Antiproliferative Properties of Papaver rhoeas Ovule Extracts and Derived Fractions Tested on HL60 Leukemia Human Cells.

Papaver rhoeas plant is common in many regions worldwide and contributes to the landscape with its red flower. In the present study we first carried out morphological investigation by optical and scanning electron microscopy of the ovules within the ovary. After ovules' isolation we prepared extracts to test possible cytotoxic activities on HL60 leukemia human cells and investigated the extracts using thin-layer chromatography (TLC) and gas-chromatography/mass spectrometry (GC-MS). P. rhoeas ovules showed an elongated, round shape and the presence of ordered sculptures on the ovule surface. The ovule extracts showed cytotoxic activity on HL60 human cells mainly found in some TLC-isolated spots. Compounds consisting of active spots were identified by GC-MS investigations. Our findings on the P. rhoeas ovule compounds open perspectives for further investigations of TLC-isolated spots on other human cancer cell lines.

Effect of Diet on Adult House Fly (Diptera: Muscidae) Injected With the Salivary Gland Hypertrophy Virus (MdSGHV).

Research to date on the salivary gland hypertrophy virus (SGHV) in three species of flies has focused on adult flies having access to and taking a proteinaceous diet. Since many studies have shown that diet affects viral infection in numerous organisms, this study examined the effect of a protein-free diet on the effect of the SGHV virus in adult house flies, Musca domestica. L. Adults infected with the virus, and maintained on a sugar diet only, showed salivary glands with a blue rather than a grayish color and mild hypertrophy compared with protein-fed flies. It was possible to retrieve the virus from these glands and successfully infect noninfected flies. When injected at various ages, female flies fed only sugar showed that regardless of age, sugar-fed flies still became infected and showed the pathology of the glands. In addition, electron microscope studies revealed at the ultrastructural level that there was no difference between viral replication in cells from salivary glands of adults fed a proteinaceous-free diet and those feeding on protein.

Ultrastructure of the salivary glands of non-infected and infected glands in Glossina pallidipes by the salivary glands hypertrophy virus.

Light, scanning electron, and transmission electron microscopy analyses were conducted to examine the morphology and ultrastructure of the salivary glands of Glossina pallidipes. Three distinct regions, each with a characteristic composition and organization of tissues and cells, were identified: secretory, reabsorptive and proximal. When infected with the salivary gland hypertrophy (SGH) virus, glands showed a severe hypertrophy, accompanied by profound changes in their morphology and ultrastructure. In addition, the muscular fibers surrounding the secretory region of the glands were disrupted. The morphological alterations in the muscular tissue, caused by viral infection, could be an important aspect of the pathology and may shed light on the mode of action of the SGH virus. Results were discussed with regard to the potential effect of viral infection on normal salivation and on the ability of infected tsetse flies to transmit a trypanosome parasite.

Sperm models in European Plecoptera.

Systematic issues regarding Plecoptera are still debated, and the molecular data seem to be unable to definitively clarify the relationships within the order. Spermatozoa are under constant evolutionary pressure, and comparative spermatology can be useful in carrying systematic and phylogenetic information. In the present paper we describe the sperm structure, using light, scanning and transmission electron and immunofluorescence microscopy, of six Euholognatha species belonging to genera not analyzed in our previous studies, i.e. Capnopsis, Amphinemura, Rhabdiopteryx, Tyrrhenoleuctra, Zwicknia and Protonemura. The spermatozoa of all the species examined are fîliform and have a flagellum characterized by an axoneme with 9 + 9+2 pattern and two mitochondrial derivatives. Their ultrastructure shows a degree of heterogeneity within the order. On the contrary, morphological features of sperm are well conserved inside a single Euholognathan family, and the species share a general family sperm model, even if different interspecific or intergeneric characters can be identified and used for systematic inferences. Among Nemouroidea, Taeniopterygidae, showing a peculiar sperm model, seems to have an isolated phylogenetic position. Nemouridae, with a mono-layered acrosome, are isolated among the remaining families, while we can hypothesize a sister taxa relationship between Leuctridae and Capniidae. As regards Perloidea, the sperm characters suggest a closer relationship between Chloroperlidae and Perlodidae, rather than between Perlidae and Perlodidae, as commonly hypothesized.

A morphological and biochemical analysis comparative study of the collagen products Biopad, Promogram, Puracol, and Colactive.

The aim of this study was to compare the capacity of the collagen products Biopad (Euroresearch, Milano, Italy), Promogran (Systagenix Wound Management, Quincy, Massachusetts), Colactive (Smith & Nephew, St Petersburg, Florida), and Puracol (Medline Industries, Mundelein, Illinois) to interact with biological tissues and to start restoring the healing process. These results demonstrate how these products can interact differently with enzymes and cells that characterize the environment of a healing wound.

The secondary metabolite euplotin C induces apoptosis-like death in the marine ciliated protist Euplotes vannus.

The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and terminal transferase dUTP nick end labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganized microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.

Ultrastructural investigation on fibroblast interaction with collagen scaffold.

Collagen-based scaffolds are used as temporary or permanent coverings to help wound healing. Under natural conditions, wound healing is affected by such factors as cell types, growth factors and several components of the extracellular matrix. Due to the complexity of the cell-to-matrix interaction, many cell based mechanisms regulating wound healing in vivo are not yet properly understood. However, the whole process can be partially simulated in vitro to determine how cells interact with the collagen scaffold in relation to such features as physico-chemical properties, matrix architecture and fiber stability. Under these conditions, cell migration into the collagen matrix can be easily assessed and causally correlated with these features. In this study, we aimed at providing a structural analysis of how NIH3T3 fibroblasts migrate and proliferate in vitro when seeded on a native type-I collagen scaffold. To this end, samples were collected at regular time intervals and analyzed by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Through this experimental approach we demonstrate that collagen is gradually frayed into progressively thinner fibrils as fibroblasts migrate into the matrix, embrace the collagen fibers with long filopodia and form large intracellular vacuoles. A key role in this process is also played by microvesicles shed from the fibroblast plasma membrane and spread over long distances inside the collagen matrix. These observations indicate that a native type-I equine collagen provides favorable conditions for simulating collagen processing in vitro and eventually for unraveling the mechanisms controlling cell uptake and intracellular degradation.

Microvesicles shed from fibroblasts act as metalloproteinase carriers in a 3-D collagen matrix.

This study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. By spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (MMP-9) to act upon the collagen fibrils. As a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. As shown by western blots and gelatin zymography, MMP-9 is secreted as a 92 kDa precursor and activated upon release of 82 kDa product into the culture medium. Activation is more efficient under three-dimensional than in two-dimensional culturing conditions. While MMP-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle's integrin ß1 marker is bound to the outer membrane. The intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. Here, we propose that fusion of the intraluminal vesicles with the outer microvesicle's membrane could work as a mechanism controlling the extent to which MMP-9 is first activated and then released extracellularly.

Reproductive biology in Anophelinae mosquitoes (Diptera, Culicidae): Fine structure of the female accessory gland.

The morphology and ultrastructure of female accessory reproductive glands of Anopheles maculipennis s.s., Anopheles labranchiae and Anopheles stephensi were investigated by light and electron microscopy. The reproductive system in these species is characterized by two ovaries, two lateral oviducts, a single spermatheca and a single accessory gland. The gland is globular and has a thin duct which empties into the vagina, near the opening of the spermathecal duct. Significant growth of the accessory reproductive gland is observed immediately after blood meal, but not at subsequent digestion steps. At ultrastructural level, the gland consists of functional glandular units belonging to type 3 ectodermal glands. The secretory cells are elongated and goblet shaped, with most of their cytoplasm and large nucleus in the basal part, close to the basement lamella. Finely fibrous electron-transparent material occupies the secretory cavity that is in contact with the end of a short efferent duct (ductule) emerging from the gland duct. The present study is the first detailed description of female accessory gland ultrastructure in Anophelinae and provides insights into the gland's functional role in the reproductive biology of these insects.

Vitellin cleavage products are proteolytically degraded by ubiquitination in stick insect embryos.

Vitellin polypeptides are proteolytically processed in ovarian follicles and embryos of the stick insect Carausius morosus. Data show that vitellin polypeptide A(3) of 54kDa is processed to yield polypeptide A(3)(*) of about 48kDa upon completion of ovarian development, whereas vitellin polypeptide A(2) of 90kDa yields polypeptide E(9) during embryonic development. As vitellin polypeptides are processed, polypeptides A(3)(*) and E(9) are transferred from the yolk granules to the cytosolic space of the vitellophages and start to express a ubiquitin reactivity. At the confocal microscope, anti-ubiquitin antibodies label specifically numerous small yolk granules and the cytosolic space of vitellophages. During embryonic development, ubiquitin carrying granules undergo acidification in much the same way as larger yolk granules. However, only these latter organelles are capable of converting a latent cysteine pro-protease into an active yolk protease upon acidification of their luminal space. These data are interpreted as indicating that ubiquitin-like polypeptides are restricted to small granules throughout ovarian and embryonic development, and that vitellin cleavage products are ubiquitinated following acidification of large yolk granules and transfer to the cytosolic space of the vitellophages.

In vitro pollen tube growth reveals the cytotoxic potential of the flavonols, quercetin and rutin.

Flavonols are phytochemicals widely found in commonly consumed foods. In spite of their beneficial effects on human health, however, cytotoxicity and even suspected genotoxicity have also been reported for the flavonol, quercetin. This points to the need for preventive studies to identify any cytotoxic effects associated with pure flavonol intake. This work was performed with the aim of verifying whether a plant-based in vitro system, the pollen tube, could be used to evaluate the cytotoxic potential of exogenous flavonols. Increasing concentrations of the aglycone, quercetin, and its glycoside, rutin, were assayed with regard to tube growth of kiwifruit pollen, determined by applying the pollen tube growth test protocol. This test, based on the photometric quantification of pollen tube mass production in suspension cultures, has already been applied in the sensitive and reliable toxicological evaluation of a wide range of chemicals. Whereas 60-800 microM rutin promoted kiwifruit pollen tube elongation, 10-50 microM quercetin strongly inhibited growth, and also produced irreversible malformations, such as screw-like tube growth, abnormal vacuolation, alteration of organelle streaming, and nuclear positioning. Thus, the cytotoxic potentials of the two flavonols have been confirmed to differ. Pollen tubes seem to afford a promising test system for a preventive, rapid in vitro biosafety assessment of antioxidant nutritional supplements, without using laboratory animals.

Targeted amino acid substitutions impair streptolysin O toxicity and group A Streptococcus virulence.

UNLABELLED: Streptolysin O is a potent pore-forming toxin produced by group A Streptococcus. The aims of the present study were to dissect the relative contributions of different structural domains of the protein to hemolytic activity, to obtain a detoxified form of streptolysin O amenable to human vaccine formulation, and to investigate the role of streptolysin O-specific antibodies in protection against group A Streptococcus infection. On the basis of in silico structural predictions, we introduced two amino acid substitutions, one in the proline-rich domain 1 and the other in the conserved undecapeptide loop in domain 4. The resulting streptolysin O derivative showed no toxicity, was highly impaired in binding to eukaryotic cells, and was unable to form organized oligomeric structures on the cell surface. However, it was fully capable of conferring consistent protection in a murine model of group A Streptococcus infection. When we engineered a streptococcal strain to express the double-mutated streptolysin O, a drastic reduction in virulence as well as a diminished capacity to kill immune cells recruited at the infection site was observed. Furthermore, when mice immunized with the toxoid were challenged with the wild-type and mutant strains, protection only against the wild-type strain, not against the strain expressing the double-mutated streptolysin O, was obtained. We conclude that protection occurs by antibody-mediated neutralization of active toxin. IMPORTANCE: We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.

The diverticulated crop of adult Phormia regina.

The crop of adult Phormia regina consists of a duct that diverges from the esophagus, just in front of the cardia, and extends ventrally and posteriorly into the thorax and abdomen where it forms a bilobed sac. Flattened epithelial cells produce the cuticular lining of the crop. When empty, or partially full, the epithelial cells and cuticular lining form folds extending into the lumen, thus providing for expansion as the crop sac fills. Covering the sac on the hemolymph side is a layer of anastomosed, intrinsic muscles connected to one another by intercellular cytoplasmic bridges. Mitochondria are located at the periphery of the sarcomere. Also inside the sarcomere are glycogen, sarcoplasmic reticula, and transverse tubular systems (T-system). I, A, and Z-bands are present and the Z-bands are not in register making the muscle-type supercontractile. Important structures, not previously researched and associated with the crop muscles, are the crop nerves. Coming off the corpora cardiaca, and running down each side of the crop duct, is a pair of nerves, each housing several axons. These nerves extend to and branch over the crop sac. Here they penetrate the muscle mass and form neuromuscular junctions where electron-dense droplets of neurosecretion are released. Based on the literature, and research in our laboratory, it has now been shown that these nerves carry adipokinetic hormone, Drosophila insulin-like peptide, and a dromyosuppressin-like neuropeptide.

Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilus-negative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three-dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild-type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GAS-mediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.

Altered morphology of rod bipolar cell axonal terminals in the retinas of mice carrying genetic deletion of somatostatin subtype receptor 1 or 2.

Somatostatin (SRIF), similar to other neuropeptides, is likely to influence the morpho-functional characteristics of neurons. We studied possible morphological alterations of mouse retinal neurons following genetic deletion of SRIF subtype receptor 1 [sst1 knockout (KO)] or 2 (sst2 KO). In sst1 KO retinas, axonal terminals of rod bipolar cells (RBCs), identified with protein kinase C immunoreactivity, were 25% larger than in controls. In contrast, in sst2 KO retinas, RBC axonal terminals were significantly smaller (-14%). No major ultrastructural differences were observed between control and KO RBCs. In sst2 KO retinas, SRIF levels decreased by about 35%, while both sst1 receptor mRNA and protein increased by about 170% and 100%, respectively. This compares to previous results reporting an increase of both retinal SRIF and sst2 receptors following sst1 receptor deletion. Together, these findings suggest that, on the one hand, sst1 receptor deletion induces over-expression of sst2 receptors, and vice versa; on the other hand, that an imbalance in sst1 and sst2 receptor expression and/or changes in the levels of retinal SRIF induced by sst1 or sst2 receptor deletion are responsible for the morphological changes in RBC axonal terminals. Similar alterations of RBC terminals were observed in KO retinas at 2 weeks of age (eye opening). In addition, reverse transcription-polymerase chain reaction analysis of the expression of sst2 and sst1 receptors in developing sst1 and sst2 KO retinas, respectively, demonstrated that these receptors are up-regulated at or near eye opening. These findings suggest that the integrity of the somatostatinergic system during development is necessary for proper RBC maturation.