Islet transplantation as a biological β-cell replacement therapy has emerged as a promising option for achieving restoration of metabolic control in type 1 diabetes patients. However, partial or ...complete loss of islet graft function occurs in relatively short time (months to few years) after implantation. The high rate of early transplant dysfunction has been attributed to poorly viable and/or functional islets and is mediated by innate inflammatory response at the intravascular (hepatic) transplant site and critical lack of initial nutrient/oxygen supply prior to islet engraftment. In addition, the diabetogenic effect of mandatory immunosuppressive agents, limited control of alloimmunity, and the recurrence of autoimmunity limit the long-term success of islet transplantation. In order to abrogate instant blood-mediated inflammatory reaction and to provide oxygen supply for the islet graft, we have developed an extravascular (subcutaneous) transplant macrochamber (the 'βAir' device). This device contains islets immobilized in alginate, protected from the immune system by a thin hydrophilized teflon membrane impregnated with alginate and supplied with oxygen by daily refueling with oxygen-CO (2) mixture. We have demonstrated successful utilization of the oxygen-refueling macrochamber for sustained islet viability and function as well as immunoprotection after allogeneic subcutaneous transplantation in healthy minipigs. Considering the current limitations of intraportal islet engraftment and the restricted indication for islet transplantation mainly due to necessary immunosuppressive therapy, this work could very likely lead to remarkable improvements in the procedure and moreover opens up further strategies for porcine islet cell xenotransplantation.
Transplantation of islet cells is an effective treatment for type 1 diabetes with critically labile metabolic control. However, during islet isolation, blood supply is disrupted, and the transport of ...nutrients/metabolites to and from the islet cells occurs entirely by diffusion. Adequate oxygen supply is essential for function/survival of islet cells and is the limiting factor for graft integrity. Recently, we developed an immunoisolated chamber system for transplantation of human islets without immunosuppression. This system depended on daily oxygen supply. To provide independence from this external source, we incorporated a novel approach based on photosynthetically-generated oxygen. The chamber system was packed sandwich-like with a slab of immobilized photosynthetically active microorganisms (Synechococcus lividus) on top of a flat light source (LEDs, red light at 660 nm, intensity of 8 μE/m(2)/s). Islet cells immobilized in an alginate slab (500-1,000 islet equivalents/cm(2)) were mounted on the photosynthetic slab separated by a gas permeable silicone rubber-Teflon membrane, and the complete module was sealed with a microporous polytetrafluorethylene (Teflon) membrane (pore size: 0.4 μm) to protect the contents from the host immune cells. Upon illumination, oxygen produced by photosynthesis diffused via the silicone Teflon membrane into the islet compartment. Oxygen production from implanted encapsulated microorganisms was stable for 1 month. After implantation of the device into diabetic rats, normoglycemia was achieved for 1 week. Upon retrieval of the device, blood glucose levels returned to the diabetic state. Our results demonstrate that an implanted photosynthetic bioreactor can supply oxygen to transplanted islets and thus maintain islet viability/functionality.
Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron acceptor, ferricyanide and the ...ΔpH indicator, 9-aminoacridine, was used to measure simultaneously electron transport rates and ΔpH solely by changes in the fluorescence of 9-aminoacridine. This method yields values for the rate of electron transport that are comparable with those obtained by established methods. Using this method a relationship between the rate of electron transport and ΔpH at various uncoupler concentrations or light intensities was obtained. In addition, the method was used to study the effect of reducing the disulfide bridge in the γ-subunit of the chloroplast ATP synthase on the relation of electron transport to ΔpH. When the ATP synthase is reduced and alkylated, the threshold ΔpH at which the ATP synthase becomes leaky to protons is lower compared with the oxidized enzyme. Proton flow through the enzyme at a lower ΔpH may be a key step in initiation of ATP synthesis in the reduced enzyme and may be the way by which reduction of the disulfide bridge in the γ-subunit enables high rates of ATP synthesis at low ΔpH values.
A large proton leak not coupled to ATP synthesis (slip) occurs at alkaline pH through the chloroplast ATP synthase (Y. Evron, M. Avron 1990 Biochim Biophys Acta 1019: 115-120). The involvement of the ...ATP synthase gamma-subunit in the regulation of proton conductance was analyzed by measuring the effect of thiolalkylating agents on proton slip. Alkylation by N-ethylmaleimide of gamma-cysteine (Cys)-89, which is exposed upon energization of thylakoids, increases the slip only at alkaline pH. The slip is partially suppressed by low concentrations of adenine nucleotides and is completely eliminated by venturicidin, a blocker of the hydrophobic polypeptide complex of the chloroplast ATP synthase (CF0). Conversely, cross-linking of gamma-Cys-89 with gamma-Cys-322 renders the ATP synthase leaky to protons and insensitive to ATP also at neutral pH. The accessibility of gamma-Cys-89 to alkylation by fluorescein maleimide is completely suppressed by N,N-dicyclohexylcarbodiimide and by venturicidin, which block proton conductance through CF0 and increase the pH gradient. These results suggest that the gamma-subunit has a dominant role in proton gating through the ATP synthase and responds to changes in pH and ligands taking place on either side of the thylakoid membrane. It is proposed that the conformational changes that induce the proton slip and the exposure of gamma-Cys-89 reflect the conversion of the enzyme from a catalytically latent to an active state, and depend on the deprotonation of a stromal site at alkaline pH and on protonation of an intrathylakoid inner site upon energization. Therefore, conditions that induce the conformational activation also provide the driving force for ATP synthesis.
The purpose of this work was to clarify the mechanism of tentoxin-induced chlorosis in Nicotiana spp. seedlings. We found that chlorosis does not correlate with the inhibition of chloroplast ATP ...synthesis in vivo, since it occurs at tentoxin concentrations far higher than that required for the inhibition of photophosphorylation measured in the same seedlings. However, tentoxin-induced chlorosis does correlate with in vivo overenergization of thylakoids. We show that tentoxin induces overenergization in intact plants and isolated thylakoids, probably via multiple interactions with ATP synthase. Furthermore, gramicidin D, a protonophore that relieves overenergization, also relieves chlorosis. Two lines of evidence suggest that reactive oxygen species may be involved in the process of chlorosis: ascorbate, a quencher of oxygen radicals, significantly protects against chlorosis, whereas transgenic Nicotiana spp. mutants overexpressing chloroplast superoxide dismutase are partially resistant to tentoxin-induced chlorosis. It is proposed that chlorosis in developing seedlings results from overenergization of thylakoids, which leads to the generation of oxygen radicals
Objectives: Current therapy for insulin-dependent patients requires frequent blood glucose testing and insulin injections. Quite often, this treatment does not result in optimized control over blood ...glucose. Islet transplantation could form the optimal solution for these patients once two key targets are met: adequate oxygenation and efficient immune protection of the donor tissue. The sub-dermal implanted beta Air registered is designed to overcome both hurdles. Methods: The beta Air registered is a two compartment device. Donor islets of Langerhans immobilized in a flat sheet hydrogel and an oxygen chamber separated from the islet module by a gas permeable membrane. The immune barrier is a three-layer system: a 0.4 mu m hydrophilyzed Teflon membrane, that prevents cell-cell contact, thus preventing cellular immunity; a small pore size alginate hydrogel Impregnated into the Teflon membrane, significantly inhibiting inward diffusion of immune macromolecules and a large flat alginate hydrogel inflicting with the capacity of cytokines and NO to affect the islets. Results: The beta Air registered was implanted in small and large animals. Average blood glucose levels were adjusted to near normal for up to 6 months in iso and allogenic diabetic rats and for one month in large xenogenic pig. Upon retrieval, blood glucose levels returned to the disease state. No pig's DNA was found within the device. Migration of IgG across the impregnated membrane was reduced by 20 fold, suggesting a strict delay in penetration of antibodies and other components of the immune system. More than 90% of the alginate was maintained with minor toss of the cross linking ion, suggesting a stable alginate gel. Conclusions: Results demonstrate the ability of the beta Air registered to treat diabetes in rats and pigs with long-term immuno protection.
Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron acceptor, ferricyanide and the ...DeltapH indicator, 9-aminoacridine, was used to measure simultaneously electron transport rates and DeltapH solely by changes in the fluorescence of 9-aminoacridine. This method yields values for the rate of electron transport that are comparable with those obtained by established methods. Using this method a relationship between the rate of electron transport and DeltapH at various uncoupler concentrations or light intensities was obtained. In addition, the method was used to study the effect of reducing the disulfide bridge in the gamma-subunit of the chloroplast ATP synthase on the relation of electron transport to DeltapH. When the ATP synthase is reduced and alkylated, the threshold DeltapH at which the ATP synthase becomes leaky to protons is lower compared with the oxidized enzyme. Proton flow through the enzyme at a lower DeltapH may be a key step in initiation of ATP synthesis in the reduced enzyme and may be the way by which reduction of the disulfide bridge in the gamma-subunit enables high rates of ATP synthesis at low DeltapH values.
A large proton leak not coupled to ATP synthesis (slip) occurs at alkaline pH through the chloroplast ATP synthase (Y. Evron, M. Avron 1990 Biochim Biophys Acta 1019: 115-120). The involvement of the ...ATP synthase gamma-subunit in the regulation of proton conductance was analyzed by measuring the effect of thiol-alkylating agents on proton slip. Alkylation by N-ethylmaleimide of gamma-cysteine (Cys)-89, which is exposed upon energization of thylakoids, increases the slip only at alkaline pH. The slip is partially suppressed by low concentrations of adenine nucleotides and is completely eliminated by venturicidin, a blocker of the hydrophobic polypeptide complex of the chloroplast ATP synthase (CF(0)). Conversely, cross-linking of gamma-Cys-89 with gamma-Cys-322 renders the ATP synthase leaky to protons and insensitive to ATP also at neutral pH. The accessibility of gamma-Cys-89 to alkylation by fluorescein maleimide is completely suppressed by N,N-dicyclohexylcarbodiimide and by venturicidin, which block proton conductance through CF(0), and increase the pH gradient. These results suggest that the gamma-subunit has a dominant role in proton gating through the ATP synthase and responds to changes in pH and ligands taking place on either side of the thylakoid membrane. It is proposed that the conformational changes that induce the proton slip and the exposure of gamma-Cys-89 reflect the conversion of the enzyme from a catalytically latent to an active state, and depend on the deprotonation of a stromal site at alkaline pH and on protonation of an intrathylakoid inner site upon energization. Therefore, conditions that induce the conformational activation also provide the driving force for ATP synthesis
Target site resistance has evolved to only those herbicides affecting a single system. Extensive resistance has evolved to photosystem II inhibitors, especially atrazine, but not to the phenolic-type ...herbicides (e.g., dinoseb), which both affect photosystem II and purportedly uncouple mitochondrial phosphorylation and photophosphorylation. Pyridate, which has been classified as a "phenolic"-type herbicide, is highly effective in controlling triazine-resistant weeds. We demonstrate here that the active de-S-octyl derivative of pyridate does not have this second target site; photophosphorylation was only affected at 100 times greater concentration than photosystem II activity. From this point of view, pyridate may be more prone to evolution of resistance than phenolic-type herbicides with two sites of action
The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 micromol/min/mg protein), but virtually inactive in the dark. ...The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the gamma and epsilon subunits of CF(1) play key roles in both regulation of activity and proton translocation.
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