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Biomedical subjects

T Nozaki

Publications and source records attributed to T Nozaki.

At least 19 recordsLinked to original sources
PubMed2002

Trophoblast functions, angiogenesis and remodeling of the maternal vasculature in the placenta.

One of the most important local adaptations to pregnancy is the change in maternal blood flow to the implantation site. In rodents and primates, new blood vessels form through angiogenesis, dilate and then become modified such that the blood enters into trophoblast cell-lined sinuses (hemochorial). Evidence from gene knockout mice suggests that factors from the placenta regulate the uterine vasculature. Consistent with this, trophoblast giant cells produce a number of angiogenic and vasoactive substances that may mediate these effects. Teratocarcinomas containing large numbers of trophoblast giant cells (derived from Parp1 gene-deficient ES cells) show similar 'hemochorial' host blood flow, implying that the effects are not specific to the uterine vascular bed. As in primates, murine trophoblast cells also invade into the uterine arteries of the mother. However, in normal pregnancy, dilation of the uterine arteries may be largely mediated by the effect of uterine natural killer cells.

Angiogenesis Inducing Agents↗
PubMed2001

Efficacy of double filtration plasmapheretic cross-circulation using a high permeability membrane between totally hepatectomized dogs and donor pigs.

BACKGROUND: Major obstacles to develop a bioartificial liver are xenogeneic immune reactions and viral infection from donor pigs. To solve these problems, we studied the effect of xenogeneic double filtration plasmapheretic cross-circulation (DFPCC) using a high performance semipermeable membrane on totally hepatectomized dogs. METHODS: Mongrel dogs, weighing 12-15 kg, underwent total hepatectomy in one stage (n=18). One hr after total hepatectomy, the femoral vein and the jugular vein were cannulated in both dogs and pigs by using the blood access catheter tubes that were connected to the DFPCC system. In the DFPCC circuit, filtrated dog plasma and pig plasma counterflowed in a hollow fiber cartilage at a rate of 25 ml/min for 6 hr and met through a semipermeable membrane with 100 kd nominal molecular weight cut-off (n=5). In control dogs, the circuit was not connected to the pig (n=13). RESULTS: In vitro mass transfer study suggested that very little immunoglobulins crossed the semipermeable membrane. During and after 6 hr of DFPCC, anhepatic dogs had significantly lower blood ammonia and aromatic amino acid levels than did controls. DFPCC-treated dogs demonstrated decreased intracranial pressure and survived significantly longer than control dogs (20.75+/-3.80 hr vs. 14.75+/-1.30 hr, P<0.05). Histology showed no xenogeneic rejection in both dogs and pigs. CONCLUSIONS: Our DFPCC systems with a high permeability membrane demonstrated detoxification-function and contributed to intracranial decompression and longer animal survivals without adverse immune reaction and the possibility of zoonosis.

Animals↗
PubMed2001

Involvement of poly(ADP-ribose) polymerase in trophoblastic cell differentiation during tumorigenesis.

Poly(ADP-ribose) polymerase (Parp) monitors DNA strand breaks and poly(ADP-ribosyl)ates nuclear proteins using NAD as a substrate. The participation of Parp in DNA damage responses has been demonstrated by recent studies using Parp knockout mice. On the other hand, accumulated evidence has shown that Parp is involved in the regulation of gene expression and cell differentiation. In this study, the role of Parp in tumorigenesis and differentiation was studied with Parp-/- embryonic stem (ES) cells. When Parp+/+, Parp+/-, and Parp-/- ES cells were injected subcutaneously into nude mice, teratocarcinoma-like tumors developed from ES cells. However, only tumors derived from Parp-/- ES cells showed trophoblast giant cells (TGCs) containing single or multiple megalo-nuclei. These TGCs are located in a large blood-lake like hemorrhage. This example suggests that Parp is not essential for tumor formation, however, it is involved in trophoblastic cell differentiation and could consequently affect tumor phenotype.

Animals↗
PubMed2001

Purification and identification of major soluble 40-kDa antigenic protein from Entamoeba histolytica: its application for serodiagnosis of asymptomatic amebiasis.

One of the major soluble antigenic proteins of Entamoeba histolytica was purified to homogeneity and identified on a molecular basis. Its recombinant protein was expressed in Escherichia coli as a fusion protein with Shistosoma japonicum glutathione S-transferase. Apparent molecular weight of the purified antigenic protein was estimated to be 40-kDa and molecular-based analysis indicated that the purified protein was NADP+-dependent alcohol dehydrogenase (EhADH1). The application of the purified protein for the serodiagnosis of amebiasis was evaluated using an enzyme-linked immunosorbent assay applied to sera obtained from patients with amebiasis and healthy human controls. The purified protein was well recognized by the sera from asymptomatic amebiasis humans (22/22, 100%), whereas, it was less recognized by the sera from symptomatic amebiasis patients (5/16, 31%) with amebic colitis or liver abscess. To confirm the antigenicity of EhADH1, the recombinant glutathione S-transferase-EhADH1 fusion protein was also evaluated by the enzyme-linked immunosorbent assay using the same sera. The recombinant protein was also recognized by the sera from asymptomatic amebiasis humans (14/22, 64%) and less recognized by the sera from symptomatic amebiasis patients (2/16, 13%). These results suggest that the purified protein is applicable antigen for serodiagnostic screening of asymptomatic amebiasis humans.

Animals↗
PubMed2001

Acridine orange induces binucleation in chondrocytes.

OBJECTIVE: Although it is well known that binuclear cells commonly appear among the chondrocytes of normal cartilages as well as among neoplastic chondrocytes of chondrosarcomas, the mechanism of binucleation is still unclear. Therefore, this study was undertaken to clarify the mechanism of binucleation in chondrocytes, using primary culture cells of growth plate cartilage. DESIGN: These chondrocytes were exposed to acridine orange (AO) which is a fluorescent dye for differentiating certain DNAs and RNAs in nuclei and cytoplasm, and which inhibits mitosis. After exposure to 0.5 microg/ml AO, for 0, 6, 24, 48, and 96 h, the following parameters were investigated: (1) cell growth rate (GR); (2) frequency of hyperdiploid cells (%HDC) by DNA cytofluorometry; (3) mitotic index (MI); (4) BrdU labeling index (LI); (5) frequency of binuclear cells (%BNC). RESULTS: Compared with the control cells, which were cultured in AO-free medium, the GR was remarkably inhibited at 24 h. MI was also decreased from 6 to 24 h, and LI decreased at 48 h. However, these parameters were recovered at 96 h. The %HDC was increased from 6 to 96 h, and the %BNC was also increased to a maximum of six times that of the control cells at 96 h. DISCUSSION: These results suggested that the binuclear cells observed among the cultured chondrocytes may be formed from G2 arrested cells by amitotic nuclear division, but not by mitosis without cytoplasmic division or cell fusion.

Acridine Orange↗
PubMed2001

DNA cytofluorometric analysis of chondrocytes in human articular cartilages under normal aging or arthritic conditions.

OBJECTIVE: Since most chondrocytes in articular cartilage are in the resting phase (G0) of the cell cycle, it has been difficult to investigate their cell kinetics using 3H-thymidine autoradiography, or immunohistochemistry. In the present study, DNA cytofluorometry, which is useful to analyse the cell kinetics even for such inactive cell populations as in the G0 phase, was applied to human chondrocytes of the articular cartilages under normal aging and pathologic conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), and aseptic necrosis (AN). DESIGN: The human articular cartilages for the study were obtained from autopsy and surgical materials. Fifty joints were used for the study of aging, 54 for the study of OA, 20 for studying RA, and 10 for AN study. The isolated chondrocytes were quickly prepared from fresh articular cartilages, using a combination method of enzymatic digestion with papain and collagenase, followed by mechanical cell separation by churning and homogenization. RESULTS: The DNA histograms obtained by cytofluorometry with propidium-iodide staining showed that most chondrocytes had diploid DNA content (2c) in all cartilages studied, suggesting that they were in the G0 phase. However, there were a few chondrocytes having tetraploid DNA content (4c) in the normally aged articular cartilages, and there were some cells having DNA content between 2c and 4c in the diseased cartilages. The former cells were considered to be G0-phase cells of the 4c chondrocytes, while the latter cells were considered to be in the DNA synthetic (S) phase or G2-phase of the 2c chondrocytes. The frequency of 4c chondrocytes in aged cartilage was significantly increased, compared to that in the young cartilage. In contrast to the normal cartilage, the frequency of S- and G2-phase cells, which was expressed as the S- G2 index, in diseased cartilages (OA, RA and AN) was significantly high (P< 0.0001). In OA cartilage, the S-G2 index was much higher in the severe or moderate stage than in the mild stage, suggesting that the chondrocytes in clusters may actively proliferate. CONCLUSION: These results showed that in normal articular cartilages most chondrocytes are in the G0 phase, while some became 4c polyploid cells, and that these G0-phase chondrocytes had a potential to proliferate under diseased conditions.

Adolescent↗
PubMed2001

Increased susceptibility of poly(ADP-ribose) polymerase-1 knockout mice to nitrosamine carcinogenicity.

The involvement of poly(ADP-ribose) polymerase-1 (Parp-1), one of the poly(ADP-ribose) polymerase family proteins, in genomic stability, DNA repair and cell death triggered by DNA damage has been well documented. However, the potential role of Parp-1 in carcinogenesis has not been well evaluated. In this study the carcinogenic activity of N:-nitrosobis(2-hydroxypropyl)amine (BHP) was studied in Parp-1(-/-) mice, generated by disrupting P:arp-1 gene exon 1. Parp-1(-/-) and Parp-1(+/+) male mice received 0, 250 and 500 p.p.m. BHP in their drinking water for 20 weeks and were then killed. The percentage of animals bearing hemangiomas and hemangiosarcomas in the liver and numbers of tumors per mouse were markedly higher in the Parp-1(-/-) groups given 250 or 500 p.p.m. BHP than in their Parp-1(+/+) counterparts. Hemangiosarcomas developed only in Parp-1(-/-) mice. In the lung the numbers of adenomas per mouse were increased in Parp-1(-/-) mice given BHP at 250 and 500 p.p.m. (P < 0.01) compared with the Parp-1(+/+) case. The results show that susceptibility to BHP is significantly elevated in Parp-1(-/-) mice, thus providing direct evidence that Parp-1 is relevant to carcinogenesis.

Adenocarcinoma↗
PubMed2001

Adenosine regulates the IL-1 beta-induced cellular functions of human gingival fibroblasts.

In this study we examined the influence of adenosine on the cellular functions of human gingival fibroblasts (HGF), such as the production of inflammatory cytokines and extracellular matrices (ECM), and the expression and function of adhesion molecules. Concerning the expression of adenosine receptors, RT-PCR analysis revealed that HGF expressed adenosine receptor A1, A2a and A2b, but not A3 mRNA. Ligation of adenosine receptors by adenosine or its related analogue, 2-chloroadenosine (2-CADO), N(6)-cyclopentyladenosine (CPA) or CGS21680 synergistically increased IL-1beta-induced IL-6 and IL-8 production. In terms of ECM expression, adenosine and the adenosine receptor agonists, 2-CADO and CPA, enhanced constitutive and IL-1beta-induced expression of hyaluronate synthase mRNA, but not the mRNA levels of other ECM, such as collagen type I, III and fibronectin. Moreover, the adherence of IL-1beta-stimulated HGF to activated lymphocytes was also inhibited by adenosine, which is in part explained by the fact that adenosine down-regulated the IL-1beta-induced expression of ICAM-1 on HGF. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses in periodontal tissues.

2-Chloroadenosine↗
PubMed2001

Orexin-A and leptin change inversely in fasting non-obese subjects.

OBJECTIVE: Leptin, neuropeptide-Y (NPY) and orexin are peptides regulating energy metabolism and appetite control. NPY and orexin are mainly found in the central nervous system and they have also recently been found in the peripheral nervous system. We investigated how fasting affects changes in circulating concentrations of these peptides and their association with nutritional and metabolic parameters in humans. DESIGN AND METHODS: Ten non-obese female patients with psychosomatic disorders fasted for 7 or 10 days. Blood samples were collected at 0800 h before fasting, on the 3rd and 7th days during the fast (with an additional sample taken on the 10th day when the fasting continued for 10 days) and on the 3rd and 7th days of refeeding. We measured blood concentrations of orexin-A, NPY, leptin, adrenocorticotropin, cortisol, insulin, C-peptide, glucose, and beta-hydroxybutyrate. RESULTS: Body mass index and plasma leptin concentrations concomitantly and significantly decreased during fasting, whereas serum orexin-A concentrations significantly increased and were negatively correlated with plasma leptin concentrations. Plasma NPY concentrations decreased slightly but were not significantly different from the prefasting values, and no significant relationship with leptin or orexin-A was found. Orexin-A and leptin concentrations showed a significant inverse correlation with serum glucose, insulin, C-peptide, and beta-hydroxybutyrate concentrations. Only changes in plasma leptin concentrations showed a significant negative correlation with serum cortisol concentrations. All the measured indices which changed during fasting returned to the prefasting concentrations by the 7th day of refeeding. CONCLUSION: Peripheral orexin-A and leptin concentrations inversely change during fasting, which is significantly correlated with energy metabolism in humans.

Adolescent↗
PubMed2001

A sensitive method for detecting Porphyromonas gingivalis by polymerase chain reaction and its possible clinical application.

BACKGROUND: It is useful for the clinical diagnosis of periodontitis to monitor the colonization of periodontopathic bacteria in periodontal pockets. In this study, we attempted to establish and possibly identify the clinical application of a sensitive method to detect Porphyromonas gingivalis (P.g.), one of the putative periodontopathic bacteria related to chronic periodontitis. METHODS: Genomic DNA extracted from cultured P.g. 381 and clinically isolated subgingival plaque samples were used as a template of polymerase chain reaction (PCR). We designed primers to amplify the genomic DNA coding 40 kDa outer membrane protein (OMP), one of the unique proteins to all strains of P.g. The efficiency and specificity of amplification were evaluated by agarose gel electrophoresis and subsequent Southern hybridization with a digoxygenin-labeled oligonucleotide probe. RESULTS: Fewer than 100 P.g. bacterial cells in the specimen were reproducibly detected by PCR-hybridization assay. This PCR-hybridization assay was at least 100 times more sensitive than the conventional indirect immunofluorescence assay (IIF). Furthermore, the imaging analysis showed that there is a linear correlation between the strength of the signal and the cell number of P.g. from which the template DNA was extracted semiquantitatively. It is noteworthy that the PCR assay could also be applied to detect P.g. from clinical plaque samples and that it was approximately 100 times more sensitive than a conventional IIF assay. CONCLUSION: The PCR assay established in this study can be a powerful tool to detect P.g. in periodontal pockets and monitor the colonization and/or recolonization of P.g. at the very early phase.

Bacterial Outer Membrane Proteins↗
PubMed2001

Comparison of dihydrofolate reductase activities for folic acid in pigs and rats using in vivo and in vitro evaluation techniques.

The activity of dihydrofolate reductase (DHFR) for folic acid (PteGlu) was evaluated in pigs by in vivo and in vitro experiments. The results were compared with those of rats. Since bile secretion of reduced folates reflects the activity of DHFR for PteGlu in the body, the bile secretion rates of reduced folates including tetrahydrofolate (H4PteGlu), 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, and 10-formyltetrahydrofolate were determined by using high-performance liquid chromatography with electrochemical detection, after the intravenous injection of PteGlu at 1 mg/kg body weight to pigs and rats. Although the PteGlu injection raised the total secretion rate of reduced folates. the total increased amount of reduced folates secreted into bile from 0 h to 2.5 h after PteGlu injection in pigs was about one-tenth of that in rats. The enzyme kinetics of DHFR for PteGlu was examined at the physiological condition (pH 7.4 and 3 7 degrees C). Affinity chromatography was applied to liver homogenates of pigs and rats to obtain DHFR. The final product of the enzyme reaction, H4PteGlu, was measured. The Km for pig enzyme was similar to that for rat enzyme, whereas the Vmax for the pig enzyme was less than 1/5 of that for the rat's. The comparison of the ratio of Vmax to Km between pig and rat enzymes suggests that PteGlu is a much less efficient substrate for pig liver DHFR. In short, these results from in vivo and in vitro experiments suggest that the role of DHFR for PteGlu in pigs is physiologically much less important than that in rats.

Animals↗
PubMed2001

[Automated analysis on urine formed element by using FCM].

Recent advances in the automated instruments for morphological tests are significant. Although the urine sediment (urine formed element) analyzer was developed 20 years after the automated leukocyte differential analyzer, remarkable advances were made in these several years in Japanese manufacturers with an excellent technology. Now accurate and precise results can be obtainable quickly, especially, in RBCs & WBCs with unspun urine. Moreover, additional clinically useful findings are available such as RBC morphological information and Glitter cells of WBCs. At the moment, detailed examination of epithelial cells and casts is difficult but from now on, the automation of these fundamental tests will be improved increasingly by a development of analytical algorithm & staining technology, supported by the current laboratory needs for higher reliability and labor savings.

Autoanalysis↗
PubMed2000

Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma.

Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine beta-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies on T. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant T. cruzi SAT showed allosteric inhibition by l-cysteine, l-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways.

Acetyltransferases↗
PubMed2000

Identification of a novel prostaglandin f(2alpha) synthase in Trypanosoma brucei.

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH(2). Among all PGs synthesized, PGF(2alpha) was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF(2alpha) synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF(2alpha) in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

Amino Acid Sequence↗
PubMed2000

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei.

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B/GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.

Animals↗