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We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrPSc) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrPC source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.

The precise mechanism underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrPSc, results in PrPSc replication that preserves the strain-specific characteristics of the input PrPSc; thus, PMCA mimics the process of in vivo PrPSc replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrPSc amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrPSc replication in vitro. Here, we investigated whether GAGs play a role in the faithful replication of PrPSc by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrPSc. These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrPSc through the sulfated groups present on HP and that the N-terminal domain of Bac-PrPSc might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrPC and/or PrPSc through their sulfate groups is critical for the faithful replication of prions.

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.

Properties of rubber products are formed through several stages of processing such as mixing, vulcanization, and combining with other materials. These properties have been found to be dependent on certain types of molecular structure, composition, morphology and so on. Analysis of these rubber and rubber-related products is useful tool for understanding the essence of materials or phenomena, which would provide appropriate suggestions to “MONODUKURI (products manufacturing)”.This article reviews analytical techniques used for each stage of rubber processing and shows their applications to “MONODUKURI”.

Natural rubber is one of the most important polymers produced by plants because it is strategic raw material used in more than 40000 products including the tire. It is focused as carbon neutral industrial material because it is synthesized from atmospheric CO2. Among over 2500 rubber producing plant species, the Para rubber tree (Hevea brasiliensis Muell. Arg.) is presently the only commercial source of natural rubber. There are some unknown points about the detail of natural rubber biosynthesis. The objective of this review is to provide readers with information on the newest trends of the natural rubber and Para rubber tree research from standpoint of biochemistry and molecular biology.

We recently established mouse microglial cells persistently infected with mouse-adapted scrapie ME7 (ScMG20/ME7) for in vitro study of prion pathogenesis. Here, we found that ScMG20/ME7 cells were hypersensitive to P2X7 receptor agonists, as demonstrated by sustained Ca2+ influx, membrane pore formation, cell death, and interleukin-1β release. P2X7 mRNA expression was upregulated in these cells, and also in scrapie-infected mice brains. Treatment with pentosan polysulfate eliminated the infectivity and disease-related forms of prion protein from ScMG20/ME7 cell cultures, however, hypersensitivity of P2X7 receptors remained. These results suggest that prion infections may strongly affect the P2X7 receptor system in mouse microglial cells.

Recently it has become possible for persons with hearing impairments in remote locations to communicate via sign language using video phones and videoconferencing systems. Video interpreting makes use of videoconferencing technology to allow remote sign language interpreting services to occur without an interpreter on site. In this paper, we describe our experimental system for the remote sign language interpreting services of lecture. And we discuss that a sign language interpreter, in a remote sign language interpreting services, can realize more effective interpretation by visualization support in lecture.

Recently it has become possible for persons with hearing impairments in remote locations to communicate via sign language using video phones and videoconferencing systems. Video interpreting makes use of videoconferencing technology to allow remote sign language interpreting services to occur without an interpreter on site. In actually attempting video interpreting services for the hearing impaired, however, critical problems must be take in account, besides the issue of video quality, picture quality and refresh rate, that is current being examined. Our approach uses dual-video transmission which gives interpreters full view of the remote place. For example, the interpreter can see the face of a hearing speaker giving a presentation and the face of the audience. with hearing impairments at the same time. In this paper, we discuss that a sign language interpreter, in a remote sign language interpreting services, can realize more effective interpretation by visualization support in a lecture scene.

Surveillance results from pediatric cases and outbreaks of viral gastroenteritis in Japan are presented. In winter, both small round structured virus (SRSV, or Norwalk-like viruses) and rotavirus were detected from infants with gastroenteritis; however, in recent years, the prevailing time of SRSV infection has preceded that of rotavirus infection. Most nonbacterial gastroenteritis outbreaks were related to SRSV infection, and >60% of the outbreaks were caused by contaminated food. In small-sized outbreaks, raw oysters were the primary source of transmission. In large-sized outbreaks, school lunches and catered meals that were served at schools, banquet halls, and hospitals were most often implicated in the transmission of foodborne gastroenteritis.

Several approximate algorithms have been reported to solve large constraint-satisfaction problems (CSPs) within a practical time. While those papers discuss techniques to escape from local optima, this paper describes a method that actively performs global searches. The present method improves the rate of search of genetic algorithms by using viral infection instead of mutation. Partial solutions of a CSP are considered to be viruses, and a population of viruses is created, as well as a population of candidate solutions. The search for a solution is conducted by crossover and infection. Infection substitutes the gene of a virus for the locus decided by the virus. Experimental results using randomly generated CSPs prove that the proposed method is faster that usual genetic algorithms at finding a solution when the constraint density of a CSP is low.

The prevalence of measles antibody in Japan was surveyed with a newly developed gelatin particle agglutination (PA) test, and the results compared with those of the hemagglutination inhibition (HI) test. The two age-distribution curves of the PA antibody-positive rates ator = 1:8 andor = 1:32 were almost the same in all the age groups, except the less-than-1-year-old group for which the rate ator = 1:8 was higher than that ator = 1:32 (p0.05, chi 2 test). In the vaccinated children, all groups older-than-1-year of age had antibody-positive levels of 96% or more. In contrast, in the unvaccinated children, there was a sharp increase in antibody-positive rates between the 1- and 4-year-old groups, indicative that about 80% of the children were infected by wild measles virus at these ages. A significant number of PA antibody-positive specimens were antibody-negative (1:8) by HI. The percentage of specimens in this category, PA (+) but HI (-), was greatest in infants less than one year old, and least in young children, but it increased with age to 97% of the HI (-) specimens from adults of more than 20 years of age. The PA test therefore detected some measles antibodies that HI could not. This test is simple and useful for making serosurveys in both developed and developing countries.

The Infectious Agents Surveillance Center, the National Institute of Health, Japan, received 17,265 reports from 1982 to 1993 on cases from whom adenovirus was isolated or detected; 85% from 57 public health institutes and the other 15% from two national hospitals and two commercial diagnostic laboratories. The followings were found. Three major diseases caused by adenovirus were upper respiratory tract infection, gastroenteritis, and conjunctivitis. Patients of upper respiratory tract infection numbered 6,837 (40% of all patients due to adenovirus), the identified serotypes being in order of frequency types 3, 2, 1, and 5. Those of gastroenteritis numbered 1,636 (9.5%). From 40% of the gastroenteritis patients, adenovirus was detected by electron microscopy or immunochemical methods without cultivation. From the remaining 60%, virus was isolated in tissue culture; the serotypes of the isolates resembled those causing upper respiratory tract infection. Patients of conjunctivitis numbered 3,437 (20%), the frequency being in order of types 3, 4, 8, 37, and 19. Conjunctivitis due to types 3 and 4 prevailed every summer; type 3 was isolated often from children with pharyngo-conjunctival fever and the other four types were mostly from adults with epidemic keratoconjunctivitis. Type 3 had a unique feature not seen in other types: it was most frequently isolated, causing upper respiratory tract infection, gastroenteritis, conjunctivitis, and pharyngo-conjunctival fever. Reports on isolation of type 7, which has been reported to cause severe pneumonia in many other countries, were as few as 28 (0.2%).

Two rages of epidemic of aseptic meningitis (AM) due to echovirus 30 (E30) in Japan were analyzed with respect to two sources of information, AM incidence and E30 isolation, both gathered through the National Epidemiological Surveillance of Infectious Diseases. The first E30 epidemic spread throughout Japan in 1983 and ceased within the year. The second epidemic, starting in 1989, continued for the three successive years, and in the last year, 1991, the total E30 reports numbered 4,061, the largest number of a single virus type ever reported. Although the epidemic showed temporal and geographical shift and lasted for one or two years in some areas, most laboratories reported the largest number of E30 isolation in 1991. Among E30-yielding cases with clinical information during 1982-1992, the associating frequency with AM was as high as 82.5%. Other central nervous system involvements such as encephalitis, myelitis, encephalomyelitis and/or paralysis were reported in 36 E30-yielding cases and their monthly and age distributions were different from those of AM cases. The proportion of such disease among E30-yielding cases (0.60%) was close to that of other enteroviruses (0.56%). During the epidemics, E30 was isolated more frequently from cerebrospinal fluid than was E4 or E9 which prevailed coincidentally. E30 was most frequently isolated from cases of 4-7 years of age, sharing the common characteristic pattern of age distribution with other enteroviral meningitis. E30-yielding cases, however, involved a large number of older age groups than those of other enterovirus infections, and this tendency was the most pronounced in the first epidemic year, 1983. The contribution of these E30 epidemics on the yearly trend of clinically reported AM incidence and on the shift of its age distribution was also analyzed.

We studied the effects of interferon gamma (IFN-gamma), a T-cell lymphokine, on the proliferation and chemotaxis of vascular smooth muscle cells (SMC). Recombinant human IFN-gamma dose-dependently inhibited the proliferation of SMC cultured in the presence of 20% fetal calf-serum. It also inhibited PDGF-induced chemotaxis of SMC. Similar concentrations of IFN-gamma induced DNA-synthesis of SMC cultured in mitogen-depleted medium for 5 days. The inhibition and the stimulation of SMC proliferation were accompanied by concomitant decrease and increase in the number of PDGF receptors. Our study indicated that IFN-gamma is a bidirectional regulator of SMC proliferation.

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.

The pathogenic isoform (PrP Sc ) of the host-encoded cellular prion protein (PrP C ) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP Sc seed catalyzes the structural conversion of endogenous PrP C into nascent PrP Sc in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP res ) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP Sc by PMCA. However, the biological properties of PrP Sc obtained by in vivo transmission of recPrP res are unique or different from those of PrP Sc used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP Sc . In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP C substrate for amplification of the mouse scrapie prion strain Chandler, and PrP Sc that accumulated in mice inoculated with Bac-PrP res had biochemical and pathological properties very similar to those of the PrP Sc seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP res , the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP Sc . Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.

Nucleotide substitutions in the viral-encoded proteinase 3C (3Cpro) region (549 nucleotides) of the RNA genome of a coxsackievirus A24 variant (CA24v), one of the agents causing acute hemorrhagic conjunctivitis (AHC), were studied using 32 isolates collected from the Eastern hemisphere in 1970-1989. Based on regression analysis of nucleotide differences among isolates, the nucleotide substitution rate of CA24v 3Cpro was estimated to be 3.7 x 10(-3)/nucleotide/year. A phylogenetic tree constructed by the modified unweighted pair group method using arithmetic averages (UPGMA) indicated that CA24v had evolved from a common ancestor which appeared in one focal place in November 1963 +/- 21 months, about 7 years before the first isolation of CA24v in Singapore. The tree also revealed that all the recent epidemic isolates in 1985-1989 including Asian and Ghanian strains diverged from each other after 1981. This finding is consistent with the evidence that AHC due to CA24v had been confined to Southeast Asia and the Indian subcontinent until 1985, then suddenly and explosively spread to other areas where no CA24v isolations had been reported.

In Japan, aseptic meningitis cases due to enterovirus infections increase every summer in various degrees with an incidence peak usually in July. During the past 11 years from 1981 through 1991, a total of 8,595 enterovirus isolations from aseptic meningitis cases were reported from 54 participating laboratories. Eight enterovirus types caused large epidemics; more than 100 isolations of each type from aseptic meningitis cases were reported for every epidemic year of the respective type. They were coxsackievirus (C) types B3 and B5, echovirus (E) types 4, 6, 7, 9, 18 and 30. Among these, the highest meningitis-associating frequency was reported for E30, representing 82.6% of the total isolations reported for the type during this period, followed by E4, 71.1%. The frequencies of E9, E7, E6 and CB5 were in a range from 54.5% to 44.4%, while that of E18 was 37.7% and that of CB3 21.0%. During the epidemics, enterovirus-associated meningitis was most frequently reported among children of 4-7 years of age. High frequencies were also shown in infants less than 1-year of age in some types. A total of 4,240 enteroviruses were isolated from cerebrospinal fluid of aseptic meningitis cases, representing 49.3% of the cases with enterovirus isolation.

This report presents an overall distribution of poliovirus isolations in Japan, where poliomyelitis has been under control over two decades as a result of legal administration of two doses of the trivalent live oral poliovirus vaccine of the Sabin strains (OPV) to children under 48 months of age. During the past 12 years from 1980 through 1991, a total of 1,126 poliovirus isolations from humans and 268 isolations from sewage/river water were reported by respectively 49 and nine of the participating laboratories. Type 2 was most frequently isolated from children after administration of one dose of OPV, followed by type 1 and type 3. On the contrary, after the second dose of OPV, the rate of isolation of type 3 exceeded those of type 2 and type 1. Seasonal and age distribution of poliovirus isolations from both humans and sewage/river water paralleled the OPV vaccination schedule in Japan. One percent of the isolations were, however, from infants younger than the vaccination-scheduled ages and 5% were from children older than those ages, including one each from 15 and 16 years olds. The data indicate that the poliovirus has silently been disseminated from vaccinated children to others and the community, thus suggesting repeated transmission of the viruses. The fact that some elder children had poliovirus colonization in their alimentary tracts indicates a potential risk of infection of such a population when exposed to a wild virus and of becoming a source of transmission to others.

We have conducted a series of experiments on recognizing emotions conveyed on the hearing of musical performances as a means of building a musical performance assistance system with which deaf and hard of hearing people could communicate through musical performance. Until recently, we had assumed that visual information provided simultaneously with musical performance was useful in helping the emotions emitted from music to be recognized, especially if the information conveyed the same emotions as those expressed in the musical performance. Currently we are not sure that this assumption is correct after having gone through experiments we conducted. In this paper, we describe the background of our plan to build a musical performance assistance system and review our past experiments by comparing the emotions recognized by recipients among several types of stimuli. Reviewing the results of our experiments made us unsure that multichannel information that supposedly helps deaf and hard of hearing people to recognize emotions actually works well. Based on a new question we pose about multichannel music information, we describe an experiment that compare the stimuli provided by music types only, music with video sequences, and video sequences only. The results we obtained do not clarify the question that we pose.

In Japan, herpangina cases increase every summer with an incidence peak in July. During the past eight years from January, 1982 to December, 1989, a total of 3,974 viruses from herpangina cases were reported from 47 participating laboratories, and coxsackie A (CA) viruses accounted for most of them, 3,055 (76.9%). The major types associated with herpangina were, in order of frequency, CA4, 10, 5, 6, 2 and 3, representing 87.2%-80.8% of total isolations reported for each respective type in this period. Eighty-four point four percent of the total virus isolations were from children at four years of age or younger. More than 80% of total CA virus isolations were from nasopharyngeal specimens and nearly 90% of the viruses were isolated in mice.

The aromatic proton peak shape in 400MHz 1H-NMR spectra for commercially available styrene-butadiene copolymers is classified into the five categories corresponding to the styrene sequence distribution obtained by ozonolysis-GPC. The relationship between the sequence length and peak splitting was also investigated.

Laboratory reports of isolation of echovirus serotype 18 (E18) slightly increased in the summer of 1987 followed by a sharp increase with a peak in July, 1988. A total of 1,094 isolations were reported during these two years from 39 laboratories participating in the National Epidemiological Surveillance of Infectious Agents in Japan. When compared with the previous E18 outbreaks, a higher proportion of children at two years of age or under (58.3%) and a much higher incidence of exanthem (46.4%) were remarkable. Meningitis-associated isolations were reported in 30.6%, less than half of the percentage of the previous epidemic.