Hisashi Fujioka

Effects of the epipodophyllotoxin VP-16-213 on herpes simplex virus type 2 replication.

It has been recently shown that VP-16-213, a semi-synthetic derivative of podophyllotoxin, inhibits the function of mammalian DNA topoisomerase II. In the present study, we examined the effects of VP-16-213 on the replication of herpes simplex virus type 2 (HSV-2). The compound did not inhibit the synthesis of early viral polypeptides at concentrations at which viral DNA synthesis was strongly suppressed, but induced double-strand breaks in newly synthesized HSV DNA. Electron microscopic examination of treated cells revealed the presence of a number of capsids with empty or partial cores. The level of topoisomerase II activity remained unaltered after infection, and all attempts to isolate VP-16-213-resistant mutants of HSV-2 have failed in spite of extensive efforts. It is suggested therefore that the mode of action of VP-16-213 may be inhibition of viral DNA synthesis by impairing the function of host cell topoisomerase II.

On the intracellular transport and the nuclear association of human cytomegalovirus structural proteins.

In cells productively infected with human cytomegalovirus (HCMV) AD169, large amounts of two viral proteins, the 150K major capsid and the 68K major matrix proteins, are continuously produced during the late phase of infection. In the present study, the mechanism for the intracellular transport of the 150K and 68K proteins was investigated. Infected cells were labelled for 30 min at 72 h post-infection with [35S]methionine, chased for various periods of time at 37 degrees C, and fractionated into cytoplasmic and nuclear fractions. Immediately after 30 min of labelling, the 68K protein was already associated with the nuclear fraction. In contrast, the major proportion of the 150K protein remained in the cytoplasm for more than 1 h; the migration of the 150K protein was much slower than that of the 68K protein. Both the 150K and the 68K proteins were associated with the perinuclear cytoskeletal fraction in the process of migration. After migration into the nucleus, these proteins were resistant to extraction with DNase and high salt, indicating that they were associated with the nuclear skeleton (nuclear matrix). Effects of various inhibitors on the migration of the 150K protein showed that cycloheximide inhibited the transport of the 150K protein, but other inhibitors such as arabinosyl cytosine, cytochalasin D, colchicine or sodium azide did not. The results suggest that the cytoskeletal structure may play a role in the intracellular transport of HCMV structural proteins from the cytoplasm into the nucleus.

Studies on the post-larval development of cestodes of the genus Mesocestoides: Shedding and further development of M. lineatus and M. corti tetrathyridia in vivo

Abstract In vivo development of Mesocestoides lineatus and M. corti in cats were compared. On day 1–2 post infection, tetrathyridia of both species shed the majority of their posterior tissue. After shedding, the post-scolex region of M. lineatus grew and eventually formed a gravid strobila. The shed worm of M. corti split the scolex and body longitudinally, resulting in two worms. These split worms were classified into two types, small and large, the latter type developed to form a strobila which divided again into two individuals by splitting the scolex after it had formed a pair of new suckers. Regeneration of two new suckers was also seen in the small worm which multiplied without strobila formation by dividing longitudinally into two at the scolex as observed in the large worms. From these and previously published results, the developmental pathway during asexual multiplication of M. corti in the host intestine is discussed.

Mesocestoides lineatus: Trypsin induced development to adult mediated by Ca2+ and protein kinase C

A mode of action of the inducible treatment with trypsin for the development of Mesocestoides lineatus tetrathyridium to adult was analyzed by administering various agents effective on Ca2+-dependent metabolic pathways in the cells: protein kinase C activators such as a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, and a tumor promoting phorbol, 12-O-tetra-decanoyl-phorbol-13-acetate, enhanced the trypsin induced developmental processes. On the contrary, a calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, cyclic adenosine 3,5-monophosphate, and adenylate cyclase activators such as forskolin and cholera toxin, inhibited the triggering action of trypsin. Furthermore, a combined administration of Ca2+ ionophore (A23187) and the phorbol showed a similar effect with trypsin treatment, and sodium taurocholate acted as a potent enhancer like the activators of protein kinase C. These results strongly suggest that the initiation of development to adult in this cestode may be regulated synergistically by Ca2+ and protein kinase C, and that a bile acid may be involved in an activation mechanism of protein kinase C.

Studies on the post-larval development of cestodes of the genus Mesocestoides: Trypsin-induced development of M. lineatus in vitro

Abstract By using a conventional monophasic culture system, the tetrathyridia of Mesocestoides lineatus were grown in vitro into the mature, strobilated adults, and the role of trypsin and sodium taurocholate in inducing adult formation was determined. By treatment with trypsin alone tetrathyridia evaginated their scolices and subsequently developed to the strobilated adults. The induction of development was apparently related with the proteolytic activity of trypsin; it was inhibited or decreased by the addition of soybean trypsin inhibitor. Pronase E was as effective as trypsin, but chymotrypsin had less activity. In the paruterine organs of the adults grown in vitro, shelled eggs containing live onchospheres were observed.

Enhancement of Newcastle Disease Virus-Induced Fusion of Mouse L Cells by Sodium Vanadate

Sodium vanadate enhanced Newcastle disease virus (NDV)‐induced cell fusion in L cells, and there was a direct correlation between the degree of cell fusion and the dose of vanadate added. When anti‐F protein of NDV monospecific antiserum was added to the culture fluid of L cells infected with NDV, the enhancement of cell fusion was suppressed. In contrast, neither anti‐HN nor anti‐M protein monospecific antiserum inhibited the enhancement. Incubation at low temperature (4 C) and addition of sodium azide to the culture fluid suppressed the enhancement. The suppression by azide was seen only when the drug was added within 5 min after the beginning of incubation of NDV‐infected L cells with vanadate. On the other hand, incubation at low temperature inhibited the enhancement at any time during incubation with vanadate. Cytochalasin D also inhibited the enhancement if it was added at any time during incubation with vanadate.

Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host‐dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C‐4 cells. The synthesis and intracellular distribution of virus‐specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1‐5C‐4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.

Removal of HA1 Subunit of HA Monomer from Influenza Virions

Methode de degradation chimique de lhemagglutinine de linfluenzavirus A/PR/8134 en vue dobtenir un antigene produisant des anticorps