The persistence of Legionella pneumophila in non-sterile, sterile and artificial hard waters and their growth pattern on tap washer fittings

Simultaneous experiments were performed with sterilized and non-sterile water and an artificial hard water. After seeding with an environmental isolate of Legionella pneumophila numbers in the sterile land hard water decreased rapidly and colonization of various tap washer fittings failed to take place. Adhesion and growth of an environmental isolate of L. pneumophila to washers in non-sterile tap water was followed over a 4-month period with fluorescein-labelled antibody and by scanning electron microscopy. After adherence the individual cells appeared to divide to form chains which spread over the surfaces. Organisms other than legionellas were also present and a complex colonization matt was formed which was embedded in a protective coat of slime and debris. The numbers of L. pneumophila recovered from the water were highest between 4 and 7 weeks but they could still be cultivated after 4 months.     

Pregnancy modulates the expression of beta-N-acetylhexosaminidase in rat serum and tissues.

1. beta-N-Acetylhexosaminidases in maternal rat serum were separated by DEAE-cellulose chromatography and compared with those of adult rat serum. 2. In pregnant serum there is an increase of the isoenzymes which are entirely composed of beta-subunits (B and intermediate forms). 3. These alterations could be compared to those already described in human pregnancy. 4. The levels of beta-N-acetylhexosaminidase and the relative expression of alpha- and beta-subunits in normal and pregnant serum correlate with the above isoenzyme expression. 5. The increase of B and intermediate forms as well as the increase of specific activity during pregnancy was not peculiar to maternal serum but was also demonstrated in several foetal tissues and in maternal tissues, in which cases the beta-N-acetylhexosaminidase isoenzyme patterns closely resemble the foetal ones rather than those of the adult rat tissues. 6. These analogies strongly suggest that the expression of beta-subunit of beta-N-acetylhexosaminidase is regulated by hormones or other factors related to pregnancy.     

Imazalil-cyclomaltoheptaose (beta-cyclodextrin) inclusion complex: preparation by supercritical carbon dioxide and 13C CPMAS and 1H NMR characterization

An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H).     

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.     

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Serosa membrane plays a key role in transferring vitellin polypeptides to the perivitelline fluid in insect embryos

In mid-embryogenesis, the stick insect Carausius morosus comes to be comprised of three distinct districts: the embryo proper, the yolk sac and the perivitelline fluid. A monolayered epithelium, the so-called serosa membrane, encloses the yolk sac and its content of vitellophages and large yolk granules. During embryonic development, the yolk sac declines gradually in protein concentration due to Vt polypeptides undergoing limited proteolysis to yield a number of Vt cleavage products of lower molecular weights. mAbs 1D1 and 5H11 are monoclonal antibodies raised against some of the Vt cleavage products generated by this process in the yolk sac. At the confocal microscope, antibody fluorescence is initially associated with a few yolk granules, while it is gradually displaced in the cytosolic spaces of the vitellophages. With the proceeding of embryonic development, label appears also in the serosa membrane in the form of clustered dots. At the ultrastructural level, gold particles are initially associated with the vitellophages that are labeled on a few yolk granules and in the cytosolic space flanking the yolk granules. Subsequently, the serosa cells become labeled on vesicles close to the yolk granules or just underneath the plasma membrane. Inside the serosa cells, label is also associated with granules budding from the Golgi apparatus, but never with the intercellular channels percolating the serosa membrane. These observations are interpreted as indicating that Vt cleavage products leak out from the yolk granules into the cytosolic spaces of the vitellophages and are eventually transferred to the perivitelline fluid via transcytosis through the serosa cells.     

Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin A and ammonium chloride administration.

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14-day-old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.