Gene polymorphism and frequencies of the NPC1L1 gene (rs2072183, rs217434 and rs217428) in Japanese patients with dyslipidemia.

WHAT IS KNOWN AND OBJECTIVE: Niemann-Pick C1-Like 1 (NPC1L1) plays a pivotal role in intestinal cholesterol absorption. Ezetimibe is known as an inhibitor for NPC1L1 and decreases concentration of low-density lipoprotein cholesterol (LDL-C) in blood. Responses of the decrease of serum LDL-C levels to ezetimibe have been reported to be different among NPC1L1 variants. However, there are still limited data concerning the genetic variation in the NPC1L1 gene, specifically, in Japanese patients with dyslipidemia. The purpose of this study is to elucidate genotype and allele frequencies of the NPC1L1 gene in Japanese patients with dyslipidemia. METHODS: Written informed consent was obtained from all participants. All patients were administered ezetimibe at the dose of 10 mg for once a day either alone or coadministered with statins. Patient's data were retrospectively obtained from their medical records. Genomic DNA was extracted from whole blood samples and analysed three NPC1L1 SNPs (rs2072183, rs217428 and rs217434) by the direct sequencing method. RESULTS AND DISCUSSION: We found that there is a significant difference of genotype frequencies between healthy Japanese and dyslipidemic subjects in rs2072183. No significant differences were observed in rs217428 and rs217434; however, comparison of our data with literature reports suggests that there are significant differences in the frequencies of rs217428 and rs217434 between Canadian and Japanese dyslipidemic patients. WHAT IS NEW AND CONCLUSION: Our study is the first report concerning the genotype and allele frequencies of the gene coding for NPC1L1 in Japanese patients with dyslipidemia. The most notable result was to demonstrate that there exists a significant difference in rs2072183 variant between healthy Japanese and dyslipidemic subjects and also found that there exists genetic variation of rs2072183 between Japanese and Canadian patients with dyslipidemia. Our results are expected to facilitate research in the proper use of ezetimibe-based mono- or combination therapies. Further studies will be required to evaluate the effects of rs2072183 on the efficacy of LDL cholesterol reduction by ezetimibe.

Establishment and characterization of a malignant melanocytic tumor cell line expressing the ret oncogene.

We established a cell line (designated Mel-ret) from a melanocytic tumor developed in a metallothionein/ret transgenic mouse. Unlike primary melanocytic tumors, which did not show malignant features, when the Mel-ret cells were transplanted into nude mice they invaded into surrounding tissues and had metastatic ability. Although the Ret proteins were expressed at similar levels in the cell line and the primary tumors, the level of tyrosine phosphorylation in the Mel-ret cells was much higher than that in the primary tumors. In particular, an 85-kDa tyrosine-phosphorylated band was specifically detected in the Mel-ret cells. These results suggest that the increase in tyrosine phosphorylation may be responsible for malignant transformation of the Mel-ret cells. Immunofluorescence and cell fractionation studies showed that the Ret proteins and most of tyrosine-phosphorylated proteins in the Mel-ret cells localized in the membrane fraction. No activation of phosphatidyl-inositol-3 kinase (PI-3 kinase), a target protein for several tyrosine kinases, was detected in the Mel-ret cells.

Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines.

We examined the basis of the all or none difference in inducing melanocytic tumor development among three transgenic mouse lines (304, 192 and 242) to which the same promoter-enhancer (metallothionein-I) and oncogene (ret) were introduced. We initially demonstrated that both skin melanosis and Ret protein expression in skin, thymus and brain first became detectable before or immediately after birth in the mice of the tumor developing lines (304 and 192), whereas they became detectable a few days after birth in the mice of the non-tumor developing line (242) by Western blotting and immunohistochemical analysis. Interestingly, the Ret protein expression in skin developed rapidly after birth as a burst with peak levels on 0.5-1.5 day newborns of lines 304 and 192 and on 7.0-7.5 day-old mice of line 242. The levels of autophosphorylation of Ret kinase in skin were, however, invariable among these three transgenic mouse lines. The mice of line 242, but not those of lines 192 and 304, responded to Ret protein immunization by increased antigen-dependent lymphocyte proliferation and T-cell-mediated tumor growth suppression in vitro. Furthermore, ret-transgenic mice of line 242, but not line 304, rejected the subcutaneously transplanted tumors that had originally developed in a mouse of line 304. These results suggest that whether oncogene product-specific-tolerance is established or not to antitumor immunity may be decided by the dynamics of ret oncogene expression before and after delivery and this is the primary factor determining development or non-development of melanoma.

Basic study on gene therapy of human malignant glioma by use of the cationic multilamellar liposome-entrapped human interferon beta gene.

For gene therapy of human malignant glioma, we adopted positively charged multilamellar liposomes entrapping the human interferon beta (hIFN-beta) gene. One week after the transplantation of human malignant glioma U251-SP cells to produce glioma in nude mouse brain, the liposomes entrapping the gene (500 ng of DNA per 25 nmol of lipids per 2 microl) were injected into the same site of the cell transplantation once every second day for a total of five injections; and by this means the tumor completely disappeared. To confirm the antiproliferative effect of hIFN-beta, we performed an in vitro study using a plasmid containing a secretion signal sequence-deleted hIFN-beta gene and one containing the hIFN-beta gene inserted in reverse. In both cases, there was no hIFN-beta release into the medium and no growth inhibition effect. On addition of anti-hIFN-beta antibody to the medium, the growth inhibition effect was abolished. As this cell line expresses IFN-alpha/beta receptor, the hIFN-beta produced in the transfected cells could be released and acted in a paracrine manner. For 120 days the body weight change of normal mice treated by the same procedure as used in the curing experiment was not significant among the groups injected with empty liposomes, plasmid only, and liposomes entrapping the gene. In all of these three groups, death, abnormal behavior, and significant histological changes were not observed.

Expression of chimeric P450 genes encoding flavonoid-3', 5'-hydroxylase in transgenic tobacco and petunia plants(1).

Flavonoid-3',5'-hydroxylase (F3'5'H), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3', 5'-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3'5'H. To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme. Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3',5'-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3',5'-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3'5'H activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3'5'H activity between tobacco and petunia is discussed.

Colocalization of neuropilin-1 and Flk-1 in retinal neovascularization in a mouse model of retinopathy.

PURPOSE: To investigate the mechanisms of the development of retinal neovascularization, the localizations of vascular endothelial (VEGF) receptors Flk-1 and neuropilin (NP)-1 mRNAs were examined. METHODS: The model of retinopathy of prematurity (ROP) was produced by ischemia-induced ocular neovascularization, by exposing postnatal day-7 mice to 75% oxygen for 5 days and then returning them to room air for 5 days. Retinal neovascularization was visualized by injection of fluorescein-dextran. Expression of Flk-1 and NP-1 mRNAs were examined by in situ hybridization with flatmount and serial sections of the retina. The localization of NP-1 was also confirmed by immunohistochemistry. Blood vessel patterns were characterized by immunohistochemical localization of von Willebrand factor (vWF). RESULTS: Flatmount in situ hybridization showed intense expression of NP-1 and Flk-1 mRNAs colocalized in the area of neovascularization. In situ hybridization of serial sections of the retina revealed that expression of Flk-1 and NP-1 was restricted to neovascularized vessels of the retina from ROP mice. CONCLUSIONS: The restricted expression of Flk-1 and NP-1 on neovascularized vessels suggests that these molecules may play important roles in retinal neovascularization. This is the first report of the colocalization of NP-1 and Flk-1 on neovascularized vessels of the retina from ROP mice.

Phenotypic changes in hepatic mast cells accumulating around the metacestodes of Taenia taeniaeformis in rats.

Staining properties, kinetics and degranulation of the hepatic mast cells (HMC) accumulating around the metacestodes of Taenia taeniaeformis in the liver of rats were studied. Two different types of HMC, designated as Type I and Type II, could be classified according to histochemical properties and response to compound 48/80. Type I cells resembled mucosal mast cells (MMC), whereas Type II did not. HMC, mostly Type I, were observed from day 14 postinfection (PI), while Type II were seen only from day 28 PI. On day 28 PI, Type II represented a transitional staining pattern between MMC and connective tissue mast cells (CTMC). The ratio of Type II to Type I increased gradually with the course of infection and was about 1 to 1 on day 70 PI. At this time, most of the HMC that constituted Type II as well as CTMC could be stained with berberine sulfate. While the phenotypic change of HMC to CTMC was found in the middle and inner capsular layers, most of the HMC in the outer capsular layer maintained the phenotype of MMC. The present results suggest that hepatic mast cells increased as the MMC phenotype, then showed the heterogeneity in which the transitional form of mast cells emerged followed by the appearance of the CTMC type.

Detection of Helicobacter pylori DNA in gastric juice by the polymerase chain reaction: comparison with findings in bacterial culture and the detection of tissue IgA and serum IgG antibodies against Helicobacter pylori.

The detection of Helicobacter pylori in gastric juice by the polymerase chain reaction (PCR) was undertaken in 124 patients with peptic ulcer or chronic gastritis. PCR products were evaluated by agarose gel electrophoresis and Southern hybridization of H. pylori-specific DNA sequences. Positive and negative results of the PCR analysis in 72 examinations were compared with those from bacterial culture, and with the detection of tissue IgA antibody against H. pylori by enzyme-linked immunosorbent assay ELISA; Serion, Wuerzburg, Germany, and detection of serum IgG antibody against H. pylori by ELISA; Radim Pomezia, Italy. Thirty-four PCR-positive samples evaluated by electrophoresis and hybridization coincided with positive samples in 56% of bacterial cultures, 59% of tissue IgA antibody identifications, and 94% of serum IgG antibody evaluations; 26 PCR-negative samples coincided with negative samples in 96% of bacterial cultures, 81% of tissue IgA antibody evaluations, and 38% of serum IgG assessments. We compared the detection achieved with the H. pylori PCR assay in gastric juice with that in biopsies taken from the antrum and upper corpus in 90 examinations, and found them to be both positive in 34 (38%) and 36 (40%) of specimens, both negative in 37 (41%) and 30 (33%) specimens, gastric juice-positive but biopsy-negative in 10 (11%) and 12 (13%) specimens, and vice versa in 9 (10%) and 12 (13%) specimens, when detected by electrophoresis and hybridization, respectively, showing equivalent detection rates. In relation to the type of disease, the positive PCR assay results with gastric juice, evaluated by electrophoresis and hybridization, respectively, were: gastric ulcer 34/53 (64%) and 39/53 (74%), duodenal ulcer 23/38 (61%) and 25/38 (66%), and chronic gastritis 20/33 (61%) and 23/33 (70%), showing no significant difference in positive rates between peptic ulcer and chronic gastritis. Of the samples of 16 patients with H. pylori-positive gastric juice by the PCR assay, 7 were negative by PCR assay analyzed by electrophoresis and hybridization after the completion of treatment H. pylori. However, after treatment, 3 were negative on electrophoresis but still had positive results with hybridization, indicating that a minimal number of bacilli may have still remained. Detection of H. pylori in gastric juice has potential advantages for examining H. pylori infection in the entire stomach and for follow up after treatment for the eradication of H. pylori.

Tallysomycin, a new antitumor antibiotic complex related to bleomycin. III. Antitumor activity of tallysomycins A and B.

The antitumor activity of tallysomycins A and B was determined in five experimental tumor systems in mice. Tallysomycins A and B were highly active against B16 melanoma, sarcoma 180 ascites tumor and Lewis lung carcinoma, and moderately active against P388 leukemia but were without effect on lymphoid leukemia L1210. The antitumor activity of tallysomycin A was 2 to 3 times that of tallysomycin B and 3 to 17 times that of bleomycin. Tallysomycin A was about 1.5 and 4 times more toxic for mice than tallysomycin B and bleomycin, respectively, in terms of subacute LD50 values.

BMY-28438 (3,7-dihydroxytropolone), a new antitumor antibiotic active against B16 melanoma. I. Production, isolation, structure and biological activity.

A new antitumor antibiotic BMY-28438 was isolated from the cultured broth of Streptomyces tropolofaciens No. K611-97. The antibiotic showed potent cytotoxicity against cultured B16 melanoma cells and remarkable prolongation of life span of mice bearing B16 melanoma. The structure of BMY-28438 was determined as 3,7-dihydroxytropolone on the basis of spectral analyses and direct comparison with the authentic compound synthesized.

Quinaldopeptin, a novel antibiotic of the quinomycin family.

Quinaldopeptin, a new type of quinomycin antibiotic, was isolated from the culture of Streptoverticillium album strain Q132-6. The antibiotic exhibited strong in vitro antimicrobial and cytotoxic activity and significantly prolonged the survival time of mice inoculated with a murine tumor. Quinaldopeptin is a symmetric cyclic peptide linked only by peptide bonds and differs from known antibiotics of the quinomycin family by the lack of ester linkage.

A new screening method for melanin biosynthesis inhibitors using Streptomyces bikiniensis.

A novel screening method for melanin biosynthesis inhibitors using Streptomyces bikiniensis NRRL B-1049 as the indicator organism has been developed. Several known compounds, including tyrosinase inhibitors, were found to inhibit melanin production of S. bikiniensis on agar plates. This screening method was applied to fermentation broths of actinomycetes and several cultures with melanin biosynthesis inhibitory activity were found.

Maduropeptin, a complex of new macromolecular antitumor antibiotics.

Maduropeptin, a complex of new macromolecular antitumor antibiotics, is a metabolite of Actinomadura madurae H710-49. The active components maduropeptins A1, A2 and B are acidic chromopeptides with MW of around 22,500 and composed of 14 types of amino acids and an unstable chromophore. The antibiotics are active in vitro against Gram-positive bacteria and highly cytotoxic to tumor cells. They produced significant prolongation of survival time of mice implanted with P388 leukemia and B16 melanoma.

BBM-928, a new antitumor antibiotic complex. I. Production, isolation, characterization and antitumor activity.

A complex of the antitumor antibiotic BBM-928 was produced by an actinomycete strain No. G455-101. Four components, BBM-928 A, B, C and D, were isolated in crystalline form and characterized. They were shown to be cyclic depsipeptide antibiotics containing a quinoline nucleus as the chromophore. BBM-928 A is a monoacetyl derivative of BBM-928 B and a diacetyl derivative of BBM-928 C. BBM-928 components exhibit antimicrobial activity against Gram-positive and acid-fast bacteria. BBM-928 A is highly active in mice against various experimental tumors including leukemia P388, leukemia L1210, melanoma B16, LEWIS lung carcinoma and sarcoma 180. BBM-928 B is less active than BBM-928 A, and BBM-928 C has no antitumor activity.

Tallysomycin, a new antitumor antibiotic complex related to bleomycin. IV. New biosynthetic derivatives of tallysomycin.

A series of new biosynthetic derivatives of tallysomycins A and B were obtained by precursor amine-feeding fermentation. Certain diamines were incorporated into tallysomycins as the terminal amine moiety affording two classes of biosynthetic derivatives, with or without the Beta-lysine moiety in the subterminal position. Among 21 derivatives prepared, tallysomycin S10b was selected for further studies in view of its favorable therapeutic indices.

Synthesis, stereochemistry, and biological properties of the depigmenting agents, melanostatin, feldamycin and analogs.

Syntheses of melanostatin and feldamycin have been completed from L-serine and L-threonine, respectively, and the configuration of unknown asymmetric carbons determined. Feldamycin analogs have also been prepared and the L-tryptophyl analog was the most potent in the depigmentation of Streptomyces bikiniensis and B16 melanoma cells.

Multiple cavernous hemangiomas of the orbits.

Intraocular tumors were detected in both orbits simultaneously by computed tomography scanning in a 45-year-old woman complaining of proptosis of the right eye, decreased visual acuity, and diplopia. The tumor in the right orbit was resected by the subfrontal extradural approach, and that in the left orbit by the Krönlein-Berke method. The right tumor was found on the side of the external ear of the muscle cone and the left tumor was located inferiorly on the side of the external ear of the muscle cone. Both tumors were cavernous hemangiomas with identical macroscopic and histologic features.

The taxonomic status of Echinococcus cruzi Brumpt and Joyeux, 1924 (Cestoda: Taeniidae) from an agouti (Rodentia: Dasyproctidae) in Brazil.

Paratype material of Echinococcus cruzi Brumpt and Joyeux, 1924, described from an agouti, Dasyprocta leporina (L.), in Brazil, was compared with Echinococcus oligarthrus (Diesing, 1863), of which the larval stage occurs also in agoutis and other rodents in South America and Central America. Comparisons of the larval cestodes (metacestodes) showed that the rostellar hooks from protoscolices of the two taxa corresponded in form, and their slightly greater lengths in E. cruzi were considered to be of no taxonomic significance. They agreed as well in other morphological characteristics. Echinococcus cruzi was compared also with the other neotropical species, E. vogeli Rausch and Bernstein, 1972, Based on these comparisons and in agreement with the earlier conclusion of Cameron (1926), E. cruzi Brumpt and Joyeux, 1924 is placed in synonymy with E. oligarthrus (Diesing, 1863).

Portal-hepatic venous shunt through a portal aneurysm complicated by hepatic encephalopathy and pulmonary hypertension.

We report a rare case of portal-hepatic venous shunt through an enormous portal aneurysm complicated by pulmonary hypertension. A 66-year-old woman was admitted to our hospital for hepatic encephalopathy. Chest roentgenography revealed pulmonary hypertension. Computed tomography and ultrasound examination demonstrated a shunt between the portal and hepatic veins through an enormous portal aneurysm. The diagnoses of portal-hepatic venous shunt and pulmonary hypertension were confirmed by hepatic venous catheterization and cardiac catheterization. Pulmonary hypertension might result from the effects of vasoconstrictive agents, which should be metabolized by the liver in normal subjects, passing through the intrahepatic shunt into the lung.

Sarcoidosis after interferon therapy for chronic active hepatitis C.

Sarcoidosis is characterized by multisystemic granulomatous lesions of unknown etiology. A 62-year-old woman developed sarcoidosis after treatment with alpha-2a interferon (IFN) for 24 weeks (total dose: 522 million units) for chronic hepatitis C. She developed complete atrioventricular block and multiple noncaseating granulomatous lesions in the lung. IFN therapy, which may disturb cellular immune activation in some patients, may have contributed to the onset and progression of sarcoidosis.