Deep-water shrimp (Pandalus borealis, Krøyer 1838) as indicator organism for fish-farm waste

A feeding experiment was carried out with deep-water shrimp (Pandalus borealis), a common benthic inhabitant of Norwegian fjords. Shrimp were reared in tanks for three months, fed either salmon feed or cod, and the fatty acid profile of their muscle tissue was monitored. The salmon feed pellets and cod had completely different fatty acid compositions, with significant differences in most of the analysed fatty acids, eight of them, 14:0, iso16:0, 16:1n7, 16:2n11, 18:3n3, 20:0, 22:0 and 22:1n11, differing between the two diets by a factor of 10 or more. The levels of the fatty acids in the diets differed also substantially from the levels in the shrimp tissue at the start of the experiment. The shrimp were fed ad libitum and both groups increased significantly in total fatty acid content in the course of the experiment. Nevertheless, the fatty acids made up no more than 6.5 mg g^− 1 muscle tissue, with 88 ± 1% of them bound in polar, membrane lipids. Their composition in the tissue did also change to a certain extent, but the resulting tissue composition did not reflect the fatty acid composition of the diets. Only two fatty acids, 18:2n6 and 18:3n3, clearly followed the dietary level. They are abundant in commercial salmon feed since they are rich in plant oils used as additives to the commercial feed. Determination of fatty acid levels in shrimp tissue may enable shrimp to be used as indicator organisms for the influence of organic fish-farm waste on the food web in adjacent areas.     

Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Background

The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized.

Methodology/Principal Findings

We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t_1/2<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited.

Conclusion

It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.     

Photochemical treatment with endosomally localized photosensitizers enhances the number of adenoviruses in the nucleus

BACKGROUND: In the present study the physical targeting technique photochemical internalization (PCI) has been used in combination with adenovirus. We have previously shown that PCI enhances transgene expression from AdhCMV-lacZ, and the aim of the present study was to further increase the understanding of photochemically mediated adenoviral transduction. METHODS: Two colorectal carcinoma cell lines, WiDr and HCT116, were pre-incubated with the photosensitizer TPPS(2a) or methylene blue derivates (MBD) followed by infection with adenovirus and light exposure. Transgene expression was measured by flow cytometry. Real-time polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) were used to quantify the level of viral DNA in the nuclei. Real-time PCR was also used to measure the level of beta-galactosidase mRNA in samples infected with AdhCMV-lacZ. RESULTS: Exposing TPPS(2a)-treated cells to light enhanced the quantity of viral DNA in the nucleus, the mRNA level of the transgene and the transgene expression compared to non-illuminated cells. The increased transgene expression was independent of the promoter used, but dependent on the time of light exposure and the cellular localization of the photosensitizer. CONCLUSIONS: The enhanced transgene expression observed after photochemical treatment is most likely not a result of one event, but more an interplay between various mechanisms. An increased level of adenoviral DNA in the nucleus and a dependency of endosomal localization of the photosensitizer to obtain enhanced transgene expression suggested that endosomal rupture facilitated the transport of adenoviruses to the nucleus.     

Regulation of cell growth and the expression of extracellular matrix proteins in colorectal adenocarcinoma: a fibroblast-tumor cell coculture model to study tumor-host interactions in vitro

The production of abundant connective tissue within malignant tumors, the so-called desmoplastic stromal reaction, is a hallmark of colorectal adenocarcinomas. This stroma is produced to a large extent by myofibroblasts and contains various amounts of collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. In this study we have established a monolayer coculture model between two different colorectal adenocarcinoma cell lines (HRT-18, and CX-2) and colonic fibroblasts (CCD-18) to investigate the mechanisms regulating (i) the production of extracellular matrix (ECM) components, (ii) the induction of myofibroblastic differentiation, and (iii) cellular proliferation. We found that TGFbeta1 and FGF-2 stimulated ECM synthesis of fibroblasts. Myofibroblastic differentiation was stimulated by TGFbeta1 but suppressed by FGF-2. There was a mutual stimulation of proliferation between fibroblasts and carcinoma cells. The analogies with ECM components expressed in cocultures and colorectal adenocarcinoma samples suggest that the coculture model used in this study is useful to study tumor cell-fibroblast interactions.     

Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell-fates in Xenopus

We analysed the role of Notch signalling during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway we found that Notch expands the floor plate domain of sonic hedgehog and pintallavis and represses the notochordal markers chordin and brachyury, with a concomitant reduction of the notochord size. We propose that within a population of the early organiser with equivalent potential to develop either as notochord or floor plate, Notch activation favours floor plate development at the expense of the notochord, preferentially before mid gastrula. We present evidence that sonic hedgehog down-regulates chordin, suggesting that secreted Sonic hedgehog may be involved or reinforcing the cell-fate switch executed by Notch. We also show that Notch signalling requires Presenilin to modulate this switch.     

Species-specific monoclonal antibodies in the assignment of the gene for human fibronectin to chromosome 2.

Using three different species-specific monoclonal antibodies we have studied, in human-mouse and human-hamster somatic cell hybrids, the correlation between the presence of different human chromosomes and the ability to release human fibronectin into the tissue culture medium. Presence of human fibronectin was determined by an affinity-radioimmunoassay. In addition, tissue culture media of the different hybrids were separated on SDS-polyacrylamide gels, the proteins were blotted onto a nitrocellulose sheet and human fibronectin visualized by an immunoenzymatic technique. Karyology and determination of isoenzyme markers of specific human chromosomes show that the ability to produce human fibronectin segregated with the presence of human chromosome 2.     

Natural Infections Caused by the Fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> as a Pathogen of <Emphasis Type="Italic">Musca domestica</Emphasis>in the Neotropic

A survey for entomopathogenic fungi of Musca domestica adults was conducted in poultry houses in La Plata, Buenos Aires province, Argentina, during the years 2002 and 2003. Adult house flies were found infected with the fungus Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) from field collections, with a natural infected prevalence between 0.4–1.45%. This is the first record of natural infections of house flies caused by B. bassiana for the neotropics. Pathogenicity assays under laboratory conditions showed 94% adult mortality at 14 days post challenge. CIC fellow     

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.     

Recent Acquisition of Helicobacter pylori by Baka Pygmies

Both anatomically modern humans and the gastric pathogen Helicobacter pylori originated in Africa, and both species have been associated for at least 100,000 years. Seven geographically distinct H. pylori populations exist, three of which are indigenous to Africa: hpAfrica1, hpAfrica2, and hpNEAfrica. The oldest and most divergent population, hpAfrica2, evolved within San hunter-gatherers, who represent one of the deepest branches of the human population tree. Anticipating the presence of ancient H. pylori lineages within all hunter-gatherer populations, we investigated the prevalence and population structure of H. pylori within Baka Pygmies in Cameroon. Gastric biopsies were obtained by esophagogastroduodenoscopy from 77 Baka from two geographically separated populations, and from 101 non-Baka individuals from neighboring agriculturalist populations, and subsequently cultured for H. pylori. Unexpectedly, Baka Pygmies showed a significantly lower H. pylori infection rate (20.8%) than non-Baka (80.2%). We generated multilocus haplotypes for each H. pylori isolate by DNA sequencing, but were not able to identify Baka-specific lineages, and most isolates in our sample were assigned to hpNEAfrica or hpAfrica1. The population hpNEAfrica, a marker for the expansion of the Nilo-Saharan language family, was divided into East African and Central West African subpopulations. Similarly, a new hpAfrica1 subpopulation, identified mainly among Cameroonians, supports eastern and western expansions of Bantu languages. An age-structured transmission model shows that the low H. pylori prevalence among Baka Pygmies is achievable within the timeframe of a few hundred years and suggests that demographic factors such as small population size and unusually low life expectancy can lead to the eradication of H. pylori from individual human populations. The Baka were thus either H. pylori-free or lost their ancient lineages during past demographic fluctuations. Using coalescent simulations and phylogenetic inference, we show that Baka almost certainly acquired their extant H. pylori through secondary contact with their agriculturalist neighbors.     

Myofibroblast expression in airways and alveoli is affected by smoking and COPD

Background

Chronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD.

Methods

α-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistochemical findings were correlated with clinical data. α-SMA protein was also analysed by Western blotting from fibroblastic cells cultured from peripheral lung of non-smokers, smokers without COPD and smokers with COPD.

Results

In many cases, the endings of the detached alveolar walls were widened, the structures of which were named as widened alveolar tips. Widened alveolar tips contained α-SMA positive cells, which were obviously myofibroblasts. There were less alveolar tips containing positive cells for α-SMA in alveoli and α-SMA positive cells in bronchioles in smokers and in COPD compared to non-smokers. The quantity of α-SMA positive cells was increased in bronchi in COPD. Tn-C was elevated in bronchi in COPD and smokers’ lung. The α-SMA protein level was 1.43-fold higher in stromal cells cultured from non-smokers than in those of smokers.

Conclusions

Myofibroblasts are localized variably in normal and diseased lung. This indicates that they have roles in both regeneration of lung and pathogenesis of COPD. The widened alveolar tips, these newly characterized histological structures, seemed to be the source of myofibroblasts at the alveolar level.