Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7)M native GIP, with an IC50 value of 2.6 muM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P <0.05 to P <0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P <0.001) and lower the glycemic excursion (1.5-fold decrease; P <0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes. (C) 2002 Elsevier Science (USA).

Uptake of carbon, nitrogen and phosphorus by phytoplankton along the 20 degrees W meridian in the NE Atlantic between 57.5 degrees N and 37 degrees N

Phytoplankton biomass and rate of production were measured along a transect from 57.54 degreesN to 37.01 degreesN in the northeast Atlantic during July 1996 and at a series of stations over a 7-day period at 37 degreesN 20 degreesW. Surface nutrient concentrations ranged from 4 mu mol l(-1) NO3-, and 0.35 mu mol l(-1) PO43- at 57.54 degreesN to <10 nmol l(-1) NO3- and similar to 10 nmol l(-1) PO43- at 37.01 degreesN. The greatest phytoplankton biomass and production were measured in the vicinity of a frontal system at 50 degreesN, and there was a general decline in total phytoplankton biomass and production to the south of the transect. Production was measured in three size fractions. At the station with the highest chlorophyll concentrations (50.34 degreesN), phytoplankton cells larger than 5 mum dominated the assemblage, accounting for 72% of the chlorophyll concentration (22.9 mg m(-2)) and 51% of primary production (54.1 mmol Cm-2 d(-1)), but picophytoplankton production was also high (43%). At 57 degreesN, carbon fixation by the > 5 mum fraction accounted for 75% of the daily production of 60.75 mmol Cm-2 d(-1). At 37 degreesN, picophytoplankton was the dominant group, accounting for similar to 58% (10 mg m(-2)) of chlorophyll and similar to 64% (46 mmol Cm-2 d(-1)), of primary production. Nitrate, ammonium and phosphate uptake rates also were determined. Although high nitrate uptake rates were measured in the surface water at similar to 50 degreesN, the greatest uptake rates of both depth-integrated nitrate and ammonium were at the south of the transect. At 37 degreesN, a deep euphotic zone was present and light penetrated through the nitracline; total nitrate uptake was enhanced because of assimilation at the base of the euphotic zone. As a consequence, high values of depth-integrated f-ratio were measured in the oligotrophic waters at the south of the transect. Phosphate was predominantly incorporated into the picoplankton fraction, which included heterotrophic and autotrophic components, at all stations and a significant proportion of phosphate uptake occurred in the dark. The C:N:P assimilation ratios were variable throughout the region; phosphate uptake was generally greater than would be expected if nutrient assimilation were in proportion to the Redfield ratio. (C) 2001 Elsevier Science Ltd. All rights reserved.

Photosensitiser delivery for photodynamic therapy. Part 2: systemic carrier platforms

Background: The treatment of solid tumours and angiogenic ocular diseases by photodynamic therapy (PDT) requires the injection of a photosensitiser (PS) to destroy target cells through a combination of visible light irradiation and molecular oxygen. There is currently great interest in the development of efficient and specific carrier delivery platforms for systemic PDT. Objective: This article aims to review recent developments in systemic carrier delivery platforms for PDT, with an emphasis on target specificity. Methods: Recent publications, spanning the last five years, concerning delivery carrier platforms for systemic PDT were reviewed, including PS conjugates, dendrimers, micelles, liposomes and nanoparticles. Results/conclusion: PS conjugates and supramolecular delivery platforms can improve PDT selectivity by exploiting cellular and physiological specificities of the targeted tissue. Overexpression of receptors in cancer and angiogenic endothelial cells allows their targeting by affinity-based moieties for the selective uptake of PS conjugates and encapsulating delivery carriers, while the abnormal tumour neovascularisation induces a specific accumulation of heavy weighted PS carriers by enhanced permeability and retention (EPR) effect. in addition, polymeric prodrug delivery platforms triggered by the acidic nature of the tumour environment or the expression of proteases can be designed. Promising results obtained with recent systemic carrier platforms will, in due course, be translated into the clinic for highly efficient and selective PDT protocols.

A role of cellular prion protein in programming T-cell cytokine responses in disease.

The cellular prion protein (PrPC) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrPC in adaptive immunity has been a particular conundrum: increased expression of cell surface PrPC has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8–24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrPC is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP0/0 T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF- and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrPC is a potentially important molecule influencing T-cell activation and effector function.

Enhanced sensitivity of protein kinase B/Akt to insulin in hypoxia is independent of HIF1 alpha and promotes cell viability

Maintenance of oxygen homeostasis is a key requirement to ensure normal mammalian cell growth and differentiation. Hypoxia arises when oxygen demand exceeds supply, and is a feature of multiple human diseases including stroke, cancer and renal fibrosis. We have investigated the effect of hypoxia on kidney cells, and observed that insulin-induced cell viability is increased in hypoxia. We have characterized the role of protein kinase B (PKB/ Akt) in these cells as a potential mediator of this effect. PKB/Akt activity was increased by low oxygen concentrations in kidney cells, and insulin-stimulated activation of PKB/Akt was stronger, more rapid and more sustained in hypoxia. Reduction of HIF1 alpha levels using antimycin-A or siRNA targeting HlF1 alpha did not affect PKB/Akt activation in hypoxia. Pharmacologic stabilization of HIF1 alpha independent of hypoxia did not increase insulin-stimulated PKB/Akt activation. Although increased insulin-stimulated cell viability was observed in hypoxia, no differences in the degree of insulin-stimulated glucose uptake were observed in L6 muscle cells in hypoxia compared to normoxia. Thus, PKB/Akt may regulate specific cellular responses to growth factors such as insulin under adverse conditions such as hypoxia. alpha 2007 Elsevier GmbH. All rights reserved.

Advances in protein kinase B signalling: AKTion on multiple fronts

The role of the serine/threonine protein kinase B (PKB, also known as Akt) is becoming increasingly more evident to researchers investigating diverse cellular processes such as glucose uptake, cell-cycle progression, apoptosis and transcriptional regulation. New roles for PKB/Akt have been described in various organisms and biological processes. From the regulation of ovarian ecdysteroid production in the humble mosquito (Aedes aegypti), through the seasonal, tissue-specific regulation of PKB/Akt during the hibernation of yellow-bellied marmots (Marmota flaviventris), to the control of glucose metabolism and insulin signalling in the mouse (Mus musculus), our knowledge of the function of this protein kinase has expanded greatly in recent years. Significant advances in all aspects of PKB/Akt signalling have occurred in the past 2 years, including biological insights, novel substrates and newly discovered regulatory mechanisms of PKB/Akt. Collectively, these data expand the current models of PKB/Akt signalling and highlight potential directions for PKB/Akt research in the future.

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.