The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34(cdc2) levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9.     

Metabolic Basis for the Self-Referential Genetic Code

An investigation of the biosynthesis pathways producing glycine and serine was necessary to clarify an apparent inconsistency between the self-referential model (SRM) for the formation of the genetic code and the model of coevolution of encodings and of amino acid biosynthesis routes. According to the SRM proposal, glycine was the first amino acid encoded, followed by serine. The coevolution model does not state precisely which the first encodings were, only presenting a list of about ten early assignments including the derivation of glycine from serine—this being derived from the glycolysis intermediate glycerate, which reverses the order proposed by the self-referential model. Our search identified the glycine-serine pathway of syntheses based on one-carbon sources, involving activities of the glycine decarboxylase complex and its associated serine hydroxymethyltransferase, which is consistent with the order proposed by the self-referential model and supports its rationale for the origin of the genetic code: protein synthesis was developed inside an early metabolic system, serving the function of a sink of amino acids; the first peptides were glycine-rich and fit for the function of building the early ribonucleoproteins; glycine consumption in proteins drove the fixation of the glycine-serine pathway.     

Speciation of the Paradeontacylix spp. (Sanguinicolidae) of Seriola dumerili. Two new species of the genus Paradeontacylix from the Mediterranean

Two new species of teleost blood fluke belonging to the sanguinicolid genus Paradeontacylix are described from the greater amberjack, Seriola dumerili, i.e. Paradeontacylix ibericus n. sp. from the Iberian Peninsula and Paradeontacylix balearicus n. sp. from the Balearic Islands. P. ibericus n. sp. and P. balearicus n. sp. show morphological similarities with Paradeontacylix kampachi and Paradeontacylix grandispinus respectively, which occur in mixed infection in S. dumerili from Japan. Multivariate analysis of morphometrical data provided statistical evidence for the separation of four species. However, component by component analysis did not show statistically significant differences between P. balearicus and P. grandispinus. Molecular data based on rITS2 and mCO1 gene sequences also supported the separation into four species. Morphological and molecular data were used to examine phylogenetic relationships between Paradeontacylix species from S. dumerili and other species in the genus. The results coincided in revealing two main branches with P. kampachi+P. ibericus and (((P. grandispinus+P. balearicus) Paradeontacylix sanguinicoloides) Paradeontacylix godfreyi). Paradeontacylix odhneri, for which little data are available, was located basal in a separate branch. This is the only species of Paradeontacylix which parasitizes a non-carangid host which might probably explain the separation from the other species. Paired similarities between the Japanese and the Mediterranean species, despite the large geographic distance, could be explained by the speciation of parasite geminate lines before host separation by tectonic events. Consequently, geographic and historical isolation support the morphological and genetic differences leading to the evolution of the new species described here.     

Effect of coffee alkaloids and phenolics on egg-laying by the coffee leaf miner Leucoptera coffeella

The recognized importance of coffee alkaloids and phenolics mediating insect-plant interactions led to the present investigation aiming to test the hypothesis that the phenolics chlorogenic and caffeic acids and the alkaloid caffeine and some of its derivatives present in coffee leaves affect egg-laying by the coffee leaf miner Leucoptera (=Perileucoptera) coffeella (Guérin-Méneville & Perrottet) (Lepidoptera: Lyonetiidae), one of the main coffee pests in the Neotropical region. These phytochemicals were, therefore, quantified in leaves from 12 coffee genotypes and their effect on the egg-laying preference by the coffee leaf miner was assessed. Canonical variate analysis and partial canonical correlation provided evidence that increased leaf levels of caffeine favour egg-laying by the coffee leaf miner. An egg-laying preference bioassay was, therefore, carried out to specifically test this hypothesis using increasing caffeine concentrations sprayed on leaves of one of the coffee genotypes with the lowest level of this compound (i.e. Hybrid UFV 557-04 generated from a cross between Coffea racemosa Lour. and C. arabica L.). The results obtained allowed the recognition of a significant concentration-response relationship, providing support for the hypothesis that caffeine stimulates egg-laying by the coffee leaf miner in coffee leaves.     

Guanylin peptide family: history, interactions with ANP, and new pharmacological perspectives

The guanylin family of peptides has 3 subclasses of peptides containing either 3 intramolecular disulfide bonds found in bacterial heat-stable enterotoxins (ST), or 2 disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity. These hormones are synthesized in the intestine and released both luminally and into the circulation, and are also produced within the kidney. Stimulation of renal target cells by guanylin peptides in vivo or ex vivo elicits a long-lived diuresis, natriuresis, and kaliuresis by both cGMP-dependent and independent mechanisms. Uroguanylin may act as a hormone in a novel endocrine axis linking the digestive system and kidney as well as a paracrine system intrarenally to increase sodium excretion in the postprandial period. This highly integrated and redundant mechanism allows the organism to maintain sodium balance by eliminating excess sodium in the urine. In addition, small concentrations of the atrial natriuretic peptide (ANP) can synergize with low concentrations of both guanylin or uroguanylin, which do not induce natriuresis per se, to promote significant natriuresis. Interestingly, the activation of the particulate guanylate cyclase receptors by natriuretic peptides can promote relaxation of animal and human penile erectile tissue and increase intracavernosal pressure to induce penile erection. These peptides can be prototypes for new drugs to treat erectile dysfunction, especially in patients with endothelial and nitrergic dysfunction, such as in diabetes.     

Biology of the first generation of a laboratory colony of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae)

The phlebotomine sand flies Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) are very close and may be involved in the transmission of Leishmania spp. Ross, 1903 in Brazil. The biology of the first laboratory-reared generations of these species, descended from insects captured in Além Paraíba (N. intermedia) and Corinto (N. neivai) in the Brazilian state of Minas Gerais, is described here. The captured females were fed on hamsters and maintained individually in rearing pots. Laboratory temperature and relative humidity were maintained at 25-26 masculineC and 80% respectively. The productivity of the first generation of N. intermedia was greater than that of N. neivai, and its development time clearly shorter, particularly for the second and third larval instars.     

Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains epsilon-(gamma-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under sigma(K) control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.     

The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of <Emphasis Type="Italic">npt</Emphasis>II-transformed leaf tissues

The effect of different concentrations of the aminoglycoside antibiotics, geneticin, paromomycin and streptomycin on adventitious regeneration from leaf explants of apricot was tested to design an alternative procedure for selecting transgenic shoots. Streptomycin and paromomycin reduced shoot regeneration percentage with increasing concentration of antibiotics. Almost a complete inhibition of regeneration was reached when 20M paromomycin was used, although up to 40M streptomycin was necessary to completely inhibit regeneration. Geneticin had a very toxic effect on apricot leaves and regeneration was inhibited at almost all concentrations tested. Addition of kanamycin hastened the development of adventitious buds although silver thiosulfate and not kanamycin was responsible for the observed increase in the consistency of the results from independent experiments. Kanamycin and paromomycin at the concentrations tested improved selection of transformed cells and resulted in a larger number of gfp-expressing regions. Paromomycin at 40 and 25.7M kanamycin improved proliferation of transformed tissues as compared with the other antibiotics used and non-selected controls.     

Evaluation of the membrane lipid selectivity of the pea defensin Psd1

Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent.