Atrial natriuretic peptide and endothelin-3 target renal sodium-glucose cotransporter

Atrial natriuretic peptide (ANP) and endothelin (ET) are endogenous vasoactive factors that exert potent diuretic and natriuretic actions. We have previously shown that ANP and ET-3 act through an NO pathway to inhibit the sodium-glucose cotransporter (SGLT) in the intestine [Gonzalez Bosc LV, Elustondo PA, Ortiz MC, Vidal NA. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 1997; 18: 1491-5; Gonzalez Bosc LV, Majowicz MP, Ortiz MC, Vidal NA. Effects of endothelin-3 on intestinal ion transport. Peptides 2001; 22: 2069-75.]. Here we address the role of ANP and ET-3 on SGLT activity in renal proximal tubules. In rat renal cortical brush border membranes (BBV), fluorescein isothiocianate (FITC) labeling revealed a specific 72-kD peptide that exhibits increased FITC labeling in the presence of Na+ and D-glucose. Using alpha-14C-methylglucose active uptake, rat BBV were shown to possess SGLT activity with an affinity constant (K(0.5) approximately 2.4 mM) that is consistent with the expression of the low-affinity, high-capacity SGLT2 isoform. SGLT2 activity in these preparations is dramatically inhibited by ANP and ET-3. This inhibition is independent of changes in membrane lipids and is mimicked by the cGMP analogue, 8-Br-cGMP, suggesting the involvement of cGMP/PKG pathways. These results are the first demonstration that both ANP and ET-3 inhibit rat cortical renal SGLT2 activity, and suggest a novel mechanism by which these vasoactive substances modulate hydro-saline balance at the proximal tubular nephron level.     

NPY receptors and opioidergic system are involved in NPY-induced feeding in goldfish

The present study evaluated the effects of both intraperitoneal (i.p. ) and intracerebroventricular administration of selective Y(1) [(Leu(31), Pro(34))-NPY] and Y(2) [(Pro(13), Tyr(36))-NPY (13-36)] receptor agonists on food intake in satiated goldfish. Food intake (FI) was significantly increased by central administration of the Y(1) agonist (1 microg), but not by the Y(2) agonist, at 2 h postinjection. The feeding increase induced by (Leu(31), Pro(34))-NPY was in a similar magnitude to that obtained after ICV injection of the neuropeptide Y, and both feeding stimulations were reversed by the NPY (27-36), a general NPY antagonist. The i.p. administration of the agonists either did not significantly modify (Y(2) agonist) or decreased (Y(1) agonist) food intake in goldfish. These data indicate that it is the Y(1)-like (similar to Y(1) and/or Y(5)) receptor, and not Y(2), that is involved in the central modulation of the feeding behavior in goldfish. We also investigated the possible involvement of opioid peptides as mediators of the NPY stimulatory action on food intake in goldfish. The ICV administration of naloxone (10 microg), a general opioid antagonist, blocked the NPY-induced feeding in goldfish, suggesting that the opioidergic system is involved in feeding regulation by NPY.     

Optimum conditions for growth in liquid medium of Oscillatoria formosa Bory used as the principal food in laboratory culture of intermediate hosts for schistosomosis and fasciolosis

The rearing of snails, intermediate hosts of Schistosoma haematobium, S. intercalatum, S. bovis and Fasciola hepatica is the first step to maintain the life cycle of these parasites in laboratory in order to have biological material for the different studies, namely on the systematic biology and immunodiagnostic of schistosomosis and fasciolosis. According to the traditional method, the alga Oscillatoria formosa Bory (Cyanobacteria), principal food source for the snails, was cultivated in soil extract (Sampaio Xavier et al., 1968). However, it was sometimes very difficult to find the proper soil extract and the material was also contaminated by protozoa and fungi. In our work, using a new medium having as a base the Mineral Medium II (modified from Hughes et al., 1958) we found that O. formosa had a better growth response than in the soil extract medium. Snails fed on O. formosa reached three times the size of others at the same age, and they also reached sex maturity earlier, having more egg-masses per snail and, in addition, the rate of survival as well as the number of generations per year under laboratory conditions significantly increased. This culture was also easier to perform, and the axenic conditions easier to maintain.     

Solid substrate fermentation of Monascus purpureus: growth, carbon balance, and consistency analysis

Solid substrate fermentation (SSF) of Monascus purpureus on rice is a promising new technology for obtaining natural pigments. However, before attempts can be made at maximizing pigment yield, all significant macroscopic compounds should be assayed. Here, Monascus purpureus has been grown on rice in batch mode, and the evolution of the main components, biomass, residual rice, O(2), CO(2), ethanol, acetic acid, and pigments, have been followed. This set of data, never previously studied for Monascus SSF, allowed both the performance of a macroscopic elemental balance, which accounted for 83-94% of the initial substrate carbon, and a check of data consistency. Standard consistency analysis showed a significant underestimation of the nitrogen fraction of biomass, but it was unable to discriminate the errors in the carbon balance as a result of the simultaneous presence of two gross errors in the system. A simple stoichiometric model in tandem with consistency analysis explained unaccounted carbon as an underestimation of CO(2) and ethanol. Using the simplified method to estimate ethanol, the macroscopic balance accounted for 87-99% of the initial carbon.     

Immunohistochemical expression of p53 in breast carcinoma is associated with the intron 1 BglII polymorphism of the p53 gene

Breast carcinoma is a public health problem worldwide. It is known that both genetic and environmental factors are important for breast carcinogenesis and that structural and/or functional alterations at p53 gene are commonly observed in breast tumors. In addition, polymorphisms of several genes in either their coding or non-coding sequences have been found related to cancer risk and/or clinicopathological characteristics of tumors. In this study we have evaluated the intron 1 BglII polymorphism of the p53 gene with a PCR-based approach in 117 cases of breast cancer and 102 healthy women and its association with the immunohistochemical expression of p53 in the tumors. The results showed that the presence of the polymorphism (allele 2) is highly associated with the tumor expression of p53 (p<0.0001) and that there is a trend for increased frequency of allele 2 in cases than in controls (p=0.2376). These data suggest that the germ-line variation in the intron 1 of the p53 gene could produce functional or structural changes of the protein that is reflected by its abnormal expression.     

DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features

Non-irradiated and X-irradiated (80 Gy) human spermatozoa were processed for in situ DNA breakage detection-FISH (DBD-FISH) of the whole genome, following two alternative variations of the basic technique. In the first, cells were initially incubated in the alkaline unwinding solution for transformation of DNA breaks into single-stranded DNA (ssDNA) to be hybridized, followed by the lysing solutions for protein removal. In the second, incubation in the lysing solutions was carried out before the denaturation step. The first approach yielded two subpopulations. While most sperm nuclei were faintly labeled and had chromocenters, a small subpopulation was strongly and homogeneously labeled, due to extensive DNA breakage. X-ray exposure increased the surface and mean fluorescence intensity. Otherwise, when the denaturation step was performed after protein extraction, all sperm nuclei yielded strong and dispersed FISH signals. Protein removal allows access of the unwinding solution to the DNA, which has abundant alkali-labile sites, and thus gives rise to large areas of ssDNA that are labeled by FISH. X-ray exposure increased the dispersion of FISH signals but decreased their mean fluorescence intensity. A linear dose-response was generated using the second experimental variant, being 30 Gy the lowest dose for detecting induction of damage by X-rays in mature sperm chromatin. These results indicate that DBD-FISH is not only useful for in situ detection of DNA breakage but also for revealing structural features of chromatin.     

Germ-line mutational analysis of the TSC2 gene in 90 tuberous-sclerosis patients.

Ninety patients with tuberous-sclerosis complex (TSC) were tested for subtle mutations in the TSC2 gene, by means of single-strand conformational analysis (SSCA) of genomic DNA. Patients included 56 sporadic cases and 34 familial probands. For all patients, SSCA was performed for each of the 41 exons of the TSC2 gene. We identified 32 SSCA changes, 22 disease-causing mutations, and 10 polymorphic variants. Interestingly, we detected mutations at a much higher frequency in the sporadic cases (32%) than in the multiplex families (9%). Among the eight families for which linkage to the TSC2 region had been determined, only one mutation was found. Mutations were distributed equally across the gene; they included 5 deletions, 3 insertions, 10 missense mutations, 2 nonsense mutations, and 2 tandem duplications. We did not detect an increase in mutations either in the GTPase-activating protein (GAP)-related domains of TSC2 or in the activating domains that have been identified in rat tuberin. We did not detect any mutations in the exons (25 and 31) that are spliced out in the isoforms. There was no evidence for correspondence between variability of phenotype and type of mutation (missense versus early termination). Diagnostic testing will be difficult because of the genetic heterogeneity of TSC (which has at least two causative genes: TSC1 and TSC2), the large size of the TSC2 gene, and the variety of mutations. More than half of the mutations that we identified (missense, small in-frame deletion, and tandem duplication) are not amenable to the mutation-detection methods, such as protein-truncation testing, that are commonly employed for genes that encode proteins with tumor-suppressor function.     

Leishmania infantum Nicolle, 1908 from southern Spain: Characterisation of the strains from human visceral and cutaneous leishmaniasis and from sandflies; with a numerical analysis of the isoenzymatic data

This study presents the results of the characterisation of 17 strains ofLeishmania by isoenzyme electrophoresis from a focus of leishmaniasis in southern Spain: two from human visceral leishmaniasis, four from human cutaneous leishmaniasis and 11 from sandflies. The 17 strains are grouped in 6 zymodemes characterised by their variability as regards to the electrophoretic mobility of the enzymes MDH, G6PD, NP and ME. Thus, we confirm the high intraspecific variability ofLeishmania (L.) infantum in a focus of southern Spain, as already suggested by previous studies. Zymodemes GR-15 and GR-17 are also described for the first time in Spain, and they characteristically possess the same relative electrophoretic mobility in the enzyme ME (93). Sixteen zymodemes of theL. infantum complex found in southern Spain were numerically analysed on the basis of the enzymatic profiles of 122Leishmania strains characterised from this area.     

Cardioprotective action of urocortin in postconditioning involves recovery of intracellular calcium handling

Ischemia/reperfusion (I/R) damage in the heart occurs mainly during the first minutes of reperfusion. Urocortin (Ucn) is a member of the corticotrophin-releasing factor that has been identified as a potent endogenous cardioprotector peptide when used in pre- and postconditioning protocols. However, the underlying mechanisms are not completely elucidated. Here, we focused on intracellular calcium ([Ca²⁺]_i) handling by Ucn when applied in early reperfusion. We used Langendorff-perfused rat hearts to determine hemodynamic parameters, and confocal microscopy to study global [Ca²⁺]_i transients evoked by electrical stimulation in isolated cardiomyocytes loaded with fluorescence Ca²⁺ dye fluo-3AM. We found that the acute application of Ucn at the onset of reperfusion, in isolated hearts submitted to ischemia, fully recovered the hearts contractility and relaxation. In isolated cardiac myocytes, following ischemia we observed that the diastolic [Ca²⁺]_i was increased, the systolic [Ca²⁺]_i transients amplitude were depressed and sarcoplasmic reticulum (SR) Ca²⁺ load was reduced. These effects were correlated to a decrease in the Na⁺/Ca²⁺ exchanger (NCX) activity. Importantly, Ucn applied at reperfusion produced a complete recovery in diastolic [Ca²⁺]_i and global [Ca²⁺]_i transient amplitude, which were due to NCX activity improvement. In conclusion, we demonstrated that [Ca²⁺]_i handling play an essential role in postconditioning action of Ucn.     

Cumanensic acid, a new chromene isolated from Piper cf. cumanense Kunth. (Piperaceae)

A new chromene, cumanensic acid (1), together with eight known compounds have been isolated from the aerial part of Piper cf. cumanense Kunth (Piperaceae). Chromene structure was identified on the basis of spectroscopic analysis and comparison with literature data. The compound showed antifungal activity against Fusarium oxysporum f. sp. dianthi and Botrytis cinerea.