Takashi Kurimura

A new method for extracting dna or rna for polymerase chain reaction

The use of glass powder suspension for the extraction of RNA or DNA was studied to simplify the procedures of polymerase chain reaction (PCR). Using this procedure, proviral DNA of human T-lymphotropic virus type-1 (HTLV-1) in the blood of an asymptomatic virus carrier and viral RNA of human immunodeficiency virus (HIV) in the blood of an AIDS patient were easily detected by PCR employing glass powder. The use of glass powder is a simple and highly efficient procedure for the extraction of DNA or RNA, and can be applied for routine PCR.

Survey of the prevalence of AIDS-associated virus (LAV) infection in Japan

An indirect immunofluorescence method was developed for the assay of antibodies to lymphadenopathy-associated virus (LAV) which is known to be associated with the aetiology of the acquired immunodeficiency syndrome (AIDS). Samples of serum or plasma from 1353 healthy volunteer blood donors, 53 homosexual males, 29 patients who had received multiple blood transfusions and 163 haemophiliacs in Japan were tested for antibody to LAV. Results showed that 47 (29%) of the haemophiliacs, who had been treated largely with factor VIII or IX produced in the USA, were anti-LAV antibody positive, whereas all other subjects were anti-LAV antibody negative. The incidence of antibodies to Adult T-cell leukaemia virus or Human T-cell leukaemia virus I (ATLV or HTLV-I) in these subjects was high.

Maturation of human immunodeficiency virus, strain LAV, in vitro

The budding process of human immunodeficiency virus (HIV), strain LAV, starts with the formation of a crescent electron-dense layer directly underneath the cell membrane of infected CCRF-CEM cells. After completion of the formation of the circle of inner dense layer, immature virions with an electron-lucent center are released from the cells. Serial thin sections and stereo observation of thick sections showed that most of the immature virions adjacent to the cell surface had already come off the cell and some still had very thin connections to the cell. However, on rare occasions, virions at an intermediate stage between immature stage and mature virions with bar-shaped electron-dense cores were observed. Virions with dense cores were never observed to be connected to the cell surface. These observations support the idea that the last step of the maturation of HIV occurs outside the cell and that the electron-dense core seems to develop by rearrangement and dispersion of the substance of the inner dense layer of immature virions.

Replication of Mouse Cytomegalovirus in Thymidine Kinase‐Deficient Mouse Cells

In an attempt to determine whether mouse cytomegalovirus (MCMV) requires thymidine kinase (TK) for replication and whether it induces TK, TK‐deficient mouse cells were isolated and used as host cells for MCMV. Mutant cells resistant to 200 μg/ml of 5‐Bromodeoxyuridine (BUdR) were selected from SV40‐transformed mouse cells, mks‐A TU‐7, by propagating the cells in the presence of varying concentrations of BUdR graded by serial 2‐fold increments. The mutant cells, designated as TU‐7 BU, showed a very low TK activity (less than 1/20 that of mks‐A TU‐7). Herpes simplex virus type 1 (HSV‐1) replicated in starved as well as in unstarved TU‐7 BU, whereas MCMV could replicate only in growing TU‐7 BU and could not form plaques in monolayers of mks‐A TU‐7 or TU‐7 BU. HSV‐1 infection enhanced TK activity equally in both mks‐A TU‐7 and TU‐7 BU. In contrast, TK activity of MCMV‐infected mks‐A TU‐7 was lower than that of uninfected cells or cells inoculated with UV‐inactivated virus. In addition, TK activity of the MCMV‐infected TU‐7 BU remained minimal without showing any increase. The replication of HSV‐1 was completely inhibited in the presence of BUdR (10 μg/ml), whereas MCMV could replicate even in the presence of 50 μg/ml of BUdR. The results indicate that MCMV neither requires TK nor induces TK activity in the infected cells.

A Clustering Outbreak of Hand, Foot, and Mouth Disease Caused by Coxsackie Virus A10

A clustering outbreak of hand, foot, and mouth disease (HFMD) occurred from July, 1981 to January, 1982 in Matsue City and Gotsu City, Shimane Prefecture. Thirty‐seven patients with clinical HFMD were virologically and serologically examined, and Coxsackie virus A10 (CA10) was isolated in 18 patients from vesicles (7/16), throat‐swabs (9/31) and feces (6/7). During the period, no CA16 or enterovirus 71 were isolated from HFMD patients or from other diseases such as pharyngitis, febrile diseases, and aseptic meningitis. Serological diagnosis was performed employing an African green monkey kidney cell (AG‐1)‐adapted CA10 which demonstrated cytopathogenic effects on the cells. Paired sera from seven patients including three cases in which isolation failed showed a significant increase of neutralizing antibody titer against CA10. Finally, an etiological diagnosis was made in 21 out of 37 patients with clinical HFMD. This is the first report of a clustering outbreak of HFMD caused by CA10 in Japan.

HETEROTRANSPLANTATION OF ARGYROPHIL SMALL CELL CARCINOMA OF THE UTERINE CERVIX INTEGRATING HPV16 DNA INTO NUDE MICE

Argyrophil small cell carcinoma of the uterine cervix (ASCC) containing human papillomavirus type 16 (HPV16) DNA has been successfully transplanted in nude mice for the first time, and we have designated the resultant cell line as YIK‐1. Histology of YIK‐1 was similar to that of the original tumor. The original and YIK‐1 tumor cells contained argyrophil granules and neurosecretory granules in the cytoplasm, and were immunohistochemically stained positive for neuron‐specific enolase, serotonin and chromogranin. Both tumors contained HPV16 DNA in a multiple‐copy integrated form. Thus, YIK‐1 maintains the characteristics of the original ASCC, and may therefore be useful as an animal system for experimental studies of ASCC.

Ultrastructural localization of concanavalin A receptors in the plasma membrane: association with underlying actin filaments

Whole‐mount cell preparations of cultured rat 3Y1 cells were examined by stereo electron microscopy to identify the ultrastructural localization of concanavalin A (Con A) receptors in the plasma membrane, and to clarify the relationship between Con A receptors and cytoskeletal components. Well spread monolayer cells were extracted with saponin, briefly fixed, and then partially broken open with shearing force to facilitate the introduction of antibodies for identification of actin filaments. Stereo electron microscopy of such treated cells revealed a 3‐dimensional image of filamentous structures such as fine filaments, microtubules (MT) and endoplasmic reticulum (ER) in the flattened areas of each cell. Just beneath the plasma membrane were meshworks of actin‐containing fine filaments, as identified by an immunogold staining method. Microtubules and ER were observed to be either directly or indirectly associated with this meshwork. The broken open part of each cell exhibited a meshwork of filaments which were associated with the cytoplasmic surface of the plasma membrane. Some of the filaments were connected to the plasma membrane either by their ends or by their lateral surfaces. The localization of Con A receptors was examined by binding colloidal gold‐labelled Con A to the surface of fixed, saponin‐extracted cells. Virtually all gold particles bound externally at the same membrane sites where intracellular actin filaments attached internally. The observations strongly suggest that the distribution of Con A receptors was regulated by the underlying meshwork of actin filaments.

In situ Electron Microscopical Observation of Cells Infected with Herpes Simplex Virus

Transport and release of herpes simplex virus (HSV) in an African green monkey kidney cell line (CV-1) was followed by electron microscopy for up to 24 h p.i. Transmission electron microscopy and scanning electron microscopy were employed. For the former approach, electron microscopical autoradiography of whole cultured cells and in situ thin section techniques were used. The following new observations were made. (1) Except in peripheral parts of the cells, where the cytoplasmic membrane could make a ruffling movement relatively freely, virus particles were found only on the dorsal surface of the cell and not on the surface facing the substratum. By observation of thin section in situ, it was confirmed that the virus particles within intracytoplasmic vacuoles were apparently released by a reverse phagocytic process from the cell surface adjacent to microvillus projections. (2) Progeny virus particles in the nucleus moved to the cell surface within 2 h after maturation.

DNA synthesis and multinucleation of mouse cells infected with SV40 in the presence of cytochalasin B.

DNA synthesis and nuclear division of mouse cells, BALB/3T3, infected with SV40 were studied and were compared with those of SV40-transformed mouse cells, mKS-A TU-7. Both SV40-infected and SV40-transformed cells behaved similarly in the presence of cytochalasin B and differently from normal noninfected cells, BALB/3T3. The chemical inhibited cytokinesis of all the cell groups but the nuclear division was inhibited only in the case of non-infected BALB/3T3. With SV40-infected BALB/3T3, multinucleation occurred with the increase of input m.o.i. by SV40. SV40-infected BALB/3T3 could enter the second S phase after release from double thymidine block in the presence of cytochalasin B, while BALB/3T3 could not enter the second S phase. In bi- or multinucleated cells of SV40-infected BALB/3T3, asynchrony of DNA synthesis among nuclei in a cell was evident, as was the case with mKS-A TU-7.

Hepatitis δ virus infection in different time periods in Japan

To investigate the prevalence of hepatitis δ virus (HDV) infection in different time periods between 1973 and 1988, antibody to hepatitis δ antigen (anti‐HD) was tested for in sera collected from 1088 cases with acute or chronic hepatitis B virus (HBV) infection treated at Kure National Hospital. Between 1979 and 1983, anti‐HD was first detected in 16% (four of 25 cases) of patients with acute hepatitis B, in 6.8% (11 of 161 cases) of asymptomatic HBV carriers and 26% (51 of 196 cases) of those with chronic liver disease. Except for this time period anti‐HD was hardly detected. These findings indicate that sporadic HDV infections existed in this area between 1979 and 1983.