A. Foriers

Prolonged exposure of pancreatic beta cells to raised glucose concentrations results in increased cellular content of islet amyloid polypeptide precursors.

Summary Most non-insulin dependent diabetic patients have amyloid deposits in their pancreatic islets. It is not known whether chronic hyperglycaemia contributes to the formation of amyloid fibrils from the islet amyloid polypeptide that is produced by the pancreatic beta cells. Since islet amyloid exhibits islet amyloid polypeptide precursors immunoreactivity, we examined whether sustained in vitro exposure to raised glucose increases the abundance of these precursors in human beta cells. After 6 days stimulation with 20 mmol/l glucose the cellular content of insulin but not islet amyloid polypeptide was decreased leading to an increase in the ratio of the latter over insulin (3.0 ± 0.6 vs 1.8 ± 0.3 after 6 mmol/l glucose culture, p < 0.05). Similar changes occurred in rat beta cells cultured for 3 days in the presence of 20 mmol/1 glucose plus 3-isobutyl-1-methylxanthine. Western blot analysis of cellular islet amyloid polypeptide after prolonged exposure to high glucose indicated the presence of higher proportions of its precursor- and intermediate forms. In human beta cells cultured in 20 mmol/l glucose, the major form corresponds to an intermediate species which exhibits an immunoreactivity for the N-flanking peptide, as is also the case in islet amyloid. We concluded that prolonged in vitro exposure of beta cells to raised glucose concentrations increases the relative proportion of islet amyloid polypeptide over insulin, as well as of its precursors over the mature form of islet amyloid polypeptide. [Diabetologia (1999) 42: 188–194]

Comparison of capillary zone electrophoresis with high-performance liquid chromatography for the determination of additives in foodstuffs

A capillary zone electrophoretic (CZE) method was developed to determine caffeine, aspartame and benzoic acid in diet cola soft drinks and in artificial sweetening powders. The effects of pH, ionic strength, organic solvents and different buffers were investigated to select the optimum conditions. These consisted of a sodium phosphate buffer at pH 11 and ionic strength 0.025. The running voltage was set at 15 kV and the injection was performed hydrostatically for 30 s. The CZE method was then compared with a previously developed high-performance liquid chromatographic (HPLC) method in terms of repeatability, reproducibility, accuracy, linearity, sensitivity and separation efficiency. Both methods gave good repeatability. The relative standard deviations for reproducibility were significantly higher in CZE than in HPLC. The main reason for this is probably the condition of the wall of the capillary, which was difficult to keep constant between the days of analysis. The separation efficiency of CZE was 65-110 times higher than that of HPLC; on the other hand, 10-20 times lower detection limits were obtained in HPLC. Both methods were linear, but the linear ranges were different owing to the lower detection limit of HPLC. In CZE, the effect of the matrix was higher.

Abnormal circulating pancreatic enzyme activities in more than twenty-five percent of recent-onset insulin-dependent diabetic patients: Association of hyperlipasemia with high-titer islet cell antibodies.

Pancreatic amylase and lipase activities were measured in sera of 307 Caucasian insulin-dependent diabetes mellitus patients (IDDM) at clinical onset, 303 nondiabetic siblings of registered patients, and 207 control subjects under age 40 years. In all subject groups lipasemia and pancreatic (but not salivary) amylasemia increased with age and were significantly correlated. Using age-dependent reference ranges, reduced pancreatic enzyme levels were measured in 18% of patients, 6% of siblings, and only 2% of control subjects (p < 0.001). Increased lipase levels were noted in 10% of patients and in only 3% of siblings and 2% of control subjects (p, < 0.001). Using both univariate and multivariate statistical analysis, elevated lipase activities at clinical onset were associated with higher titers of autoantibodies against islet cell cytoplasmic antigens and glucagon, but not against insulin or the 65-kDa isoform of glutamic acid decarboxylase (GAD65-Ab), or with markers of genetic predispovsition or metabolic dysregulation. These findings indicate the presence of modest, but statistically significant, variations in circulating pancreatic enzyme levels in 28% of IDDM patients at clinical onset (p < 0.001 vs. 5% in control subjects). Increased lipase levels may express a form or a stage of the disease with exocrine cell damage; their association with higher titers of islet cell and glucagon autoantibodies is not yet explained. Lower lipase and isoamylase levels are thought to result from the reduced acinar cell function in the vicinity of insulin-depleted islets. It must be tested whether pancreatic enzyme activities in serum can also be altered during the preclinical stage and can thus be considered as an additional marker for the disease process in the pancreas.

N-terminal sequences of the α and β subunits of the lectin from the garden pea (Pisum sativum)

Lectins constitute a group of proteins mainly found in the seeds of a wide variety of plants and in certain invertebrates. These proteins have proven to be useful tools for the study of protein-carbohydrate interactions and the topography of cell surfaces. #en immobilised on Sepharose, lectins may be used for the fractionation of cells and for the isolation of glycoproteins and cell receptors. Certain lectins are able to induce mitogenesis in lymphocytes a phenomenon analogous to the stimulation of immune cells by a specific antigen [l-3] . Although lectins have been extensively studied for their biological properties, few structural data are available to relate their activity to their structure, except for Concanavalin A [4]. In this paper we describe the subunit structure and N-terminal sequences of the oand &subunits of the phytohemagglutinin of the garden pea (Pisum sutivum), a protein with sugar binding properties similar to those of concanavalin A.

Effect of phenobarbital on the expression of glutathione S-transferase isoenzymes in cultured rat hepatocytes

Cultured adult rat hepatocytes were treated daily with 3.2 mM phenobarbital (PB) in order to study its effect on the expression of cytosolic glutathione S‐transferase isoenzymes. Glutathione S‐transferase (GST) activities, using 1‐chloro‐2,4‐dinitrobenzene and 1,2‐dichloro‐4‐nitrobenzene as substrates, were increased when PB was present in the culture medium. After purification and separation of GST on glutathione Sepharose 6 B and reversed‐phase HPLC, respectively, it was observed in vitro that PB caused an increase in the relative amounts of subunits 1, 3 and 7 compared to subunits 2 and 4. Using Northern blot technique, elevated levels of GST subunit 1/2 and 7 mRNA were measured, after addition of PB to the cultures.

Differential Expression of Rat Insulin I and II Messenger Ribonucleic Acid after Prolonged Exposure of Islet β-Cells to Elevated Glucose Levels1

Prolonged exposure of rat islet beta-cells to 10 mmol/liter glucose has been previously shown to activate more cells into a glucose-responsive state (>90%) than has exposure to 6 mmol/liter glucose (50%). The present study demonstrates that this recruitment of more activated cells results in 4- to 6-fold higher levels of proinsulin I and proinsulin II messenger RNA (mRNA). However, only the rate of proinsulin I synthesis is increased. Failure to increase the rate of proinsulin II synthesis in the glucose-activated cells results in cellular depletion of the insulin II isoform, which can be responsible for degranulation of beta-cells cultured at 10 mmol/liter glucose. Higher glucose levels (20 mmol/liter) during culture did not correct this dissociation between the stimulated insulin I formation and the nonstimulated insulin II formation. On the contrary, the rise from 10 to 20 mmol/liter glucose resulted in a 2-fold reduction in the levels of proinsulin II mRNA, but not of proinsulin I mRNA; this process further increased the ratio of insulin I over insulin II to 5-fold higher values than those in freshly isolated beta-cells. The present data suggest that an elevated insulin I over insulin II ratio in pancreatic tissue is a marker for a prolonged exposure to elevated glucose levels. The increased ratio in this condition results from a transcriptional and/or a posttranscriptional failure in elevating insulin II formation while insulin I production is stimulated in the glucose-activated beta-cells.

Detection of autoantibodies against islet amyloid polypeptide in human serum. Lack of association with Type 1 (insulin-dependent) diabetes mellitus, or with conditions favouring amyloid deposition in islets

SummaryA radiobinding assay for the detection of autoantibodies against islet amyloid polypeptide was developed, analytically validated, and -in parallel with a similar assay for the detection of autoantibodies against insulin — applied to sera from recent-onset Type 1 (insulin-dependent) diabetic patients and from age- and sex-matched control subjects. There was no difference in islet amyloid polypeptide autoantibody titres between patient groups and matched control subjects, nor within subject groups according to age. At onset of Type 1 diabetes, elevated islet amyloid polypeptide-autoantibody levels (> 97th percentile of control subjects) were only detected in 1 of 30 patients aged 0–19 years and in 2 of 35 patients aged 20–39 years. By contrast, insulin autoantibodies were frequently demonstrated, in particular at onset of diabetes under age 20 (0–19 years: 18 of 30 patients; 20–39 years: 10 of 35 patients; p < 0.01 vs matched control subjects). Islet amyloid polypeptide autoantibodies were not detectable in 3 insulinoma patients nor in 37 patients (aged 33–70 years) with Type 2 diabetes (vs 1 of 40 in matched control subjects). In positive serum, adsorption onto protein A-Sepharose removed islet amyloid polypeptide binding activity, hereby confirming its antibody nature. In conclusion, Type 1 diabetes is associated with an age-dependent autoantibody reaction against insulin but not against islet amyloid polypeptide. Conditions associated with amyloid deposition in islets (Type 2 diabetes, insulinoma and ageing) do not favour the formation of autoantibodies against islet amyloid polypeptide.

Use of high-performance size-exclusion chromatography for the separation of poliovirus and subviral particles

The size-dependent separation of viral and subviral particles in the range 10(5)-10(7) daltons was undertaken by high-performance liquid chromatography. A combination of Ultrahydrogel 2000 and 1000 size-exclusion columns, equilibrated and developed with Tris buffer (pH 7.4), was used to fractionate extracts of cells infected with radiolabelled poliovirus. Poliovirions (30 nm) and subviral particles (20 nm) were separated according to size with full retention to their biological activities. Procapsids (same size as virions, but devoid of RNA) could not be separated from virions. Sample recoveries as determined with radiolabelled material constantly exceeded 70%. The method was successfully applied to the separation of viral and subviral particles from complex mixtures.

The subunit structure and N-terminal sequences of the α- and gb-subunits of the lentil lectin (Lens culinaris)

Lectins are proteins or glycoproteins present in many plants and are usually recognized by their ability to agglutinate erythrocytes. Agglutination of cells is in many cases inhibited by specific sugars, suggesting that the binding is to sugar residues on the cell surface. Very little is known of the mechanism by which the lectin leads to agglutination or cellsurface alteration. In order to understand the relationship between the activity and the structure of lectins, we have undertaken the determination of the amino acid sequence of several of these proteins. The lectin studied in this work is present in seeds of the lentil, Lens culinaris [ 1,2] . The protein has a molecular weight of approximately 49 000 and is composed of two types of subunits with molecular weights of 18 000 and 8000 [3] . The isolation and characterization of this hemagglutinin was described in our previous communication [4]. We report here the N-terminal sequences of the (Yand P-subunits of the lentil lectin. This protein has the same sugar-binding specificity as two other mitogenic lectins, Concanavalin A [5] and pea lectin [6], for which sequence studies were reported previously [7,8]. The comparison of these three proteins reveals surprising homologies.

Purification of poliovirus 14 S subunits by sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography

Purification of 14 S subunits from extracts of poliovirus-infected HeLa cells was achieved by a combination of sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography. The particles were free of admixtures of other subviral particles, of nonstructural viral proteins, and of host cell proteins. The purified material retained the physical and antigenic properties of native 14 S subunits fully, as well as their ability to assemble to empty capsids in vitro.