Showing papers in "Japanese journal of medical science & biology in 1960"

Fractionation of Habu snake venom by chromatography on cm-cellulose with special reference to biological activities.

Abstract: One of the most prominent pathological changes evoked by the parenteral injection of Crotalidae venoms, including Habu venom, is hemorrhage (Taube et al., 1937; Fidler et al., 1940; Mitsuhashi et al., 1959; Kondo et al., 1960; Ohsaka et al., 1960 a, c). Hemorrhage is claimed to be due, at least in part, to the action of proteolytic enzymes (Houssay, 1930; Kellaway, 1939; Zeller, 1948; Porges, 1953; van Heyningen, 1954; Slotta, 1955; Kaiser et al., 1958; Maeno et al., 1958). Recently Ohsaka et al. (1960 a, b) reported that zone electrophoretic fractionation of Habu venom revealed the pre sence of at least two hemorrhagic principles, designated as HR1 and HR2, both of which were associated with proteolytic activity on casein. It is also well-known that Habu venom manifests lethal toxicity. It has been reported that the lethal toxicity of this venom was separable from such enzyme activities as phos pholipase A, phosphodiesterase, 5'-nucleotidase and proteinase (with casein as substrate), which had been claimed to relate to the toxic actions (Ohsaka, 1958, 1960). It is also interesting to show whether or not the principle responsible for the local effects also has the lethal toxicity. In our recent study it was indicated that one of the hemorrhagic principles (HR2) could be separated from at least the main part of lethal toxicity, while the other (HR1) associated with it (Ohsaka et al., 1960 a). In the present work, a study was undertaken on chromatographic fractionation of venom of Habu (Trimeresurus flavoviridis), a Crotalidae, in an attempt to obtain fur ther informations about the relationships among hemorrhagic activity, lethal toxicity and proteolytic activity.

Isolation of arbor viruses from mosquitoes collected at live-stock pens in Gumma Prefecture in 1959.

Abstract: Hitherto Japanese B encephalitis (JBE) virus has been isolated in many instances from men, animals and mosquitoes and it has been thought probable that JBE virus is the only prevalent arbor virus in Japan. In the meantime, Ando et al. (1952) reported the isolation of a virus (Negishi virus) from the autopsied brain of a patient clinically diagnosed as JBE. By our recent studies, evidences have been accumulated which indicate that Negishi is a member of Group B virus bearing antigen closely related with Russian Spring Summer Encephalitis virus. However, neither virus isolation nor discovery of a human infection has been reported ever since 1949. More recently, Scherer (1958) reported that he could isolate a new Group A virus (Sagiyama virus) from mosquitoes in Saitama, Japan. Taking those facts in account, it seems important to pursue the distribution of arbor viruses present in Japan over a period of years, hoping that such an investigation might give a clue to learn the ecology of arbor virus in nature. In a limited area, Gumma Prefecture, an investigation was started to gain detailed informations on the yearly appearance of arbor virus in arthropod. The present paper reveals the resuls obtained in the first year on the infection of mosquitoes in summer. The details of the isolation of 3 arbor viruses including a new one will be presented.

Two hemorrhagic principles derived from Habu snake venom and their difference in zone electrophoretical mobility.

Abstract: In the separate paper (Ohsaka et al., 1961) it was reported that zone electrophoresis provided evidence for the presence in Habu venom of at least two hemorrhagic principles, designated as HR1 and HR2, both of which were associated with proteolytic activity on casein. The present experiment is directed to confirm the difference between HR1 and HR2 in the zone electrophoretical mobility and to examine further the possible correlation between hemorrhagic and proteolytic activities by repeat electrophoresis.

Contributions to the morphology of cercariae obtained from a snail host,Semisulcospira libertina in Japan.

Abstract: Fresh water snails, Semisulcospira libertina, including S reiniana and S. japonica, are known as the first intermediate hosts of pathogenic trematodes, e. g. Paragonimus, Metagonimus, etc. These snails are also infected with many other non-pathogenic cercariae, the majority of which have been described up to now incompletely. Therefore, in the case of epidemiological studies, research workers used to be confused on the differentiation of cercariae. In order to correct such meagre descriptions, the author have already carried out many investigations regarding the morphology of carcariae. More than 18 species of cercariae were detected from Semisulcospira libertine by the author in the past ten years, 1949 to 1959. Ten species of them were already described in details (Ito, 1950, 1952 a, 1952 b, 1953 a, 1953b, 1956, 1958, 1959, 1960). In this paper, another seven species of cercariae are presented and described on their morphological differences; i. e, two furcocercous-, one echinostome-, one xiphidio-, two tailless-, and one cysticercous-cercaria. Among them, four are the redescriptions, while the other three are given a new name respectively.

The occurrence of three flagellar phases in arizona serotypes

Abstract: Since Andrewes (1922) described •gspecific•h and •gnonspecific•h flagellar antigens in several Salmonella types, the diphasic state has been recognized in hundreds of Salmo nella serotypes and has been recognized also in the Arizona group (Edwards and West, 1945). Similar variations have been found in the Escherichia freundii (Citobacter) group by Edwards (1946), in the Hafnia group by Deacon (1952), and in the Aerobacter clo acae (Cloaca) group by Sakazaki and Namioka (1960). Salmonella types which had complex phases and which gave rise to simpler types through loss variation were reported. Edwards and Bruner (1942) described S. salinatis (4, 12:d, e, h:d, e, n, z15) which could be changed irreversibly to S. san diego (4, 12:e, h:e, n, z15) while Edwards, Kauffmann and Huey (1957) found that S. mont gomery (11:d, a:d, e, n, z15) was changed to S. luciana (11:a:e, n, z15). Both of the changes resulted from the permanent loss of the common major antigenic constitu ent of phases 1 and 2 through cultivation in semisolid medium which contained d serum. Further, Edwards (1950) observed spontaneous irreversible segregation of e, h phases from more complex d, e, h phases. •gArtificial•h or induced phases often have been obtained by cultivation of salmonellae in homologous flagellar antisera. Thus, Kauffmann (1936) obtained j phases by cultivation of S, typhi in d serum and Edwards and Bruner (1939) and Bruner and Edwards (1941) obtained induced phases from S, abortus equi and S.

Enzymic inactivation of vaccinia virus with a bacterial protease.

Abstract: pepsin (Dawson and McFarlane, 1948 ; Peter and Stoeckenius, 1954). For the purpose of finding out any enzyme to affect the surface structure of vaccinia virus under less radical conditions than with pepsin (pH 3.0), attempts were made to test various enzymes for the effect on the infectivity of the virus. Virus suspension was prepared from the homogenate of infected chorioallantoic membranes (CAM) by one cycle of differential centrifugation at 7.500•~g for 40min. and at 500•~g for 10min. To 1.0cc of virus suspension in 1/150M phosphate-buffered saline (pH 7.4), 0.5cc of enzyme solution was added and the final volume of the mixture was made to 2.0cc with phosphate-buffered saline. The mixture was incubated at 37•Ž for specified period being followed by a high speed centrifugation to separate virus from enzyme. The following enzymes were investigated ; snake venom •glecithinase•h1), bacterial protease •gNagarse•h2), pepsin3), and streptokinase-streptodornase •gVaridase•h4). Titration of infectivity was carried out by pock count on the CAM of 12-day old fertile hen's eggs. Nagarse, a crystalline protease prepared from Bacillus subtilis, was the sole enzyme which inactivated significantly vaccinia virus. Table 1 shows the effect of Nagarse at different concentrations on the infectivity of vaccinia virus. After 2 hours' incubation with 250ƒÊg per cc of Nagarse the infectivity dropped from 8.8 log10 to 6.3 log10. The inactivation power of this solution is 2.5 in log difference (‡TM log), which is considered to be statistically significant for the titration method employed. In the next experiment an approach was made to elucidate the mode of action of Nagarse on vaccinia virus, employing DFP (Diisopropylfluorophosphate), which is known as a specific inhibitor of the protease and cholinesterase. The results are summarized in Table 2, indicating that the inactivation effect of Nagarse was inhibited by the co existence of DFP in the reaction mixture. The data led the authors to consider that the digestion of the protein-moiety which might be surface-constituent of virus particle accounted for the loss of infectivity of vaccinia virus. The other interesting finding was that no further inactivation was demonstrated by repeated digestions, as shown in Table 3. One possibility may be that two types of viruses are mixed in the preparation, one of which, Nagarse resistant virus, occupies about 1% of the total virus population. Another possibility that infectious nucleic acid might be released by enzymic digestion, to which the remaining infectivity may be due, should be considered as well. Based on the following observations, however, the latter

Survival of Ancylostoma duodenale in vitro.

Abstract: The lack of knowledge concerning the metabolism and general physiology of adult hookworms is mainly due to difficulties in keeping them alive for prolonged observation outside the body of their host. In a previous paper (Komiya et al., 1956), it was shown that the adult of dog hookworm, A. caninum, can be kept alive for 6 weeks in the case of male and for 12 weeks in the case of female worms in dog serum. Since the filariform larvae of human hookworm, A. duodenale, were found to develop to the adult stage in the dog body experimentally (Yoshida et al., 1959), the following experiment was con ducted to maintain this adult worm in vitro. On the other hand, in an attempt to know whether eggs laid in vitro could be developed to filariform larvae, the cultivation of the free-living stages was performed in the absence of living bacteria.

Chemical and biological properties of a lipopolysaccharide of BCG responsible for hemagglutination reaction.

Abstract: In the previous paper the author and her collaborators reported the isolation and purifi cation of hemagglutination antigen of Mycobacterium tuberculosis Aoyama B (virulent human type) by zone electrophoresis and ultracentrifugation techniques (Tsumita, Matsumoto and Mizuno, 1959, 1960). They isolated a macromolecular lipopolysaccharide which was electrophoretically and ultracentrifugally homogenous (26 S). The component sugars were mannose and arabinose. It gave a high titer of hemagglutination reaction against antiAoyama B horse serum as well as against anti-BCG rabbit serum. The present author intended to isolate and purify a hemagglutination antigen from BCG similar to that of Aoyama B strain and to elucidate as to whether it crosses with anti-Aoyama B serum or not. A lipopolysaccharide composed of mannose, arabinose and glucose was isolated, giving a high titer in hemagglutination reaction both for anti-Aoyama B and anti-BCG sera.

Chemistry of phosphatide and wax d of mycobacterium tuberculosis bcg in one week culture

Abstract: •g Purified wax•h as isolated by Anderson (1929), is a complex lipid extracted with chloroform from cultures of acid fast bacilli. Extracting the purified wax with boiling acetone, Asselineau (1951) obtained a soluble portion termed •gWax C•h and an insoluble residue, the •gWax D•h fraction. Likewise, the alcohol-ether soluble part of acid fast bacilli was classified into several fractions. •gPhosphatide•h is a boiling acetone insoluble residue in the alcohol-ether soluble fraction, corresponding to Wax D of the alcohol-ether insoluble and chloroform soluble fraction. Therefore, the substances contained in Wax D can be migrated from •gphosphatide•h depending on the conditions of extraction and on the metabolic stage of bacterial cells.

A mutant of herpes simplex virus possessing an attenuated pathogenicity for baby mice obtained by egg passage of a newly isolated strain.

Abstract: It has been attempted in this laboratory for the past years to isolate and passage herpes simplex virus by the chorioallantoic inoculation of embryonated eggs and pursue the changes in pathogenicities for various host animals occurring during the course of the passage. Among the pathogenicities examined were included those for baby mice intracerebrally or intraperitoneally inoculated, since Kilbourne and Horsfall (1951) stated that one-day old mice were equally susceptible to herpes virus inoculated by these two routes. A strain designated SK was studied most extensively. A fact came to light thereby that the SK strain after passaged through eggs about 50 times was hardly lethal to baby mice when given by the intraperitoneal route, whereas the classical HF strain experiencing more than 450 egg passages still possessed a high infectivity to baby mice even by the peripheral inoculation. This paradoxical result stimulated a quantitative analysis of the infectivities to baby mice of SK strain in comparison with HF strain, and it was eventually disclosed that a mutant had appeared incidentally in an early egg passage of SK strain, which subsequently had replaced the parent type virus after several more passages. Details of those experiments are reported in the present communication.