Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome.
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Dubin-Johnson syndrome (DJS) is an autosomal recessive disease characterized by conjugated hyperbilirubinemia. Previous studies of the defects in the human canalicular multispecific organic anion transporter gene (MRP2/cMOAT) in patients with DJS have suggested that the gene defects are responsible for DJS. In this study, we determined the exon/intron structure of the human MRP2/cMOAT gene and further characterized mutations in patients with DJS. The human MRP2/cMOAT gene contains 32 exons, and it has a structure that is highly conserved with that of another ATP-binding-cassette gene, that for a multidrug resistance-associated protein. We then identified three mutations, including two novel ones. All mutations identified to date are in the cytoplasmic domain, which includes the two ATP-binding cassettes and the linker region, or adjacent putative transmembrane domain. Our results confirm that MRP2/cMOAT is the gene responsible for DJS. The finding that mutations are concentrated in the first ATP-binding-cassette domain strongly suggests that a disruption of this region is a critical route to loss of function. (+info)
Allele-specific differences in apolipoprotein C-III mRNA expression in human liver.
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BACKGROUND: Sequence variations at the apolipoprotein (apo)C-III gene locus have been associated with increased plasma triglycerides. In particular, the S2 allele of an SstI polymorphism in the 3' untranslated region has been associated with hypertriglyceridemia in many populations. The aim of this study was to determine whether the variant S2 allele is related to increased mRNA expression in vivo. METHODS: We measured allele-specific apoC-III expression in liver biopsies of five obese subjects, using restriction isotyping and a primer extension method, both based on the SstI polymorphism. RESULTS: The expression of mRNA by the S1 and S2 alleles was similar in two patients, whereas the mRNA encoded by the S2 allele was 14%, 26%, and 29% more abundant than the wild-type mRNA in the remaining three patients. Because other polymorphisms at the apoC-III gene locus have been implicated in the S2-associated hypertriglyceridemia, we determined apoC-III haplotypes comprising promoter polymorphisms at -935, -641, -630, -625, -482, -455, as well as the SstI sites and a BbvI site, both located in the 3' untranslated region. None of these polymorphisms nor any haplotype exhibited a perfect association with allele-specific expression, but variation at the T-482C site correlated in four of five subjects with the relative allele abundance. CONCLUSION: These data provide preliminary evidence for allele-specific differences in apoC-III mRNA expression in vivo and suggest that such differences may contribute to associations of apoC-III gene polymorphisms with hypertriglyceridemia. (+info)
cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium.
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Bovine corneal epithelium contains arachidonate 12- and 15-lipoxygenase activity, while human corneal epithelium contains only 15-lipoxygenase activity. Our purpose was to identify the corneal 12- and 15-lipoxygenase isozymes. We used cDNA cloning to isolate the amino acid coding nucleotide sequences of two bovine lipoxygenases. The translated sequence of one lipoxygenase was 82% identical with human 15-lipoxygenase type 2 and 75% identical with mouse 8-lipoxygenase, whereas the other translated nucleotide sequence was 87% identical with human 12-lipoxygenase of the platelet type. Expression of 15-lipoxygenase type 2 and platelet type 12-lipoxygenase mRNAs were detected by Northern analysis. In addition to these two lipoxygenases, 12-lipoxygenase of leukocyte (tracheal) type was detected by polymerase chain reaction (PCR), sequencing, and Northern analysis. Finally, PCR and sequencing suggested that human corneal epithelium contains 15-lipoxygenase types 1 and 2. (+info)
Sequence, expression in Escherichia coli, and characterization of lysophospholipase II.
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Here we report the sequence, expression in Escherichia coli cells, and characterization of a new small-form lysophospholipase named lysophospholipase II from mouse embryo. The cDNA clone was found and identified among mouse expressed sequence tags in the database search for the homologue of lysophospholipase I previously cloned from rat liver (H. Sugimoto et al., J. Biol. Chem. 271 (1996) 7705-7711). The predicted amino acids sequence contained 231 residues with a calculated molecular weight of 24794, and showed 64% identity to that of lysophospholipase I with the Gly-X-Ser-X-Gly esterase/lipase consensus. The lacZ fusion protein expressed in E. coli cells exhibited lysophospholipase activity and reacted with antibody raised against previously purified pig gastric lysophospholipase II (H. Sunaga et al., Biochem. J. 308 (1995) 551-557), but not with antibody against rat liver lysophospholipase I. The expressed enzyme was purified to a specific activity of 0.15 micromol/min per mg by DEAE-Sepharose A-500 chromatography. The enzyme preferentially utilized zwitterionic lysophospholipids in the order of lysophosphatidylcholine>lysophosphatidylethanolamine, but poorly acidic lysophospholipids, such as lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid. Not only the 1-acyl isomer, but also the 2-acyl isomer were deacylated. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that lysophospholipase II transcript as well as lysophospholipase I transcript was widely distributed in mouse tissues. (+info)
A novel 53-kDa nodulin of the symbiosome membrane of soybean nodules, controlled by Bradyrhizobium japonicum.
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A nodule-specific 53-kDa protein (GmNOD53b) of the symbiosome membrane from soybean was isolated and its LysC digestion products were microsequenced. cDNA clones of this novel nodulin, obtained from cDNA library screening with an RT-PCR (reverse-transcriptase polymerase chain reaction)-generated hybridization probe exhibited no homology to proteins identified so far. The expression of GmNOD53b coincides with the onset of nitrogen fixation. Therefore, it is a late nodulin. Among other changes, the GmNOD53b is significantly reduced in nodules infected with the Bradyrhizobium japonicum mutant 184 on the protein level as well as on the level of mRNA expression, compared with the wild-type infected nodules. The reduction of GmNOD53b mRNA is related to an inactivation of the sipF gene in B. japonicum 184, coding for a functionally active signal peptidase. (+info)
In-vitro fertilization and culture of mouse embryos in vitro significantly retards the onset of insulin-like growth factor-II expression from the zygotic genome.
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In this study, the effect of in-vitro fertilization (IVF) and culture of mouse embryos in vitro on the normal expression of insulin-like growth factor-II (IFG-II) ligand and receptor was examined. The expression of IGF-II increased in a linear fashion at least up to the 8-cell stage of development. IGF-II expression in embryos collected fresh from the reproductive tract was significantly (P < 0.001) greater than in embryos fertilized in the reproductive tract and cultured in vitro (in-situ fertilized: ISF), and its expression was further reduced (P < 0.001) in IVF embryos at all development stages tested. The expression of IGF-II was significantly (P < 0.001) lower when embryos were cultured individually in 100 microl drops compared with culture in groups of 10 in 10 microl drops of medium. The addition of platelet activating factor to culture medium partially overcame this density-dependent decline of expression. Culture of ISF and IVF zygotes also caused the onset of new IGF-II mRNA transcription from the zygotic genome to be significantly (P < 0.001) retarded, until at least the 8-cell stage of development. This effect was greater (P < 0.05) for IVF than for ISF embryos. Neither IVF nor culture had any obvious effect on IFG-II/mannose-6-phosphate receptor (IGF-IIr) mRNA expression. (+info)
Tumour necrosis factor-alpha (TNF-alpha) in human endometrium and uterine secretion: an evaluation by immunohistochemistry, ELISA and semiquantitative RT-PCR.
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Tumour necrosis factor-alpha (TNF-alpha is a pleiotropic cytokine synthesized throughout the female reproductive tract. Even though evidence has accumulated that supports its role in autocrine and paracrine processes, its expression and function in the human endometrium are still not completely understood. To gain a better understanding of the synthesis and release of TNF-alpha in the endometrium and how this relates to concentrations in uterine secretion, its expression throughout the menstrual cycle was investigated by three different techniques. Samples of endometrial tissue and uterine secretions were collected from patients undergoing abdominal and vaginal hysterectomy for benign reasons. The mRNA expression of TNF-alpha was investigated in homogenized endometrial tissue by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) (n = 18). An assessment of the cellular TNF-alpha protein localization in the endometrial glands was performed immunohistochemically (n = 39). The concentrations of the secreted TNF-alpha protein in endometrial secretion were determined by enzyme-linked immunosorbent analysis (n = 30). All three methods gave similar results on the temporal expression of TNF-alpha mRNA and TNF-alpha protein during the cycle. Concentrations of endometrial TNF-alpha mRNA in tissue samples and TNF-alpha protein in uterine secretion were quite low at the beginning of the cycle, rose sharply in the mid- to late proliferative phase and decreased towards the end of the cycle. The concentrations of TNF-alpha protein in the endometrial glands, as shown by immunohistochemical investigation, stayed high throughout the secretory phase at values slightly below those of the late proliferative phase. (+info)
CD9 is expressed in extravillous trophoblasts in association with integrin alpha3 and integrin alpha5.
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The CD9 molecule is a 24-27 kDa cell surface glycoprotein, which may be related to Schwann cell migration and adhesion. In this study, we examined the expression of CD9 in human extravillous trophoblasts, which invade into the endometrium during implantation and placentation. CD9 was detected immunohistochemically on the extravillous trophoblasts in the cell columns of first trimester placentae, but not on villous trophoblasts. In the second and third trimester, CD9 was highly expressed on the extravillous trophoblasts in the basal plate of placentae, and in the chorion laeve in the fetal membrane of term placentae. The molecular mass of CD9 in the chorion laeve was shown to be 27 kDa by Western blotting. The mRNA of CD9 was also detected in the chorion laeve by reverse transcription-polymerase chain reaction (RT-PCR). Proteins were purified from chorion laeve by affinity chromatography with anti-integrin alpha3 and alpha5 monoclonal antibodies and Western blotting, revealed that CD9 was associated with both integrins. These findings indicate that CD9 is a differentiation-related molecule present in the extravillous trophoblasts. Since it is associated with integrin alpha5 which has been proposed to regulate trophoblast invasion, CD9 may be implicated in trophoblast invasion at the feto-maternal interface. (+info)