Search PubMed⌕ Search

Biomedical subjects

Yasuhiro Yoshikawa

Publications and source records attributed to Yasuhiro Yoshikawa.

At least 73 records · Page 4Linked to original sources
PubMed2003

Spontaneous T-cell-rich B-cell lymphoma in a cynomolgus monkey (Macaca fascicularis).

A spontaneous T-cell-rich B-cell lymphoma (TCRBCL) occurred as a subcutaneous mass in the buccal region and enlarged submandibular lymph node in a 6-year-old female cynomolgus monkey (Macaca fascicularis). The constituent cells were examined by histology, immunohistochemistry and the double labeled-immunofluorescence method (dl-IF). Further, in situ hybridization (ISH) was employed to detect the gene expression of Epstein Barr virus (EBV). Histologically, the mass was comprised mainly of neoplastic large lymphoid cells and reactive small mononuclear cells. Immunohistochemically, the neoplastic large lymphoid cells were positive for CD20, CD79 alpha, MHC class II, and either IgG, IgM, or IgA. Polyclonal Ig production by the neoplastic large lymphoid cells was demonstrated by dl-IF, although IgG-positive ones predominated in number. On the other hand, most of the small mononuclear cells were positive for CD3 and were regarded as reactive T lymphocytes, while the remaining cells appeared to be histocytes or reactive B-cells. Transcripts of EBV gene were not demonstrated in these neoplastic or reactive cells by ISH. This is the first reported case of spontaneous TCRBCL in the cynomolgus monkey.

Animals↗
PubMed2003

Immunohistochemical analysis of protein gene product 9.5, a ubiquitin carboxyl-terminal hydrolase, during placental and embryonic development in the mouse.

Protein gene product 9.5 (PGP9.5) is expressed at high level in the neural and neuroendocrine systems. We investigated the localization and degree of expression of PGP9.5 in the developing mouse placenta and embryo at 6.5, 10.5 and 14 days of gestation using an immunohistochemical technique. At 6.5 days of gestation PGP9.5 was detected at various levels in decidual and primary trophoblast giant cells in the placenta, and in embryonic ectodermal cells in the embryo. At 10.5 and 14 days of gestation PGP9.5 was expressed at moderate to strong levels in neurons in the embryo, but rarely in the placenta. These findings suggest that the protein may play a significant role in implantation and placental development, and differentiation of embryonic ectoderm.

Animals↗
PubMed2003

Molecular evolution inferred from immunological cross-reactivity of immunoglobulin G among Chiroptera and closely related species.

We examined the relationships between Megachiroptera and Microchiroptera, and between Chiroptera and other closely related species by the cross-reactivity of immunoglobulin epitopes. Rabbit polyclonal antibody to bat IgG was used for determining the cross-reactivity by a competitive ELISA method. Megachiroptera and Microchiroptera showed high cross-reactivity, over 95.3%, with each other. However, primates and insectivores showed very low cross-reactivity, 8.6 to 20.2% and 5.3 to 12.7%, respectively. These results suggest that suborders of Chiroptera are monophyletic and Chiroptera have a relatively closer relationship to primates than to insectivores.

Animals↗
PubMed2003

D-galactosamine induced hepatocyte apoptosis is inhibited in vivo and in cell culture by a calcium calmodulin antagonist, chlorpromazine, and a calcium channel blocker, verapamil.

Studies were conducted in C57BL/6N Crj male mice and in cultured hepatocytes to clarify the relationship between galactosamine (GaIN) induced apoptosis and [Ca2+]i kinetics. Chlorpromazine (CPZ), a Ca(2+)-calmodulin antagonist, and verapamil (VR), a Ca(2+)-channel blocker each inhibited GaIN-induced DNA fragmentation and the appearance of apoptotic bodies. The kinetics of calcium uptake were evaluated using a calcium analyzer with the acetoxymethyl ester of fura-PE3 (fura-PE3/AM, 2.5 microM) as the calcium reporter. An increase in [Ca2+]i was detected in the cultured hepatocytes within 3 hours after treatment with 20 mM GaIN; this increase was inhibited by pretreatment with either 20 microM CPZ or 30 microM VR. Ca2+ imaging by confocal laser scanning microscopy showed that increase in [Ca2+]i after treatment with GaIN was initially localized around nuclei, while [Ca2+]i signals were later diffuse and observed throughout the cytoplasm. The activities of lactate dehydrogenase (LDH) and serum glutamate-pyruvate transaminase (sGPT), used as indicators of plasma membrane damage and leakage, however, were not reduced by pretreatment with CPZ or VR. From these findings, we infer that the DNA fragmentation in GaIN-induced hepatocyte apoptosis is associated with an elevation in the perinuclear concentration of Ca2+, but GaIN-induced necrotic cell death is triggered through pathway(s) that are insensitive to blockage of Ca2+ influx and therefore appear to occur independently of elevation in [Ca2+]i. These results help to clarify the role of calcium flux in hepatocyte apoptosis and necrosis induced by exposure to hepatotoxins in vivo and in vitro.

Animals↗
PubMed2003

Differences between BALB/c and C57BL/6 mice in mouse hepatitis virus replication in primary hepatocyte culture.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.

Animals↗
PubMed2002

Cryopreservation and primary culture of cerebral neurons from cynomolgus monkeys (Macaca fascicularis).

We established the procedures for cryopreservation and primary culture of fetal cerebral neurons of cynomolgus monkeys (Macaca fascicularis). Three developmental stages of fetuses (80, 93, and 102 days of gestation) were compared to determine the optimal stage of cerebrum development for primary culture. Among the three fetuses, the 80-day-old fetus produced the most process-rich neurons with the highest survival. The number of total recovery cells from the cryopreserved 80-day-old fetus corresponded to 83.4% of that from fresh tissue. Besides, synchronous oscillations of intracellular calcium were first seen in primate cerebral neurons, which suggested the formation of synapse-networks. Cultured neurons expressed synaptophysin protein. Successful cryopreservation and subsequent cell culture of primate neurons would be useful tools for neuroscience research with species specificity.

Animals↗
PubMed2002

The effects of diabetes with hyperlipidemia on P450 expression in APA hamster livers.

The effect of chronic hyperglycemia and hyperlipidemia induced by streptozotocin (SZ) on the expression of P450 in the liver of APA hamsters was studied in this experiment. No effect on the total activity of P450 was seen in SZ-induced diabetic hamsters throughout the experimental period. At 1 and 6 months after SZ-injection, the levels of CYP1A, 2C6, and 3A of SZ-injected hamsters were much lower than those of age-matched control hamsters. CYP2B expression tended to decrease and CYP2E1 and 4A expression tended to increase in SZ-injected hamsters, although the results were not significant. At 3 months after SZ-injection, however, no significant difference between SZ-injected and normal hamsters was seen in these P450 isozymes. On the other hand, CYP2C11 expression was slightly depressed in SZ1M and SZ6M, and almost equivalent to control hamsters in SZ3M. Immunohistochemistry by the use of each isozyme antibody revealed that SZ-induced diabetes affected the localization of CYP2C6, 3A, and 4A in the hepatic acinus. The expression of CYP2C6 and 3A was depressed mainly in the periportal region of the acinus, and CYP4A expression was induced mainly in the perivenous region by SZ-induced diabetes. On the other hand, the expression pattern of CYP1A, 2B, 2C11, and 2E1 were not affected. These results demonstrate that the effects of SZ-induced diabetes on hepatic P450 differ for each isozyme in APA hamsters and also differ from those of other experimental diabetic animals, including golden hamsters.

Animals↗
PubMed2002

Age-related telomere length dynamics in peripheral blood mononuclear cells of healthy cynomolgus monkeys measured by Flow FISH.

Telomere length is a good biomarker to study the cellular senescence as well as aging of an organism, because it regulates the replicative capacity of vertebrate somatic cells. To demonstrate age-related telomere length dynamics in the peripheral blood mononuclear cells (PBMC) of the cynomolgus monkey, we introduced a novel method of measuring telomere length by fluorescence in situ hybridization with a Peptide Nucleic Acid (PNA) labelled probe and flow cytometry (Flow FISH). A highly significant correlation was observed between the intensity of telomere-specific fluorescence by Flow FISH and telomere length by Southern blot analysis (R = 0.923, n = 22). The intensity of telomere fluorescence in PBMC significantly decreased with age in 55 monkeys aged from 0 to 34 years and this decrease corresponded to the loss of 62.7 base pairs per year (R = - 0.52, P < 0.00004). We also analysed the expression of naive cell-associated markers, CD28, CD62L and CD45RA/CD62L in T lymphocytes of 47 cynomolgus monkeys. An age-related increase in the CD28- subset was observed in CD8+ T lymphocytes in monkeys less than 11 years old and in CD4+ T lymphocytes in monkeys over 23 years old, respectively. The percentage of CD62L+ subsets was significantly decreased with age in both CD4+ (R = - 0.55) and CD8+ T lymphocytes (R = - 0.73). From the comparison of telomere length among PBMC, CD62L+ and CD62L- T lymphocytes, it was clearly evident that loss of naive subsets results in the shortening of telomere length in vivo. These results show that this method can be applicable to studying the turnover and precursor-progeny of PBMC in cynomolgus monkeys as an animal model of aging.

Aging↗
PubMed2002

Development of an immunofluorescence method for the detection of antibodies to Ebola virus subtype Reston by the use of recombinant nucleoprotein-expressing HeLa cells.

An indirect immunofluorescent assay (IFA) to detect Ebola virus subtype Reston (EBO-R) antibodies was developed by the use of a HeLa cell line stably expressing EBO-R nucleoprotein (NP). This IFA has a high specificity for the detection of EBO-R IgG antibodies in both hyperimmune rabbit sera and monkey sera collected during an EBO-R outbreak in the Philippines in 1996. Furthermore, this IFA showed a higher sensitivity for the detection of EBO-R antibodies than did the IFA using HeLa cells expressing the NP of Ebola virus subtype Zaire. These results suggest that this new IFA is useful for seroepidemiological studies of EBO-R infection among monkeys.

Animals↗
PubMed2002

Relationship between body weight and hematological and serum biochemical parameters in female cynomolgus monkeys (Macaca fascicularis).

Obesity is a risk factor triggering a variety of metabolic diseases. Cynomolgus monkeys (Macaca fascicularis) exhibit spontaneous onset of obesity in adulthood, similar to such onset exhibited by humans. To clarify the characteristics accompanying obesity in female cynomolgus monkeys, we used simple and multiple regression analyses to determine the relationship between body weight and hematological and serum biochemical parameters as well as obesity-related hormones, namely, leptin and insulin. Simple regression analysis showed that body weight was significantly (P < 0.05) correlated with leptin level, insulin level, hemoglobin concentration, hematocrit values, mean corpuscular volume, glucose concentration, and triglyceride concentration. In addition, a multiple regression model containing leptin level, insulin level, mean corpuscular volume, and red blood cell count explained 66.9% of the variance in body weight. Therefore, female cynomolgus monkeys show similar obesity characteristics to humans, i.e., obesity is associated with enhanced synthesis and excretion of leptin in adipocytes, high risk of diabetes mellitus, and high levels of hematocytes. Our results indicate that female cynomolgus monkeys are good models for studying obesity in humans.

Animals↗
PubMed2002

Analysis of cDNA coding MHC class II beta chain of the chimpanzee (Pan troglodytes).

The chimpanzee (Pan troglodytes, Patr) is the closest zoological living relative of humans and shares approximately 98.6% genetic homology to human beings. Although major histocompatibility complex (MHC) plays a critical role in T cell-mediated immune responses in vertebrates, the information on Patr MHC remains at a relatively poor level. Therefore, we attempted to isolate Patr MHC class II genes and determine their nucleotide sequences. The cDNAs encoding Patr MHC class II DP, DQ and DR beta chains were isolated from the cDNA library of a chimpanzee B lymphocyte cell line Bch261. As a result of screening, the clone 6-3-1 as a representative of Patr DP clone, clone 30-1 as a Patr DQ clone, and clones 4-7-1 and 55-1 having different sequences as Patr DR clones were detected. The clone 6-3-1 consisted of 1,062 nucleotides including an open reading frame (ORF) of 777 bp. In the same way, clone 30-1 consisted of 1,172 nucleotides including ORF of 786 bp, clones 4-7-1 and 55-1 consisted of 1,163 nucleotides including ORF of 801 bp. Except for five nucleotide changes, clones 4-7-1 and 55-1 were the same sequence. By comparison with the nucleotide sequences already reported on chimpanzee MHC class II beta 1 genes, clones 6-3-1, 30-1, 4-7-1 and 55-1 were classified as PatrDPB1*16, PatrDQB1*0302, PatrDRB1*0201 and PatrDRB1*0204, respectively. This is the first report to describe complete cDNA sequences of Patr DP and DQ molecules. The nucleotide sequence data of Patr MHC class II genes obtained in this study will be useful for the genotyping of Patr MHC class II genes in individual chimpanzees.

Amino Acid Sequence↗
PubMed2002

Chronological and spatial analysis of the 1996 Ebola Reston virus outbreak in a monkey breeding facility in the Philippines.

To describe the transmission pattern of natural infection with Ebola Reston (EBO-R) virus in a breeding colony, the chronological and spatial analysis of mortality during the 1996 EBO-R virus outbreak was done in this study. The EBO-R virus infection among monkeys in the facility was widespread. Over a period of 3 months, 14 out of 21 occupied units were contaminated with antigen positive animals. A large number of wild-caught monkeys were involved in this outbreak suggesting that wild-caught monkeys have a high susceptibility to EBO-R virus infection. In this outbreak, morbidity patterns for individual animal units were very different regardless of the type and size of cages, individual or gang cages. The results suggest that not only the cage size but also poor animal husbandry practices may be risk factors for the spread of EBO-R infection.

Animal Husbandry↗
PubMed2002

Expression of lipoprotein receptors in the aortic walls of diabetic APA hamsters.

Syrian hamsters of the APA strain (APA hamsters) have recently been demonstrated to develop atheromatous lesions in the aortic arches under the diabetic condition induced by a single injection of streptozotocin (SZ). Various lipoprotein receptors are reported to play important roles in atherogenesis mainly in vitro, while there are few reports on the relative expressions of these receptors in vivo. In this study, we therefore examined messenger RNA (mRNA) expressions of several lipoprotein receptors on the aortic arches of diabetic APA hamsters at 6, 14 and 26 weeks after the injection (WAI) of SZ. In semi-quantitative RT-PCR, scavenger receptor (SR)-AI, macrosialin (MS)/CD68, and receptor for advanced glycation end-products (RAGE) mRNAs showed significant increases at 6 WAI of SZ, and SR-AI and CD36 mRNA obviously increased until 26 WAI, as compared with the control. Low-density lipoprotein receptor mRNA showed a significant decrease at 14 and 26 WAI, and SR-BI mRNA significantly decreased at 6 and 14 WAI, as compared with the control. Very low-density lipoprotein receptor mRNA was at the same level as the control. By means of in situ hybridization, SR-AI, MS/CD68 and RAGE mRNA were detected in the foam cells of the fatty streaks at 6 WAI, which suggested that SR-AI, MS/CD68 and RAGE play crucial roles in the formation of the fatty streaks, the initial lesions of atherogenesis in diabetic APA hamsters. SR-AI and CD36 were also believed to be related to the progression of atherogenesis in this model.

Animals↗
PubMed2002

Cryopreservation of brain tissue for primary culture.

Factors affecting recovery of brain cells from cryopreserved cerebral tissues of fetal rats were examined based on yields of viable cells on cell culture. Favorable preservation was obtained with freezing small pieces (less than 1 mm cube) of brain tissues rather than whole tissues or dissociated single cells, and use of 10% dimethylsulfoxide as a cryoprotectant in liquid nitrogen. As for cell preparation procedures, cell survival was improved when tissues were heated at 32 degrees C during papain digestion and centrifugation. Under favorable conditions, the number of brain cells recovered from cryopreserved tissues corresponded to 20-30% of those from fresh control tissues. Immunocytochemical characteristics of cultured neurons, astrocytes, and oligodendrocytes from cryopreserved and fresh tissues were indistinguishable. Semi-quantitive analyses of microtubule-associated protein-2 (MAP-2) and synaptophysin revealed that there was no difference in the amounts of these markers between cultures from both fresh and cryopreserved tissues. These results suggest that most of all cell types including neurons were equally susceptible to the cryopreservation procedures. We concluded that cryopreservation in liquid nitrogen is an effective method for preservation of embryonic brain tissues for later use in cell culture studies.

Animals↗
PubMed2002

Changes in mRNA expression in mouse postnatal cochlea by differential display method.

We compared mRNA expression by mRNA differential display method in postnatal day 11 (P11), P13 and adult C3H/HeJ mouse cochlea. Forty-seven bands were differentially displayed on polyacrylamide gel when 27 patterns of PCR primer sets were used, and 24 of them showed a remarkable increase within only two days (P11 and P13). DNA sequences of the bands were analyzed for homology to known genes using the BLAST search. Most of the clones were identical to sequences of functions unknown. However, one of the clones showing an increase of mRNA expression between P11 and P13 was identified as mouse TIS7 which is known to markedly increase during differentiation of PC-12 cells to neurons by NGF-treatment. TIS7 expression may be involved in differentiation of neuronal progenitor cells to spiral ganglion cells by the initial sound input. The comparison among P11, P13 and adult mouse cochlear mRNA expressions investigated the genes involved in the various growing stages of the postnatal cochlea.

Acoustic Stimulation↗
PubMed2002

Renal function tests on diabetes-induced and non-induced APA hamsters.

Although it has been said that Syrian hamsters of the APA strain (APA hamsters) spontaneously develop glomerulosclerosis with age, more prominent and severe glomerulosclerosis with proteinuria as well as arteriosclerosis is induced in diabetic APA hamsters. In this study, in order to supply new information on APA hamsters, tests on renal function and histology were done on non-diabetic and streptozotocin (SZ)-induced diabetic APA hamsters (APA-N and APA-D, respectively), and the data were compared with those of normal Syrian (golden) hamsters (GOL). At 4, 8, 12, 20, and 32 weeks of age, the markers indicating renal function, serum urea nitrogen and creatinine levels and the urinary total protein level were measured and thereafter histological studies were done. Although there were no remarkable differences between APA-N and GOL in serum urea nitrogen and creatinine levels, APA-N excreted more urinary total protein from the early weeks of age. In APA-D, an apparent worsening in these markers indicating renal function was detected and diabetic nephropathy in this model was confirmed also in terms of renal function. In the histological studies, the major lesion observed in APA-D was diffuse glomerulosclerosis. This may mean that renal dysfunction in APA-D was mainly caused by the glomerular change and that it is similar to other experimental diabetic animals and human diabetic patients. These data show that the diabetic APA hamster is a desirable model of human diabetic nephropathy.

Animals↗
PubMed2002

Histopathology of natural Ebola virus subtype Reston infection in cynomolgus macaques during the Philippine outbreak in 1996.

We investigated the livers, spleens, kidneys and lungs collected from 24 cynomolgus macaques (Macaca fascicularis) naturally infected with Ebola virus subtype Reston (EBO-R) during the Philippine outbreak in 1996, in order to reveal the histopathologic findings. These macaques showed necrotic hepatocytes with inclusions, slight to massive fibrin deposition in splenic cords, depletion of lymphoid cells in the white pulp of the spleen, and fibrin thrombi in some organs. Immunohistochemical analysis using anti-leukocyte antigen L1 antibody revealed an increase in blood-derived macrophages/monocytes in the livers, kidneys and lungs of EBO-R infected macaques. EBO-R NP antigens were detected in the macrophages/monocytes, endothelial cells and fibroblasts in the liver, spleen, kidney and lung. These results indicate that EBO-R infection is characterized by systemic coagulopathy and an increase in blood-derived macrophages/monocytes in accordance with the EBO-R propagation in macrophages/monocytes.

Animals↗