Peritoneal endometriotic lesions differentially express a haptoglobin-like gene.
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A unique glycoprotein, called endometriosis protein-I (ENDO-I), has previously been shown to be synthesized and secreted by rat and human endometriotic explants. Furthermore, the N-terminal amino acid sequence analysis showed that ENDO-I shares homology with haptoglobin. The present study was designed to determine this sequence of ENDO-I cDNA from human peritoneal endometriotic lesions and to determine the relative expression of the ENDO-I gene in several human tissues. Poly A-enriched RNA was isolated and reverse-transcribed. To determine the sequence of ENDO-I cDNA, a polymerase chain reaction was performed on cDNA from human endometriotic lesions and a gene-specific primer based on the human haptoglobin sequence. A similar procedure was followed to assess the relative expression of the ENDO-I gene in various tissues. Glyceraldehyde-3-phosphate-dehydrogenase was used as an internal control. ENDO-I gene expression was quantified by densitometry. Sequence analysis of ENDO-I cDNA identified 873 nucleotides that displayed 99.4% homology with the human haptoglobin beta-chain. Relative expression of ENDO-I mRNA by peritoneal endometriotic lesions was 19-fold greater than peritoneum, 28-fold greater than endometrioma and 37-fold greater than eutopic endometrium (P<0.01). Haptoglobin-like ENDO-I may be associated with localized angiogenesis and altered immune response involved with the aetiology/pathophysiology of endometriosis. (+info)
Ialpha exon-replacement mice synthesize a spliced HPRT-C(alpha) transcript which may explain their ability to switch to IgA. Inhibition of switching to IgG in these mice.
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Antibody class switching is regulated by transcription of unrearranged C(H) genes to produce germline (GL) transcripts which direct the choice of isotype and are required for switching. However, their role is unknown. GL transcripts are initiated at the I exons located upstream of each switch region. Although deletion of the I exon by gene targeting prevents switch recombination to that CH gene, the Ialpha exon can be replaced by an entirely different DNA segment, a minigene driven by the phosphoglycerate kinase (PGK) promoter and encoding hypoxanthine phosphoribosyl transferase (HPRT), oriented in the sense direction, without reducing antibody class switching to IgA. To understand why HPRT substitution of the Ialpha exon does not disrupt switch recombination, we have analyzed the structure of the transcript from the targeted allele in these mice. We identify a spliced transcript in which the HPRT exons are spliced to the C(alpha) gene segments, resulting in a structure similar to normal GL transcripts. The abundance of this transcript is similar to that of the normal alpha GL RNA. We also demonstrate that switching to the four IgG subclasses in B cells from these mice is reduced in comparison to wild-type mice. We discuss the possibility that the strong PGK promoter inserted at the Ig alpha locus may interfere with interaction of the promoters for gamma GL transcripts with the 3' IgH enhancer. (+info)
Synechocystis sp. slr0787 protein is a novel bifunctional enzyme endowed with both nicotinamide mononucleotide adenylyltransferase and 'Nudix' hydrolase activities.
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Synechocystis sp. slr0787 open reading frame encodes a 339 residue polypeptide with a predicted molecular mass of 38.5 kDa. Its deduced amino acid sequence shows extensive homology with known separate sequences of proteins from the thermophilic archaeon Methanococcus jannaschii. The N-terminal domain is highly homologous to the archaeal NMN adenylyltransferase, which catalyzes NAD synthesis from NMN and ATP. The C-terminal domain shares homology with the archaeal ADP-ribose pyrophosphatase, a member of the 'Nudix' hydrolase family. The slr0787 gene has been cloned into a T7-based vector for expression in Escherichia coli cells. The recombinant protein has been purified to homogeneity and demonstrated to possess both NMN adenylyltransferase and ADP-ribose pyrophosphatase activities. Both activities have been characterized and compared to their archaeal counterparts. (+info)
The pro-phenoloxidase of coleopteran insect, Tenebrio molitor, larvae was activated during cell clump/cell adhesion of insect cellular defense reactions.
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To characterize the proteins involved in cell clump/cell adhesion of insect cellular defense reactions, we induced the cell clump/cell adhesion reaction in vitro with the hemolymph of larvae of the coleopteran insect, Tenebrio molitor. The 72 kDa protein was specifically enriched in the residues of cell clump/cell adhesion and was purified to homogeneity. A cDNA clone for the 72 kDa protein was isolated. We found that the 72 kDa protein was an activated phenoloxidase from Tenebrio pro-phenoloxidase. We suggest that activated phenoloxidase is involved in the cell clump/cell adhesion reaction as well as in the synthesis of melanin. (+info)
A common MSH2 mutation in English and North American HNPCC families: origin, phenotypic expression, and sex specific differences in colorectal cancer.
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The frequency, origin, and phenotypic expression of a germline MSH2 gene mutation previously identified in seven kindreds with hereditary non-polyposis cancer syndrome (HNPCC) was investigated. The mutation (A-->T at nt943+3) disrupts the 3' splice site of exon 5 leading to the deletion of this exon from MSH2 mRNA and represents the only frequent MSH2 mutation so far reported. Although this mutation was initially detected in four of 33 colorectal cancer families analysed from eastern England, more extensive analysis has reduced the frequency to four of 52 (8%) English HNPCC kindreds analysed. In contrast, the MSH2 mutation was identified in 10 of 20 (50%) separately identified colorectal families from Newfoundland. To investigate the origin of this mutation in colorectal cancer families from England (n=4), Newfoundland (n=10), and the United States (n=3), haplotype analysis using microsatellite markers linked to MSH2 was performed. Within the English and US families there was little evidence for a recent common origin of the MSH2 splice site mutation in most families. In contrast, a common haplotype was identified at the two flanking markers (CA5 and D2S288) in eight of the Newfoundland families. These findings suggested a founder effect within Newfoundland similar to that reported by others for two MLH1 mutations in Finnish HNPCC families. We calculated age related risks of all, colorectal, endometrial, and ovarian cancers in nt943+3 A-->T MSH2 mutation carriers (n=76) for all patients and for men and women separately. For both sexes combined, the penetrances at age 60 years for all cancers and for colorectal cancer were 0.86 and 0.57, respectively. The risk of colorectal cancer was significantly higher (p<0.01) in males than females (0.63 v 0.30 and 0.84 v 0.44 at ages 50 and 60 years, respectively). For females there was a high risk of endometrial cancer (0.5 at age 60 years) and premenopausal ovarian cancer (0.2 at 50 years). These intersex differences in colorectal cancer risks have implications for screening programmes and for attempts to identify colorectal cancer susceptibility modifiers. (+info)
Analysis of spinocerebellar ataxia type 2 gene and haplotype analysis: (CCG)1-2 polymorphism and contribution to founder effect.
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Spinocerebellar ataxia type 2 is a familial spinocerebellar ataxia with autosomal dominant inheritance. The gene responsible was recently cloned and this disorder was found to be the result of a CAG expansion in its open reading frame. We analysed 13 SCA2 patients in seven unrelated families in Gunma Prefecture, Japan. In four of the seven families, we detected CCG or CCGCCG interruptions in only the expanded alleles. Cosegregation of these polymorphisms with SCA2 patients was established within each family. Together with the results of haplotype analyses, we considered that at least two founders were present in our area and that these (CCG)1-2 polymorphisms may make analysis of founder effects easier. By sequencing analysis we found that although the number of the long CAG repeat varied in each subclone of expanded alleles, these polymorphisms did not change their configuration. This finding suggests that CCG or CCGCCG sequences are stable when surrounded by the long CAG repeat and a single CAG. Moreover, the presence of these polymorphisms may lead to miscounting the repeat size by conventional estimation using a size marker such as an M13 sequencing ladder. Therefore we should consider these polymorphisms and accurately determine the repeat size by sequencing. (+info)
The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15.
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Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components. (+info)
Identification of a novel Arg-->Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia in pigs.
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We previously carried out genetic and metabolic studies in a partially inbred herd of pigs carrying cholesterol-elevating mutations. Quantitative pedigree analysis indicated that apolipoprotein (apo)B and a second major gene were responsible for the hypercholesterolemia in these animals. In this study, we assessed LDL receptor function by three different methods: ligand blots of liver membranes using beta-very low density lipoprotein (VLDL) as a ligand; low density lipoprotein (LDL)-dependent proliferation of T-lymphocytes; and direct binding of 125I-labeled LDL to cultured skin fibroblasts. All three methods demonstrated that LDL receptor ligands bound with decreased affinity to the LDL receptor in these animals. In skin fibroblasts from the hypercholesterolemic pigs, the Kd of binding was about 4-fold higher than in cells from normal pigs. The cDNA of the pig LDL receptor from normal and hypercholesterolemic pigs was isolated and sequenced. We identified a missense mutation that results in an Arg'Cys substitution at the position corresponding to Arg94 of the human LDL receptor. The mutation is in the third repeat of the ligand binding domain of the receptor. By single-stranded conformational polymorphism (SSCP) analysis, we studied the relationship between LDL receptor genotype and plasma cholesterol phenotype. In contrast to humans, the hypercholesterolemia associated with the LDL receptor mutation in pigs was expressed as a recessive trait. The LDL receptor mutation made a far more significant contribution to hypercholesterolemia than did the apoB mutation, consistent with observations made in human subjects with apoB mutations. Within each genotypic group (mutated apoB or mutated receptor), there was a wide range in plasma cholesterol. As the animals were on a well-controlled low-fat diet, this suggests that there are additional genetic factors that influence the penetrance of cholesterol-elevating mutations. (+info)