Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines.

Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated.

Highly blood perfused, highly metabolically active pancreatic islets may be more susceptible for immune attack.

Differences in pancreatic islet susceptibility during type 1 diabetes development may be explained by interislet variations. This study aimed to investigate if heterogeneities in vascular support and metabolic activity in rat and human islets may explain why some islets are attacked earlier than other islets. In rats, highly blood perfused islets were identified by injection of microspheres into the ascending aorta, whereas a combination of anterograde and retrograde injections of microspheres into pancreas was used to determine the islet vascular drainage system. Highly blood perfused islets had superior function and lower glucose threshold for insulin release when compared with other islets. These islets had a preferential direct venous drainage to the portal vein, whereas other islets mainly were incorporated into the exocrine capillary system. In BioBreeding rats, the hypothesis that islets with high islet blood perfusion was more prone to immune cell infiltration was investigated. Indeed, highly blood perfused islets were the first affected by the immune attack. In human subjects, differences in glucose threshold for insulin (C-peptide) secretion was evaluated in individuals recently diagnosed for type 1 diabetes and compared to control subjects. A preferential loss of capacity for insulin release in response to low glucose concentrations was observed at debut of type 1 diabetes. Our study indicates that highly blood perfused islets with direct venous drainage and lower glucose threshold for insulin release are of great importance for normal glucose homeostasis. At the same time, these highly metabolically active islets were the primary target of the immune system.

Anti-Bacterial Effect and Cytotoxicity Assessment of Lipid 430 Isolated from Algibacter sp.

Two bacterial isolates from the Barents Sea, both belonging to the genus Algibacter, were found to yield extracts with anti-bacterial bioactivity. Mass spectrometry guided dereplication and purification of the active extracts lead to the isolation of the same active principle in both extracts. The structure of the bioactive compound was identified via mass spectrometry and nuclear resonance spectroscopy and it turned out to be the known lipopeptide Lipid 430. We discovered and determined its previously unknown anti-bacterial activity against Streptococcus agalactiae and revealed a cytotoxic effect against the A2058 human melanoma cell line at significantly lower concentrations compared to its anti-bacterial concentration. Flow cytometry and microscopy investigations of the cytotoxicity against the melanoma cell line indicated that Lipid 430 did not cause immediate cell lysis. The experiments with melanoma cells suggest that the compound functions trough more complex pathways than acting as a simple detergent.

Decreased ß-Cell Proliferation and Vascular Density in a Subpopulation of Low-Oxygenated Male Rat Islets.

Low-oxygenated and dormant islets with a capacity to become activated when needed may play a crucial role in the complex machinery behind glucose homeostasis. We hypothesized that low-oxygenated islets, when not functionally challenged, do not rapidly cycle between activation and inactivation but are a stable population that remain low-oxygenated. As this was confirmed, we aimed to characterize these islets with regard to cell composition, vascular density, and endocrine cell proliferation. The 2-nitroimidazole low-oxygenation marker pimonidazole was administered as a single or repeated dose to Wistar Furth rats. The stability of oxygen status of islets was evaluated by immunohistochemistry as the number of islets with incorporated pimonidazole adducts after one or repeated pimonidazole injections. Adjacent sections were evaluated for islet cell composition, vascular density, and endocrine cell proliferation. Single and repeated pimonidazole injections over an 8-hour period yielded accumulation of pimonidazole adducts in the same islets. An average of 30% of all islets was in all cases positively stained for pimonidazole adducts. These islets showed a similar endocrine cell composition as other islets but had lower vascular density and ß-cell proliferation. In conclusion, low-oxygenated islets were found to be a stable subpopulation of islets for at least 8 hours. Although they have previously been observed to be less functionally active, their islet cell composition was similar to that of other islets. Consistent with their lower oxygenation, they had fewer blood vessels than other islets. Notably, ß-cell regeneration preferentially occurred in better-oxygenated islets.

Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats.

AIMS/HYPOTHESIS: Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available. METHODS: We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells. RESULTS: DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5-7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet-1 h-1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 µm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 µm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 µl min-1 [g pancreas]-1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats. CONCLUSIONS/INTERPRETATION: The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

Islet amyloid deposits preferentially in the highly functional and most blood-perfused islets.

Islet amyloid and beta cell death in type 2 diabetes are heterogeneous events, where some islets are affected early in the disease process, whereas others remain visibly unaffected. This study investigated the possibility that inter-islet functional and vascular differences may explain the propensity for amyloid accumulation in certain islets. Highly blood-perfused islets were identified by microspheres in human islet amyloid polypeptide expressing mice fed a high-fat diet for three or 10 months. These highly blood-perfused islets had better glucose-stimulated insulin secretion capacity than other islets and developed more amyloid deposits after 10 months of high-fat diet. Similarly, human islets with a superior release capacity formed more amyloid in high glucose culture than islets with a lower release capacity. The amyloid formation in mouse islets was associated with a higher amount of prohormone convertase 1/3 and with a decreased expression of its inhibitor proSAAS when compared to islets with less amyloid. In contrast, levels of prohormone convertase 2 and expression of its inhibitor neuroendocrine protein 7B2 were unaltered. A misbalance in prohormone convertase levels may interrupt the normal processing of islet amyloid polypeptide and induce amyloid formation. Preferential amyloid load in the most blood-perfused and functional islets may accelerate the progression of type 2 diabetes.

Pancreatic islet blood flow and its measurement.

Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting ß-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future.

Vascular heterogeneity between native rat pancreatic islets is responsible for differences in survival and revascularisation post transplantation.

AIMS/HYPOTHESIS: Highly blood-perfused islets have been observed to be the most functional islets in the native pancreas. We hypothesised that differences in vascular support of islets in donor pancreases influence their susceptibility to cellular stress and capacity for vascular engraftment after transplantation. METHODS: Highly blood-perfused islets in rats were identified by injection of microspheres into the ascending aorta before islet isolation. Cell death was evaluated after in vitro cytokine or hypoxia exposure, and 2 days post transplantation. One month post transplantation, islet engraftment, including vascular density, blood perfusion and oxygen tension (pO2) in the tissue, was evaluated. RESULTS: Microsphere-containing islets had a similar frequency of cell death during standard culture conditions but increased cell death after exposure to cytokines and hypoxia in comparison with other islets. Two days after transplantation the percentage of apoptotic or necrotic cells was also higher in grafts of such islets and 1 month post transplantation these grafts were composed of substantially more connective tissue. Grafts of highly blood-perfused islets in the native pancreas regained a higher vascular density, blood perfusion and pO2 in comparison with grafts of other islets. CONCLUSIONS/INTERPRETATION: Native islets that are highly blood-perfused regained this feature after transplantation, indicating a superior capacity for revascularisation and post-transplant function. However, the same group of islets was more vulnerable to different kinds of cellular stress, which limited their early survival post transplantation. Preferential death of these most active islets may contribute to the high number of islets needed to provide cure with islet transplantation.

Adsorbed cationic polymer coatings for enhanced capillary electrophoresis/mass spectrometry of proteins.

The combination of capillary electrophoresis (CE) with mass spectrometry (MS) constitutes a powerful microanalytical system for the analysis of biological samples. The anionic and hydrophobic surface of the fused-silica capillary is, however, known to cause severe analyte-wall interactions in protein analysis. In order to control surface properties and eliminate protein adsorption, a capillary coating can be applied. A fast and simple strategy is to coat the anionic capillary with a cationic polymer via multisite electrostatic interaction. This generates a stable deactivation layer, without the need for addition of coating agent to the background electrolyte solution. This chapter reviews the present knowledge of capillary coatings and especially cationic polymers in CE-MS, and describes the synthesis of a cationic polymer, PolyE-323, for deactivation of fused-silica capillaries. The capillary coating procedure is a simple three-step rinsing protocol comprising deprotonation of surface silanol groups using a base, adsorption of polymer, and a final rinse to remove excess polymer not adsorbed to the surface. As a result of the simplicity of the coating procedure, highly reproducible coatings can be prepared with little or no expert skills. Some practical aspects on using cationic-coated capillaries in CE-MS protein analysis are also discussed.

Urine profiling using capillary electrophoresis-mass spectrometry and multivariate data analysis.

This work presents the development of a general and fast method for metabolic profiling of urine, using capillary electrophoresis-electrospray ionisation mass spectrometry (CE-ESIMS) and multivariate data analysis (DA). Human urine samples collected before and after ingestion of paracetamol were analysed at acidic and basic CE conditions, using both positive and negative ESI-MS detection. Analysis of the entire resulting data set, with no prior knowledge of the target compounds, using pair-wise 'fuzzy' correlation and eigenvalue analysis enabled the samples to be discriminated between on the basis of blank urine and urine collected after drug intake. By generating two-dimensional loadings plots, it was also possible to identify the m/z values of the substances responsible for the differentiation between control and dosed samples.

Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries.

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.

A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides.

A procedure for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) of proteins is presented. The use of a newly presented capillary coating, PolyE-323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE-323 showed high stability over a range of pH 2-10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE-ESI-MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE-323-coated capillaries in bioanalysis using CE-ESI-MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)-MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on-line CE-ESI-TOF system using PolyE-323-coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).

Quaternary ammonium substituted agarose as surface coating for capillary electrophoresis.

A novel positively charged polymer of quaternary ammonium substituted agarose (Q-agarose) has been synthesized and explored for use as a coating in capillary electrophoresis. The fast and simple coating procedure is based on a multi-site electrostatic interaction between the polycationic agarose polymer and the negatively charged fused-silica surface. By simply flushing fused-silica capillaries with hot polymer solution a positively charged, hydrophilic deactivation layer is achieved. The polymer surface provides an intermediate electroosmotic flow of reversed direction, over a range of pH 2-11, compared to unmodified fused-silica. The coating procedure was highly reproducible with an RSD of 4%, evaluated as the electroosmotic flow mobility for 30 capillaries prepared at 10 different occasions. The application of Q-agarose coated capillaries in separation science was investigated using a set of basic drugs and model proteins and peptides. Due to the intermediate electroosmotic flow generated, the resolution of basic drugs could be increased, compared to using bare fused-silica capillaries. Moreover, the coating enabled separation of proteins and peptides with efficiencies up to 300.000 plates m(-1).

Novel polyamine coating providing non-covalent deactivation and reversed electroosmotic flow of fused-silica capillaries for capillary electrophoresis.

A new polycationic coating for use in capillary electrophoresis has been developed that enables chemical modification of fused-silica capillary surfaces for analysis of compounds like basic proteins. The cationic polyamine, containing short aliphatic blocks of combined 2 and 3-carbon length, was physically adsorbed onto the negatively charged fused-silica surface through ionic interaction by flushing the capillary with a polyamine solution, followed by a self-stabilization step. The polyamine coated capillaries generated an anodal electroosmotic flow that was independent of pH in the investigated range of pH 4-8. The capillary performance was demonstrated by fast separations of basic proteins with peak efficiencies in the range of 265,000-584,000 plates.

Method for immobilization of liposomes in capillary electrophoresis by electrostatic interaction with derivatized agarose.

A novel procedure for immobilization of liposomes inside fused-silica capillaries is demonstrated. First, the inner wall of the capillaries was coated with a positively charged polymer, composed of derivatized agarose. Subsequently, negatively charged liposomes were immobilized by electrostatic interaction on the polymer coating. The developed liposome coated capillaries were used as a nanoseparation tool for studying interactions between small drug compounds and liposomes. Part of this work was presented at the 15th International Symposium on Microscale Separations and Analysis, HPCE 2002, Stockholm, Sweden, April 2002.