Diabetes and mitochondrial bioenergetics: alterations with age

Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats. However, some of the results reported are controversial, since experimental conditions, such as aging, and/or strain of animals used were different. The purpose of this study was to evaluate the metabolic changes in liver mitochondria, both in the presence of severe hyperglycaemia (STZ-treated rats) and mild hyperglycaemia (Goto-Kakizaki (GK) rats). Moreover, metabolic alterations were evaluated both at initial and at advanced states of the disease. We observed that both models of type 1 and type 2 diabetes presented alterations on respiratory chain activity. Because of continual severe hyperglycaemia, 9 weeks after the induction of diabetes, the respiratory function declined in STZ-treated rats, as observed by membrane potential and respiratory ratios (RCR, P/O, and FCCP-stimulated respiration) assessment. In contrast, GK rats of 6 months age presented increased respiratory ratios. To localize which respiratory complexes are affected by diabetes, enzymatic respiratory chain activities were evaluated. We observed that succinate dehydrogenase and cytochrome c oxidase activities were significantly augmented both in STZ-treated rats and GK rats of 6 months age. Moreover, H(+)-ATPase activity was also significantly increased in STZ-treated rats with 3 weeks of diabetes and in GK rats of 6 months age as compared to controls. Therefore, these results clearly suggest that both animal models of diabetes present some metabolic adjustments in order to circumvent the deleterious effects promoted by the high glucose levels typical of the disease.     

Cytokine control of memory T-cell development and survival

Evidence has accumulated that cytokines have a fundamental role in the differentiation of memory T cells. Here, we follow the CD8+ T cell from initial activation to memory-cell generation, indicating the checkpoints at which cytokines determine the fate of the T cell. Members of the common cytokine-receptor gamma-chain (gammac)-cytokine family--in particular, interleukin-7 (IL-7) and IL-15--act at each stage of the immune response to promote proliferation and survival. In this manner, a stable and protective, long-lived memory CD8+ T-cell pool can be propagated and maintained.     

Report of a Scientific Working Group on Serious Adverse Events following Mectizan<Superscript>®</Superscript> treatment of onchocerciasis in <Emphasis Type="Italic">Loa loa</Emphasis> endemic areas

The occurrence of Serious Adverse Experiences (SAEs) following Mectizan^® treatment of onchocerciasis in Loa loa endemic areas has been increasingly reported over the past decade. These SAEs include a severely disabling, and potentially fatal, encephalopathy, which appears to correlate with a high load of L. loa microfilariae (> 30,000 mf/ml).Previous consultations organized by the Mectizan^® Donation Program (MDP) in 1995 and 1999 have developed useful "case" definitions of encephalopathic SAEs following Mectizan^® treatment and have summarized available evidence on its pathogenesis and optimal clinical management. At both meetings, the need for better understanding of the pathogenesis of the encephalopathy was emphasized, including the need for biological and autopsy specimens from the affected cases.Following a recommendation at the Joint Action Forum of the African Programme for Onchocerciasis Control in December 2001, the MDP, on behalf of the Mectizan^® Expert Committee, organized a Scientific Working Group on L. loa associated SAEs following Mectizan^® treatment in May 2002. The present report includes the background, new evidence, conclusions and recommendations from that Scientific Working Group. The following points represent a summary of the present status:1. Although there are more and better quality clinical and epidemiological data on L. loa, the pathogenesis of the Mectizan^®-related L. loa encephalopathy remains obscure.2. Very limited progress has been made in research on the pathogenesis of encephalopathy, because of the lack of specimens from cases, and the lack of animal models.3. There has been no particular breakthrough in terms of the medical management of patients with L. loa encephalopathy; however, a favorable outcome usually results from prompt general nursing and nutritional care which remain the major interventions.The main recommendations for future actions are as follows:1. Validate and update the mapping of L. loa with a combination of remote sensing and RAPLOA techniques.2. Conduct an expert analysis of the apparent clustering of encephalopathic SAEs reported so far.3. Investigate a possible "pre-treatment" scheme with high-dose albendazole in L. loa endemic communities at high risk of encephalopathic SAEs if treated with Mectizan^®; this study will be conducted in collaboration with WHO/TDR.4. Establish a post of Loiasis Technical Advisor for research and operational support in Cameroon, to conduct population surveys and to facilitate better data collection from SAE cases, including postmortem studies as appropriate.5. Investigate the possibility of developing an animal model of L. loa encephalopathy; this activity would be linked to the above-mentioned research agenda in Cameroon.6. Investigate the best care model for encephalopathic SAEs, including identification of early warning signs and therapeutic interventions.7. Develop further models for health education messages needed for community compliance with Mectizan^® treatment, and family support for SAE cases.8. Conduct research studies on the safety of combination therapy of Mectizan^® and albendazole in areas co-endemic for L. loa and lymphatic filariasis (LF) with coordination from the relevant technical bodies that oversee these issues.The above recommendations will be implemented through a continuing collaboration between the interested parties represented at the Scientific Working Group, involved in onchocerciasis control and/or the Global Programme to Eliminate Lymphatic Filariasis.     

In vivo and in vitro effects of some plant hormones on rat erythrocyte carbonic anhydrase and glucose-6-phosphate dehydrogenase activities

The present study was undertaken to determine in vivo and in vitro effects of some plant growth regulators on rat erythrocyte carbonic anhydrase (CA) and glucose-6-phosphate dehydrogenase (G6PD) activities. Both in vivo and in vitro, spermidine and kinetin did not affect enzymatic activities of CA and G6PD, whereas putrescine decreased these activities, and abscisic acid increased them. Since plants use such growth regulators, their effects should be considered on mammals consuming them since they may possess important biological effects.     

Intracerebroventricular injections of noradrenaline affect brain energy metabolism of rainbow trout

To assess the role of noradrenaline (NA) as a possible regulator of brain energy metabolism in teleost fish, the impact of increased noradrenaline levels within the brain on several parameters of energy metabolism was assessed in rainbow trout brain. Accordingly, two different doses of noradrenaline, producing increases in brain NA levels comparable to those occurring in several physiological processes in nature, were selected. In a subsequent set of three different experiments, fish were intracerebroventricularly injected with 1 microL 100 g(-1) body weight of Cortland saline alone (control) or containing NA (5 nmol NA and 10 nmol NA); after 30 min, brain and plasma samples were taken to assess changes in parameters of energy metabolism due to NA treatment. The results obtained clearly show dose-dependent changes in NA-treated fish in several parameters, including decreased glycogen and ATP levels, increased lactate and pyruvate levels, decreased fructose 1,6-bisphosphatase activity, and increased pyruvate kinase and lactate dehydrogenase activities. Altogether, the present experiments show for the first time in a teleost fish evidence supporting that increased noradrenaline levels in the brain elicit metabolic changes in the brain (enhanced glycogenolysis and glycolysis), resulting in an increased energy demand. These metabolic changes may be related to those occurring under several physiological conditions in nature such as hypoxia, in which increased energy demand and increased noradrenaline levels occur in the brain simultaneously.     

Bioactive coatings of endovascular stents based on polyelectrolyte multilayers

Layer-by-layer self-assembly of two polysaccharides, hyaluronan (HA) and chitosan (CH), was employed to engineer bioactive coatings for endovascular stents. A polyethyleneimine (PEI) primer layer was adsorbed on the metallic surface to initiate the sequential adsorption of the weak polyelectrolytes. The multilayer growth was monitored using a radiolabeled HA and shown to be linear as a function of the number of layers. The chemical structure, interfacial properties, and morphology of the self-assembled multilayer were investigated by time-of-flight secondary ions mass spectrometry (ToF-SIMS), contact angle measurements, and atomic force microscopy (AFM), respectively. Multilayer-coated NiTi disks presented enhanced antifouling properties, compared to unmodified NiTi disks, as demonstrated by a decrease of platelet adhesion in an in vitro assay (38% reduction; p = 0.036). An ex vivo assay on a porcine model indicated that the coating did not prevent fouling by neutrophils. To assess whether the multilayers may be exploited as in situ drug delivery systems, the nitric-oxide-donor sodium nitroprusside (SNP) was incorporated within the multilayer. SNP-doped multilayers were shown to further reduce platelet adhesion, compared to standard multilayers (40% reduction). When NiTi wires coated with a multilayer containing a fluorescently labeled HA were placed in intimate contact with the vascular wall, the polysaccharide translocated on the porcine aortic samples, as shown by confocal microscopy observation of a treated artery. The enhanced thromboresistance of the self-assembled multilayer together with the antiinflammatory and wound healing properties of hyaluronan and chitosan are expected to reduce the neointimal hyperplasia associated with stent implantation.     

The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum

IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments.     

Inward and outward potassium currents through the same chimeric human Kv channel

Voltage-gated ion channels are among the most intensely studied membrane proteins today and a variety of techniques has led to a basic mapping of functional roles onto specific regions of their structure. The architecture of the proteins appears to be modular and segments associated with voltage sensing and the pore lining have been identified. However, the means by which movement of the sensor is transduced into channel opening is still unclear. In this communication, we report on a chimeric potassium channel construct which can function in two distinct operating voltage ranges, spanning both inward and outward currents with a non-conducting intervening regime. The observed changes in operating range could be brought about by perturbing either the direction of sensor movement or the process of transducing movements of the sensor into channel opening and closing. The construct could thus provide a means to identify the machinery underlying these processes.     

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.     

Contribution of plasma proteins to splanchnic and total anabolic utilization of dietary nitrogen in humans

Splanchnic tissues are largely involved in the postprandial utilization of dietary amino acids, but little is yet known, particularly in humans, about the relative contributions of different splanchnic protein pools to splanchnic and total postprandial anabolism. Our aim was to develop a compartmental model that could distinguish dietary nitrogen (N) incorporation among splanchnic constitutive, plasma (splanchnic exported), and peripheral proteins after a mixed-protein meal in humans. Eight healthy subjects were fed a single mixed meal containing 15N-labeled soy protein, and dietary N postprandial kinetics were measured in plasma free amino acids, proteins, and urea and urinary urea and ammonia. These experimental data and others previously obtained for dietary N kinetics in ileal effluents under similar experimental conditions were used to develop the compartmental model. Six hours after the mixed-meal ingestion, 31.5, 7.5, and 21% of ingested N were predicted to be incorporated into splanchnic constitutive, splanchnic exported, and peripheral proteins, respectively. The contribution of splanchnic exported proteins to total splanchnic anabolism from dietary N was predicted to be approximately 19% and to remain steady throughout the simulation period. Model behavior and its predictions were strongly in line with current knowledge of the system and the scarce, specific data available in the literature. This model provides the first data concerning the anabolism of splanchnic constitutive proteins in the nonsteady postprandial state in humans. By use of only slightly invasive techniques, this model could help to assess how the splanchnic anabolism is modulated under different nutritional or pathophysiological conditions in humans.