The different banding patterns produced by restriction endonuclease digestion in mitotic chromosomes of the American and European eel

The detection of three classes of C-heterochromatin by in situ restriction endonuclease digestion allowed a karyotype differentiation between the American and the European eel.     

Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.     

A conformational study of the opioid peptide dermorphin by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy

Dermorphin, a natural peptide opioid containing a D-Ala2 residue, has been studied in dimethyl sulfoxide (DMSO) solution by means of several one-dimensional and two-dimensional 1H nuclear magnetic resonance (NMR) methods at various fields from 80 to 600 MHz. The combined use of conventional NMR parameters and of nuclear Overhauser effect effects points to an essentially extended structure. This conformation may be, in part, the result of strong interaction of the amide groups with DMSO molecules.     

Prolactin releasing and luteinizing hormone inhibiting activity of dermorphin shorter homologues in the rat

Dermorphin, a heptapeptide isolated from the skin of the frogs Phillomedusa sauvagei and Phillomedusa rhodei, is endowed with potent peripheral and central opioid-like activity. Intracerebroventricular (icv) injection of dermorphin (31.2, 62.5 and 125 pmol/100g) induced in ovariectomized (OVX) rats dose related rises and decreases in prolactin (PRL) and luteinizing hormone (LH) levels, respectively. The aim of this work was to evaluate the same endocrine responses after administration of shorter peptide amide homologues, related to the N-terminal sequence of dermorphin. These compounds retain a substantial analgesic activity although the latter decreases with the decrease in the number of amino acid residues. Icv administration of the hexapeptide homologue (dermorphin 1-6 amide) to OVX rats did not induce any PRL rise or LH inhibition, even at the high dose of 250 pmol/100g. The pentapeptide (dermorphin 1-5 amide), instead, increased PRL and decreased LH secretion, although the effect was significant only at the dose of 250 pmol/100g. Administration of the tetrapeptide (dermorphin 1-4 amide) induced a significant PRL rise and LH inhibition at both the doses of 125 and 250 pmol/100g. The tetrapeptide was the smallest fragment of the dermorphin moiety which caused endocrine responses while the tripeptide (dermorphin 1-3 amide) was completely ineffective in this context. These data indicate that a complete dissociation exists between the behavioral and endocrine effects of the dermorphin homologues examined. In fact, shorter dermorphins whose analgesic potency was directly related to the number of amino acids, exhibited an opposite pattern in evoking endocrine effects.     

Dermorphin decreases plasma LH levels in human: evidence for a modulatory role of gonadal steroids

The purpose of this study was to evaluate the effects of dermorphin, a new synthetic powerful opiate-like heptapeptide, on plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in fertile and postmenopausal women. In fertile subjects, dermorphin (5.5 micrograms/kg min for 30 min) decreases plasma LH (p less than 0.01 vs. baseline and placebo values), but not plasma FSH. The area under the curve during dermorphin infusion was significantly lower than during placebo infusion (p less than 0.01). Pretreatment with the opioid receptor antagonist naloxone, blocked the decrease of plasma LH levels. In postmenopausal women not subjected to any treatment, dermorphin infusion did not significantly modify plasma LH and FSH levels. On the contrary, its administration to postmenopausal subjects treated with conjugated estrogens and medroxyprogesterone acetate significantly decreased plasma LH levels (p less than 0.01, vs. baseline, placebo and area under the curve). Considering the modulatory role exerted by ovarian steroids on the activity of such receptors, these data also indicate that opioid systems play a very important part in the hypothalamus-pituitary-ovarian axis.     

Purification of Tissue Forms (Amastigotes) of Trypanosoma cruzi by Immunoaffinity Chromatography1

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Scpharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

Distribution and Pharmacological Properties of the GABAA/ Benzodiazepine/Chloride Ionophore Receptor Complex in the Brain of the Fish Anguilla anguilla

In the present study, we characterized the distribution and the pharmacological properties of the different components of the GABAA receptor complex in the brain of the eel (Anguilla anguilla). Benzodiazepine recognition sites labeled "in vitro" with [3H]flunitrazepam ([3H]FNT) were present in highest concentration in the optic lobe and in lowest concentration in the medulla oblongata and spinal cord. A similar distribution was observed in the density of gamma-[3H]aminobutyric acid ([3H]GABA) binding sites. GABA increased the binding of [3H]FNT in a concentration-dependent manner, with a maximal enhancement of 45% above the control value, and, vice versa, diazepam stimulated the binding of [3H]GABA to eel brain membrane preparations. The density of benzodiazepine and GABA recognition sites and their reciprocal regulation were similar to those observed in the rat brain. In contrast, the binding of the specific ligand for the Cl- ionophore, t-[35S]butylbicyclophosphorothionate ([35S]TBPS), to eel brain membranes was lower than that found in the rat brain. In addition, [35S]TBPS binding in eel brain was less sensitive to the inhibitory effects of GABA and muscimol and much more sensitive to the stimulatory effect of bicuculline, when compared with [35S]TBPS binding in the rat brain. Moreover, the uptake of 36Cl- into eel brain membrane vesicles was only marginally stimulated by concentrations of GABA or muscimol that significantly enhanced the 36Cl- uptake into rat brain membrane vesicles. Finally, intravenous administration of the beta-carboline inverse agonist 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (20 mg/kg) and of the chloride channel blocker pentylenetetrazole (80 mg/kg) produced convulsions in eels that were antagonized by diazepam at doses five to 20 times higher than those required to produce similar effects in rats. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Prerequisite for His4 in deltorphin A for highδ opioid receptor selectivity

Summary Analysis of deltorphin A position 4 analogues included: backbone constrained N MeHis, spinacine (Spi), N MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofl- andd-His⁴ enantiomers. High selectivity was lost with the following replacements: N MeHis⁴, N MePhe⁴ and Phg⁴ reduced binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi⁴ or Tic⁴ increasedµ affinity. Imidazole methylation of His⁴ marginally affected opioid binding and doubled selectivity; alkylatedd-His⁴-derivatives generally maintained selectivity in spite of decreased affinities. Thus, His⁴ imidazole preserves selectivity by facilitating high binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His⁴ imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr¹ suggestive that this conformation might contribute to high affinity and selectivity.     

The legacy of forest logging on organic matter inputs and storage in tropical streams

Riparian forests play an important role in stream ecosystems, as they support biodiversity, reduce water erosion, and provide litter that fuels aquatic biota. However, they are affected by great array of anthropogenic threats (e.g., fire, logging, and organic pollution), which alter species composition and their physical structure. Although forest recovery after disturbance such as logging can take decades, the legacy of forest clear-cut logging on key processes in tropical riparian ecosystems is mostly unknown. Here, we investigated how litter inputs (leaves, twigs, and reproductive parts) and storage, key processes for carbon and nutrient recycling and for forest and stream biota, are influenced by riparian vegetation undergoing succession (after 28 years from logging) through the comparison of reference and logged forest sites in the Cerrado biome. Litterfall was overall similar between forest types, but litterfall of twigs was twofold higher at logged than reference sites. Similarly, litter inputs from the bank to the stream (i.e., lateral inputs) and streambed storage were 50–60% higher at logged than reference sites. The higher litterfall observed in logged forests could be related to higher proportion of tree species that are characteristic of primary and secondary successional stages, including fast-growing and liana species, which often are more productive and common in anthropogenic areas. Our results showed that the legacy impact of clear-cut logging, even if residual woody vegetation is maintained in riparian buffers, can shift the type, quantity, and seasonality of litter subsidies to tropical streams. This knowledge should be considered within the context of management and conservation of communities and ecosystem processes in the forest-stream interfaces.