OBJECTIVE RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic beta-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether alteri...

A complementary DNA (cDNA) library was prepared from messenger RNA (mRNA) isolated from Syrian hamster islets. Bacterial colonies containing hamster preproinsulin cDNA were identified by cross-hybridization with the human preproinsulin gene. The sequences of two of these established the complete sequence of Syrian hamster preproinsulin mRNA and predicted the sequence of the protein. Hamster pre...

Short-term (less than 2 h) glucose stimulation of isolated pancreatic islets specifically increases the biosynthesis of proinsulin and its converting enzymes PC2 and PC3 at the translation level. To determine whether gene expression of PC2 and PC3 was also regulated by longer-term (more than 6 h) glucose stimulation along with that of preproinsulin, studies were performed with the beta TC3 insu...

OBJECTIVE A restricted region of proinsulin located in the B chain and adjacent region of C-peptide has been shown to contain numerous candidate epitopes recognized by CD8(+) T-cells. Our objective is to characterize HLA class I-restricted epitopes located within the preproinsulin leader sequence. RESEARCH DESIGN AND METHODS Seven 8- to 11-mer preproinsulin peptides carrying anchoring residue...

We have recently determined through standard molecular cloning methods that Xenopus laevis contains two different nonallelic preproinsulin genes which are very similar to each other (i.e. 94% identity in the coding region) (1). In order to obtain the remaining sequence of the 5'-untranslated regions we used the anchored polymerase chain reaction method as described by others (2,3). We first rev...

Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure we...

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (</=2h) this is due to increases in the translation of pre-existing mRNA. However, prolonging glucose stimulation (24 h) also increases preproinsulin mRNA levels. It has been proposed that secreted insulin from the pancreatic beta-cell regulates its own synthesis through a positive autocrine f...

A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized protein was sequestered in an insoluble form in i...

The 5' terminus of the rat preproinsulin II gene exhibits a tissue-dependent DNase I sensitivity. Only in the chromatin froma pancreatic beta-cell tumor, but not in liver, spleen, kidney, or brain chromatin, is a region at and before the 5' end of the gene exposed to cleavage. The region of exposure extends 250-300 base pairs upstream from the 5' terminus of the preproinsulin mRNA. Such a regio...

Guinea pig insulin exhibits an unusually high degree of divergence from the conserved insulins of other mammals. cDNA clones encoding guinea pig preproinsulin were isolated, and their nucleic acid sequences were determined. Comparisons of the nucleic acid sequence and its predicted amino acid sequence with sequences encoding insulins of other species revealed that the gene encoding guinea pig p...