The escape of the mink embryo from obligate diapause

The obligate embryonic diapause that characterizes gestation in mink engenders a developmental arrest at the blastocyst stage. The characteristics of escape from obligate diapause were investigated in embryos reactivated by treatment of the dams with exogenous prolactin. Protein and DNA synthesis showed marked increases within 72 h after the reinitiation of development, and embryo diameter increased thereafter. Trophoblast cells from embryos at Day 5 after activation proliferated more readily in vitro than trophoblasts from diapause or from Day 9 after activation, while in Day 9 embryos, cells from the inner cell mass (ICM) replicated comparatively more readily in vitro. There was evidence of expression of fibroblast growth factor-4 (FGF4) in both diapause and activated embryos and in ICM, but not the trophoblast. FGF receptor-2 was present in embryos from Day 5 after reactivation in both trophoblast and ICM cell lines. Trophoblast cell lines established from mink embryos proliferated in culture in the presence of FGF4 with a doubling time of 1.4 days, while in its absence, the doubling time was 4.0 days. We conclude that, during reinitiation of embryogenesis in the mink after diapause, embryo growth is characterized by gradual increases in protein synthesis, accompanied by mitosis of the trophoblast and ICM. There appears to be a pattern of differential proliferation between cells derived from these embryonic compartments, with the trophoblast phase of replication occurring mainly in the early reactivation phase, while the ICM proliferates more rapidly nearer to the time of implantation.     

Adaptation to hypoosmotic challenge in brachyuran crabs: a microanatomical and electrophysiological characterization of the intestinal epithelia

Besides its role in digestion and nutrient absorption, the crustacean gut participates in osmo/ionic regulation. We investigate microanatomy, ionic permeability and transepithelial electrophysiological parameters in the mid- and hindguts of three hyperosmoregulating crabs that inhabit estuarine waters (Chasmagnathus granulata), brackish mangrove swamp (Sesarma rectum) or freshwater (Dilocarcinus pagei). The abdominal hindguts are cuticle lined, the single-layered epithelia consisting of narrow, columnar cells exhibiting apically dense, unvesiculated cytoplasm. In the saltwater species, the thoracic midgut epithelium consists of tall, narrow, columnar cells displaying numerous, apical microvilli above dense apical cytoplasm. However, the corresponding gut segment in the hololimnetic species, D. pagei, consists of squat cells lacking apical microvilli, overlain by a heavy cuticle, constituting a thoracic or anterior hindgut. The midgut/thoracic hindgut epithelia in all three crabs, and abdominal (posterior) hindgut of D. pagei, exhibit similar, small, lumen-negative voltages when perfused symmetrically with hemolymph-like salines. The hindguts of the saltwater species show similar, small, lumen-positive voltages. Small short-circuit currents are detectable after voltage clamping. Washout and/or addition of luminal glucose or amino acids do not alter current or conductance, suggesting the absence of active, electrogenic nutrient absorption. Ion substitution did not disclose active, electrogenic absorption or secretion of Na+ and/or Cl-. The midguts of the saltwater species exhibit similar conductances, greater than in D. pagei, but no ion selectivity; hindgut conductance is low, the epithelia showing moderate anion selectivity. The thoracic (anterior) and abdominal (posterior) hindgut epithelia of D. pagei, the freshwater species, exhibit similar, low conductances, and are ion selective. These findings reveal that active, electrogenic, salt and nutrient transport is undetectably low or absent. The reduced transepithelial conductances and notable ion selectivities in the abdominal and thoracic hindguts of D. pagei may reduce passive salt losses in fresh water, contributing to osmotic and ionic regulation.     

Synaptophysin: leading actor or walk‐on role in synaptic vesicle exocytosis?

Synaptophysin (Syp) was the first synaptic vesicle (SV) protein to be cloned. Since its discovery in 1985, it has been used by us and by many laboratories around the world as an invaluable marker to study the distribution of synapses in the brain and to uncover the basic features of the life cycle of SVs. Although single gene ablation of Syp does not lead to an overt phenotype, a large body of experimental data both in vitro and in vivo indicate that Syp (alone or in association with homologous proteins) is involved in multiple, important aspects of SV exo-endocytosis, including regulation of SNARE assembly into the fusion core complex, formation of the fusion pore initiating neurotransmitter release, activation of SV endocytosis and SV biogenesis. In this article, we summarise the main results of the studies on Syp carried out by our and other laboratories, and explain why we believe that Syp plays a major role in SV trafficking.     

Laryngeal chemosensory clusters

The expression of molecules involved in the transductory cascade of the sense of taste (TRs, alpha-gustducin, PLCbeta2, IP3R3) has been described in lingual taste buds or in solitary chemoreceptor cells located in different organs. At the laryngeal inlet, immunocytochemical staining at the light and electron microscope levels revealed that alpha-gustducin and PLCbeta2 are mainly localized in chemosensory clusters (CCs), which are multicellular organizations differing from taste buds, being generally composed of two or three chemoreceptor cells. Compared with lingual taste buds, CCs are lower in height and smaller in diameter. In laryngeal CCs, immunocytochemistry using the two antibodies identified a similar cell type which appears rather unlike the alpha-gustducin-immunoreactive (IR) and PLCbeta2-IR cells visible in lingual taste buds. The laryngeal IR cells are shorter than the lingual ones, with poorly developed basal processes and their apical process is shorter and thicker. Some cells show a flask-like shape due to the presence of a large body and the absence of basal processes. CCs lack pores and their delimitation from the surrounding epithelium is poorly evident. The demonstration of the existence of CCs strengthens the hypothesis of a phylogenetic link between gustatory and solitary chemosensory cells.     

Growth and metabolic control during puberty in girls with X-linked hypophosphataemic rickets

OBJECTIVE: X-linked hypophosphataemic rickets (XLH) results in defective bone mineralization and impaired growth. Treatment with oral phosphate (Pi) and calcitriol improves but does not normalize growth. This study assessed whether pubertal growth and metabolic control contribute to the height deficit. METHODS: Study included patients with XLH who were treated with Pi-calcitriol from diagnosis to adult height; their hospital records, biochemistry and radiographs were reviewed. RESULTS: Six females with XLH were included. Their mean peak height velocity and total height gain during puberty were nearly normal despite deteriorating metabolic control. CONCLUSIONS: In treated girls with XLH, the pubertal growth is nearly normal despite suboptimal metabolic control. The major height loss occurs prior to puberty and is not recovered during the pubertal growth spurt.     

Modification of malachite green by Fomes sclerodermeus and reduction of toxicity to Phanerochaete chrysosporium

Malachite green (MG) is a triphenylmethane dye used as a fungicide but also possesses a high toxicity to mammalian cells. The toxicity of MG to Fomes sclerodermeus and Phanerochaete chrysosporium was assessed. P. chrysosporium was highly sensitive to the dye and it was unable to grow on solid media containing 64 microM of MG, lower concentrations caused a delay in growth. The radial growth of F. sclerodermeus was not affected at this concentration and up to 128 microM. In liquid media both fungi were more sensitive. F. sclerodermeus not only was able to grow in the presence of high concentrations of MG, but also it was able to decolorize and detoxify the dye. MG treated with supernatants containing high laccase activity in the presence or absence of 1-hydroxybenzotriazole (1-HBT) gave a colorless product (DMG) that was not toxic to P. chrysosporium and other white rot fungi tested. On the basis of the data of maximal absorbance, it is probable that the mechanism involved in the modification of the dye was different if 1-HBT was added to the reaction.     

Aberrant intracellular cholesterol transport disrupts pituitary and ovarian function

Cholesterol is imported and processed to provide substrate for ovarian steroidogenesis. The Niemann Pick type C-1 gene codes for a glycoprotein that processes low-density lipoproteinimported cholesterol. Mutation of this gene causes marked impairment of export of low-density lipoprotein-derived cholesterol from endosomes, and consequent lysosomal accumulation of the sterol. The BALB/c npc(nih-/-) mouse line, bearing spontaneous mutation of the NPC-1 gene, provides a model for investigation of aberrant endosomal cholesterol transfer in the ovary. Female homozygote mutant mice are infertile, with underdeveloped ovarian follicles, reduced steroidogenesis, no ovulation, and no corpora lutea. Mutant ovaries transplanted under wild-type kidney capsules display both ovulation and formation of corpora lutea. Gonadotropin treatment induces ovulation and restores expression of steroidogenic proteins. Pituitary glands of mutants are hypoplastic, and prolactin expression is dramatically reduced compared with wild-type mice. Both long and short splice variants of the dopamine-D2 receptors are overexpressed in the pituitary of BALB/c npc(nih-/-) mice. Chronic treatment of mutant mice with 17beta-estradiol restores pituitary volume, prolactin expression, and folliculogenetic capability. We conclude that inactivating mutation of Niemann Pick C-1 perturbs the hypothalamic-pituitary-ovarian feedback loop. Reduced estrogens attenuate prolactin expression and alter gonadotropin secretion patterns and interfere with normal ovarian follicular development and ovulation.     

Evaluation of antioxidant effect of different extracts of Myrtus communis L

Oxidative stress is involved in the pathogenesis of numerous diseases. Nevertheless, no optimal natural antioxidant has been found for therapeutics, therefore polyphenol antioxidants have been looked for in myrtle leaves, a plant that in folk medicine has been used as anti-inflammatory drug. Antioxidant-rich fractions were prepared from myrtle (Myrtus communis L.) leaves liquid-liquid extraction (LLE) with different solvents. All myrtle extracts were very rich in polyphenols. In particular, hydroalcoholic extracts contain galloyl-glucosides, ellagitannins, galloyl-quinic acids and flavonol glycosides; ethylacetate extract and aqueous residues after LLE are enriched in flavonol glycosides and hydrolysable tannins (galloyl-glucosides, ellagitannins, galloyl-quinic acids), respectively. Qualitative and quantitative analysis for the single unidentified compound was also performed. Human LDL exposed to copper ions was used to evaluate the antioxidant activity of the myrtle extracts. Addition of these extracts did not affect the basal oxidation of LDL but dose-dependently decreased the oxidation induced by copper ions. Moreover, the myrtle extracts reduce the formation of conjugated dienes. The antioxidant effect of three myrtle extracts decreased in the following order: hydroalcoholic extracts, ethylacetate and aqueous residues after LLE. The extracts had the following IC₅₀: 0.36, 2.27 and 2.88 μM, when the sum of total phenolic compounds was considered after the correction of molecular weight based on pure compounds. Statistical analysis showed a significant difference among hydroalcoholic extracts vs. the ethylacetate and aqueous residues after LLE. These results suggest that the myrtle extracts have a potent antioxidant activity mainly due to the presence of galloyl derivatives.     

Rare genera of actinomycetes as potential producers of new antibiotics

A literature survey covering more than twenty-three thousand bioactive microbial products including eight thousand antiinfectives demonstrated the increasing relevance of the so called 'rare' actinomycetes as a source of new antibiotics. Past and present efforts in the isolation of rare actinomycetes have enriched the Biosearch Italia Strain Collection with more than twenty thousand strains, showing that, when selective isolation methods are developed and extensively applied, some genera, such as Actinomadura, Actinoplanes, Micromonospora, Microtetraspora, are not rare at all and can be recovered from many soil samples. The current focus is on the isolation of members of Streptosporangiaceae family, given their promising chemical diversity.     

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K(m), but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25°C but concealed at 37°C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25°C, but not at 37°C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.