The occurrence of ecdysteroids in the cestode, Moniezia expansa

The occurrence of free ecdysteroids in the sheep cestode, Moniezia expansa, was demonstrated. Significant amounts of conjugated ecdysteroids were not detected. Characterization of the free hormones by high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by gas chromatography/mass spectrometry (selected ion monitoring) indicated the presence of ecdysone, 20-hydroxyecdysone and 20,26-dihydroxyecdysone. Analysis of the ecdysteroids by radioimmunoassay in segments along part of the strobila indicated that the anterior parts contained the greatest amount of hormone. GC/MS (SIM) analysis of the hormones in a strobilar segment containing the most mature proglottids suggested the presence of several ecdysteroid metabolites.     

Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture

Ketoconazole, a clinically effective antimycotic agent active in vitro against the amastigote stage of Leishmania mexicana Walter Reed 227 in human monocyte-derived macrophages, was found to inhibit growth and impair sterol biosynthesis of the cultured promastigote stage by approx. 50% at a concentration of approx. 10(-8)M. Sterol biosynthesis was interfered with at the level of the removal of the 14 alpha-methyl group of lanosterol, as judged by changes in the distribution of [2-14C]mevalonate radioactivity among desmethyl sterol and methyl sterol thin-layer chromatography fractions, by the loss of 4-desmethyl sterols (mainly 5-dehydroepisterol), and by the accumulation of 14 alpha-methyl sterols. The growth inhibition and sterol changes were evident in promastigotes cultured in a cholesterol-rich medium and in a cholesterol-poor medium, even though promastigotes incorporated cholesterol. The mechanism of action of ketoconazole against promastigotes may be that postulated for Candida albicans: interference with membrane permeability secondary to loss of desmethyl sterols and accumulation of 14 alpha-methyl sterols.     

In vivo release of met-enkephalin in the cat brain

Push-pull cannulae were implanted into the globus pallidus (anterior part), the caudate nucleus (medio-dorsal part) and the substantia nigra of halothane-anaesthesized cats to study the characteristics of the in vivo release of met-enkephalin. Met-enkephalin was measured by radioimmunoassay (sensitivity: 0.1 pg) and identified by Bio-Gel P₂ chromatography and high-pressure liquid chromatography. Under resting conditions, met-enkephalin was detected in perfusates from these three regions; in the globus pallidus, the rate of spontaneous release of met-enkephalin was 5–6 times higher than that observed in the caudate nucleus and the substantia nigra. The local application of either an excess (60 mM) of K⁺ or of glutamate (10 μM) produced a marked increase in the rate of met-enkephalin release both from the globus pallidus and the caudate nucleus. Further analyses in the globus pallidus indicated that the depolarizing agents, veratridine (50 μM) and batrachotoxin (0.1 μM), produced a large increment in the rate of met-enkephalin release; this effect was completely prevented by the local application of tetrodotoxin (2 μM).The chemical (with 60 mM K⁺ or 10 μM glutamate), electrical or mechanical stimulation of the dorsomedial part of the caudate nucleus failed to significantly alter the rate of met-enkephalin release in the ipsilateral globus pallidus, in spite of the high sensitivity of enkephalinergic nerve terminals in the globus pallidus itself to local stimuli. This observation argues against the existence of a major caudatopallidal enkephalinergic pathway.     

Sterols of Leishmania species. Implications for biosynthesis

The major sterol of promastigotes of stocks of Leishmania tropica, L. donovani and 3 subspecies of L. mexicana has been identified as ergosta-5,7,24(28)-trien-3 beta-ol; and of an L. major stock as ergosta-7,24(28)-dien-3 beta-ol. 24-Methylcholesta-5,7,22-trien-3 beta-ol and 24-ethylcholesta-5,7,22-trien-3 beta-ol were minor constituents, and traces of ergosta-5,7,22,24(28)-tetraen-3 beta-ol and a C27-diene were also recognized in some species. Lanosterol and 4,4-dimethylcholesta-8,24-dien-3 beta-ol were detected in all species studied, and squalene was identified in a stock of L. tropica. The sterol composition of members of the genus Leishmania and the sterol biosynthetic pathways it implies are characteristic of yeast and other fungi.     

Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes

Leishmania mexicana mexicana promastigotes grown with cholesterol, supplied in natural products as the free sterol and as cholesteryl esters, were exposed to [2-14C]mevalonate and to the antimycotic drug ketoconazole. Growth was inhibited and cholesterol and 14 alpha-methyl sterols accumulated in free and esterified forms (cholesterol much greater than 4 alpha,14 alpha-dimethylcholesta-8,24-dien-3 beta-ol much greater than 14 alpha-methylcholesta-8,24-dien-3 beta-ol congruent to 14 alpha-methylergosta-8,24(28)-dien-3 beta-ol much greater than 4 alpha,14 alpha-dimethylergosta-8,24(28)-dien-3 beta-ol; identified by capillary gas chromatography/mass spectrometry, and by 1H and 13C nuclear magnetic resonance spectrometry). The 14 alpha-methyl sterols were preferentially labelled with 14C. The cholesterol was unlabelled and substituted for a substantial fraction of the major product of sterol biosynthesis, ergosta-5,7, 24(28)-trien-3 beta-ol (5-dehydroepisterol), but did not replace it and did not offer remarkable protection against either growth inhibition or alteration of sterol biosynthesis. Promastigotes grown with [6-2H]cholesterol or [4-14C]cholesterol did not contain labelled forms of Leishmania sterols, or other sterols. The chromatographic and spectrometric sterol analyses and the isotopic tracer findings suggested that ketoconazole impaired the cytochrome P-450 dependent 14 alpha-demethylation of lanosterol, that cholesterol was neither biosynthesized nor metabolized, and that the physiological functions of 5-dehydroepisterol had sterol structural requirements not entirely met by cholesterol. In all these studies, L. mexicana mexicana demonstrated a sterol biochemistry remarkably similar to that of fungi. This recommends an increase in interest in antimycotic drugs as chemotherapeutic agents for leishmanial infections.     

Eicosapentaenoic acid inhibits prostaglandin D2 generation by inhibiting cyclo-oxygenase-2 in cultured human mast cells

BACKGROUND: Eicosapentaenoic acid (EPA) is catalysed by cyclo-oxygenase (COX), as is arachidonic acid, and is a competitive inhibitor of arachidonate metabolism. OBJECTIVES: We examined the effect of EPA on prostaglandin (PG) D2 generation in the cultured human mast cells with IgE-anti-IgE challenge incubation. METHODS: Cultured human mast cells were incubated with EPA (1 micromol/L) for 20 h, then challenged with anti-IgE incubation after treatment with IgE. At the same time, COX inhibitors were tested to identify COX-1 and COX-2 activity. PGD2 synthetic activity was also assayed in a cell-free homogenate of cultured mast cells with COX inhibitors and EPA. Histamine in the culture medium and in cells was assayed with the HPLC-fluorescent method. PGD2 and PGD3 were assayed with gas chromatography-mass spectrometry and the stable isotope dilution method. RESULTS: Although EPA incubation did not affect histamine release by cultured human mast cells in response to IgE-anti-IgE challenge incubation, it did decrease PGD2 generation by inhibiting the COX-2 pathway. In contrast, in the cell-free homogenate of cultured human mast cells, EPA inhibited both COX-1 and COX-2 activities. CONCLUSION: Pre-incubation with EPA primarily affects the COX-2 pathway in cultured human mast cells and reduces PGD2 generation in response to IgE-anti-IgE challenge incubation. These findings suggest that COX-1 and COX-2 have different substrate flow systems in mast cells. They also suggest that endogenous EPA diet supplementation would reduce PGD2 production and could serve as an anti-inflammatory substrate in human mast cells.     

Cod cathelicidin: isolation of the mature peptide, cleavage site characterisation and developmental expression

Cathelicidin antimicrobial peptides are multifunctional peptides that are important in the innate immune system of mammals. Cathelicidins have been identified in several fish species. In this study we have isolated cathelicidin from Atlantic cod (Gadus morhua) and identified the cleavage site from the cathelin propart. This is the first isolation of a cathelicidin from teleost fish. The mature cathelicidin was found to be a 67-residues peptide, highly cationic with a pI of 13. Reversed phase chromatographic fractions containing the purified peptide had pronounced antimicrobial activity and the activity of the mature peptide was confirmed using a synthetic peptide. We examined the expression of cathelicidin during cod larvae early development using real-time PCR and detected expression that varied in the course of the first 68 days post hatching (dph). Two groups of larvae having a different food regime were compared. Cathelicidin expression was found to differ between the two groups and this could be linked to their food input. The presence and rapid adjustment of cathelicidin expression in the larvae indicate that the immune system of cod is active from early on in development and responds to external stimuli by the production of antimicrobial peptides.     

Functional roles of endogenous d-serine in the chronic pain-induced plasticity of NMDAR-mediated synaptic transmission in the central amygdala of mice

The amygdala is implicated in chronic pain-induced emotional changes. Chronic pain induces plastic changes of the N-methyl-d-aspartate receptor (NMDAR) functions in the brain including the amygdala. d-Serine is synthesized endogenously by serine racemase and modulates NMDAR-mediated synaptic transmission as a coagonist of glycine binding site. To clarify the functional roles of endogenous d-serine in chronic pain-induced plasticity of NMDAR mediated synaptic transmission, we investigated the NMDAR-mediated excitatory synaptic current (EPSC) of neurons in the latero-capsular division of the central amygdala (CeLC) using brain slices from serine racemase knockout (SR-KO) mice with chronic pain induced by monoarthritis. The decay time of NMDAR-mediated EPSC was significantly elongated by monoarthritis in wild type (WT) mice, but not in SR-KO mice. The d-serine application-induced increase of NMDAR-mediated EPSC was significantly facilitated by monoarthritis in WT mice, but not in SR-KO mice. These results suggest that endogenous d-serine facilitates chronic pain-induced plastic changes of NMDAR mediated synaptic transmission in CeLC.     

Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates

Promastigotes of Leishmania adleri were submitted to an extraction procedure providing different carbohydrate-containing extracts. The purified aqueous extract showed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a complex peptide pattern but carbohydrate was present only in bands of M_r ≈ 45 000–50 000 and 13 500. Methylated derivatives of the hexose components in this extract, analysed by mass spectrometry, suggest the presence of short sugar chains of α-d-mannopyranose and a branched α-d-mannan. The phenol extract, released in the aqueous layer a chloroform/methanol/water soluble complex contained 25% protein, 17% phosphate, 11% glucosamine, uronic acid and 61% neutral carbohydrate, and a chloroform/methanol/water insoluble fraction consisting of a glycoprotein M_r ≈ 22 000 and a proteic doublet M_r ≈ 58 000–66 000. A polysaccharide, showing galactose as predominant sugar, was released through alkaline extraction corresponding to a branched, mainly 1→3 linked galactan associated with α-d-mannopyranosyl units.     

Neuropeptidergic signaling in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans joins the menagerie of behavioral model systems next to the fruit fly Drosophila melanogaster, the marine snail Aplysia californica and the mouse. In contrast to Aplysia, which contains 20,000 neurons having cell bodies of hundreds of microns in diameter, C. elegans harbors only 302 tiny neurons from which the cell lineage is completely described, as is the case for all the other somatic cells. As such, this nervous system appears at first sight incommensurable with those of higher organisms, although genome-wide comparison of predicted C. elegans genes with their counterparts in vertebrates revealed many parallels. Together with its short lifespan and ease of cultivation, suitability for high-throughput genetic screenings and genome-wide RNA interference approaches, access to an advanced genetic toolkit and cell-ablation techniques, it seems that this tiny transparent organism of only 1mm in length has nothing to hide. Recently, highly exciting developments have occurred within the field of neuropeptidergic signaling in C. elegans, not only because of the availability of a sequenced genome since 1998, but especially because of state of the art post genomic technologies, that allow for molecular characterization of the signaling molecules. Here, we will focus on endogenous, bioactive (neuro)peptides and mainly discuss biosynthesis, peptide sequence information, localization and G-protein coupled receptors of the three major peptide families in C. elegans.     

Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency

The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency.     

Protein methionine content and MDA-lysine adducts are inversely related to maximum life span in the heart of mammals

Aging affects all organisms and its basic mechanisms are expected to be conserved across species. Oxidation of proteins has been proposed to be one of the basic mechanisms linking oxygen radicals with the basic aging process. If oxidative damage to proteins is involved in aging, long-lived animals (which age slowly) should show lower levels of markers of this kind of damage than short-lived ones. However, this possibility has not been investigated yet. In this study, steady-state levels of markers of different kinds of protein damage--oxidation (glutamic and aminoadipic semialdehydes), mixed glyco- and lipoxidation (carboxymethyl- and carboxyethyllysine), lipoxidation (malondialdehydelysine) and amino acid composition--were measured in the heart of eight mammalian species ranging in maximum life span (MLSP) from 3.5 to 46 years. Oxidation markers were directly correlated with MLSP across species. Mixed glyco- and lipoxidation markers did not correlate with MLSP. However, the lipoxidation marker malondialdehydelysine was inversely correlated with MLSP (r2=0.85; P<0.001). The amino acid compositional analysis revealed that methionine is the only amino acid strongly correlated MLSP and that such correlation is negative (r2=0.93; P<0.001). This trait may contribute to lower steady-state levels of oxidized methionine residues in cellular proteins. These results reinforce the notion that high longevity in homeothermic vertebrates is achieved in part by constitutively decreasing the sensitivity of both tissue proteins and lipids to oxidative damage. This is obtained by modifying the constituent structural components of proteins and lipids, selecting those less sensitive to oxidative modifications.     

The early detection of Salla disease through second-tier tests in newborn screening: How to face incidental findings

We describe here a 34 months child, practically asymptomatic which presented with high levels of free sialic acid in urine by biochemical detection in second-tier tests newborn screening and with two disease causing mutations in SLC17A5 gene. SLC17A5 mutation analysis showed p.Tyr306* previously decribed and the novel mutation p.Leu167Pro. This early onset diagnosis allowed us to perform a fast and accurate genetic counseling to the family, helped to better understanding the natural history of this rare disease and probably it could promote cost reduction in future diagnostic tests in the hypothetic case of starting symptoms without diagnosis established. Moreover, an early diagnosis could save family from a long period of time until achieving a definitive diagnostic and to develop an early symptomatic and supportive management of patient to attenuate, as much as possible, disease complications. But, above all, this case illustrates the huge ethical dilemma which arises from any secondary finding (second tier) in newborn screening.