Biomedical subjects

K Josefsen

Publications and source records attributed to K Josefsen.

At least 19 recordsLinked to original sources

PubMed2002

The (ProB27, ThrB28) human insulin analogue is more potent and more rapidly absorbed from subcutis than human insulin.

OBJECTIVE: To test the physiological properties of human insulin in which the amino acids Thr (B27) and Pro (B28) are interchanged (PT insulin). This was hypothesised to prevent dimerisation and accelerate the absorption from s.c. tissue without altering the affinity for the insulin receptor. DESIGN: PT insulin was expressed in Pichia pastoris and processed in vitro. The purified compound was used for physiological investigations. METHODS: Receptor binding activity to insulin and IGF receptors was evaluated in a competition assay using iodinated PT insulin and recombinant receptors while growth induction properties were evaluated by thymidine incorporation. Absorption kinetics from pig subcutis was investigated by measuring the disappearance of iodinated PT insulin. The potency was evaluated by measuring the blood glucose lowering activity in mice. RESULTS: The absorption of PT insulin was accelerated compared with human insulin, although still slower than Asp (B28) insulin. Human and PT insulin had similar affinities for the human insulin receptor (K(d)=3.6 x 10(-12) vs 5.2 x 10(-12) mol/l) while the affinity for the IGF receptor was four times higher for PT insulin than for human insulin (K(d)=3.4 x 10(-8) vs 1.3 x 10(-7) mol/l). This resulted in a slightly higher DNA synthesis when assayed in intermediary insulin concentrations. The blood glucose lowering effect in mice exceeded the effect of human insulin (integral 0-60 min: 61.4+/-7 vs 30+/-4, n=6, P=0.046). CONCLUSIONS: PT insulin is absorbed faster and is more potent than human insulin. Although PT insulin stimulates growth more than human insulin, this will not prevent its use in the clinic, but the main interest will probably focus on investigations to clarify the paradox of full biological activity in connection with the recently described lack of structure in the B-chain.

Absorption↗

PubMed2001

Flexibility and bioactivity of insulin: an NMR investigation of the solution structure and folding of an unusually flexible human insulin mutant with increased biological activity.

The structure and folding of a novel human insulin mutant, [Thr(B27) --> Pro, Pro(B28) --> Thr]insulin (PT insulin), in aqueous solution and in mixtures of water and 2,2,2-trifluoroethanol (TFE) have been studied by NMR spectroscopy. It was found that PT insulin has a highly flexible structure in pure water and is present in at least two different conformations, although with an overall tertiary structure similar to that of native insulin. Furthermore, the native helical structures are poorly defined. Surprisingly, the mutant has a biological activity about 50% higher than native insulin. In contrast, in TFE/water solution the mutant reveals a propensity of forming a well-defined structure at the secondary structure level, similar to monomeric native insulin. Thus, as shown by a detailed determination of the structure from 208 distance restraints and 52 torsion angle restraints by distance geometry, simulated annealing, and restrained energy minimization, the native insulin helices (A2-A7, A13-A19, and B10-B19) as well as the beta-turn (B20-B23) are formed in 35% TFE. However, the amount of tertiary structure is decreased significantly in TFE/water solution. The obtained results suggest that only an overall tertiary fold, as observed for PT insulin in pure water, is necessary for expressing the biological activity of insulin, as long as the molecule is flexible and retains the propensity to form the secondary structure required for its receptor binding. In contrast, a compact secondary structure, as found for native insulin in solution, is unnecessary for the biological activity. A model for the receptor binding of insulin is suggested that relates the increased bioactivity to the enhanced flexibility of the mutant.

Amino Acid Sequence↗

PubMed2000

Ligand binding characteristics of a glycosylphosphatidyl inositol membrane-anchored HeLa cell folate receptor epitope-related to human milk folate binding protein.

The folate receptor (FR) in HeLa cells was characterized as to ligand binding mechanism, antigenic properties and membrane anchor in order to obtain information to be used for the design of biological agents targeting FR in malignant tumors. The receptor displayed the following binding characteristics in equilibrium dialysis experiments (37 degrees C, pH 7.4) with [3H] folate: a high-affinity type of binding that exhibited positive cooperativity with a Hill coefficient > 1.0 and an upward convex Scatchard plot, a slow radioligand dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of other folates. The molecular size of the receptor was 100 kDa on gel filtration with Triton X-100, or similar to that of high molecular weight human milk folate binding protein (FBP). The latter protein represents a 25 kDa molecule which equipped with a hydrophobic glycosylphosphatidyl inositol (GPI) membrane anchor susceptible to cleavage by phosphatidylinositol specific phospholipase C (PI-PLC) forms micelles of 1kDa size with Triton X-100. The HeLa cell FR immunoreacted with antibodies against purified human milk FBP in ELISA, and in a fluorescence activated cell sorting system, where HeLa cells exposed to increasing concentrations of antibody showed a dose-dependent response. Exposure to PI-PLC decreased the fraction of immunolabeled cells indicating a linkage of FR to cell membranes by a GPI anchor. HeLa cells incubated with radiofolate showed a continuous uptake with time, however, with a complete suppression of uptake in the presence of an excess of cold folate. Prewash of cells at acidic pH to remove endogenous folate increased the uptake. Binding and uptake of [3H] folate was increased in cells grown in a folate-deprived medium. The HeLa FR seems to be epitope related to human milk FBP.

Binding Sites↗

PubMed2000

The glycosphingolipid sulfatide in the islets of Langerhans in rat pancreas is processed through recycling: possible involvement in insulin trafficking.

In previous studies we have shown that sulfatide (galactosylceramide-3-O-sulfate), in various species, is present in the insulin-producing cells in pancreatic islets of Langerhans. In this study the synthesis of sulfatide in the islets has been investigated by pulse chase labeling at varying glucose levels and in the presence or absence of the glycosphingolipid synthesis inhibitory agents, Brefeldin A, fumonisin B1 and chloroquine and the distribution of sulfatide by immune-electronmicroscopy. The data showed that (1) sulfatide was produced in islets of Langerhans, (2) the main pathway for synthesis was through recycling involving partial degradation in the lysosome, and that (3) high glucose levels, although not primarily reflected in an increased synthesis of sulfatide, lead to an increased expression of mRNA for the UDP-galactose:ceramide galactosyltransferase, producing the immediate precursor of sulfatide. Furthermore, mass spectrometry analyses revealed a high proportion of short chain fatty acids, C16:0 (50%) and no hydroxylated forms and thus special physicochemical properties, indicating important differences between pancreatic and brain/neural sulfatide. Immune electron microscopy revealed an intracellular expression of sulfatide in the secretory granules, the Golgi network and the lysosomes of the islets. These results indicate that sulfatide follows the same intracellular route as insulin and suggest a functional association between these molecules. We have raised the hypothesis that sulfatide possibly plays a role in the trafficking of insulin in the islets of Langerhans in rat pancreas.

Animals↗

PubMed2000

Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta.

Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line derived from rat pancreatic beta-cells, before and after challenge with 30 and 1,000 pg/ml of recombinant human IL-1beta. The highest concentration resulted in decreased insulin production and cell death over a period of 4 days. Using three different time points, 2, 4 and 24 hours after challenge, we found that 146 full-length genes and a large number of expressed sequence tags were differentially regulated 3-fold or more. Most of the differentially regulated transcripts have not previously been described to be regulated by IL-1beta in beta-cells. We have analysed the expression data and sorted the genes into groups according to functional relations on the basis of knowledge of the structure or function ascribed to the individual genes. Many of the differentially regulated genes are known to play a role in immune- and stress-related pathways as well as in insulin secretion and vesicle trafficking, e.g. alpha-endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated.

Animals↗

PubMed2000

Neonatal treatment with beta-cell stimulatory agents reduces the incidence of diabetes in BB rats.

The aim of the study was to investigate whether various beta-cell stimulatory drugs, given neonatally, influence the incidence of diabetes in BB rats. Newborn BB rats were treated twice daily for 6 days and diabetes development was observed during the following 200-day study period. Compared to a diabetes incidence of 63.8% in 163 control BB rats which received saline or were untreated, the percentage of experimental BB rats that developed diabetes was as follows in the different subgroups: arginine-glucose: 47% (n = 73, p < 0.02); glucagon: 37% (n = 93, p < 0.0001); tolbutamide-glucose: 36% (n = 58, p < 0.0005); and theophylline-glucose: 39% (n = 41, p < 0.005). A long-term arginine-glucose treatment was not superior to the shorter neonatal treatment. Histological examination revealed a higher degree of insulitis in diabetic than in non-diabetic animals but no difference according to the kind of treatment was observed. Finally, we found that the diabetes incidence in BB rats was higher in the first litter compared to subsequent litters (p = 0.04). Thus, neonatal treatment with various beta-cell stimulatory agents reduces diabetes incidence in BB rats. The theory behind the study, that the treatment accelerates beta-cell maturation leading to increased immunological tolerance towards beta cells, is discussed.

Animals↗

PubMed1999

Glucose induces early growth response gene (Egr-1) expression in pancreatic beta cells.

A copy deoxyribonucleic acid (cDNA) clone of the immediate early growth response gene, egr-1 (Krox-24, Zif268, NGFI-1), was isolated through subtractive hybridization screening to identify glucose-induced genes in pancreatic beta cells. Glucose rapidly and transiently induced egr-1 mRNA in the SV40-transformed murine beta-cell line, MIN6. Glucose also increased egr-1 mRNA expression in INS-1, betaTC3 and RINm5F beta-cell lines, although with different kinetics. Expression of the 82 kDa Egr-1 protein was induced both in MIN6 cells stimulated with glucose in vitro and in primary rat islet cells stimulated in vivo or in vitro. This response is unique to beta cells since glucose did not affect egr-1 expression in NIH-3T3 fibroblasts or glucose-sensitive hepatocytes. In beta cells egr-1 induction is specifically associated with insulin secretion, as it was not observed after stimulation with serum or insulin but was elicited by insulin secretagogues, including membrane depolarizing agents and cAMP agonists. Moreover, induction of egr-1 by glucose was inhibited by EDTA, indicating dependence on influx of extracellular Ca2+. Other immediate early response genes, c-fos and junB, were also induced following glucose stimulation with kinetics similar to egr-1, whereas c-jun and junD expression were not affected. Since the zinc-finger protein encoded by egr-1 is highly homologous to transcription factors that control expression of glucose-regulated genes in yeast, Egr-1 could mediate delayed adaptive responses of beta cells to sustained glucose stimulation through transcriptional regulation.

3T3 Cells↗

PubMed1998

Glucose stimulation of pancreatic beta-cell lines induces expression and secretion of dynorphin.

To investigate adaptive responses of pancreatic beta-cells to hyperglycemia, genes induced by glucose stimulation were identified by subtraction cloning. Among 53 clones representing differentially expressed genes, 20 encoded the endogenous opioid precursor, prodynorphin. The amino acid sequence of murine prodynorphin is identical to the rat protein in sequences comprising the opioid peptides and 86% identical in the remainder of the molecule. Stimulation of MIN6 cells increased prodynorphin RNA levels to more than 20-fold in proportion to physiological glucose concentrations. Similar induction levels were observed in murine betaTC3 and rat Rinm5F beta-cell lines. Prodynorphin RNA expression increased within 1 h of glucose stimulation, achieved maximal levels by 4 h, and remained elevated for at least 24 h. By using RIA, MIN6 cells were shown to contain and secrete increased amounts of dynorphin-A following glucose stimulation. Treatment of MIN6 cells with KCl, forskolin, or isobutyl-methyl-xanthine strongly induced prodynorphin RNA expression, suggesting that induction may be related to secretion-coupled signaling pathways. The induction of prodynorphin in several beta-cell lines is consistent with previous demonstrations of beta-cell synthesis of other endogenous opioids, including beta-endorphin, and suggests that opioids may have a potentially significant role in regulating beta-cell secretion.

Amino Acid Sequence↗

PubMed1996

Presence of sulphatide (3'-sulphogalactosylceramide) in pericytes in the choroid layer of the eye: sharing of this glycolipid autoantigen with islets of Langerhans.

The aim of the study was to investigate the distribution in the eye of sulphatide, an acid glycolipid which has previously been demonstrated in islets of Langerhans, nervous tissue and in kidney glomeruli of diabetic patients, and against which antibodies have been found in patients with newly diagnosed insulin-dependent diabetes mellitus. A specific monoclonal antibody, Sulph I, was used for detection of sulphatide by thin-layer chromatography, and light and electron microscope immunohistochemistry. A distinct, patchy staining was found in the choroid layer and the ciliary processes. The antigen was confirmed to be sulphatide and its concentration in human eyes was 30 nmol sulphatide/g wet tissue. By electron microscopy, anti-sulphatide choroid labelling was demonstrated in pericytes and in smooth muscle cells surrounding vessels. No Sulph I-negative pericytes were seen. Double labelling with Sulph I and anti-smooth muscle actin revealed that only pericytes in the eye contained sulphatide and not those in heart, lung, liver, adrenal, spleen, lymph node, thymus, or pancreatic tissue. Thus, sharing of the autoantigen sulphatide has been demonstrated between islets of Langerhans and pericytes in the choroid layer of the eye.

Aged↗

PubMed1996

Fluorescence-activated cell sorted rat islet cells and studies of the insulin secretory process.

Studies of individual cell types in the islets of Langerhans are complicated by the cells' functional coupling by gap junctions and paracrine interaction. Access to purified alpha and beta cells is therefore desirable. We present a simplified and optimized method for fluorescence-activated cell sorting of endocrine pancreatic rat islets. For dispersion of the islets, dispase was superior to trypsin, as the number of vital single cells was higher (1.1 +/- 0.1 x 10(3) vs 0.6 +/- 0.1 x 10(3)/islet, P < 0.05). The purity of the sorted cells was 96.7 +/- 1.2% for the non-beta cells and 97.8 +/- 0.6% for the beta cells (numbers in percentages of endocrine cells). In culture, isolated beta cells, non-beta cells and mixtures of beta and non-beta cells formed aggregates, but not at low temperature (4 degrees C) and not in medium with low serum content (2%). Finally, in pure beta cell aggregates, glucose stimulated changes in cytoplasmic free Ca2+ concentration although both glucose- and arginine-induced insulin secretion was much reduced. We conclude that alpha cells are necessary for insulin secretion but not for glucose sensing.

Animals↗

PubMed1995

Tissue culture supernatants from human islets of Langerhans activate the oxidative burst response of human monocytes in vitro.

Macrophages play a major role in the pathogenesis of insulin-dependent diabetes mellitus in animals. These cells are the first to invade the pancreas and macrophage-eradicating treatments reduce the incidence of the disease. In humans, however, their role is less clear. In this study we investigated the hypothesis that the pancreatic environment per se could activate macrophages. Tissue culture supernatants from human islets of Langerhans were tested for chemotactic activity and oxidative burst response in monocytes isolated from healthy adults. Preincubation with the supernatants enhanced the oxidative burst response evoked by fMLP (up to 379%) and opsonized zymosan (up to 173%). The activity decreased by dilution and was no longer detectable at 1:16. No increased activity was seen in supernatants from a number of other human endocrine and non-endocrine primary cells, suggesting a factor specific for islet tissue. The increased oxidative burst response could partially be eliminated by heat- and proteinase K treatment, suggesting that the activity could be of polypeptide nature. The factor could not be absorbed by polyvalent rabbit antibodies directed towards a variety of cytokines not by a mixture of high-titer anti-cytokine antibodies. It is possible that islet factors could also promote such monocyte activation in vivo in monocytes attracted to the islets of Langerhans by other means. This could contribute to the development of insulin-dependent diabetes in humans.

Adolescent↗

PubMed1994

Sulphatide in islets of Langerhans and in organs affected in diabetic late complications: a study in human and animal tissue.

Sulphatide has been found in rat islets of Langerhans and anti-sulphatide antibodies have been demonstrated in patients with insulin-dependent diabetes mellitus. Using a specific monoclonal antibody, Sulph I, directed against sulphatide, we investigated the in situ distribution of this glycolipid immunohistochemically; furthermore, the sulphatide concentration was determined in several organs and cells by thin-layer chromatography. The islets of Langerhans in all species examined, mouse, rat, pig, and monkey were intensively stained but exocrine tissue remained unlabelled. The sulphatide concentration in human islets was 150 +/- 46 pmol/100 islets. The only glycolipid-antigen detected was sulphatide. Regarding other tissues, sulphatide was found to be located in distal tubules in the kidney, peripheral nerves, distinct scattered spot-like structures in the choreoid layer of the eye, the ovum, and peripheral granulocytes. Sulph I injection in mice showed homing to kidney tubules, Lung, heart, liver, adrenal, spleen, lymph node and thymus were not stained by Sulph I. Thus, the distribution of sulphatide shows an association with organs known to be affected in diabetes, either initially or in late complications.

Adult↗

PubMed1994

Circulating monocytes are activated in newly diagnosed type 1 diabetes mellitus patients.

Investigations in the BB rat and the non-obese diabetic (NOD) mouse have provided substantial evidence for the involvement of the monocyte/macrophage system in the development of type 1 diabetes mellitus. However, it is not known whether monocytes play the same role in the pathogenesis of human type 1 diabetes. We investigated this problem in a longitudinal study of 29 recent-onset type 1 diabetes mellitus patients. Monocyte chemotaxis, phagocytosis and superoxide production as well as metabolic and haematological parameters were studied immediately after diagnosis and 6 months later. At diagnosis the patients had activated casein and C5a chemotaxis (casein 70 +/- 9 versus 150 +/- 5 (mean +/- s.e.m.), P < 0.001; C5a 137 +/- 10 versus 158 +/- 5, P < 0.05 (activation immobilizes monocytes, reducing the measured values)), and activated superoxide production (3.6 +/- 0.3 versus 3.0 +/- 0.3, P < 0.05). After 6 months casein chemotaxis (115 +/- 16 versus 150 +/- 5, P < 0.05) and Candida phagocytosis (3.3 +/- 0.1 versus 2.8 +/- 0.2, P < 0.001) were still activated. There was no correlation with other clinical or paraclinical parameters. We conclude that the circulating monocytes in newly diagnosed type 1 diabetes patients are activated. It is reasonable to expect that monocytes at the local site of inflammation in pancreas are even further activated. This could play a pathogenic role in beta cell destruction.

Adult↗

PubMed1993

Sulphatide and sulphatide antibodies in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is associated with neurological disorders. Sulph I, a monoclonal antibody to sulphatide (a neural epitope), stained secretory granules in alpha and beta cells of rat islets of Langerhans, but not exocrine tissue. Sera from 88% of 57 newly diagnosed IDDM patients was anti-sulphatide positive, and 76% were positive 6 months later. All 135 healthy controls were negative. Sulphatide antibody may be an IDDM marker.

Adult↗

PubMed1993

An alternative approach to 32P radiation protection: source shielding.

32P is the most widely used isotope in molecular biology laboratories and a high-energy beta-emitter. Local doses are often underestimated and plexiglass shielding converts some energy into penetrating gamma radiation. We describe a series of equipment that reduces local doses and gamma radiation more efficiently than traditional equipment and at the same time facilitates the handling of the isotopes.

Beta Particles↗

PubMed1993

Neonatal stimulation of beta-cells reduces the incidence and delays the onset of diabetes in a barrier-protected breeding colony of BB rats.

The off-spring in a barrier maintained colony of spontaneously type 1 diabetic BB/Wor/Mol-BB rats was treated with a daily injection of either saline, forskolin, arginine, glucose or both glucose and arginine for the first six days after birth. The incidence was reduced from 88% to 72% by the neonatal stimulation with arginine and glucose in combination, which also delayed the onset time from 76.0 +/- 2.2 days to 88.1 +/- 2.3 days. No such effect was observed after stimulation with either one of the compounds. Neonatal stimulation with forskolin also delayed the onset to 87.9 +/- 3.8 days, however without reducing the incidence. A higher incidence of diabetes was observed in the barrier-protected rats taking part in this study than in an earlier study using BB rats of the same origin, but kept under conventional conditions.

Animals↗

PubMed1993

Sulphatide antigen in islets of Langerhans and in diabetic glomeruli, and anti-sulphatide antibodies in type 1 diabetes mellitus.

Clinical coincidence between diabetes and neurological disorders, and sharing of antigen determinants between islets of Langerhans and neural tissue, has been suggested. Sulphatide is a neural epitope which can be visualized with a monoclonal antibody Sulph I. Different tissues were examined by immunohistological methods. Sulphatide and anti-sulphatide antibodies were determined by thin-layer chromatographic techniques. IgG was isolated using protein A columns. A specific staining by Sulph I was found of rat islets, assigned to the secretory granules of both alpha and beta cells. No labelling of the exocrine tissue or other body tissues was seen, except for nerve and kidney structures. The latter showed staining of the distal tubules and, in addition, but only in the diabetic kidney, of glomeruli located in the subendothelial area in the capillary loops and the mesangial space. Sera from 38% of 40 spontaneously diabetic BB rats displayed anti-sulphatide antibodies, mainly IgG, whereas all 30 control Lewis rats were negative. Most recently we have demonstrated anti-sulphatide antibodies in 88% of 57 patients with newly diagnosed Type 1 diabetes (titres of > 1:400); all 135 healthy control persons were negative. The sulphatide antibody reactivity was present in the IgG fractions of the patients' sera. Thus, sulphatide is demonstrated in islets of Langerhans and in kidney related to the diabetic lesion, and, furthermore, anti-sulphatide antibodies exist in Type 1 diabetes mellitus.

Adolescent↗

PubMed1993

Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors.

Since Epstein-Barr virus (EBV) infection of Burkitt's lymphoma (BL) cells in vitro reproduces many of the activation effects of EBV infection of primary B lymphocytes, mRNAs induced in BL cells have been cloned and identified by subtractive hybridization. Nine genes encode RNAs which are 4- to > 100-fold more abundant after EBV infection. Two of these, the genes for CD21 and vimentin, were previously known to be induced by EBV infection. Five others, the genes for cathepsin H, annexin VI (p68), serglycin proteoglycan core protein, CD44, and the myristylated alanine-rich protein kinase C substrate (MARCKS), are genes which were not previously known to be induced by EBV infection. Two novel genes, EBV-induced genes 1 and 2 (EBI 1 and EBI 2, respectively) can be predicted from their cDNA sequences to encode G protein-coupled peptide receptors. EBI 1 is expressed exclusively in B- and T-lymphocyte cell lines and in lymphoid tissues and is highly homologous to the interleukin 8 receptors. EBI 2 is most closely related to the thrombin receptor. EBI 2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. EBI 2 is also expressed at lower levels in a promyelocytic and a histiocytic cell line and in pulmonary tissue. These predicted G protein-coupled peptide receptors are more likely to be mediators of EBV effects on B lymphocytes or of normal lymphocyte functions than are genes previously known to be up-regulated by EBV infection.

Actins↗