the lactate concentration in early neonatal period was arrived at 6.20 days and the equilibrium level was estimated to 1.29 mmol/l. As the results, the normal ranges were calculated to the value of 0.49-3.95 mmol/l as initial (age in 0-3 days) and that of 0.29-2.92 mmol/l as final (age in 6-7 days) period, by the truncation method, respectively.

A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5 x 10(4) cells/mL (10(4) cells/well) with test samples in 96-well tissue culture plates for 4 days at 37 degrees C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C-serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples--including a wide variety of cytokines and unknown growth factors, alone or in combinations--and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway.

Hydrophobic fractions isolated from cyst fluid of Taenia hydatigena metacestodes, obtained from naturally infected goats from Tanzania, were used in an indirect ELISA and in immunoblot (Western or dot blot) procedures. The use of the most hydrophobic fractions of the cyst fluid proved to result in a better diagnostic discrimination in ELISA than the more hydrophilic fractions. Best diagnostic ratio between responses from T. saginata infected and non-infected cattle was achieved when using horse-radish peroxidase-labelled sheep antibovine IgG1 as the conjugate. When the hydrophobic fractions of T. hydatigena cyst fluid were employed as antigen in ELISA, no cross-reaction took place with sera from cattle infected with Fasciola hepatica, Ostertagia ostertagi, Cooperia oncophora, Dictyocaulus viviparus, Sarcocystis cruzi or a mixture of D. viviparus, Trichostrongylus axei and Eimeria. Using the Western blot technique, specific low molecular weight components (M(r) 10-18 kDa) of the hydrophobic fractions of the cyst fluid were reactive with sera from 14 of the 21 calves infected with T. saginata for 60 days. Also, hydrophobic fractions of T. hydatigena cyst fluid were dotted onto strips of filter membranes and reacted specifically with sera from 21 calves infected with T. saginata. This technique resulted in the highest sensitivity, detecting 17 of the 21 calves infected with T. saginata. Calves harbouring as few as 16, 26, 43, 53 and 74 cysts could be detected. Thirteen calves harbouring between 74 and 2,545 cysts were all detected. However, 4 calves harbouring 2, 22, 52 and 66 cysts, respectively, were not detected. The potential of using a dot blot technique in large scale investigations in the tropics is present. However, before accepting any of the three tests in the tropical environment, a broader spectrum of helminth infections should be evaluated for their eventual cross-reactivity.

A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1,100 rats by measuring the production of megakaryocytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49 x 10(8), respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro, and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl.

Diffuse axonal injury (DAI) is defined as widespread damage to axons in the white matter of the brain without focal injury such as contusion and acute subdural hematoma. A case of traumatic subdural hematoma from arterio-venous malformation accompanied by DAI is reported. A 58-year-old man was assaulted, and immediately lost consciousness, and remained unconscious during about 44 hours until his death. The autopsy revealed acute subdural hematoma (about 160 g) on left temporal lobe and left cingular, uncal and cerebellar tonsillar herniation, and tear and hemorrhage of the corpus callosum. Under this subdural hematoma, gray-whitish vascular lesion with subarachnoid hemorrhage was found. Histologically, this lesion was diagnosed as the arterio-venous malformation. Neuropathological examination of the corpus callosum, dorsolateral part of midbrain and superior cerebellar peduncle revealed DAI findings, such as swelling and ballooning of the myelin fibers, swelling and waving of axons, and retraction balls. Axon degenerations were also observed immunohistochemically by anti-200 kD neurofilament antibody. From the results, his unconsciousness from the moment of impact might be occurred from not only subdural hematoma but also DAI.

In order to investigate changes of morphology and metabolism of living protoplasts, cytochemical methods are very important. For this purpose, it is necessary to stain protoplasts with various dyes in the living state. In the staining procedures of plant materials in usual histochemical (cytochemical) studies, almost all tissues or cells are already fixed (dead) before staining. Moreover, many kinds of dyes are toxic to living cells. Therefore, there is a limitation to staining living cells by the methods used for fixed sections in histochemistry. Since fluorescent dyes were introduced into botany at the end of the 19th century, a large number of new fluorescent compounds have been used as vital dyes in cytology and cytochemistry (Gahan 1984).

188/187Re nuclear quadrupole resonance (NQR) and ac susceptibility measurements in Cd2Re2O7 have been performed to study correlation between superconductivity and structural transition (Ts1 = 200 K and Ts2 = 120 K at ambient pressure) under high pressure, using a hybrid piston cylinder-type high-pressure cell. Tc is almost pressure independent below 1 GPa, while, above 1 GPa, Tc increases monotonously and reaches up to 2.3 K at 2.5 GPa. The 188/187Re-NQR spectrum for ±3/2 ↔ ±5/2 transition broadens and shifts to lower frequency with applying pressure. Furthermore, Re NQR spectrum broadens suddenly around 2GPa, suggesting that electric field gradient at Re sites distributes extendedly, which may be related to the disorder state with suppressing Ts2 down to ~ 0 K.