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Two variants of the adipose hormone resistin are generated by alternative splicing in Wistar rats. Here we analyzed the expression of these resistin variants in 2 main visceral adipose depots, epididymal and retroperitoneal, as well as the resistin serum concentration during aging and food restriction. Total protein levels of resistin were also analyzed in extracts from both visceral adipose depots. Resistin variants show similar patterns of relative expression in visceral adipose tissues in 3-month-old rats, representing the short variant, s-resistin, which is 15% of the full-length transcript. However, only epididymal, but not retroperitoneal, fat pad shows a decrease in both messenger RNA and protein levels of resistin isoforms with aging. Food restriction decreases adiposity index in 8- and 24-month-old animals to values even lower than those of 3-month-old animals. Food restriction decreases resistin expression in both adipose tissues in 8-month-old but not in 24-month-old rats. Interestingly, concomitant with the improvement of insulin sensitivity asserted by homeostasis model assessment, resistin serum levels decrease only in food-restricted 8-month-old animals. In contrast, food restriction up-regulates s-resistin messenger RNA in epididymal adipose tissue, whereas no significant changes are appreciated in retroperitoneal adipose tissue. These data indicate that both forms of resistin are differentially regulated by fat depot location, aging, and even nutritional status, suggesting that alternative splicing plays a key role in this differential regulation.

Energy balance in mammals is modulated by peripheral signals that inform the brain about the magnitude of fat stores, the amount of food in the gastrointestinal tract, and the level of nutrients such as glucose in the circulation. Among these, insulin and leptin are considered adiposity signals involved in the long-term maintenance of fat stores. Here we review the mechanisms of action of leptin and insulin in the hypothalamus and how these mechanisms are altered during aging in rat models. Aged rats are characterized by increased fat mass, central leptin and insulin resistance, and hyperleptinemia. Leptin resistance is manifested by its failure to inhibit food intake, deplete fat stores, down regulate its own expression in adipose tissue, and increase energy expenditure. Moreover, leptin and insulin signaling are decreased in hypothalamus from aged rats. Calorie restriction and fasting studies provide controversial data on the cause-effect interrelationship between increased adiposity and development of central leptin resistance. Although in the absence of obesity leptin resistance seems to be a characteristic of aged animals, adiposity could either reinforce it or cause an early onset of this resistance. More studies are necessary to clarify the role of the hypothalamus in the development of age-associated obesity and insulin resistance.

Mitochondrial DNA (mtDNA) mutations are an important cause of human disease. Most mtDNA mutations are found in heteroplasmy, in which the proportion of mutant vs. wild-type species is believed to explain some of the observed high phenotypic heterogeneity. However, homoplasmic mutations also observe phenotypic heterogeneity, which may be in part due to undetected low levels of heteroplasmy. In the present report, we have developed two assays, using DHPLC and Pyrosequencing (Biotage AB, Uppsala, Sweden), for reliably and accurately detecting low-level mtDNA heteroplasmy. Using these assays we have identified a three-generation family segregating two mtDNA mutations in heteroplasmy: the deafness-related m.1555A>G mutation in the 12S rRNA gene (MTRNR1) and a new variant (m.15287T>C) in the cytochrome b gene (MTCYB). Both heteroplasmic mtDNA mutations are transmitted through generations in a random manner, thus showing differences in mutation load between siblings within the family. In addition, the developed assays were also used to screen a group of deaf subjects of unknown etiology for the presence of heteroplasmy for both mtDNA variants. Two additional heteroplasmic m.1555A>G samples, previously considered as homoplasmic, and two deaf subjects carrying m.15287T>C variant were identified, thus confirming the high specificity and reliability of the approach. The development of assays for reliably detecting low-level heteroplasmy, together with the study of heteroplasmic mtDNA transmission, are essential steps for a better knowledge and clinical management of mtDNA diseases., ((c) 2007 Wiley-Liss, Inc.)

Insulin, like leptin, is considered as a lipostatic signal acting at a central level. Aging and age-associated adiposity have been related to the development of leptin resistance in Wistar rats. In the present article, hypothalamic insulin response during aging has been studied in Wistar rats. Thus, the effects of intracerebroventricular infusion of insulin during a week on food intake and body weight as well as insulin signal transduction after acute intracerebroventricular insulin administration have been studied in 3-, 8-, and 24-month-old rats. To explore the possible role of age-associated adiposity, these experiments were also performed in 8- and 24-month-old rats after 3 months of food restriction to reduce visceral adiposity index to values below those of young animals. Intracerebroventricular administration of insulin during a week was more efficient at reducing food intake and body weight in 3-month-old rats than in 8- and 24-month-old rats. Hypothalamic insulin-stimulated insulin receptor, GSK3, AKT, and p70S6K phosphorylation decreased with aging. Insulin receptor and IRS-2 phosphoserine was increased in 24-month-old rats. Food restriction improved both insulin responsiveness and insulin signaling. These data suggest that Wistar rats develop hypothalamic insulin resistance with aging. This can be explained by alterations of the signal transduction pathway. The fact that food restriction improves central insulin response and signal transduction points to the age-associated adiposity as a key player in the development of central insulin resistance.

Catastrophic collapse of a mine tailings dam released several million cubic meters of toxic mud and acidic water into the Guadiamar River valley, southern Spain, in 1998. Remediation efforts removed most of the sludge from the floodplain, but contamination persists. Clean-up activities also produced clouds of aerosolized materials that further contaminated the surrounding landscape. Whole-body concentrations of 21 elements in the Moorish wall gecko, Tarentola mauritanica, a common inhabitant of both rural and urban areas, were compared among seven locations. Locations spanned an expected contamination gradient and included a rural and an urban non-mine-affected location, two mine-affected towns, and three locations on the contaminated floodplain. Multivariate analyses of whole-body concentrations identified pollutants that increased across the expected contamination gradient, a trend particularly evident for As, Pb, and Cd. Additionally, higher contaminant concentrations occurred in prey items eaten by geckos from mine-affected areas. Comparison of element concentrations in tails and whole bodies suggests that tail clips are a viable nondestructive index of contaminant accumulation. Our results indicate that areas polluted by the mine continue to experience contamination of the terrestrial food chain. Where abundant, geckos represent useful taxa to study the bioavailability of some hazardous pollutants.

In rodents, soluble leptin receptor (SLR) may be generated by alternative splicing of ObR mRNA and/or as a cleavage product of ObR membrane-anchored receptors. In this study, we investigated the contribution of both processes on the generation of SLR in 3-, 8-, and 24-month-old Wistar rats fed ad libitum (AL) or under food restriction (FR). To this end, we determined serum SLR levels and analyzed ObRa and ObRe mRNA expression under these physiological conditions. Additionally, we studied the cellular distribution of ObRa and the generation of SLR by N-ethyl-maleimide-induced shedding from ObRa membrane receptors in isolated adipocytes. Serum SLR levels were significantly increased in 8- and 24-month-old rats under FR, whereas similar amounts were found in rats of different ages fed AL. ObRa and ObRe mRNA expression in epididymal adipose tissue increased with aging. In contrast, after FR, ObRe mRNA expression decreased, whereas ObRa mRNA expression further increased compared with 8- and 24-month-old rats fed AL. Additionally, FR promoted a change in the distribution of ObRa between internal and plasma membranes in isolated adipocytes, increasing its presence at the cell surface. Finally, the generation of SLR by N-ethyl-maleimide-induced shedding from ObRa was also increased under FR. These data suggest that shedding of ObRa membrane-anchored receptors, rather than ObRe expression, might preferentially contribute to the generation of the increased levels of SLR in serum under conditions of FR.

Leptin modulates glucose homeostasis by acting as an insulin-sensitizing factor in most insulin target tissues. Nevertheless, insulin-dependent glucose uptake in white adipose tissue decreases after in vivo treatment with leptin. Moreover, elevated leptin concentrations inhibit insulin metabolic effects in adipocytes. Here we studied both, direct and centrally mediated effects of leptin on insulin signaling in rat adipocytes. Adipocyte incubation with low leptin concentrations did not modify the insulin stimulation of mitogen-activated protein kinase (MAPK). However, at elevated concentrations, leptin impaired insulin-stimulated MAPK activity, glycogen synthase kinase (GSK)3beta phosphorylation, and insulin receptor tyrosine phosphorylation without altering vanadate stimulation. An increase of suppressor of cytokine signaling-3 protein was also observed. Central administration of leptin decreased insulin effects on adipocyte MAPK and GSK3beta phosphorylation. In insulin-resistant aged rats with hyperleptinemia and central leptin resistance, insulin poorly stimulated MAPK and central leptin infusion did not further deteriorate adipocyte insulin responsiveness. Food restriction increased MAPK stimulation by insulin and restored the ability of centrally infused leptin to attenuate adipocyte insulin signaling in aged rats. We conclude that leptin can modulate, in an inhibitory manner, adipocyte insulin signaling by two different ways: as an autocrine signal and, indirectly, through neuroendocrine pathways. These mechanisms may be of relevance in situations of hyperleptinemia, such as aging and/or obesity.

Genotoxic compounds can act at various levels in the cell (causing gene, chromosome, or genome mutations), necessitating the use of a range of genotoxicity assays designed to detect these different types of mutations. The production of melanoidins during the processing and cooking of foods is associated with changes in their nutritional character, and the discovery of mutagenic substances in pyrolyzed protein and amino acids has raised concern about the safety of these foods. The aim of this work was to test melanoidin fractions in three different in vitro assays (Ames test, Vitotox test, and micronucleus test). These melanoidin fractions were produced from the condensation of glucose with glycine and their separation was conducted by dialysis. The crude reaction mixture (before dialysis) and both the LMW and HMW fractions obtained by dialysis showed no genotoxicity in these assays, despite being tested at concentrations much higher than those naturally found in food products. The LMW fraction, however, showed toxicity at these high concentrations. The volatile fraction produced in this reaction showed genotoxicity only in the Vitotox test, at high concentrations.

Resistin is a secreted adipose tissue hormone that belongs to the resistin-like molecule family. We report here a new alternatively spliced isoform of the rat resistin gene, named S-resistin (short resistin), detected in adipose tissue by reverse transcription-polymerase chain reaction (RT-PCR). A comparison of this cDNA variant and genomic sequences indicates the lack of the second exon containing the secretory consensus signal. Both cDNAs, resistin and S-resistin, were carboxy-tagged with FLAG epitope and transiently expressed in cultured cell lines. While the resistin-FLAG construct gives the expected pattern for a secretion protein, the S-resistin-FLAG construct yielded a predominant nuclear staining. These results indicate that this splicing event regulates the fate and probably the function of the mature protein.

On 5 July 2003, the Tour de France (TDF) has celebrated 100th running. Instead of a chimney sweep competing during his free time (as in 1903), the recent winner is a highly trained, professional cyclist whose entire life-style has been dedicated to reach his pinnacle during this event. The TDF has been held successfully for 100 years, but the application of the physiologic sciences to the sport is a relatively recent phenomenon. Although some historical reports help to understand the unique physiological characteristics of this race, scientific studies were not available in Sports Science/Applied Physiology journals until the 1990s. The aim of this article is to review the history of the TDF. Special emphasis is placed on the last decade where classic physiology has been integrated into applied scientific cycling data.

Aged Wistar rats are characterized by leptin and insulin resistance. The expression of SOCS-3 in hypothalamus increases with ageing. Food restriction during 3 months decreases obesity Lee index in aged rats with respect to their ad libitum aged-mates and brings serum leptin concentrations to values close to those of young rats. Food restriction partially reverts the increases in SOCS-3 mRNA levels associated with ageing. These results suggest that SOCS-3 may be a mediator of hypothalamic leptin resistance in the aged Wistar rat and that the hyperleptinemia associated with ageing is, at least in part, responsible for the increase of SOCS-3 expression in hypothalamus.

Aims/hypothesis: Ageing is associated with insulin and leptin resistance in mammals. These alterations might be caused by the increased adiposity associated with ageing, by ageing alone or both. We studied whether leptin resistance occurs at the central level in the Wistar rat and we aimed to discriminate between the effects of ageing from those of the increased adiposity associated with ageing., Methods: Leptin was infused intracerebroventricularly at a constant rate in young adult, old and old Wistar rats fasted for 3 months, using osmotic pumps. The effects on body weight, daily food intake, Lee index, adiposity and serum leptin values were analysed. The effect of food restriction on the expression of the long form of leptin receptor in the hypothalamus was also studied., Results: Leptin decreased daily food intake and body weight in young and old Wistar rats. With a dose of 10 microg/day similar responses were obtained in young and old rats but with a dose of 0.2 microg/day, only young rats showed decreases in these parameters. Food-restriction in old rats lowered adiposity and serum leptin to values close to those of young rats, recovered responsiveness to i.c.v. administration of leptin at the dose of 0.2 microg/day and increased leptin receptor expression in the hypothalamus., Conclusion/interpretation: Our data show that old Wistar rats have a decreased response to leptin at the central level. Food-restriction recovers leptin responsiveness and increases leptin receptor in the hypothalamus suggesting that adiposity plays a key role in the development of leptin resistance associated with ageing.

Leptin interacts with specific receptors in hypothalamic nuclei and modulates energy balance. Growing evidence has shown the association of obesity and hyperleptinaemia with non-insulin-dependent diabetes mellitus and insulin resistance. The aged Wistar rat shows peripheral insulin resistance in the absence of obesity and alterations of glucose homeostasis. However, it is not known whether, in these animals, the leptin action is altered. Here we studied the effect of ageing on plasma leptin concentration and the ability of hypothalamic nuclei to capture i.c.v.-injected digoxigenin-labelled leptin. Our data indicate that 24-month-old animals are hyperleptinaemic. However, daily food intake was greater in old animals, suggesting that they are leptin resistant. Leptin uptake in the hypothalamus was reduced in old rats. This uptake was a receptor-mediated process as demonstrated by displacement. Leptin accumulation in hypothalamic nuclei was partially colocalized with neuropeptide Y fibres. Immunohistochemical and western blot analyses showed a lower amount of the long form of leptin receptors in the hypothalamus of aged rats. Analysis by RT-PCR also demonstrated a decreased expression of leptin receptor mRNA in old animals. We conclude that the lower leptin uptake may be explained, at least in part, by a decreased amount of receptors in hypothalamic neurones of the aged rats.

Hetero Diels-Alder (HDA) cycloaddition of chiral 1-p-tolylsulfinyl-1,3-pentadiene with benzyl nitrosoformate, under mild conditions, yields 2H-1,2-oxazine 3 with complete regioselectivity and pi-facial diastereoselectivity. Sequential osmylation and protection of the resulting glycol gives the oxazine 5 which is directly transformed into enantiomerically pure 1,4,5-trideoxy-1,4-imino-L-ribitol 8 by reduction under Pd/C.

We describe a case of prolonged neuromuscular block (longer than 8 hours) after administration of mivacurium. The patient was shown to be homozygous for the atypical butyrylcholinesterase gene. We discuss our treatment of the patient as well as other cases described in the literature, emphasizing the need for neuromuscular monitoring.

We have done a study in order two know the prevalence of anti-hepatitis C virus antibodies in polytransfused patients with hemophilia, leukemia and hemolytic anemia, along with 17 healthy donors, without previous history of transfusions. We analyzed samples from 10 hemophilic patients and 12 from leukemia, lymphoma and hemolytic anemia, all of them had received blood or blood products, at least six months before the study. Using a second generation ELISA, 4 positive sample (3 hemophilic and 1 lymphoma) were detected (10.26%), which represent a prevalence of 30% in the hemophilic group, in contrast with the prevalence detected in other countries. A very significant statistic association was demonstrated, between the positive ELISA, the amount of the transfused product (P < 0.0004) and the type of blood product used for transfusion (crioprecipited, P = 0.000, plasma P = 0.000).

In the fruitfly Drosophila, as in all eukaryotes examined so far, some ubiquitin-coding sequences appear fused to unrelated open reading frames. Two of these fusion genes have been previously described (the homologues of UBI1-UBI2 and UBI4 in yeast), and we report here the organization and expression of a third one, the DUb80 gene (the homologue of UBI3 in yeast). This gene encodes a ubiquitin monomer fused to an 80-amino-acid extension which is homologous with the ribosomal protein encoded by the UB13 gene. The 5' regulatory region of DUb80 shares common features with another ubiquitin fusion gene, DUb52, and with the ribosomal protein genes of Drosophila, Xenopus and mouse. We also find helix-loop-helix protein-binding sequences (E-boxes). The DUb80 gene is transcribed to a 0.9 kb mRNA which is particularly abundant under conditions of high protein synthesis, such as in ovaries and exponentially growing cells.

Using a combination of polymerase chain reaction and single-strand conformation polymorphism techniques (PCR-SSCP) we have analyzed 78 brain tumor samples (70 primary and 8 metastatic) for the presence of mutations in the conserved regions of the Tp53 (tumor p53) gene. We have found that only two groups, gliomas (exclusively in astrocytomas) and metastases, displayed Tp53 mutations. Three of eight (37.5%) metastases showed a mutant Tp53 allele accompanied by loss of the normal one. In contrast, the frequency of Tp53 mutations in the primary brain tumors examined was lower (5.7%). Although we have examined different types of primary brain tumors, Tp53 mutations were exclusively observed in both, low and high-grade astrocytomas (four of 24). The Tp53 mutations detected in astrocytic tumors appear to be correlated with the malignancy grade. The low-grade astrocytomas were heterozygous for the mutation, whereas the high-grade astrocytomas had affected the two Tp53 alleles, suggesting a two-steps model for inactivation of the p53 gene in astrocytomas. Thus, single p53 mutation seems to occur in initial stages of astrocytoma tumorigenesis; the later lost of the remaining wild-type allele appears associated with the progression towards a more malignant stage.

Ubiquitin belongs to a multigene family. In Drosophila two members of this family have been previously described. We report here the organization and expression of a third member, the DUb52 gene, isolated by screening a Drosophila melanogaster genomic library. This gene encodes an ubiquitin monomer fused to a 52-amino acid extension protein. There are no introns interrupting the coding sequence. Recently, it has been described that this extension encodes a ribosomal protein in Saccharomyces, Dictyostelium, and Arabidopsis. The present results show that the 5' regulatory region of DUb52 shares common features with the ribosomal protein genes of Drosophila, Xenopus and mouse, including GC- and pyrimidine-rich regions. Moreover, sequences similar to the consensus Ribo-box in Neurospora crassa have been identified. Furthermore, a sequence has been found that is similar to the binding site for the TFIIIA distal element factor from Xenopus laevis. The DUb52 gene is transcribed to a 0.9 kb mRNA that is expressed constitutively throughout development and is particularly abundant in ovaries. In addition, the DUb52 gene has been found to be preferentially transcribed in exponentially growing Drosophila cells.

In a sample of 20 healthy men (ASA I) we studied the thermal effects induced after epidural anesthetic blockade with bupivacaine (0.625%) and their relationship with the level of sensitive blockade to puncture or to cold. After 30 min of epidural injection of bupivacaine the level of cephalic analgesia was D IX (D IX +/- 2 segments) and that of cold discrimination D VII (D VII +/- 2 segments) being the thermo-algesic differential blockade of 2 to 3 segments. During epidural anesthesia there was a significant increase in foot skin temperature (4.1 +/- 1 centigrade degrees, p less than 0.001) without any appreciable skin temperature change at the thorax, abdomen, thigh, and calves. It is concluded that epidural anesthesia with bupivacaine (0.625%) at a sensitive analgesic level D IX produces significant increases in skin temperature only at the foot. This indicates that the extension level of sympathetic blockade is lower that of the analgesia.

We have isolated by molecular cloning techniques a structural gene that maps to subregion 63F of Drosophila melanogaster chromosome 3L. This locus being one of the early chromosomal targets for ecdysone stimulation, may be induced to puff by the hormone. The gene is on a plasmid vector and it has been designated as pDm 63F. This recombinant molecule also maps to the early ecdysone inducible subregion 90B of Drosophila hydei chromosome 4. The cytological inspection of large number of chromosomal sets after in situ hybridization of the cloned DNA, locates the cloned sequence between bands 63F 2-4 according to Bridges map. Similarly, in Drosophila hydei the cloned DNA maps between subdivisions 90B 2-4 according to Berendes' map. In situ hybridization of the pDm 63F cloned DNA, directed to the nascent RNA rather than to the DNA, shows two to three times more silver grains over the corresponding regions when puffed than in the resting stage. There are however quantitative differences attending to the transcriptional activity of homologous loci in both species. By RNA excess hybridization we have found that the cellular concentration of the 63F cloned mRNA is bout 2 times higher in hormone stimulated total larval tissues than in non-stimulated ones.

We have studied the presence of a cloned fragment of DNA from Drosophila melanogaster in other organisms by means of nucleic acid hybridization analysis. The isolated region is localized in polytene chromosomes at the 63F subdivision. This region includes a puff that responds within minutes to ecdysone stimulation. We have found that 63F DNA from D. melanogaster hybridizes 'in situ' to both DNA and RNA from D. simulans, D. teissieri, and D. hydei. In all these species the isolated DNA remains associated with one early-ecdysone stimulated puff. The isolated Drosophila recombinant DNA is also complementary to polyadenylated RNA from foetal and adult rat liver but fails to hybridize to the nonpolyadenylated RNA classes from both sources and to polyadenylated RNA from rat mammary glands.