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Defining apoptosis-regulatory cascades of the epithelium is important for understanding carcinogenesis, since cancer cells are considered to arise as a result of the collapse of the cascades. We previously reported that a novel gene GASDERMIN (GSDM) is expressed in the stomach but suppressed in gastric cancer cell lines. Furthermore, in this study, we demonstrated that GSDM is expressed in the mucus-secreting pit cells of the gastric epithelium and frequently silenced in primary gastric cancers. We found that GSDM has a highly apoptotic activity and its expression is regulated by a transcription factor LIM domain only 1 (LMO1) through a sequence to which Runt-related transcription factor 3 (RUNX3) binds, in a GSDM promoter region. We observed coexpression of GSDM with LMO1, RUNX3 and type II transforming growth factor-beta receptor (TGF-betaRII) in the pit cells, and found that TGF-beta upregulates the LMO1- and GSDM-expression in the gastric epithelial cell line and induces apoptosis, which was confirmed by the finding that the apoptosis induction is inhibited by suppression of each LMO1-, RUNX3- and GSDM expression, respectively. The present data suggest that TGF-beta, LMO1, possibly RUNX3, and GSDM form a regulatory pathway for directing the pit cells to apoptosis.

The Gamma Ray Large Area Space Telescope (GLAST) Large Area Telescope (LAT) is a pair-production high-energy (>20 MeV) gamma-ray telescope being built by an international partnership of astrophysicists and particle physicists for a satellite launch in 2006, designed to study a wide variety of high-energy astrophysical phenomena. As part of the development effort, the collaboration has built a Balloon Flight Engineering Model (BFEM) for flight on a high-altitude scientific balloon. The BFEM is approximately the size of one of the 16 GLAST-LAT towers and contains all the components of the full instrument: plastic scintillator anticoincidence system (ACD), high-Z foil/Si strip pair-conversion tracker (TKR), CsI hodoscopic calorimeter (CAL), triggering and data acquisition electronics (DAQ), commanding system, power distribution, telemetry, real-time data display, and ground data processing system. The principal goal of the balloon flight was to demonstrate the performance of this instrument configuration under conditions similar to those expected in orbit. Results from a balloon flight from Palestine, Texas, on August 4, 2001, show that the BFEM successfully obtained gamma-ray data in this high-background environment., To be published in IEEE Transactions on Nuclear Science, Vol 49 Replacement to include an author who was omitted

The phase properties and structural characteristics of stratum corneum (SC) lipid lamellae have been a subject of considerable interest. To clarify the individual role of the stratum corneum constituent lipids, such as ceramides, free fatty acids, and cholesterol, we investigated the thermotropic properties and aggregation structures of a pseudo-ceramide/stearic acid (1/1 mole ratio)-cholesterol system, which is a simplified model for the natural lipids. Differential scanning calorimetry (DSC) detected decreases of melting entropies (delta Sm) by the incorporation of cholesterol into both anhydrous and hydrated equimolar mixture of pseudo-ceramide (SLE) and stearic acid. Moreover, there was a linear relationship between the cholesterol content and the melting entropies in the region of 0-33 mol% cholesterol for both the anhydrous and hydrate lipids. In addition, as the concentration of cholesterol increased, a liquid lateral packing (4.5 A) appeared in the wide-angle X-ray diffraction and the intensity of a hexagonal packing (4.15 A) decreased. The results from the present study strongly follow the idea that cholesterol can regulate the mobility of hydrocarbon chains of the natural stratum corneum lipid bilayer, which is primarily responsible for the barrier properties.

Two proteinase activities, encoded by hepatitis C virus (HCV), Cpro-1 and Cpro-2. Cpro-1 and Cpro-2 appear to process the precursor polyprotein from which they originate. Mutant HCV polypeptides containing the region for these proteinases were produced in Escherichia coli as fusion proteins. The N- and C-terminal ends of the HCV polypeptides were fused with the E. coli maltose-binding protein (MBP) and E. coli dihydrofolate reductase (DHFR), respectively. The proteinase activities cleaved the fusion polypeptides by the same processing pathway used in eukaryotic protein production systems. The N-terminal amino acid (aa) sequences of the processed fusion proteins were determined. A comparison of those N-terminal sequences with the aa sequence of the HCV precursor polyprotein showed that the N-terminal and C-terminal cleavage sites of p70 (NS3), one of the HCV nonstructural (NS) proteins, were the same as those identified in other processing studies: cleavages were estimated to be between aa 1026 and 1027 and between aa 1657 and 1658 of the HCV precursor protein, which are known to be cleaved by Cpro-1 and Cpro-2, respectively. Cpro-1 and Cpro-2 both functioned in E. coli and possessed authentic characteristic features.

We determined the partial amino (N)-terminal amino acid sequence of hepatitis C virus p21 (nonstructural protein 2 [NS2]). Cleavage at the p21 (NS2) N terminus depended on the presence of microsomal membranes. The amino-terminal position of p21 (NS2) was assigned to amino acid 810 of the hepatitis C virus strain IIJ precursor polyprotein. Mutation of the alanine residue at position P1 of the putative cleavage site inhibited membrane-dependent processing. This alteration in processing together with the fact that hydrophobic amino acid residues are clustered upstream of the putative cleavage site suggested the involvement of a signal peptidase(s) in the cleavage. Furthermore, mutation analysis of this possible cleavage site revealed the presence of another microsome membrane-dependent cleavage site upstream of the N terminus of p21 (NS2).

By using a plasmid-based transient protein expression system in cultured cells and an in vitro transcription/translation system, we analyzed the proteolytic processing of the putative nonstructural protein region of the precursor polyprotein from a Japanese type of hepatitis C virus. In addition to the previously reported viral proteins, p21 and p70, we identified products of 4 kDa (p4), 27 kDa (p27), 56 kDa (p56), 58 kDa (p58), and 66 kDa (p66). These products were produced in a viral serine proteinase (proteinase 2)-dependent manner from the region downstream of p70 in the precursor polyprotein and were arranged as NH2-p70-p4-p27-p58(p56)-p66-COOH as determined with region-specific antibodies. We showed that p56 was an N-terminally truncated form of p58, which suggested that a small polypeptide of 2 kDa (p2) was produced from the N-terminal part of p58. Cleavage between p4 and p27 was inefficient in vitro and we saw the 31-kDa precursor polypeptide (p31) accumulate. Furthermore, efficient cleavage at this site in vivo required the presence of p58/p56. Immunoprecipitation analysis in vitro also suggested the mutual interaction of those nonstructural protein products. An especially close association of p4 with p70 may contribute to association of p70 with microsomal membranes.

Gene products of hepatitis C virus (HCV), a possible major causative agent of posttransfusion non-A, non-B hepatitis, are considered to be produced from a precursor polyprotein via proteolytic processing mediated by either host cell or viral proteinases. The presence of HCV serine proteinase has been proposed from analyses of amino acid sequence homology. To examine the processing mechanism of the HCV precursor polyprotein, the amino-terminal region of the putative nonstructural protein region of the HCV genome, containing the serine proteinase motif, was expressed and analyzed by using an in vitro transcription/translation system and a transient expression system in cultured cells. Two distinct proteinase activities which function in the production of a 70-kDa protein (p70) from the precursor polyprotein were detected. One of these proteinase activities, which cleaved the carboxyl (C)-terminal side of p70, required the presence of the serine proteinase motif, which is located in the amino (N)-terminal region of p70. That suggested that the predicted HCV serine proteinase was functional. The other activity, which was responsible for the cleavage of the N-terminal side of p70, required the expression of the region upstream and downstream of that cleavage site, including the p70 serine proteinase domain. From the results of pulse-chase analysis, using proteinase inhibitors coupled with a point mutation analysis, the latter activity was proposed to be a novel zinc-dependent metalloproteinase.

Although underwater vent fields are of great scientific interest, accurate visual mapping is difficult because of the presence of bubble plumes that degrade the accuracy of conventional acoustic positioning systems such as long base line (LBL) and super short base line (SSBL). The authors had proposed a visual mapping method of shallow vent fields with an autonomous underwater vehicle (AUV) equipped with a profiling sonar, where positioning is based on vertical rod-shaped acoustic reflectors and bubble plumes. Although performance was verified through a series of experiments, there remain two challenges as follows. Firstly, observation is terminated if the vehicle was surrounded by bubble plumes, since the vehicle tries to avoid collision with not only artificial reflectors but also collision-safe bubble plumes. Secondly, the observation area drifts by disturbance since the waypoints are defined relative to the vehicle's position after descending close to seafloor. This paper proposes a real-time path planning method of an AUV as a part of the proposed observation method. The path of the vehicle is defined based on the types of landmarks as well as the geometric relationship between the vehicle and the landmarks. The vehicle can distinguish landmark types using a sheet laser and a camera. The proposed method was implemented on the AUV Tri-Dog 1 and a series of experiments were carried out in order to verify its performance.

Although underwater vent fields are of great scientific interest, little is understood concerning their influence on the underwater ecosystem due to the great difficulties involved in their underwater survey. The authors propose an underwater survey method using AUVs. In previous works, the authors proposed a practical method to survey the "Tagiri" vent area in Kagoshima, in southern Japan [1] [2], and carried out experiments. The field experiments in Tagiri were at some degree successful, but the system also encountered some difficulties. One of the problems was that the AUV could not identify the type of landmarks used to support accurate positioning. In this paper, we propose an autonomous recognition method of bubble plumes using a sheet laser and a camera by image processing. The performance of the proposed method was verified with data obtained in tank experiments, and then implemented in the AUV Tri-Dog 1. Using the proposed method, Tri-Dog 1 performed seafloor observations of the Tagiri vent area in March 2007. Tri-Dog 1 succeeded in distinguishing between bubble plumes and artificial acoustic reflectors, and localizing itself with sufficient accuracy to create a vast, high resolution visual map of the area.

While visual observation is an ideal method for detailed survey of seafloors, it is still difficult for an autonomous underwater vehicle (AUV) to track rugged terrains at low altitudes of ideal 1 to 2 meters. This paper proposes a real-time navigation architecture for hovering class AUVs equipped with perceptional sensors looking forwards, sideways and downward, to operate under such conditions. The method controls vertical motion while placing almost no restrictions on motion in the horizontal plane, achieving high compatibility with existing navigation scheme. The algorithm is a hybrid of sensor and map based approaches where the vehicle continually updates a local geometrical map of hazard areas using its perceptional measurements while performing conventional auto-altitude tracking. If the vehicle enters any of the hazard areas, sensor based control is invoked with depth as the control reference to elevate the AUV until it passes the hazard area. The method was verified through tank experiments using the AUV Tri-Dog 1. The AUV successfully followed a lawn-mower trajectory over a rugged terrain consisting of knolls and slopes while maintaining an extremely low altitude of 1.2 meters.

In vitro studies and the multiple applications of an oxybutynin (OXY) transdermal delivery system to Japanese healthy volunteers were conducted to characterize the stereoselectivity in the pharmacokinetics of OXY and its metabolite, N-desethyloxybutynin (DEOB). In human liver microsomes, (R)-OXY and (R)-DEOB were eliminated slightly slower than the corresponding (S)-enantiomers. The production of DEOB from OXY for the (R)-enantiomer was also slower than that for the (S)-enantiomer. In human P450-expressing liver microsomes, OXY was metabolized mainly by CYP3A4 among five cytochrome P450s (CYPs) tested (CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5) and the kinetics were slightly different for the enantiomer. The unbound fraction of (R)-OXY in plasma was almost two times higher than that of (S)-OXY, whereas (R)-DEOB was bound to plasma protein more than (S)-DEOB. No differences were observed in the blood-plasma concentration ratios for the enantiomers. After multiple applications of the transdermal delivery system, the plasma concentrations of (R)-OXY were lower than those of (S)-OXY. These data indicate that for the stereoselectivity of OXY, the unbound fraction of each OXY enantiomer was a major factor and the metabolism in liver had a minimal effect.

In 1997, an Internet-based static image telepathology system was built at Sapporo National Hospital, Japan. We can exchange high-resolution microscopical images through a file transfer protocol server and discuss cytological findings and diagnosis on an electronic mailing list. We applied the system to primary telecytodiagnosis. From May 1997 to April 1999 we have made diagnoses of 614 daily cases only by looking at the video monitor images transmitted from the cytotechnologist of Wakkanai Municipal Hospital 300 km distant from Sapporo. The concordance between telecytodiagnosis and glass slide diagnosis was 88.6%. Kappa statistics for cervical smears was 0.919 and that for specimens other than uterine cervix was 0.810. The accuracy of telecytodiagnosis was 91.4%, and was not substantially different from that of the conventional mail-based cytology in a previous year. We had five cases with a severely inappropriate diagnosis in telecytology, all of which however were quickly corrected by follow-up histological or cytological specimens. With the use of an electronic mailing list the participants had quick and sufficient discussions. We conclude that telecytology is very useful for primary cytodiagnosis in regional medicine and that it may raise the accuracy of cytodiagnosis in future, if we make consistent efforts to reflect the benefits of telecytology in daily practices. This is the first report of clinical results of telecytology from Japan.

Heme oxygenase-1 (HO-1) is a member of the heat shock protein family (HSP-32). It responds to thermal stress in cultured glial cells. To our knowledge. nothing is known about the expression and response of the HO-1 in cerebral ischemia. Therefore, we show here the induction of HO-1 in the brain of mice after global cerebral ischemia. HO-1-like immunoreactivity was detected at 12, 24, and 48 hours after ischemia recirculation. The HO-1-like immunoreactive cells were observed in astrocytes in the hippocampal dentate gyrus and CA1. The peak level of HO-1-like immunoreactivity was found 48 hours after the recirculation. HO-1-like immunoreactivity was observed in GFAP-positive astrocytes by use of a double immunostaining method. These results provide direct evidence for the induction and localization of HO-1 immunoreactivity in vivo in a mouse cerebral ischemia. We suggest that HO-1, produced in astrocytes after ischemia-recirculation, may directly affect neurons to protect from cell death.

Immunohistochemical distribution of laminin gamma2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin gamma2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5-6 weeks of gestation. Between 6-7 and 12-13 weeks, laminin gamma2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin gamma2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin gamma2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin gamma2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its gamma2 chain in the morphogenesis of human tissues.

An inversion method is proposed to obtain vibration characteristic of an immersion ultrasonic transmitter from radiated waveforms in water. A basic equation is the Rayleigh surface integral, which gives the relationship between wave pressures in water and the surface velocity of the transmitter. The inverse problem is to obtain the velocity distribution on the vibration surface to minimize the difference between Fourier amplitudes in theory and experiment in conjunction with the Tikhovon’s regularization scheme. The inversion method is applied to simulated water pressures and measured waveforms. It is shown that the Tikhonov’s regularization method is very effective to suppress excessive oscillations in the inverse analysis and the velocity distribution on the transducer’s surface is well reconstructed. It is, however, suggested that in practical application, more detailed mathematical modeling is necessary to obtain local variations of the velocity distribution.

We designed two new concept of computer aided molecular modeling system using virtual reality technologies. Although it is still a prototype, the most characteristic function of the system is enabling its user to "touch" and feel the electrostatic potential field of a protein or a drug molecule. User can scan surface of a protein using a globular probe, which is given an electrostatic charge, controlled by a force feedback devise. The electrostatic force between the protein and the probe is calculated in real time, and immediately fed back into the force feedback device. The user can easily search interactively for positions where the probe is strongly attracted to the force field. Such positions can be regarded as candidate sites where small chemical groups corresponding to the probe, functional parts of lead compounds, can bind to the target protein. We also developed a binding simulator which we can bind a small molecule (drug) to a large molecule (protein). In this system, we placed a camera, so that we can not only "feel" but also "see" how drug fits into the binding site. Our prototype system has the potential to serve as a new application method as well as being applicable to conventional VR technologies, especially to force feedback technologies.

Impotence commonly occurs after events such as acute myocardial infarction, coronary bypass, head trauma, and cerebral bleeding, including subarachnoid hemorrhage. We hypothesize that the hypoxia accompanying these events could damage the blood-testis barrier (BTB) and so cause testicular dysfunction, a possible cause of impotence. We examined the effect of cardiac arrest in mice on testis weight and various aspects of BTB function. Testis weight was decreased by about 24% 12 hours after cardiac arrest but had recovered fully by day 7. The testis/serum ratio for albumin was increased 12 hours after arrest, showing a disruption in the vascular BTB with recovery by 24 hours. The testis/serum ratio for sucrose was not consistently elevated, showing that the Sertoli cell BTB remained intact. The testis/serum ratio for verapamil was increased on day 3 of cardiac arrest, suggesting impaired function of the BTB's p-glycoprotein efflux transporter. Transporters for pituitary adenylate cyclase activating polypeptide and tumor necrosis factor-alpha were not affected by cardiac arrest. These results show that cardiac arrest affects testis weight and some aspects of BTB function. Such changes might have long-term effects on testicular function.

Laminin-5 (LN-5), an important basement membrane (BM) protein consisting of laminin alpha3, beta3 and gamma 2 chains, has been suggested to be involved in tumor cell invasion and tissue repair. In this study, the distribution of the LN-5 subunits in atypical adenomatous hyperplasia (AAH) and different types of adenocarcinomas of the lung was examined by immunohistochemical analysis. In AAH and non-sclerosing, well-differentiated adenocarcinomas, the LN gamma 2 chain was frequently detected along with the continuous BMs. These BMs were also positive for both LN alpha3 and beta3 chains, suggesting that LN-5 had been deposited. In contrast, the cytoplasmic staining for the LN gamma 2 chain was frequently observed in tumor cells of sclerosing, well-differentiated adenocarcinomas, as well as of moderately and poorly differentiated adenocarcinomas, without any evidence of co-expression of the LN alpha3 and beta3 chains. This staining pattern of the LN gamma 2 chain was prominent in carcinoma cells invading into interstitial stroma and was associated with the formation of a central scar in the tumor tissues. These results suggest that the LN gamma 2 chain monomer could be an important indicator of progression of lung adenocarcinoma.

The National Cancer Center Japan started to provide free, computer-based cancer information, through a service called the National Cancer Center--Cancer Information Service (NCC-CIS), to patients and their families, physicians and other health professionals via fax (telephone) and the Internet in 1996. NCC-CIS in Japanese is modeled on the Physician Data Query service produced by the National Cancer Institute in the United States and is produced from an independent standpoint adopted to the medical and social environment in Japan. NCC-CIS provides up-to-date cancer treatment information (staging, prognosis and state-of-the-art treatment by stage of disease), supportive care for each type of cancer, an image reference database, and a directory of organizations and lecture meeting reports in Japan in order to facilitate better understanding of cancer among people and support the decision-making process for physicians in order to achieve a reduction in cancer deaths.

Laminin (LN)-5, a heterotrimer of alpha3, beta3, and gamma2 chains, has been suggested to be involved in tumor cell invasion. The present immunohistochemical study investigated the distribution of the LN gamma2 chain in 48 different human gastric adenocarcinomas. The immunohistochemical analysis showed two distinct patterns of LN gamma2 chain expression: (a) extracellular deposition; and (b) cytoplasmic accumulation. The extracellular deposition of the LN gamma2 chain was typically observed at neoplastic basement membranes of well-differentiated adenocarcinomas. The immunoreactivity was continuous along tumor basement membranes in these tumors but was irregular and diffuse in poorly differentiated carcinomas. These tumor cells coexpressed the LN alpha3 and beta3 chains, suggesting that the LN gamma2 chain was deposited as the LN-5 complex. In contrast, tumor cells at the invading fronts showed strong cytoplasmic staining for the LN gamma2 chain without any detectable signal for the LN alpha3 or beta3 chain in both well- and poorly differentiated carcinomas. On the other hand, in vitro analysis by two-dimensional SDS-PAGE demonstrated that human gastric carcinoma cells secrete a high level of LN gamma2 chain monomer in addition to the LN-5 complex into culture medium. These results indicate that the LN gamma2 chain can be secreted as a single subunit and might be involved in tumor cell invasion.

We defined a new concept of microscope system for telepathology, named World Wide Microscope (WWM), and implemented its prototype. WWM is constructed by the following three units; (1) microscope unit, (2) control unit, and (3) internet unit. The microscope unit is a conventional light microscope equipped with a motor drive X-Y stage and an objective lens revolver, auto focus and auto iris functions, and a CCD camera. The internet unit is a World Wide Web homepage in which a Java applet and a communication server are installed. The applet is implemented several methods that realize to make a PC client on the Internet as a telepathology terminal. The control unit relays request commands generated from the applet to the microscope unit, and captures the microscopic images. We think WWM will probably become the all round telepathology tool of the next generation.

The Internet has been widely used by medical institutes and hospitals around the world, however; its use for telemedicine is still low. The main reason for this is the availability of bandwidth and poor security through the net. Meanwhile, we have established and have been operating 'Cancer Information Network' among 11 Cancer Centers in Japan, mainly for Multipoint TV Conference using HDTV image. There are also similar projects among 9 cardiovascular centers in Japan. By March, all 240 national hospitals will have been connected by an IP network using an ATM backbone. The above network projects are operated independently, and have an 'Intranet' characteristics within them. There are also many hospitals and clinics connected to the Internet by commercial internet providers. To make a secure and efficient network between these medical networks and medical sites, we started the Medical Internet eXchange project (MDX project) constructing a Medical Network Operation Center to create a link between them. To provide the administrative policy of this project, we established the Medical Internet eXchange Association. We are planning to expand this project to Asian-Pacific countries using the Asian-Pacific Advanced Network (APAN), and also expand it to worldwide connections in the future. For this purpose, we are currently asking other countries to form a structure similar to MDX-Japan. The concept, hardware system, software system, firewall configuration, and routing policy will be also discussed.

Laminin-5 is an isoform of laminin that consists of alpha 3, beta 3, and gamma 2 chains and has potent cell adhesion- and cell migration-promoting activities. In this study, five subdomains in the COOH-terminal globular (G) domain of human laminin alpha 3 chain were individually expressed in Escherichia coli, and their biological activities were investigated. Recombinant G2, G4, and G5 domains promoted adhesion to plastic plates of HT1080 fibrosarcoma cells, A431 epidermoid carcinoma cells, and ECV304 vascular endothelial cells. For the cell adhesion activity, the G2 domain required a divalent cation and heat-sensitive conformation more strongly than G4 and G5. The cell adhesion to G2 but not G4 and G5 was effectively inhibited by an anti-integrin alpha 3 antibody. A cell adhesion sequence of 22 amino acids, alpha 3G2A, that was homologous to the integrin alpha 3 beta 1-binding sequence GD-6 of laminin alpha 1 chain was identified within the G2 structure. The cell adhesion to alpha 3G2A peptide was also inhibited by the anti-integrin alpha 3 antibody. The cell adhesion to G2, alpha 3G2A, G4, and G5 was strongly inhibited by heparin, but that to native laminin-5 was inhibited less effectively. Moreover, G5 potently stimulated chemotactic migration of rat liver epithelial cells in Boyden chambers, but G2 and G4 did not. These results indicate that the G domain of laminin alpha 3 contains multiple cell binding sites with different mechanisms and different functions. The G2 domain seems to recognize integrin alpha 3 beta 1, whereas G4 and G5 may interact with heparin-like molecules on cell surface.

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Behavior of pore and bond formation in sinter cake on iron ore sintering has been studied by means of computer simulation to which a percolation model based on fractal has been applied. Simulations have revealed that pore size increases with increasing coke content. On the other hand, the excess of it results in a huge void, as well as dense and massive clusters below which an unfired region is formed. This change of sinter cake is basically confirmed by the direct observation by X-ray CT.

Proteolytic processing of the putative nonstructural (NS) region of hepatitis C virus (HCV) precursor polyprotein was analyzed. The polyprotein precursors were expressed in cultured cells, in E. coli cells and in an in vitro transcription/translation system. Seven viral products of 21kDa (p21), 70kDa (p70), 4kDa (p4), 27 kDa (p27), 56kDa (p56), 58kDa (p58), and 66kDa (p66) were immunologically identified. These products were arranged as NH2-p21-p70-p4-p27-p58(p56)-p66-COOH in the precursor polyprotein. The cleavage between p21 and p70 was suggested to be mediated by a viral metalloproteinase. Mutation analysis suggested that all other cleavages were dependent on the serine proteinase activity located in the amino-terminal portion of p70. These proteinase-dependent cleavage sites were analyzed by determination of N-terminal amino acid sequences of processed products and mutation analysis at the putative cleavage sites.

Summary The effect of aging on expression of a natural glutamine suppressor tRNA (tRNA Gln(UmUG) ) was studied in different tissues of mice; this tRNA recognizes UAG and inserts glutamine at the site of the termination codon. The level of tRNA Gln(UmUG) was found to be strongly increased in aged mice, compared to newborn and mature animals. An elevated expression of tRNA Gln(UmUG) has also been found in retrovirus-infected cells; in Moloney virus-infected cells the suppressor tRNA allows to read-through the UAG codon within the retroviral protease gene. We suggest that the increase in the level of tRNA Gln(UmUG) may influence retroviral gene expression with age.

Processing of HCV viral precursor protein requires at least two viral proteinases, Cpro-1 and Cpro-2, in addition to cellular proteinases. The HCV polypeptide that covers the region for the two viral proteinase domains was expressed in insect cells using a baculovirus expression system. The two proteinase activities were demonstrated in the infected cells. The Cpro-1-dependent cleavage site was estimated from the amino acid sequence of the N-terminus of the processed product. Analyses of the susceptibilities of various mutants altered at position P 1 and P 1′ of the putative cleavage site suggested that amino acid residues at these positions is not essential for recognition and cleavage by Cpro-1-dependent activity.

The 5S rRNAs from Sulfolobus solfataricus and Pyrodictium occultum were digested to nucleosides and analyzed using directly-combined HPLC/mass spectrometry. P. occultum 5S rRNA contains two modified nucleoside species, N4-acetylcytidine (ac4C) and N4-acetyl-2'-O-methylcytidine (ac4Cm). Oligonucleotides were generated from P. occultum 5S rRNA by RNase T1 hydrolysis, and their molecular weights were determined using electrospray mass spectrometry and compared with those predicted from the P. occultum 5S RNA gene sequence. Deviation in mass between expected and observed molecular weights permitted ac4Cm to be located at position 35, in the nonanucleotide CAA-CACC[ac4Cm]G, and the ac4C in one or both of two (C,U)G trinucleotides. 2'-O-Methylcytidine is unambiguously characterized in S. solfataricus 5S rRNA, confirming earlier tentative assignments at the analogous sequence position (Stahl, D.A., Luehrsen, K.R., Woese, C.R., and Pace, N.R. (1981) Nucleic Acids Res., Vol. 9, pp. 6129-6137; Dams, E., Londei, P., Cammarano, P., Vandenberghe, A., and De Wachter, R. (1983) Nucleic Acids Res. Vol. 11, pp. 4667-4676). Potential effects of the presence of ac4C and ac4Cm on thermal stabilization of 5S rRNA in thermophiles are discussed.

After screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain. Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies. The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography. The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate. The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E. coli strains bearing its cloned structural gene, pldB [Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K.,Nojima, S. (1985) J. Biochem, 98, 1117-1125]. The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C). Its restriction enzyme map was also different from that of cloned pldB. These results further confirmed that, in E. coli, there are two lysophospholipases with distinct characteristics.

Like rat platelets, rabbit platelets contain a secretory 14-kDa group II phospholipase A2 [Mizushima, H., Kudo, I., Horigome, K., Murakami, M., Hayakawa, M., Kim, D.K., Kondo, E., Tomita, M.,Inoue, K. (1989) J. Biochem. 105, 520-525]. The present study was undertaken to determine whether or not, in addition to that of the 14-kDa group II enzyme, rabbit platelets exhibit another phospholipase A2 activity. A rabbit platelet soluble fraction was prepared by sonication and centrifugation. When this soluble fraction was subjected to heparin-Sepharose column chromatography, phospholipase A2 activity was detected in both heparin-binding and heparin-non-binding fractions. The activity detected in the heparin-binding fraction appeared to belong to the secretory 14-kDa phospholipase A2, because it bound to anti-human 14-kDa group II phospholipase A2 monoclonal antibody. The activity found in the heparin-non-binding fraction did not appreciably react with the same antibody. When platelets were gently disrupted by the nitrogen cavitation method, the heparin-non-binding activity was mainly recovered in the platelet cytosolic fraction. The heparin-non-binding phospholipase A2 hydrolyzed a phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than one with a linoleoyl residue. The biochemical features of the activity observed in the heparin-non-binding fraction generally resembled those of human platelet soluble phospholipase A2 [Kim, D.K., Kudo, I.,Inoue, K. (1988) J. Biochem. 104, 492-494].(ABSTRACT TRUNCATED AT 250 WORDS)

A comprehensive survey of Japanese patients with adult Still's disease was made by questionnaire which was sent to major institutions with rheumatology units in Japan. Of 146 cases from 32 institutions, 90 were judged to be definitely diagnosed as adult Still's disease. The major clinical features in these 90 patients consisted of high fever, polyarthralgia, rash, increased erythrocyte sedimentation rate, negative autoantibodies, leukocytosis, liver dysfunction, and hyperferritinemia. The incidence of several features showed significant differences between these cases and previous nonJapanese cases.

Mutant cats were developed with non-goitrous primary hypothyroidism. They were clinically characterized by severely retarded growth, mild anaemia and high mortality in the young. They responded markedly to thyroid hormone replacement. Thyroid glands in the mutants were normal in position but slightly reduced in size. Laboratory studies revealed low serum concentrations of thyroxine (T4) and tri-iodothyronine (T3), and increased serum concentrations of TSH. Administration of TRH induced no further increase in TSH. Administration of exogenous TSH after suppression of endogenous TSH by T3 did not increase the serum concentration of T4 in the mutants, in sharp contrast with the threefold increase in serum T4 observed in the normal litter-mates. These findings suggest that the underlying pathogenesis of this disorder is unresponsive to TSH. Moreover, we found that the mutants were transmitted in an autosomal recessive manner. Journal of Endocrinology (1991) 129, 245–251