Genetic analysis of an influenza B virus isolated from a patient with encephalopathy in Japan.

An influenza B virus, B/Saga/S172/99 (SAG99), was isolated from the nasopharynx of a patient with encephalopathy/encephalitis in Japan in 1999. To clarify the molecular characteristics of this virus, detailed analysis of the gene segments coding for the hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M) and non-structural protein (NS) was undertaken. All five genes of SAG99 showed high nucleotide and predicted amino acid similarities with those of recent non-encephalopathic strains isolated in the same epidemic season. Subsequent phylogenetic analysis revealed that all five gene segments of SAG99 analyzed in the present study were most similar to those of the recent Yamagata/16/88-like viruses. The hemagglutinin and neuraminidase proteins of SAG99 were each distinguished from those of recent epidemic strains by one characteristic amino acid substitution. These substitutions were not found in the previously reported encephalopathy/encephalitis-derived influenza B viruses, and we could not find any common characteristic amino acid changes in SAG99 and these viruses. Similarly, among the internal proteins studied, only the M2 protein of SAG99 was found to contain a single novel amino acid change when compared with other recent isolates. Thus, it was apparent that SAG99 contained very few amino acid differences when compared with other epidemic viruses. The association of recent B/Yamagata/16/88-like viruses with encephalitis/encephalopathy observed in the present study and previously suggest that these viruses may have a higher potential for causing neurological complications in certain individuals.

Serotypes of human rotaviruses in 7 regions of Japan from 1984 to 1997.

Human rotavirus (HRV) serotypes were studied from diarrheal stool specimens in children in 7 regions of Japan (Sapporo, Tokyo, Maizuru, Osaka, Kagawa, Kurume, and Saga) from 1984 to 1997 by enzyme immunoassay (EIA) with serotype-specific monoclonal antibodies against serotypes 1, 2, 3, and 4. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was conducted for analysis of "others" which included nonserotypable and mixed-serotype rotavirus specimens by EIA. In 3756 rotavirus-positive specimens, serotype 1 was detected in 2649 (70.5%), serotype 2 in 362 (9.6%), serotype 3 in 232 (6.2%) and serotype 4 in 196 (5.2%). Overall, serotype 1 was predominant from 1984 to 1997, although there were a few cases in which serotype 2, 3 and 4 became predominant based on area and year. The frequency of serotype 1 has gradually increased since 1993. Twenty two, 2, 3 and 1 among 57 specimens of "others" by EIA from Tokyo, Maizuru, Sapporo and Kurume in 1995-1996 and 1996-1997 were determined as serotypes 1, 2, 3, and 9 by RT-PCR, respectively.

Survey of human group C rotaviruses in Japan during the winter of 1992 to 1993.

Fecal specimens from patients with acute diarrhea were collected from 10 prefectures in Japan over a 6-month period (November 1992 to April 1993), and the specimens that were negative for human group A rotaviruses were screened for the presence of human group C rotaviruses (CHRVs) by the reverse passive hemagglutination test. Of 784 specimens examined, 53 samples (6.8%) that were collected in 7 of 10 prefectures were positive for CHRV, indicating that CHRVs are widely distributed across Japan. Most of the CHRV isolates were detected in March and April, and CHRVs mainly prevailed in children ages 3 to 8 years. The genome electropherotypes of eight strains isolated in five individual prefectures were surprisingly similar to each other and were different from those of CHRV strains isolated to date. The outer capsid glycoprotein (VP7) gene homologies of the isolates retrieved in 1993 were subsequently analyzed by the dot blot hybridization method. As a result, the VP7 genes of the isolates revealed very high levels of homology not only with each other but also with the VP7 gene of the OK118 strain isolated in 1988. These results suggest that a large-scale outbreak of CHRV occurred during the winter of 1992 and 1993 in Japan.

[Epidemic of influenza in Kyushu-Okinawa District (April 1994-March 1995)].

The prevalence of influenza in Kyushu-Okinawa District in April 1994- March 1995 was studied as the prevalence of influenza virus, to determine the sero-type of influenza viruses isolated in Kyushu- Okinawa District prefectures and cities. As a result, three sero-types of influenza viruses, i.e. type A/H1N1, type H3N2 and type B, were isolated in Kyushu-Okinawa District in this season, but most of the isolates were type A/H3N2 and type B. Weekly changes of reported influenza patients and period of virus isolation at local public health institutes revealed that influenza epidemics of the earlier part in this season was caused by type A/H3N2 and the latter part due to type B. Type A/H3N2 spread all over Kyushu-Okinawa District in a shorter period (about 2 weeks) through the westside of Kyushu and down south, and type B stayed about one month in northern Kyushu and took about 7 weeks to spread all over Kyushu-Okinawa District. Based on these results, the spread of influenza virus in Kyushu-Okinawa District was visualized on the isopleth maps.

Effects of various types of extracellular matrices on adhesion, proliferation, differentiation, and c-fos protein expression of porcine thyroid follicle cells.

Thyroid follicles in vivo are embedded in extracellular matrix (ECM). The composing epithelial cells are in close contact with ECM at the basal side. To examine cell-to-ECM interactions, we studied adhesion, proliferation and differentiation of porcine follicle cells monolayer-cultured on type I and IV collagen, fibronectin or laminin. At 3 h in culture, laminin had the lowest rate of cell adhesion. In proliferation, type IV collagen induced the highest level of nuclear bromodeoxyuridine intake. In a functional differentiation, laminin had about 3 times as much triiodothyronine production as the other ECM molecules. In confluent culture cells, we also examined an expression of c-fos protein, a transcription factor that plays crucial roles in signal transduction. Immunocytochemistry detected the protein mainly in the nuclei. Western blot showed that laminin induced the highest level of its expression. Thyrotropin (TSH, 10 mU/ml) did not affect adhesion of the cells on any of the substrata or proliferation of the cells on fibronectin; nor did TSH affect c-fos protein expression of the cells on the substrata except for fibronectin. Our results suggest that type IV collagen and laminin, major components of basement membrane, play positive roles in proliferation and differentiation of follicle cells, respectively, while laminin has no positive effect on adhesion of the cells at early culture; that the cells express c-fos protein even in contact inhibition of growth and its expression is regulated in part by ECM; and that ECM controls some behaviors of the cells in a TSH-dependent or TSH-independent manner.

Cellular structure and function of rat fat cells in the primary culture.

A primary culture of undigested fat tissue fragments was used to obtain fat cells in vitro. On day 2 of culture, immature fat cells, which are fibroblast-like fat cells containing fine lipid droplets, appeared, surrounding the fat tissue fragments, and began to proliferate extensively. Afterwards, these fibroblast-like fat cells grew to become multilocular fat cells containing larger intracytoplasmic lipid droplets, and differentiated further into unilocular fat cells containing a single large intracytoplasmic lipid droplet. Treatment with dibutyryl-cAMP, which is a second messenger of the lipolytic factor, caused the cultured fat cells to retract, and the intracytoplasmic lipid droplets of those fat cells became finely granulated and decreased along with an increase of hormone-sensitive lipase activities. Conversely, administration of insulin caused the lipid droplets in the fat cells to increase and become larger along with an increase of alpha-glycerophosphate dehydrogenase activities. These findings indicate the occurrence of lipolysis and lipogenesis of fat cells in vitro. Immuno-cytochemistry revealed that vimentin surrounded intracytoplasmic lipid droplets, and became distinct with an increase of lipid droplets through lipogenesis in the fat cells. Vimentin seems to be correlated to the behavior of lipid droplets in the fat cells. Fat cells in this study showed the appropriate cellular structures and functions in response to stimulation of lipolysis and lipogenesis under culture conditions. It is expected that in vitro culture of fat cells will facilitate cell biological elucidation of obesity in the future.

A simple culture method of fat cells from mature fat tissue fragments.

To obtain immature fat cells in vitro, we used a primary culture of undigested mature fat tissue fragments. The immature fat cells, i.e., fibroblast-like fat cells, proliferated extensively from the fat tissue and differentiated after reaching confluence. The process of differentiation was assumed by the development of intracytoplasmic lipid droplets and by the triglyceride content in the cells. Cellular differentiation was induced in high percentages (over 70-80%) of the cells in the medium containing high glucose concentrations (200 mg/dl) supplemented with 10-20% newborn calf serum. The intracellular accumulation of triglyceride was also enhanced by insulin administration. In these cells, a reciprocal relationship was observed between proliferation and differentiation. Fibroblast-like fat cells derived from mature fat tissue in this simple culture system are suitable for the study of the proliferation and differentiation of immature fat cells.

Testicular tumors in non-twin brothers from a consanguineous marriage.

Malignant testicular tumors occurring in non-twin brothers are reported. Both of the brothers suffered from amentia and epilepsy and were the product of a consanguineous marriage. One brother presented with teratocarcinoma and the other seminoma. With a review of the literature, genetic roles in the etiology of testicular neoplasia are discussed.

Unilocular fat cells in three-dimensional collagen gel matrix culture.

Three-dimensional culture with collagen gel, developed recently for the in vitro study of some mammalian cells in a more physiological condition than a monolayer culture, was applied for a biological study of unilocular fat cells. Successfully embedded in the gel, the unilocular fat cells were shown to be able to keep their cellular function and actively proliferate. These findings confirm that unilocular fat cells do undergo proliferation under in vitro conditions as demonstrated in monolayer culture.