Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.
A novel tissue-specific cDNA, PDIp, was previously isolated from human pancreas. It encodes a protein that is structurally and functionally related to protein disulfide isomerase (PDI). To compare the expression pattern of PDI and PDIp in human pancreas and liver tissues, we prepared rabbit polyclonal antiserum against a recombinant glutathione-S-transferase-coupled PDIp fusion protein. Western blot analysis revealed that pancreas expresses both PDI and PDIp, whereas liver only expresses PDI. Rabbit antiserum raised against recombinant PDIp immunostained specifically to the acinar cells of human pancreas. Treatment of PDIp with peptide:N-glycosidase F caused PDIp down shift in the NaDodSO4-PAGE gel, indicating that PDIp is a glycoprotein. A 2.0-kb message was detected from mouse pancreas using a human PDIp cDNA probe. Similarly, PDIp glycoprotein was detected in mouse pancreas extract by anti-human PDIp antiserum, suggesting that PDIp is highly conserved in human and mouse pancreas. From these studies, we conclude that the pancreas expresses two members of PDI and that PDIp is a glycoprotein specifically expressed in pancreatic acinar cells.
The effects of repeated s.c. transplantation of clonal insulin-secreting RINm5F cells in NEDH rats on tumour morphology, insulin-glucose homeostasis and the function of RINm5F cells re-established in culture were examined after maintenance in vivo for periods of up to 308 days. Transplantation of RINm5F cells for ten consecutive passages consistently produced a single encapsulated vascularized tumour associated with the development in recipient rats of hyperinsulinaemia, hypoglycaemia and eventual neuroglycopenic coma. At the tenth passage, tumour-bearing rats exhibited a markedly enhanced rate of insulin-stimulated glucose uptake by 19 days, with evidence of a large and variable insulin response to i.p. glucose. Survival was 22 +/- 2 days, and resected RINm5F cell tumours exhibited weak immunostaining for insulin in scattered cells, with strands of fibrous tissue separating clusters of tumour cells many of which had distinct polarity. There was no detectable immunostaining for glucagon, somatostatin or pancreatic polypeptide. The insulin content and insulin secretory output of RINm5F cells re-established in culture after 20, 146, 259 or 308 days propagation in vivo were generally enhanced compared with non-passaged RINm5F cells. The magnitude of the effect was not appreciably affected by the duration of maintenance in vivo, but it was critically dependent upon the subsequent period of culture in vitro. Thus, whereas 2-day cultured RINm5F cells from the eighth tumour passage exhibited a greater than 100-fold increment of insulin content and release, with enhanced secretory responsiveness to leucine, arginine, theophylline, K+ (25 mmol/l) or Ca2+ (7.6 mmol/l), RINm5F cells cultured for a further 19 days had almost completely lost the attributes resulting from 259 days of maintenance in vivo. The results indicate substantial enhancement of the functional capabilities of RINm5F cells in vivo, and suggest that the resulting tumours afford a novel model in NEDH rats of responsive trabecular-type insulinomas.
Autoantibodies to IA-2 not detected in NOD mice or BB rats Dear Sir, IA-2 m R N A is expressed in both human pancreatic islets and brain [1]. It encodes a 979 amino acid protein that is a major autoantigen in insulin-dependent diabetes mellitus (IDDM) [2, 3]. Up to 70 % of IDDM patients have antibodies to IA-2 [4]. In clinically non-diabetic subjects, the appearance of autoantibodies to IA-2 is highly predictive in identifying individuals at high risk of subsequently developing clinical disease [5]. The diabetic syndrome in NOD mice and BB rats has features which both resemble and differ from those of IDDM in humans [6]. The present investigation was initiated to see whether IA-2 was also an autoantigen in these animal models. Mouse IA-2 is expressed in both mouse pancreatic islets and brain [7]. It is also 979 amino acids in length. Its intracellular domain is 98.4 % similar to the intracellular domain of human IA-2, and its extraceUular domain is 87.2 % similar to the extracellular domain of human IA-2. The intracellular and extracellular domains of rat IA-2 [8] are 99 % and 87 % similar, respectively, to the intracellular and extracellular domains of human IA-2. Sera from normal, prediabetic and diabetic mice and rats were collected and tested for autoantibodies by immunoprecipitation of radiolabelled recombinant mouse IA-2. Sera from humans strongly positive for autoantibodies to IA-2 and mouse monoclonal antibody-161 to IA-2 served as controis. As seen in Figure 1, human diabetic sera immunoprecipitated mouse recombinant IA-2. Similarly, mouse monoclonal antibody also immunoprecipitated mouse recombinant IA-2. Pilot studies (Fig. 1) showed that sera from diabetic NOD/Lt mice (kindly provided by Dr. E. H. Leiter, Bar Harbor, Maine, USA) failed to immunoprecipitate mouse IA-2. To further investigate the ability of NOD mouse and BB rat sera to immunoprecipitate mouse recombinant IA-2, a total of 103 NOD mouse sera (13 normal, 61 prediabetic and 29 diabetic) and 102 DP-BB rat sera (12 normal, 12 prediabetic and 78 diabetic) were tested. Antibody to IA-2 was not detected in any of the 205 sera tested. NOD mice and BB rats have been extensively studied as autoimmune models for human IDDM and have been widely
Background Effective opioid agonist therapy (OAT) depends on good patient adherence. However, the daily, supervised administration of standard OAT represents a significant burden to patients and often drives poor adherence. Prolonged-release buprenorphine (PRB) formulations may mitigate some of this burden, enabling clinic visits to be substantially reduced. For treatment guidelines to be effective, the likely benefit of a transition to PRB therapy in different patient populations must be established. Methods The aim was to determine the feasibility of assessing PRB as an alternative to daily OAT in two groups: those currently adhering well to daily OAT (group 1, N = 5) and those not currently showing adherence or a positive response to daily OAT (group 2, N = 10). This open-label, prospective, non-controlled pilot study was conducted at the Kaleidoscope Drug Project in South Wales, UK. Participants were assessed for history, drug use, psychosocial assessment scores, and clinical severity at baseline and after 6 months of treatment. Primary outcomes were the feasibility of assessing PRB as an alternative to daily OAT and the acceptability of PRB therapy in each group. Secondary outcomes were treatment response, on-top drug use, psychosocial measures, and assessment of clinical severity. Results Participants from both groups demonstrated high levels of participation with assessment protocols at both baseline and 6-month follow-up, indicating study feasibility. PRB treatment was acceptable to the majority of participants, with all of group 1 and 70% of group 2 adhering to PRB therapy for the duration of the study and opting to persist with PRB therapy over other OAT options after study completion. All participants who remained on treatment demonstrated marked improvements in psychosocial and clinical severity assessment scores, with some returning to employment or education. On-top drug use remained absent in group 1 and was reduced in group 2. Conclusions Evaluation of transition of participants from daily OAT to PRB therapy was shown to be feasible, acceptable, and effective across both groups. A larger randomised controlled trial is warranted, particularly to assess PRB therapy in participants with a history of poor treatment engagement, as the need for therapy is greater in this group and their management is associated with higher costs of care.
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