Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes.

Enzymatic catalysis and homogeneous catalysis offer complementary means to address synthetic challenges, both in chemistry and in biology. Despite its attractiveness, the implementation of concurrent cascade reactions that combine an organometallic catalyst with an enzyme has proven challenging because of the mutual inactivation of both catalysts. To address this, we show that incorporation of a d(6)-piano stool complex within a host protein affords an artificial transfer hydrogenase (ATHase) that is fully compatible with and complementary to natural enzymes, thus enabling efficient concurrent tandem catalysis. To illustrate the generality of the approach, the ATHase was combined with various NADH-, FAD- and haem-dependent enzymes, resulting in orthogonal redox cascades. Up to three enzymes were integrated in the cascade and combined with the ATHase with a view to achieving (i) a double stereoselective amine deracemization, (ii) a horseradish peroxidase-coupled readout of the transfer hydrogenase activity towards its genetic optimization, (iii) the formation of L-pipecolic acid from L-lysine and (iv) regeneration of NADH to promote a monooxygenase-catalysed oxyfunctionalization reaction.

Helminth parasites in faecal samples from the endangered Iberian lynx (Lynx pardinus).

The Iberian lynx is the most endangered felid in the world. Enteropathogens may threaten its survival, and therefore we analysed faecal samples from 66 different individuals (37 males and 29 females), the largest population representation studied to date. The samples were obtained from November 2005 to October 2008 in the two areas where the Iberian lynx survives: Sierra Morena and Doñana (Andalusia, southern Spain). A total of 56.1% samples were parasitized with at least 6 species of helminths, including two cestodes (Hymenolepis spp. and Taenia spp.) and four Nematodes (Ancylostoma spp., Toxocara spp., Toxascaris leonina, and Capillaria sp.). In this work, the presence of Hymenolepis is reported for the first time in Lynx pardinus. The relevance of our findings is discussed focussed on the conservation of this endangered felid.

Therapeutic potential of stem cells in diabetes.

Stem cells possess the ability to self-renew by symmetric divisions and, under certain circumstances, differentiate to a committed lineage by asymmetric cell divisions. Depending on the origin, stem cells are classified as either embryonic or adult. Embryonic stem cells are obtained from the inner cell mass of the blastocyst, a structure that appears during embryonic development at day 6 in humans and day 3.5 in mice. Adult stem cells are present within tissues of adult organisms and are responsible for cell turnover or repopulation of tissues under normal or exceptional circumstances. Taken together, stem cells might represent a potential source of tissues for cell therapy protocols, and diabetes is a candidate disease that may benefit from cell replacement protocols. The pathology of type 1 diabetes is caused by the autoimmune destruction or malfunction of pancreatic beta cells, and consequently, a lack of insulin. The absence of insulin is life-threatening, thus requiring diabetic patients to take daily hormone injections from exogenous sources; however, insulin injections do not adequately mimic beta cell function. This results in the development of diabetic complications such as neuropathy, nephropathy, retinopathy and diverse cardiovascular disorders. This chapter intends to summarize the possibilities opened by embryonic and adult stem cells in regenerative medicine for the cure of diabetes.

In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells.

AIMS/HYPOTHESIS: We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. METHODS: Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice. RESULTS: The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols. CONCLUSIONS/INTERPRETATION: These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.

Ingeniería celular y diabetes mellitus / Cell engineering and diabetes mellitus

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Stem cells and diabetes.

Diabetes mellitus is a metabolic disorder affecting 2-5% of the population. Transplantation of isolated islets of Langerhans from donor pancreata could be a cure for diabetes; however, such an approach is limited by the scarcity of the transplantation material and the long-term side effects of immunosuppressive therapy. These problems may be overcome by using a renewable source of cells, such as islet cells derived from stem cells. Stem cells are defined as clonogenic cells capable of both self-renewal and multilineage differentiation. This mean that these cells can be expanded in vivo or in vitro and differentiated to produce the desired cell type. There exist several sources of stem cells that have been demonstrated to give rise to pluripotent cell lines: 1) embryonic stem cells; 2) embryonic germ cells; 3) embryonic carcinoma cells; and 4) adult stem cells. By using in vitro differentiation and selection protocols, embryonic stem cells can be guided into specific cell lineages and selected by applying genetic selection when a marker gene is expressed. Recently, differentiation and cell selection protocols have been used to generate embryonic stem cell-derived insulin-secreting cells that normalise blood glucose when transplanted into diabetic animals. Some recent reports suggest that functional plasticity of adult stem cells may be greater than expected. The use of adult stem cells will circumvent the ethical dilemma surrounding embryonic stem cells and will allow autotransplantation. These investigations have increased the expectations that cell therapy could be one of the solutions to diabetes.

Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.

Embryonic stem (ES) cells display the ability to differentiate in vitro into a variety of cell lineages. Using a cell-trapping system, we have obtained an insulin-secreting cell clone from undifferentiated ES cells. The construction used allows the expression of a neomycin selection system under the control of the regulatory regions of the human insulin gene. The chimeric gene also contained a hygromycin resistance gene (pGK-hygro) to select transfected cells. A resulting clone (IB/3x-99) containing 16.5 ng/microg protein of total insulin displays regulated hormone secretion in vitro in the presence of various secretagogues. Clusters obtained from this clone were implanted (1 x 10(6) cells) in the spleen of streptozotocin-induced diabetic animals. Transplanted animals correct hyperglycemia within 1 week and restore body weight in 4 weeks. Whereas an intraperitoneal glucose tolerance test showed a slower recovery in transplanted versus control mice, blood glucose normalization after a challenge meal was similar. This approach opens new possibilities for tissue transplantation in the treatment of type 1 and type 2 diabetes and offers an alternative to gene therapy.

Engineering pancreatic islets.

Pancreatic islets are neuroendocrine organs that control blood glucose homeostasis. The precise interplay of a heterogeneous group of cell populations (beta, alpha, delta and PP cells) results in the fine-tuned release of counterbalanced hormones (insulin, glucagon, somatostatin and pancreatic polypeptide respectively). Under the premises of detailed knowledge of the physiological basis underlying this behaviour, two lines of investigation might be inferred: generating computational and operational models to explain and predict this behaviour and engineering islet cells to reconstruct pancreatic endocrine function. Whilst the former is being fuelled by new computational strategies, giving biophysicists the possibility of modelling a system in which new "emergent" properties appear, the latter is benefiting from the useful tools and strategic knowledge achieved by molecular, cell and developmental biologists. This includes using tumour cell lines, engineering islet cell precursors, knowledge of the mechanisms of differentiation, regeneration and growth and, finally, therapeutic cloning of human tissues. Gaining deep physiological understanding of the basis governing these processes is instrumental for engineering new pancreatic islets.

Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor.

Impaired insulin secretion is a hallmark in both type I and type II diabetic individuals. Whereas type I (insulin-dependent diabetes mellitus) implies ss-cell destruction, type II (non-insulin dependent diabetes mellitus), responsible for 75% of diabetic syndromes, involves diminished glucose-dependent secretion of insulin from pancreatic beta-cells. Although a clear demonstration of a direct effect of 17beta-estradiol on the pancreatic ss-cell is lacking, an in vivo insulinotropic effect has been suggested. In this report we describe the effects of 17beta-estradiol in mouse pancreatic ss-cells. 17beta-Estradiol, at physiological concentrations, closes K(ATP) channels, which are also targets for antidiabetic sulfonylureas, in a rapid and reversible manner. Furthermore, in synergy with glucose, 17beta-estradiol depolarizes the plasma membrane, eliciting electrical activity and intracellular calcium signals, which in turn enhance insulin secretion. These effects occur through a receptor located at the plasma membrane, distinct from the classic cytosolic estrogen receptor. Specific competitive binding and localization of 17beta-estradiol receptors at the plasma membrane was demonstrated using confocal reflective microscopy and immunocytochemistry. Gaining deeper knowledge of the effect induced by 17beta-estradiol may be important in order to better understand the hormonal regulation of insulin secretion and for the treatment of NIDDM. receptor.

Altered activity of the autonomous nervous system as a determinant of the impaired beta-cell secretory response after protein-energy restriction in the rat.

Glucose-induced insulin secretion in vivo is known to be severely blunted in the rat as a consequence of protein-energy restriction starting early in life. We have recently reported in such malnourished rats (M rats) that the release of the counterregulatory hormones that defend against hypoglycemia was severely disturbed, and their plasma levels of epinephrine and norepinephrine were prominently increased. Knowing that the autonomic nervous system has the potential to play a major role in the control of insulin secretion in response to glucose in vivo, we therefore determined whether protein-energy restriction starting after weaning could alter sympathetic and/or parasympathetic nerve activities, and whether these changes could be responsible for the lack of response to glucose of their beta-cells in vivo. When tested in the basal postabsorptive state, the malnourished rats exhibited profound alterations of both parasympathetic and sympathetic nerve activities; the firing rates of the vagus nerve and the superior cervical ganglion were dramatically decreased and increased, respectively. Under the same conditions, insulin secretion in vivo in response to a glucose load (deltaI/deltaG) was severely decreased in M rats compared with that in control (C) rats. When evaluated after administration of acetylcholine, deltaI was amplified to the same extent in M rats as in C rats. After administration of the alpha2A-adrenergic agonist oxymetazoline, glucose-induced insulin release in M rats was not significantly affected, whereas it was sharply decreased in C rats. Finally, administration of yohimbine, an alpha2-adrenergic antagonist, partially restored the lack of reactivity of the beta-cells to glucose in the M rats, as deltaI/deltaG was amplified by 6-fold in the M group and by 3.3-fold in the C group. We conclude that protein-energy restriction starting early in life in rats brings about changes in the overall activity of the autonomic nervous system that, in turn, are responsible at least in part for the acquisition/maintenance of decreased beta-cell reactivity to glucose in vivo.

Alteration of the counterregulatory hormones in the conscious rat after protein-energy restriction.

We have recently reported that in rats submitted to protein-energy restriction early in life, an increased insulin efficiency upon the whole-body glucose utilization rate may be one reason for their chronic mild basal hypoglycaemia. However, the basis for their low plasma glucose level may also lie in the impaired activation of one or several of the counterregulatory hormones that prevent or correct hypoglycaemia. Our study was therefore designed to compare glucose counterregulatory mechanisms in restricted and control rats, both in the basal postabsorptive state and at controlled high plasma insulin level and standardized low glycaemic level (hypoglycaemic-hyperinsulinaemic glucose clamps performed in conscious rats). When tested in the basal postabsorptive state, the restricted rats exhibited prominent increases in the plasma levels of epinephrine (4.5 fold), norepinephrine (3.4 fold) and glucagon (1.7 fold). This was in the presence of significant decreases of plasma growth hormone and corticosterone levels (by 59 and 32%, respectively). With respect to the responses to acute severe hypoglycaemia (2.5 mmol/l), the glucagon, epinephrine and norepinephrine plasma levels in the restricted rats increased to values similar to those in controls. Also, the corticosterone level increased but remained significantly lower (p < 0.001) compared to the control response. The plasma growth hormone level was not significantly affected by acute hypoglycaemia in the restricted or in the control groups. We conclude that protein-energy restriction, starting early in life in the rat, severely impairs the release of counterregulatory hormones that defend against hypoglycaemia.

Insulin secretion and glucose tolerance after islet transplantation in rats with noninsulin-dependent diabetes induced by neonatal streptozotocin.

The present study was designed to identify in a model of noninsulin-dependent diabetes induced by neonatal streptozotocin (n0-STZ), the long-term consequences of an islet graft upon 1) glucose handling of the recipient and, 2) glucose response of the residual beta cells in the recipient pancreas. We have examined, 4 and 8 wk after islet implantation under the kidney capsule of syngeneic diabetic n0-STZ rats, their tolerance to glucose administered in vivo, together with their insulin release in response to glucose in vivo (oral glucose tolerance test) as well as in vitro (perfused pancreas). The results in the islet-grafted n0-STZ rats, were compared to those obtained in nongrafted nondiabetic rats and nongrafted n0-STZ rats. Our study shows that transplanting a limited number (900) of adult islets under the kidney capsule reverses to normal, many parameters of the noninsulin-dependent diabetic state in the n0-STZ rat model: these include body weight, basal plasma glucose in both the nonfasted and postabsorptive states, and basal plasma insulin in the postabsorptive state. Furthermore, tolerance to oral glucose administration was greatly improved in the transplanted rats and it was correlated with restoration of a manifest glucose-induced insulin secretion in vivo as evaluated (delta 1) during an oral glucose tolerance test. Our data clearly show that the insulin response to glucose from the endogenous pancreas of n0-STZ diabetic rat was not really improved by long-term (8 wk) basal normoglycemia. More precisely, we were able to detect a slight but significant improvement of the early phase of insulin release in vitro in response to glucose; however, the overall insulin response remained 15 times lower than the normal one with no reappearance of the late phase of insulin release. After cessation of glucose stimulation in vivo, off-response of insulin, which is also a landmark of the impaired insulin release by the beta cells of n0-STZ rats, was still detectable in the perfused pancreas of the transplanted n0-STZ rats. Finally, because the reactivity to glucose of the endogenous residual beta cells was not regained, the insulin released in vivo during the oral glucose test in the graft-bearing n0-STZ rats can be attributed mainly to functioning of the grafted islets population.