Biomedical subjects

N Fukunaga

Publications and source records attributed to N Fukunaga.

At least 19 recordsLinked to original sources

PubMed2001

Crystallization and preliminary X-ray diffraction studies of monomeric isocitrate dehydrogenase by the MAD method using Mn atoms.

NADP(+)-dependent isocitrate dehydrogenase (E.C. 1.1.1.42; IDH) is an enzyme of the Krebs cycle and catalyzes the oxidative decarboxylation reaction from DL-isocitrate to alpha-ketoglutarate, with a concomitant reduction of the coenzyme NADP(+) to NADPH. Single crystals of monomeric IDH from Azotobacter vinelandii in complex with DL-isocitrate and Mn(2+) were obtained by the hanging-drop vapour-diffusion method at room temperature. One crystal diffracted to a resolution of 2.9 A and was found to belong to the orthorhombic system; the space group was determined to be P2(1)2(1)2(1), with unit-cell parameters a = 108.4, b = 121.7, c = 129.7 A. The asymmetric unit contains two molecules of monomeric IDH, corresponding to a V(M) value of 2.66 A(3) Da(-1). The crystals were frozen in a capillary by a flash-cooling technique and MAD data were collected using Mn atoms as anomalous scatterers on beamline BL41XU at SPring-8, Japan. The positions of two Mn atoms binding to two independent IDH molecules were located from Bijvoet difference Patterson maps.

Azotobacter vinelandii↗

PubMed2001

Isolation of virulent Rhodococcus equi from native Japanese horses.

R. equi was isolated from soil samples obtained from the environment of seven native Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, Miyako and Yonaguni) and from fecal samples collected from three native horse breeds (Hokkaido, Kiso and Misaki). Virulent R. equi at various levels (ranging from 0.5 to 12.9%) was isolated from the feces or soil environment of Hokkaido, Kiso and Misaki horses. Isolates were investigated both for the presence of 15- to 17-kDa antigens (virulence-associated protein antigens; VapA) by colony blotting, using the monoclonal antibody 10G5, and the gene of VapA by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases, and the digestion patterns of the plasmids of virulent isolates were divided into three types. Two of the three types (87-kb type II and 90-kb type I) had already been reported in Japanese isolates, and a new type (tentatively designated as 90-kb type II) had been found in isolates from Kiso horses. Six virulent R. equi isolates from the Hokkaido horses contained an 87-kb type II plasmid. Eight of 24 isolates from the Kiso horses contained an 87-kb type II plasmid, and the remaining 16 contained a 90-kb type II (a new type) plasmid. Two isolates from the Misaki horses contained a 90-kb type I plasmid. These results demonstrate the geographic difference in the distribution of virulence plasmids in R. equi isolates among native Japanese horses.

Actinomycetales Infections↗

PubMed2001

Efficiency of using frozen-thawed testicular sperm for multiple intracytoplasmic sperm injections.

PURPOSE: To compare fertilization and pregnancy rates of fresh and frozen-thawed testicular sperm injections (TESE-ICSI). METHODS: Sperm collected from the testes of 28 azoospermic patients by an open testicular biopsy technique was used for initial ICSI or cryopreserved. RESULTS: Fresh-sperm ICSI treatment (28 cycles) resulted in a 58.1% fertilization rate and a 32.1% clinical pregnancy rate per embryo transfer, while frozen-thawed sperm (24 subsequent cycles) had rates of 54.5 and 29.2%, respectively. The PR was lower using frozen-thawed sperm from nonobstructive azoospermia patients (9.1%) than from obstructive azoospermia patients (46.2%). PR declined to 0% upon the fourth ICSI attempt. CONCLUSIONS: Fertilization, embryo cleavage, and pregnancy rates were unaffected by fresh or frozen-thawed sperm use. A 57.1% cumulative clinical PR was achieved using the latter. The PR was significantly lower using frozen-thawed sperm from nonobstructive azoospermia patients than from obstructive azoospermia patients.

Biopsy↗

PubMed2001

Pregnancy achieved following ICSI from a man with Klinefelter's syndrome and spinal cord injury.

Klinefelter's syndrome and spinal cord injury are major causes of male infertility. Intracytoplasmic sperm injection (ICSI) is a relatively new method of assisted reproduction. A testicular biopsy was obtained from a patient with the double complications of non-mosaic 47,XXY Klinefelter's syndrome and spinal cord damage, and motile spermatozoa were collected. ICSI was then performed. Of the four sperm-injected oocytes, three became fertilized and cleaved. Two embryos were implanted, resulting in a single pregnancy with visible evidence of a heartbeat appearing at 6 weeks gestation. The pregnancy is now entering its 20th week. To the best of our knowledge, this is the first case of a pregnancy resulting from the sperm of a patient with double complications.

Adult↗

PubMed2001

Purification and characterization of a cold-adapted isocitrate lyase and a malate synthase from Colwellia maris, a psychrophilic bacterium.

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20 degrees C and was rapidly inactivated at the temperatures above 30 degrees C, but the optimum temperature for the activity of MS was 45 degrees C. NaCl was found to protect ICL from heat inactivation above 30 degrees C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.

Cold Temperature↗

PubMed2000

Resistance induction in barley coleoptile cells by intracellular pH decline.

Cytoplasmic acidification in suspension-cultured plant cells has been characterized as a common intracellular response of some kinds of plant cells to elicitors. Expression of various defense genes in these cells has been increased by the cytoplasmic acidification itself without treatment by elicitors. It is not evident, however, whether or not cells with acidified cytoplasm actually exhibit resistance to the pathogen because of the lack of an adequate infection system between cultured plant cells and some pathogens. Using barley coleoptiles rather than suspension-cultured cells, we demonstrated both detection of cellular pH decline and increased resistance to Blumeria graminis. The cytoplasmic pH of barley coleoptile cells floated on 1 mM citrate buffer (CB), pH 4.0, became 0.5 unit lower than that of cells floated on 1 mM CB, pH 8.0, within 30 min after treatment. The penetration efficiency of B. graminis into the coleoptile was decreased in a pH-dependent manner; that is, when the coleoptiles were floated on 1 mM CB, pH 8.0, the penetration efficiency of the fungi was about 80%. In contrast, when the coleoptiles were floated on acidic buffers, the penetration efficiency decreased in parallel the decline of pH and the penetration efficiency reached 0% when coleoptiles were floated on 1 mM CB, pH 4.0. Morphogenesis of appressoria on the coleoptiles floating on CB was not influenced. The lowered penetration efficiency at lower pH was partially cancelled when the barley coleoptiles were irradiated with UV for 5 min prior to B. graminis inoculation. These findings suggest that the decline in cytoplasmic pH in barley coleoptile cells increases resistance to the pathogenic fungus B. graminis.

Cytoplasm↗

PubMed2000

Separation of lactoferrin-a and -b from bovine colostrum.

Bovine lactoferrin was separated into lactoferrin-a and lactoferrin-b from bovine colostrum. Lactoferrin-a was eluted at 0.38 M NaCl and lactoferrin-b was eluted at 0.43 M NaCl by carboxymethyl cation-exchange chromatography at pH 7.7, 0.05 M phosphate buffer. The molecular weights were estimated at 84,000 for lactoferrin-a and 80,000 for lactoferrin-b. Lactoferrin-a contents were 258.0 mg/L and lactoferrin-b contents were 524.3 mg/L of colostrum for cow 19. From colostrum to normal milk, total lactoferrin was from 17.1 to 129.4 mg/L during the normal lactational period; however, lactoferrin did not separate clearly into lactoferrin-a and lactoferrin-b. The lactoferrin-a measured from six cows was 258.0, 114.0, 112.8, 64.0, 59.7, and 22.4 mg/ L and the lactoferrin-b 524.3, 331.8, 184.7, 170.7, 129.3, and 44.0 mg/L, respectively. The average was 105.2 mg (31.3%) for lactoferrin-a and 230.8 mg (68.7%) for lactoferrin-b.

Animals↗

PubMed1999

NH4+ transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1.

NH4(+) transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [14C]methylammonium ion (14CH3NH3+) into the intact cells. 14CH3NH3+ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH4Cl instead of glutamate. Vibrio ABE-1 did not utilize CH3NH3+ as a carbon or nitrogen source. NH4Cl and nonradiolabeled CH3NH3+ completely inhibited 14CH3NH3+ uptake. These results indicate that 14CH3NH3+ uptake in this bacterium is mediated via an NH4+ transport system and not by a specific carrier for CH3NH3+. The respiratory substrate succinate was required to drive 14CH3NH3+ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H+ and/or Na+ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated 14CH3NH3+ uptake. The 14CH3NH3+ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0 degrees and 15 degrees C, and the apparent Km value for CH3NH3+ of the uptake did not change significantly over the temperature range from 0 degrees to 25 degrees C. Thus, the NH4+ transport system of this bacterium was highly active at low temperatures.

Ammonium Chloride↗

PubMed1999

cis-Acting elements responsible for low-temperature-inducible expression of the gene coding for the thermolabile isocitrate dehydrogenase isozyme of a psychrophilic bacterium, Vibrio sp. strain ABE-1.

Transcriptional control of the low-temperature-inducible icdII gene, encoding the thermolabile isocitrate dehydrogenase of a psychrophilic bacterium, Vibrio sp. strain ABE-1, was found to be mediated in part by a transcriptional silencer locating at nucleotide positions -560 to -526 upstream from the transcription start site of icdII. Deletion of the silencer resulted in a 20-fold-increased level of expression of the gene at low temperature (15 degrees C) but not at high temperature (37 degrees C). In addition, a CCAAT sequence located 2 bases upstream of the -35 region was found to be essential for the low-temperature-inducible expression of the gene. By deletion of this sequence, low-temperature-dependent expression of the gene was completely abolished. The ability of the icdII promoter to control the expression of other genes was confirmed by using a fusion gene containing the icdII promoter region and the promoterless icdI open reading frame, which encodes the non-cold-inducible isocitrate dehydrogenase isozyme of Vibrio sp. strain ABE-1. Escherichia coli transformants harboring icdII acquired an ability to grow rapidly at low temperature.

Adaptation, Biological↗

PubMed1998

Pathogenicity of Rhodococcus equi strains possessing virulence-associated 15- to 17-kDa and 20-kDa antigens: experimental and natural cases in pigs.

The pathogenic role of Rhodococcus equi in pigs remains controversial. Small numbers of pigs were inoculated intravenously (i.v.), or intramuscularly (i.m.) around the mouth, with a virulent, an intermediately virulent, or an avirulent strain of R. equi and killed 14 days later. None showed clinical signs other than transient fever and weight loss. The virulent and intermediately virulent strains were recovered in culture from various organs and lymph nodes of pigs inoculated i.v., but only from the mandibular lymph nodes of pigs inoculated i.m. The avirulent strain was not recovered from any site. None of the pigs developed macroscopically visible lesions, but they showed reactive hyperplasia of the mandibular lymph nodes. The latter contained scattered phagocytic cells, which were labelled immunohistochemically for virulence-associated antigens (15- to 17-kDa antigens or 20-kDa antigen). Intermediately virulent and virulent strains of R. equi were isolated from mandibular lymph nodes of 5.5% of apparently healthy abattoir pigs (n = 1615). Virulence-associated antigens were detected in phagocytic cells of culture-positive nodes, but the latter showed no lesions other than reactive lymphoid hyperplasia. The results would seem to question the pathogenic role of R. equi in pigs, and it is speculated that the organism survives in the lymph nodes without causing pathognomonic lesions.

Actinomycetales Infections↗

PubMed1997

Downregulation of DNA topoisomerase I in old versus young human diploid fibroblasts.

DNA topoisomerase I (Topo I) is an enzyme that alters the superhelicity of DNA. It has been implicated in such critical cellular functions as transcription, DNA replication, and recombination. Roles for Topo I in DNA repair following DNA damage have also been studied extensively. In the present investigation, we examined the regulation of Topo I expression and activity during cellular replicative senescence. We found that the capacity of Topo I to relax supercoiled DNA molecules is significantly decreased in senescent diploid fibroblasts when compared to young (early passage) fibroblasts. We also found that the steady-state expression level of Topo I mRNA is correlated with enzyme activity, i.e., decreased in early vs. late passage cells. We also treated early and late passage cells with agents that may modulate the process of cellular senescence: UV light, retinoic acid, and interleukin-1 beta. We found that all three agents decreased the activity of Topo I in young fibroblasts and increased the activity of Topo I in senescent fibroblasts. This effect was most striking following exposure of the cells to retinoic acid, so to analyze this effect, we postulated an age-dependent kinetics of Topo I mRNA induction in response to retinoic acid. Consistent with this postulate, we found that whereas exposure of early passage cells to retinoic acid results, in a matter of hours, in a decrease in the expression of Topo I mRNA, exposure of the senescent cells to retinoic acid results in an increased expression. These observations suggest that processes that are altered in senescent fibroblasts, such as DNA replication and repair, may be due, in part, to alteration in the expression and activity of DNA Topo I.

Cells, Cultured↗

PubMed1996

Low-grade fibromyxoid sarcoma.

A low-grade fibromyxoid sarcoma arising in the thigh of a 16-year-old Japanese girl is described. The patient had a well-circumscribed mass measuring 3.5 cm in its greatest diameter within her left vastus medialis muscle and a 6-month history of pain. Microscopically, the tumour was not encapsulated and filtrated into adjacent skeletal muscle. The tumour was characterized by poor to moderate cellularity, a proliferation of bland-appearing spindle tumour cells, and alternating fibrous and myxoid areas with a whorled pattern of the tumour cells. Neither cellular atypia nor mitotic figures were observed. There was no tumour necrosis. Immunohistochemically, the tumour cells were diffusely and strongly positive for vimentin and desmin. Some cells contained alpha smooth muscle actin. They were uniformly negative for CAM5.2, epithelial membrane antigen, muscle-specific actin (HHF35), factor-VIII-related antigen, S-100 protein, neurofilament, CD34, and CD31. The tumour had a diploid DNA content with S-phase fractions of 6.6% by flow cytometry. The patient was alive with no evidence of disease 11 months after excision.

Actins↗

PubMed1996

Expression of virulence-associated antigens of Rhodococcus equi is regulated by temperature and pH.

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10(6) cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10(7) cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.

Antibodies, Bacterial↗

PubMed1996

Oncocytic hepatocellular carcinoma with numerous globular hyaline bodies.

Two well circumscribed tumors, oncocytic and non-oncocytic, were removed from the non-cirrhotic liver of a 67 year old male. The large oncocytic tumor (OCT), occupying the entire left lobe, was multilobulated with focal coagulation necrosis and areas of hemorrhage. Light microscopy revealed that it consisted of exclusively large, granular oxyphilic cells with moderate nuclear atypia and occasional mitotic figures, which were trabecular and/or pseudoglandular in structure, but no lamellar fibrosis was seen. Characteristically, the OCT cells included numerous globular hyaline bodies (GHB) of various sizes which were stained red with acid fuchsin and deep blue or magenta with phosphotungstic acid hematoxylin (PTAH), but negative for periodic acid Schiff (PAS), orcein, rhodamine and Grimelius methods. Immunohistochemically, alpha-fetoprotein (AFP), alpha-1-antitrypsin, alpha-1-antichymotrypsin, fibrinogen and ferritin were all negative. On ultrastructural examination, tumor cells were mitochondria-rich, including electron dense, ovoid or polyhedral inclusions, with the delineated membrane identical with that of the GHB. In contrast, the small tumor in the right lobe (Segment 7) was a solid adenoma with no oncocytic transition. Based on these findings, it was postulated that OCT consists of heterogenous proliferation of mitochondria-rich hepatocytes which tend to induce lysosomal GHB closely associated with mitochondrial abnormalities.

Adenocarcinoma↗

PubMed1996

Identification of intermediately virulent Rhodococcus equi isolates from pigs.

We recently reported the existence of Rhodococcus equi isolates with at least three virulence levels, isolated from AIDS patients: virulent R. equi having 15- to 17-kDa antigens that kills mice with 10(6) cells, intermediately virulent R. equi having a 20-kDa antigen that kills mice with 10(7) cells, and avirulent R. equi that does not kill mice with 10(8) cells or more (S. Takai, Y. Imai, N. Fukunaga, Y. Uchida, K. Kamisawa, Y. Sasaki, S. Tsubaki, and T. Sekizaki, J. Infect. Dis. 172:1306-1311, 1995). Virulent R. equi having the 15- to 17-kDa antigens has been isolated frequently from horses and their environment, but the source of intermediately virulent R. equi having the 20-kDa antigen is poorly understood. There are many reports of the isolation of R. equi from the lymph nodes of pigs with and without lesions resembling those of tuberculosis. Therefore, we analyzed antigens of R. equi isolates from the submaxillary lymph nodes of pigs by immunoblotting with monoclonal antibodies against these virulence-associated antigens. Immunoblots of whole-cell antigen preparations of R. equi pig isolates revealed the presence of the 20-kDa antigen in almost all the pig isolates studied, and these isolates were intermediately virulent for mice. We also demonstrated that the expression of the 20-kDa antigen and its pathogenicity in mice were associated strongly with the presence of five large, distinct plasmids of 70 to 95 kb; two of the five plasmids from pig isolates were the same sizes as those from human isolates. These results suggest that R. equi having the 20-kDa antigen exists in the submaxillary lymph nodes of pigs and that the source of infection in some human cases might be associated with pigs and their environment.

AIDS-Related Opportunistic Infections↗

PubMed1996

Purification of membrane-bound ATPase of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 and characterization as a F0F1-type enzyme.

The ATPase bound to the inner membrane of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1) was extracted with Triton X-100 and purified by fractionation with polyethylene glycol, sucrose density gradient centrifugation, and DEAE-Toyopearl 650M column chromatography. The molecular masses of subunits constituting the purified ATPase were estimated as 54, 49, 33.5, 27, 23.5, 18.5, and 15 kDa by SDS-PAGE. The composition and molecular masses of the subunits of the purified ATPase were similar to those of Escherichia coli F0F1-ATPase (EF0F1). The 54-, 49-, and 18.5-kDa polypeptides of the Vibrio ABE-1 ATPase strongly cross-reacted with the antibodies against the EF0F1 alpha, beta, and b subunits, respectively. However, the Vibrio ABE-1 ATPase contained no cross-reactive polypeptide with the antibodies against A and B subunits of V-type H(+)-ATPase from mung bean tonoplasts. The ATPase activity showed two pH optimum peaks at pH 5.3 and 8.0 and was strongly inhibited by N,N'-dicyclohexyl carbodiimide (DCCD) and NaN3. It hydrolyzed ATP, GTP, and ITP at similar rates. These properties confirm that the purified ATPase is a F0F1-type. The optimum temperature for the ATP-hydrolyzing activity of the enzyme was observed at 50 degrees C, but the DCCD-sensitivity of the enzyme was markedly decreased above 30 degrees C, suggesting that the F1-moiety is released from the enzyme complex at high temperatures. This characteristic is compatible with the psychrophilic nature of Vibrio ABE-1.

Cross Reactions↗

PubMed1996

Isolation of virulent and intermediately virulent Rhodococcus equi from soil and sand on parks and yards in Japan.

Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. However, little is known about the distribution of virulent and intermediately virulent R. equi in human environment. In the present study, R. equi was isolated from 173 of 234 (73.9%) samples collected from soil and sand on 115 parks and 49 yards in Japan. The numbers of R. equi from soil and sand ranged from 2.5 x 10(1) to 1.2 x 10(5) per gram of sample. None of 1,294 isolates from those samples showed virulence-associated 15- to 17-kDa antigens and a 20-kDa antigen. These results suggest that avirulent R. equi is widespread in parks and yards, but the human environment has not been contaminated with virulent and intermediately virulent R. equi strains yet.

Antigens, Bacterial↗

PubMed1995

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Adenoviruses, Human↗