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M Sakaguchi

Publications and source records attributed to M Sakaguchi.

At least 235 records · Page 13Linked to original sources
PubMed1996

Evaluation of the radiosensitizing effects of RK28 intravenous, intraarterial, and intratumoral injections on the rabbit VX2 tumor system.

PURPOSE: To evaluate the differences in the radiosensitizing effects of intravenous (i.v.) injection, intraarterial (i.a.) injection, and intratumoral (i.t.) injections of the hypoxic cell radiosensitizer RK28 ([1-(4'-hydroxy-2'-butenoxy)methyl-2-nitroimidazole], a 2-nitroimidazole with an acyclic sugar analogue substituted at the N-1 position of the imidazole ring) using an animal experimental system. METHODS AND MATERIALS: Rabbit VX2 tumors, which were implanted in the muscle of left hind legs and grown to 3 cm in diameter, were treated with RK28 (80 mg/kg x b.wt.) before 15 Gy of local x-ray irradiation. The auricular vein and the left saphenous artery were used for systemic injection and regional injection, respectively. For i.t. injection, a 21-gauge needle with three lateral holes was positioned in the central area of the tumor. Tumor regression was precisely evaluated by computed tomograpy (CT), and survival time was also studied. Using high-performance liquid chromatography (HPLC), pharmacokinetic studies for RK28 and its seven major metabolites were performed in tumor and serum at 0, 10, 20, 30, and 60 min after drug injection was completed. RESULTS: Radiosensitizing effects of RK28 were considered present after i.a. injection (p < 0.05) and i.t. injection (p < 0.05) after analyzing tumor volumes on day 21 after treatment. Increased survival was not observed in any group with RK28 injection compared with survival in the group treated by x-ray irradiation alone. Pharmacokinetic studies showed the average concentration of RK28 in the tumor during x-ray irradiation was 1.3 times higher after i.a. injection and 3.5 times higher after i.t. injection than that after i.v. injection. The time modifying factor50 (TMF50: ratio of time for tumor to decrease by 50%, radiation alone vs. radiation plus drug) was calculated to be 1.5 after i.v. injection, 1.7 after i.a. injection, and 2.3 after i.t. injection. The values of TMF50 correlated to the average concentrations of RK28 in the tumor. As to metabolites of RK28, beta-glucuronated compound and cysteine conjugate were highly detected. The concentrations of cysteine conjugate were higher in the tumor than in serum via i.v. injection. CONCLUSIONS: Radiosensitizing effects of RK28 were observed on the rabbit VX-2 tumor system after i.a. or i.t. injection. Pharmacokinetic studies proved that radiosensitizing effects depended on the concentration in the tumor, though the administration routes were different. Combined forms with nonprotein thiols were detected. However, survival benefits were not obtained by RK28. For clinical applications of RK28, i.a. or i.t. injection could facilitate better local control of cancer.

Animals↗
PubMed1996

Effects of the 7 21 Robertsonian translocation on fertilization rates and preimplantation development of bovine oocytes in vitro.

A study was conducted to investigate the effect of the 7/21 Robertsonian translocation on fertilization and subsequent development of bovine oocytes matured in vitro. Semen from Japanese Black bulls, 2 with a normal karyotype (Bulls A and B) and 2 that were heterozygous for the 7/21 translocation (Bulls C and D), was used in this study. In vitro matured bovine oocytes were inseminated with frozen-thawed sperm capacitated with heparin. After insemination, oocytes were cultured at 38.5 degrees C on a monolayer of cumulus cells in TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate in an atmosphere of 2% CO2 in air. Cleavage rate was evaluated at 54 h after insemination, and development of embryos to the blastocyst stage was observed 7 to 10 d post insemination. There was no difference in the fertilization rate among the 4 bulls. Although the cleavage rate of oocytes inseminated with semen from Bull C (heterozygote) was lower (P < 0.05) than that obtained with semen from Bull B (normal), the blastocyst formation rate did not differ among the 4 bulls. These results indicate that the 7/21 Robertsonian translocation had no effect on the fertilization and blastocyst formation rates of bovine in vitro-matured oocytes.

Journal Article↗
PubMed1996

Structure, development, and maintenance of the nerve net of the body column in Hydra.

The anatomy and developmental dynamics of the nerve net in the body column of Hydra viridissima were examined immunocytochemically with a monoclonal antibody (CC04) that recognizes an antigen in nerve cells and with an antiserum against vasopressin. CC04+ neuron cell bodies, their neurites, and vasopressin-like-immunoreactive (VLI+) neurites could be clearly visualized on whole-mount preparations. All neurites of the CC04+ neurons in the body column were VLI+. However, only half of the VLI+ neurites in the body column were CC04+. Immunocytochemical analysis of macerated preparations showed that half of the neurons in the gastric region of the body column were CC04+. These results suggest that most of the neurons in the gastric region are VLI+. The density of the VLI+ neurites was uniform along the entire length of the body column. The CC04+ neuron density in the gastric region remained constant at all stages of asexual development and during foot regeneration. After pulse-labeling with 5-bromo-2'-deoxyuridine (BrdU), CC04+ neurons with labeled nuclei appeared in the body column. We conclude that neuron density in the gastric region is maintained at a constant value by insertion of new neurons in parallel with continuous epithelial cell division.

Animals↗
PubMed1996

Transgenic expression of L-gulono-gamma-lactone oxidase in medaka (Oryzias latipes), a teleost fish that lacks this enzyme necessary for L-ascorbic acid biosynthesis.

Transfer of the gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis in scurvy-prone animals, into medaka (Oryzias latipes) was successfully done. The expression plasmid pSVL-GLO, carrying rat liver L-gulono-gamma-lactone oxidase cDNA, was microinjected into the cytoplasm of fertilized eggs during the one-cell stage. Four male F0 fish having the transgene in their germ cells came to maturity, and F1 progeny derived from one of the F0 fish possessed L-gulono-gamma-lactone oxidase activity, indicating that the transgene was functionally expressed in the fish. Genomic Southern blot analysis demonstrated that the transgene existed in both chromosome-integrated and extrachromosomal forms.

Animals↗
PubMed1996

Reinitiation of protein translocation across the endoplasmic reticulum membrane for the topogenesis of multispanning membrane proteins.

The reinitiation of the translocation of the growing nascent chain across the endoplasmic reticulum membrane is essential for the topogenesis of multispanning membrane proteins. We investigated the requirements for the reinitiation process using model proteins in which systematically designed sequences were inserted after two preceding topogenic sequences, namely the N-terminal signal sequence (S) and stop transfer sequence (St). The model proteins were translated in vitro in the presence of rough microsomes, and the final topology of the proteins in the microsomal membrane was examined by proteolytic digestion. The structural requirements for S and the reinitiation sequence (R) overlapped to some extent, but substantial differences were noticed. When St and R were separated by a short cytoplasmic segment (58 amino acids), the efficiency of the reinitiation was not affected by the concentration of the signal recognition particle (SRP) in the translation system, even though the sequence inserted as R was an SRP-dependent signal sequence. However, when the cytoplasmic segment was longer (100 amino acids), the reinitiation efficiency was reduced, and the SRP improved the overall efficiency as well as impaired the accessibility of the processing site after the R to the signal peptidase.

Amino Acid Sequence↗
PubMed1996

Inversely correlated expression of p16 and Rb protein in non-small cell lung cancers: an immunohistochemical study.

Cdk4-mediated phosphorylation of Rb protein is inhibited by p16, a product of a possible tumor suppressor gene. We examined the expression of p16 and Rb protein by means of immunohistochemistry in 61 non-small cell lung cancers and have demonstrated an inverse relationship between the expression of p16 and Rb protein: 28/30 specimens that did not stain for p16 stained for Rb and 21/31 p16-positive specimens did not stain for Rb. Only 1 of the p16-negative specimens had a mutation of exon 2 of the CDKN2 gene. Our results indirectly support the theory that p16 expression is negatively regulated by the functional Rb protein.

Base Sequence↗
PubMed1996

Enhancement of delayed rectifier K+ current by P2-purinoceptor stimulation in guinea-pig atrial cells.

1. We studied the effects of P2-purinoceptor stimulation on the delayed rectifier K+ current (IK) in guinea-pig atrial myocytes using a whole-cell voltage-clamp technique. 2. External application of ATP increased IK, evoked by a 500 ms depolarizing pulse from a holding potential of -40 mV, under conditions in which the L-type Ca2+ channel was blocked; the effect was dose dependent with a half-maximal concentration (K1/2) of 0.95 microM. ATP (50 microM) produced a maximal increase of IK of about a factor of 2. 3. External ADP also enhanced IK in a dose-dependent manner with a K1/2 of 3.65 microM, whereas adenosine (100 microM) failed to evoke this response. Theophylline (500 microM), a blocker of the Pi-purinoceptor, did not antagonize the stimulating action of ATP on IK. These results indicate that IK was enhanced via P2-purinoceptors. 4. External ATP or ADP did not produce a significant change in the current kinetics of IK. 5. Pre-incubation of the atrial myocytes with pertussis toxin (PTX, 5 micrograms ml-1) did not affect the stimulating action of ATP on IK, indicating that PTX-sensitive G proteins did not mediate the ATP action. 6. The enhancement of IK by ATP developed slowly; the effects usually reached a maximum approximately 30-60 s after the application of ATP. This suggests the involvement of a diffusible cytosolic second messenger(s) in the response. ATP could further increase IK after maximal enhancement by isoprenaline (0.5-1.0 microM), suggesting that the intermediate steps were independent of cyclic AMP-dependent protein kinase (protein kinase A). 7. Potentiation of IK by ATP was not attenuated by either (i) pretreatment of the cells with 5 microM 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H-7) or (ii) intracellular perfusion of 20 mM 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that protein kinase C and intracellular Ca2+ did not mediate the response. 8. It is concluded that the activation of P2-purinoceptors increases IK through intracellular mechanisms independent of protein kinase A, protein kinase C or intracellular free Ca2+ in guinea-pig atrial myocytes.

Adenosine Triphosphate↗
PubMed1996

Activation of the muscarinic K+ channel by P2-purinoceptors via pertussis toxin-sensitive G proteins in guinea-pig atrial cells.

1. Whole-cell voltage clamp and cell-attached patch-clamp techniques were applied to single atrial myocytes enzymatically dissociated from adult guinea-pig hearts. 2. In whole-cell clamp conditions, external applications, of ATP activated the muscarinic K+ (KACh) current, identified by its inward rectification, its reversal potential near the calculated K+ equilibrium potential (EK) and its relaxation properties during step changes of whole-cell membrane potential. Theophylline, an antagonist for Pi-purinoceptors, did not affect the action of ATP on the KACh current, indicating that the response was evoked through P2-purinoceptors. 3. The concentration-response relationship for ATP was well described by a Hill equation with a half-maximal concentration of 1.84 microM and a Hill coefficient of 0.94. ATP (100 microM) produced a maximal increase of the KACh current to 10.92 microA microF-1, which corresponds to 44.9 and 80.9% of the maximal increases evoked by ACh (10 microM) and adenosine (100 microM), respectively. 4. The activation of KACh current gradually declined to a steady level despite the continuous presence of ATP (desensitization). Recovery from the desensitization was relatively rapid with a half-time of approximately 1.5 min. 5. The activation of KACh current by ATP was completely abolished by pre-incubating myocytes with pertussis toxin (PTX, 5 micrograms ml-1), indicating that P2-purinoceptors are coupled to PTX-sensitive G proteins to activate the KACh channel. 6. In the cell-attached patch recording, ATP (5 microM) applied to the pipette solution enhanced the activity of a channel with single-channel conductance of 52.7 +/- 0.9 pS (mean +/- S.E.M., n = 10), reversal potential near EK and mean open time of 1.1 +/- 0.1 ms. These conductance and kinetic properties are identical to those of the KACh channel in the heart. In contrast, ATP applied to the bath solution did not significantly affect the basal activity of KACh channel openings. These observations suggest that the mechanism coupling the P2-purinoceptor to the activation of the KACh channel involves membrane-delimited component(s) rather than soluble second messenger(s). 7. These results strongly suggest a direct coupling of the P2-purinoceptor to the KACh channel through PTX-sensitive G proteins, analogous to the coupling mechanism of the muscarinic ACh receptor and Pi-purinoceptor to this channel.

Adenosine Triphosphate↗
PubMed1996

Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria.

Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP. The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins.

14-3-3 Proteins↗
PubMed1996

Acetylcholine activates latent pseudorabies virus in pigs.

Pseudorabies virus (PrV) was isolated from the nasal swabs and the cultured trigeminal ganglia of latently infected pigs after they were treated with acetylcholine (ACH). These results indicate that ACH activates latent infections of PrV.

Acetylcholine↗
PubMed1996

Lack of a kinetic interaction between fluconazole and mexiletine.

OBJECTIVE: To investigate the effect of fluconazole on the kinetics of mexiletine. METHODS: Six healthy male volunteers participated in a crossover study. On the 1st day, the subjects received 200 mg mexiletine alone. On days 2-7 they received 200 mg fluconazole orally, and on day 8 they received 200 mg mexiletine and 200 mg fluconazole concomitantly. In a third phase two subjects received 400 mg fluconazole daily. RESULTS: No differences in concentrations were observed between the three phases. The area under the concentration curves (AUC) after administration of mexiletine alone and in combination with fluconazole 200 mg/day were 6.63 and 7.31 micrograms.h.ml-1, respectively. CONCLUSION: These findings suggest that fluconazole does not inhibit mexiletine metabolism.

Adult↗
PubMed1996

Asymmetric deletion of the junction between the short unique region and the inverted repeat does not affect viral growth in culture and vaccine-induced immunity against Marek's disease.

To construct an effective recombinant Marek's disease virus type 1 (MDV1), we localized a stable insertion site for expression of the Escherichia coli lacZ gene near or within the short inverted repeats of MDV1 strain K554 DNA. A stable recombinant MDV1 was obtained by deleting the junction region between the short unique sequence (Us) and the internal short inverted repeat (IRs). The recombinant MDV1 replicated in cultured cells as well as the parental viral DNA. Antibodies against both MDV1 antigen and beta-galactosidase encoded by the lacZ gene were detected in the sera of chickens immunized with the virus, and persisted for at least 16 weeks. Moreover, the recombinant virus conferred protection upon chickens against a challenge with virulent MDV1. These results demonstrated that the Us-IRs junction region is an effective site for the insertion of foreign genes from which to construct a polyvalent live vaccine for poultry. Analysis of the Us-IRs junction region which was deleted from the parental MDV1 indicated that there is a tandem direct repeat of a 220-bp exists within the short internal and terminal inverted repeats of avirulent MDV1 K554 strain DNA. The 220-bp sequence was well conserved among DNAs from various strains. The number of the repeat units may differ between the IRs and TRs or among various MDV1 strain DNAs.

Animals↗
PubMed1996

Protection of chickens from Newcastle disease by vaccination with a linear plasmid DNA expressing the F protein of Newcastle disease virus.

To evaluate the usefulness of a DNA vaccine for chickens, we constructed a plasmid vector expressing the Newcastle disease virus F protein (NDV-F) under the control of the human cytomegalovirus immediate early enhancer and chicken beta-actin gene promoter. One-week-old chickens injected intramusculary with the circular plasmid DNA did not produce significant levels of antibody against NDV-F. However, two of five birds injected with the linearized plasmid DNA produced high levels of the antibody. Moreover, four of five birds injected with a mixture of the linearized-plasmid and Lipofectin produced the antibody efficiently. At 9 weeks post-injection, chickens were challenged with the velogenic NDV Sato strain. These chickens that had the antibody against NDV-F were protected from lethal NDV challenge. These results demonstrate that the DNA vaccine conferred efficient protection against the disease.

Animals↗
PubMed1996

Measurement of airborne mite allergen exposure in individual subjects.

To evaluate the extent of personal exposure to airborne mite allergens, subjects were asked to carry a personal air sampler when in their houses. The level of Der 1 allergen trapped by the sampler was measured with a highly sensitive immunoassay. There were great variations in airborne Der 1 exposure in each subject. When used bedding was replaced with new allergen-free bedding, we detected a decrease in the allergen level. The use of new bedding seems to be an effective measure for reducing airborne mite allergen exposure.

Air Pollutants↗
PubMed1996

Identification of the first major allergen of a squid (Todarodes pacificus).

BACKGROUND: In Japan, squid is an important seafood, and some patients with food allergies are sensitive to squid. There has been no report, however, describing the major allergens of squid. OBJECTIVE: To characterize squid allergens, we isolated a major allergen from the Pacific flying squid (Todarodes pacificus) and compared it with a major allergen from a shrimp (Penaeus orientalis). METHODS: The major squid and shrimp allergens were isolated by column chromatography on diethylaminoethyl-Sepharose (Pharmacia, Uppsala, Sweden), hydroxylapatite, and Sephacryl S-300 (Pharmacia). The IgE reactivity of the isolated allergens was assessed by immunoblotting. The cross-reactivity between the squid and shrimp allergens was examined by use of mouse polyclonal and monoclonal antibodies to the major allergens. Amino acid sequence analyses of the isolated allergens were done. RESULTS: The isolated squid allergen is a 38 kd, heat-stable protein. IgE antibody binding to the purified squid allergen was demonstrated by immunoblotting. Cross-reactivity between major squid and shrimp allergens was demonstrated with sera from patients allergic to squid or shrimp or with allergen-specific monoclonal antibodies. The amino acid sequence analysis of the major squid allergen showed a marked homology with tropomyosin from blood fluke planorbid (Biomphalaria glabrata), which is a common vector snail of Schistosoma mansoni. CONCLUSION: This 38 kd protein is a major allergen of the squid, Todarodes pacificus, and is believed to be squid muscle protein tropomyosin. We named it Tod p 1 according to International Union of Immunological Societies allergen nomenclature regulation.

Allergens↗
PubMed1996

Food allergy to gelatin in children with systemic immediate-type reactions, including anaphylaxis, to vaccines.

BACKGROUND: Anaphylaxis to measles-mumps-rubella vaccines has been reported. We have suspected that most such reactions are caused by gelatin contained in the vaccines. OBJECTIVE: To confirm the relation between systemic allergic reactions to vaccines and the presence of anti-gelatin IgE, we measured anti-gelatin IgE in children who demonstrated allergy to gelatin-containing vaccines. Furthermore, to clarify the relation between allergic reactions to gelatin in vaccines and foods, we surveyed the occurrence of allergic reactions to gelatin-containing foods in the same children. METHODS: Serum samples were taken from 26 children who had systemic immediate-type reactions, including anaphylactic shock, to vaccines and the same number of children without allergic reactions. Specific IgE to gelatin in these samples was measured. We then surveyed whether these children had allergic reactions to gelatin-containing foods before and after vaccination. RESULTS: Twenty-four of the 26 children with allergic reactions to vaccines had anti-gelatin IgE ranging from 1.2 to 250 Ua/ml. Seven had allergic reactions on ingestion of gelatin-containing foods. Of these, two had reactions before vaccination, and five had reactions after vaccination. All the control children without allergic reactions to vaccines had no anti-gelatin IgE. CONCLUSION: We reconfirmed a strong relationship between systemic immediate-type allergic reactions, including anaphylaxis, to vaccines and the presence of specific IgE to gelatin. Moreover, some of the children also had allergic reactions to food gelatin before or after vaccination.

Anaphylaxis↗
PubMed1996

Seasonal changes in mite allergen (Der I and Der II) concentrations in Japanese homes.

BACKGROUND: There has been no report on seasonal changes in Der II allergen in floor and bedding dust. OBJECTIVE: The purpose of this study was to examine the extent of seasonal changes in Der I and Der II allergens in the floor and bedding dust found in houses. METHODS: We measured the absolute concentrations of mite allergens in dust collected monthly for 1 year from both the floors and bedding of eight houses in Tokyo. Dust samples were obtained from eight families without regard to their allergy histories. The concentrations of the Der p I, Der f I, and Der II allergens were measured by fluorometric sandwich ELISA. RESULTS: We found seasonal changes in the concentrations of these mite allergens. The highest concentrations of Der I (Der p I plus Der f I) and Der II (Der p II plus Der f II) were present from August to October, and the lowest ones from March to April. In floor dust, the mean highest concentrations of Der I and Der II (35.0 and 20.2 microgram of fine dust) were sevenfold and fivefold respectively, times the mean lowest concentrations. In bedding dust, the mean highest concentrations of Der I and Der II (51.3 and 29.6 microgram/g of fine dust) each were fourfold times the mean lowest concentrations. CONCLUSIONS: The patterns of seasonal changes in Der II in floor and bedding dust were similar to those of Der I.

Allergens↗