Cloning of mouse islet amyloid polypeptide gene and characterization of its promoter.

Islet amyloid polypeptide (IAPP) was isolated from islet amyloid deposits in patients with insulinoma and pancreatic islets of non-insulin-dependent diabetes mellitus (NIDDM) and several reports suggested that it may contribute to the development of NIDDM. IAPP is mainly expressed and synthesized in pancreatic B cells and cosecreted with insulin, so analysis of the transcriptional regulation of the IAPP gene would be helpful for the elucidation of pancreatic B cell specific gene expression. The mouse IAPP gene spans about 5.8 kb and, like the human and rat genes, it consists of three exons, and analysis of the promoter/enhancer activity of mouse IAPP gene reveals the region from -171 to -87 bp to be essential. Within this region, an E-box like sequence, CACCTG (-122 to -117 bp), and a TAAT-box like sequence, TTAATG (-139 to -134 bp), are thought to be important. The disruption of each sequence resulted in a severe decrease in promoter activity, although the decrease was less in the disruption of the E-box than that of TAAT-box like sequence, suggesting the latter is more important for IAPP gene transcription. Like the rat IAPP gene, the CCAAT-box, which does not exist in the human gene, was identified in the mouse gene, indicating the possibility of species difference in the IAPP gene transcriptional mechanism. An enhancer-like activity was also identified within intron 1, although further elucidation is necessary.

Restriction fragment length polymorphisms near the islet amyloid polypeptide gene in Japanese subjects.

Two restriction fragment length polymorphisms (RFLPs) near the human islet amyloid polypeptide (IAPP) gene were examined in 50 Japanese patients with non-insulin-independent diabetes mellitus (NIDDM) and 54 non-diabetic controls. RFLPs were identified with the enzymes PvuII (A1 = 21 kb and A2 = 18 kb) and BglII (B1 = 9 kb and B2 = 7 kb). These RFLPs were in complete linkage disequilibrium with A1 which was in disequilibrium with B2, as was A2 with B1. Since these two RFLPs map to different locations in the 5'-flanking region of the IAPP gene, they are most likely due to changes in the sequence of the sites recognized by PvuII and BglII rather than to an insertion/deletion-type DNA polymorphism. There were no differences in the genotypic or allelic frequencies of these RFLPs between Japanese subjects with NIDDM and non-diabetic controls implying that these RFLPs do not play a major role in the development of NIDDM in this population.

Molecular biology of islet amyloid polypeptide.

We investigated the relationship between non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid polypeptide (IAPP) gene by restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-direct sequencing analysis. Endonuclease BglII and/or PvuII RFLP analysis revealed no positive correlation of IAPP gene with NIDDM. In PCR-direct sequencing of 25 NIDDM patients, no nucleotide sequence differences were found. These data do not support the view that IAPP plays an important role in the pathogenesis of NIDDM. cDNAs encoding cat, rat, mouse, guinea pig and degu IAPP precursors were also cloned, and comparison of these predicted amino acid sequences clarified the species difference, especially between amyloid-forming and non-amyloid-forming species. Amino acid residues 25-28 of mature IAPP might be responsible for their amyloidogeneity. The alternative splicing transcripts of guinea pig IAPP gene were identified by using PCR. If these types of transcripts are translated, N-terminal mutated IAPP might be produced and act as an antagonist. The signal peptide cleavage site of rat IAPP precursor was also identified by an in vitro translation and processing system.

Beta-thalassemia (beta 90 GAG-->TAG): genetic diagnosis by allele-specific polymerase chain reaction and estimation of mutant mRNA expression by reverse transcription polymerase chain reaction.

Genetic diagnosis of a family of beta-thalassemia (beta 90 GAG-->TAG) was carried out by allele-specific polymerase chain reaction (AS-PCR). The proband, her daughter and granddaughter were proved to be heterozygotes of normal and mutant alleles. As some nonsense mutations express a decreased amount of MrNAs, we determined the expression level of the mutant mRNA of beta-thalassemia (beta 90 GAG-->TAG), by application of a combination method of reverse transcription PCR (RT-PCR) and dot-blot hybridization with allele-specific oligonucleotides. The mutant mRNA was not markedly reduced. In conclusion, 1) individuals with the mutant beta-globin gene were diagnosed successfully by AS-PCR, and 2) a significant amount of the mutant beta-globin mRNA was synthesized.

[A case of so-called neoplastic angioendotheliosis with fever of unknown origin and temporary loss of consciousness].

A 65-year-old man was hospitalized with prolonged fever. The erythrocyte sedimentation rate was 62 mm/hour and the c-reactive protein was 5+. Serum lactic dehydrogenase was 1005 IU with elevation of isozyme types II and III. A computerized tomography scan of the abdomen showed large, bilateral adrenal masses. An aspirated specimen of the bone marrow revealed some clustered atypical cells. These findings suggested the patient had metastatic carcinoma, but the primary lesion could not be found. The patient had an episode of transient unconsciousness in mid course and died of bleeding from the gastrointestinal tract. A postmortem examination was performed. Microscopic examination showed an accumulation of neoplastic cells in the vascular system throughout the body and their extravascular proliferation in several organs. Immunohistochemical studies were performed on paraffin-embedded fixed tissues. The neoplastic cells were positive with the monoclonal antibodies LCA, LN 1, LN 2 and N 26 but did not show positive staining for factor VIII-related antigen, a marker for endothelial cells. These studies defined the neoplastic cells as being of a germinal-center B lymphocyte origin. On the basis of the above-mentioned results, we suggest that this case may be diagnosed as angiotrophic lymphoma.