Use of freeze-cracking in ontogenetic research in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Macrostomida,Rhabditophora)

A method for studying whole mount flatworm embryos based on freeze-cracking of the eggs is described. This method allows successful immunohistological and immunocytological studies of whole mount embryos. It does not require the use of sharpened needles or a microinjection system to puncture the eggshell. Moreover, this method is more practical and less time-consuming than classical puncturing and much cheaper than the use of a microinjection system. The advantages of this method are illustrated by results of several immunolocalisation experiments in the macrostomid flatworm Macrostomum lignano. The optimal procedure and crucial steps for this method are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low species diversity and high interindividual variability in faeces of preterm infants as revealed by sequences of 16S rRNA genes and PCR-temporal temperature gradient gel electrophoresis profiles

Little information regarding the composition of the gut microbiota in preterm infants is available. The purpose of this study was to investigate the bacterial diversity in faeces of preterm infants, using analysis of randomly cloned 16S rRNA genes and PCR-TTGE (temporal temperature gradient gel electrophoresis) profiles, to determine whether noncultivated bacteria represented an important part of the community. The 288 clones obtained from faecal samples of 16 preterm infants were classified into 25 molecular species. All but one molecular species had a cultivated representative in public databases: molecular tools did not reveal any unexplored diversity. The mean number of molecular species per infant was 3.25, ranging from one to eight. There was a high interindividual variability. The main groups encountered were the Enterobacteriaceae family and the genera Enterococcus, Streptococcus and Staphylococcus. Seven preterm infants were colonized by anaerobes and only four by bifidobacteria. TTGE profiles were composed of one to nine bands (mean value: 4.3). Furthermore, 51 of 59 clones (86%) comigrated with a band of the corresponding faecal sample. This study will form a comparative framework for other studies, e.g. on the faecal microbiota of preterm infants with different pathologies or the impact of diet on colonization.     

Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus <Emphasis Type="Italic">Mannheimia</Emphasis>

Background  

The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data.     

Serglycin constitutively secreted by myeloma plasma cells is a potent inhibitor of bone mineralization in vitro

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n=2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.     

Synthesis and biological evaluation of new heterocyclic quinolinones as anti-parasite and anti-HIV drug candidates

We have synthesized quinolinones with potential antiparasitic and anti-HIV activities by an original two-step method involving microwave irradiation and have evaluated their activities against Plasmodium falciparum, Leishmania donovani, Trichomonas vaginalis, and HIV. None of the tested compounds had been previously described using this method of synthesis. One of the compounds had interesting antiparasitic and anti-HIV activity, which could be improved by substitution with different radicals.     

Autoradiographic study on the localization of estradiol-17β in neonatal mouse uterus and cervix

Summary The uptake of ³H-estradiol-17 in the neonatal mouse uterus and cervix has been studied by an autoradiographic method. When the radio-active hormone is administered in vivo and in vitro, grains are found to be concentrated above the nuclei both in the uterine and cervical epithelium and stroma. Grain counts revealed that the nuclear concentration of grains is higher at 4 h than at 2 h after isotope injection. The cervical epithelium has a higher nuclear concentration than the uterine epithelium both in vivo and in vitro. In the stroma, this situation is reversed except after in vitro treatment of the tissues.In the cervix, more of the hormone seems to be located within the nucleus while in the uterus a higher proportion of the grains are found in the vicinity of the nuclear periphery.Although the nuclear concentration of grains is higher at 4 h than at 2 h, the number of grains above the sections is lower at 4 h. Both in vivo and in vitro, the number of grains is higher above the stromal than above the epithelial compartments of the uterus and cervix.Five days old animals showed the same labeling pattern. The differences in uptake and distribution of ³H-estradiol are discussed in relation to other known differences in the hormone responsiveness in these tissues.We are greatly indebted to Professor W.E. Stumpf and the Laboratories for Reproductive Biology, University of North Carolina Medical School for the opportunity to study the method of dry mount autoradiography. The work has been supported by the Norwegian Research Council for Science and the Humanities and by the Norwegian Cancer Society (Landsforeningen mot Kreft)     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor

The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.     

Stage- and weather-dependent dispersal in the brown garden snail Cornu aspersum

Dispersal decisions are often condition-dependent, influenced by the interaction of individual phenotype and environmental conditions. Terrestrial Gastropods are simultaneous hermaphrodites, a reproductive system rarely studied in the context of dispersal. Moreover, the energetic cost of their movement is one of the highest among animals. Despite these features, which make them valuable models to understand the trade-offs between dispersal and other life-history traits, their dispersal strategies have been barely explored. We studied the movements of subadults and adults of the brown garden snail Cornu aspersum in a semi-natural 4-patch network, for 2 months in 2011 (a dry year) and 1 month in 2012 (a wet year). We assessed the effects of life-history stage (subadult/adult) and weather conditions on dispersal propensity and dispersal speed. Snails were more mobile under humid and warm weather, but nearly all individuals left patches when the relative humidity was close to 100 % in 2012. Because such humidity levels are potentially lethal to C. aspersum, we argue these extreme emigration rates might be an emergency escape response to harmful conditions. Despite a theoretically higher cost of movement, we found that subadults emigrated more, and dispersed faster and further, than adults. Thus, and contrary to what was expected, direct costs of movement do not play the main role in shaping dispersal in C. aspersum. Observed differences between subadults and adults in dispersal behaviour are discussed in the context of intraspecific competition, inbreeding avoidance and relative costs of male and female reproduction.