Fouling of gastropods: a role for parasites?

A sample of mud snails Hydrobia ulvae (Prosobranchia) from an intertidal population revealed that the shells of trematode-infected specimens were especially likely to be fouled with epibionts. Experimentally trematode-infected Biomphalaria glabrata (Pulmonata) appeared to be especially prone to develop epigrowth in comparison with uninfected conspecifics as well. These findings suggest an interaction between trematode infections and epibiosis in aquatic gastropods. The two most likely explanations for this are (1) that trematode infections weakens the snails' natural defences against epibionts, or (2) that the defences against epibionts also are effective against invading trematodes, causing snail specimens with a particularly good fouling defence to be less likely to become infected.     

Construction of a BAC Contig for a 3 cM Biologically Significant Region of Mouse Chromosome 1

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.     

Is there a fixed number of niches for endoparasites of fish?

Kennedy and Guégan, based on Cornell and Lawton (J Anim Ecol 1992;61:1–12), found a curvilinear relationship (the fundamental form of the relationship for parasite communities) between infracommunity and component community richness, and interpreted this as meaning that only processes acting within the infracommunities can explain the limitation in the number of parasite species in a given host. The research described here shows that an asymptotic relationship is the consequence of the differential likelihoods of parasite species to appear in an infracommunity as determined by transmission rates and intrinsic lifespans. Processes operating at the infracommunity level are not necessary to explain the curvilinear relationship. Even communities much richer than those found in European freshwater fishes cannot be assumed to be saturated in the sense that further species cannot be added over evolutionary time.     

Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.     

Synthesis of a small library of bivalent α-D-mannopyranosides for lectin cross-linking

A small library of bivalent α-D-mannopyranosides having rigid linkers was constructed in order to evaluate the effects of inter-saccharide distances upon multivalent binding interactions with plant and bacterial lectins. To this end, iodoaryl and propargyl α-D-mannopyranosides were synthesized and the former treated with TMS-acetylene under palladium chemistry to provide their corresponding ethynylaryl derivatives. A library of 15 dimeric members was then obtained using Lewis acid catalyzed glycosidation, aryl-aryl homocoupling, transition metal catalyzed Sonogashira cross-coupling reactions, and oxidative Glaser homocoupling.     

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.     

?-tubulin Paralogs Provide a Qualitative Test for a Phylogeny of Cyst Nematodes

Evolutionary relationships among cyst nematodes based on predicted ß-tubulin amino acid and DNA sequence data were compared with phylogenies inferred from ribosomal DNA (ITS1, 5.8S gene, ITS2). The ß-tubulin amino acid data were highly conserved and not useful for phylogenetic inference at the taxonomic level of genus and species. Phylogenetic trees based on ß-tubulin DNA sequence data were better resolved, but the relationships at lower taxonomic levels could not be inferred with confidence. Sequences from single species often appeared in more than one monophyletic clade, indicating the presence of ß-tubulin paralogs (confirmed by Southern blot analysis). For a subset of taxa, good congruence between the two data sets was revealed by the presence of the same putative ß-tubulin gene paralogs in monophyletic groups on the rDNA tree, corroborating the taxon relationships inferred from ribosomal DNA data.     

Fat stores in a migratory bird: a reservoir of carotenoid pigments for times of need?

Carotenoids are well known for their immune-stimulant function in birds and other vertebrates. Moreover, they have potential antioxidant capacity, scavenging free radicals and protecting cell compartments from oxidation. Most essential carotenoids are fat soluble and could be stored for times of need especially in adipose tissues, built up by migratory birds as the main source of energy on long-distance flights. In an exclusive diet experiment, garden warblers (Sylvia borin) were fed ad libitum with an experimental diet, enriched with two different dose rates of carotenoids, or with control food, during the period of their first autumn migration. Plasma carotenoid content was measured via HPLC and chroma of plasma and fat examined with a spectrophotometer. Birds were infected with Isospora spp. and intensity of infection determined by oocyst counts 3 days post infection. Plasma lutein levels and chroma of subcutaneous fat stores were positively correlated and chroma values of these fat stores increased in the birds that got the higher dose rate, whereas they decreased significantly in the control group after infection with Isospora spp. Chroma of subcutaneous fat deposits in vivo and intensity of Isospora infection were negatively correlated. By measuring the chroma of fat deposits in vivo, we show that fat can be a reservoir for carotenoids. These colorful antioxidants are stored in the fat and taken from there in times of a higher demand, e.g. when mounting an immune response to parasites.     

Is there a basis for novel pharmacotherapy of autism?

No medication has yet been shown to consistently alter the symptoms or the course of autism in the majority of patients. The present pharmacotherapy is mainly palliative and sometimes effective in attenuating specific behaviors. The search for better treatment involves examination of the underlying pathophysiology, the genetic or environmental etiology (including possible iatrogenic causes), and assessment of the clinically-generated evidence of efficacy, including serendipitous or unexplained findings. Subtle neuroanatomic and neurochemical changes are being explored and there are anecdotal reports or limited clinical trials that suggest some therapy might be possible. Secretin is a surprising recent addition to the list of candidates. The pharmacologic mechanism by which these agents might provide such effect is not clear, but hypotheses are beginning to emerge. In addition, the prevention of some uncertain number of autism cases is being investigated by examination of certain vaccinations as putative causative or contributory factors. These topics are reviewed in this article, which has the additional purpose of stimulating novel drug discovery efforts for this enigmatic disorder.     

Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERα-induced proliferation of breast cancer cells

Estrogens, acting through estrogen receptor α (ERα), stimulate breast cancer proliferation, making ERα an attractive drug target. Since 384-well format screens for inhibitors of proliferation can be challenging for some cells, inhibition of luciferase-based reporters is often used as a surrogate end point. To identify novel small-molecule inhibitors of 17β-estradiol (E(2))-ERα-stimulated cell proliferation, we established a cell-based screen for inhibitors of E(2)-ERα induction of an estrogen response element (ERE)(3)-luciferase reporter. Seventy-five "hits" were evaluated in tiered follow-up assays to identify where hits failed to progress and evaluate their effectiveness as inhibitors of E(2)-ERα-induced proliferation of breast cancer cells. Only 8 of 75 hits from the luciferase screen inhibited estrogen-induced proliferation of ERα-positive MCF-7 and T47D cells but not control ERα-negative MDA-MB-231 cells. Although 12% of compounds inhibited E(2)-ERα-stimulated proliferation in only one of the ERα-positive cell lines, 40% of compounds were toxic and inhibited growth of all the cell lines, and ~37% exhibited little or no ability to inhibit E(2)-ERα-stimulated cell proliferation. Representative compounds were evaluated in more detail, and a lead ERα inhibitor was identified.     

Design of new reaction conditions for characterization of a mutant thermophilic α-l-fucosidase

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.     

Consequences of protecting flowers in a fig: a one-way trip for pollinators?

Abstract. Fig trees ( Ficus ) have closed inflorescences. Closure is an efficient protection of flowers against non-specialist predators and harsh external environmental conditions. Each Ficus species is pollinated by a single insect species, an agaonid wasp, capable of forcing its way through a bract-covered pore, the ostiole, to gain access to the flowers. Figs also provide oviposition sites for the wasps. The fig/pollinator interaction is a classic example of mutualism. It has been widely assumed that, once pollinators have entered a fig, oviposited and pollinated, they die trapped within the fig. In this paper, we present observations under natural conditions and results of field experiments on three very different fig species ( Ficus aurea Nutt., F. carica L. and F. microcarpa L.) showing that some pollinators do exit or try to exit from the fig after pollination and oviposition. Moreover, experimental results demonstrate that in at least one species ( F. carica ), the pollinator is able to oviposit successively in two different figs.
The frequency of re-emergences from figs after pollination varies among species and this may be related to variations in pollination dynamics depending on environmental constraints such as the abundance of trees and tree phenology. Several factors that may favour pollinators that leave figs after pollination and oviposition are discussed. They include competition between pollinators for oviposition sites, and minimising of the risk of vertical transmission of parasites and pathogens.     

Cell Surface Engineering of a β-Galactosidase for Galactooligosaccharide Synthesis

A novel gene encoding transglycosylating β-galactosidase (BGase) was cloned from Penicillium expansum F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25°C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.Galactooligosaccharides (GOS) are beneficial for human health as prebiotics that maintain the balance of normal flora in the intestine, enhance lactose tolerance and the digestibility of milk products, reduce serum cholesterol levels, increase Ca²⁺ absorption, synthesize B-complex vitamins, and reduce the risk of cancer (15, 18). Recently, a great deal of attention has been devoted to GOS synthesis, especially via enzymatic transglycosylation, since chemical synthesis of GOS is very tedious (16). GOS can be synthesized by β-galactosidase (BGase) from lactose by glycosyl transfer of one or more galactosyl units onto a galactose moiety of lactose or other structurally related galactosides (10). Both free and immobilized BGases from different microorganisms have been employed for GOS synthesis (12). Based on previous studies, using free enzymes has been associated with limitations, such as low stability and nonreusability of the enzymes. Using immobilized enzymes could overcome these problems, but there are still some drawbacks, including low recovery rates of enzyme activity, the gradual loss of enzyme during the reaction process, finite immobilized carriers, and large mass transfer resistance between some immobilized enzymes and substrates.Recently, an alternative strategy to conventional enzyme immobilization was proposed in which the enzyme is anchored on the cell surfaces of engineered microorganisms, such as Escherichia coli and Saccharomyces cerevisiae (8, 13). S. cerevisiae is a highly advantageous host for cell surface display, as it may allow the accurate folding and glycosylation of recombinant proteins. It is generally regarded as safe in its applications in different fields. Yeast cell surface engineering has been demonstrated using the α-agglutinin receptor of S. cerevisiae to display foreign proteins on the cell surface. There were certain advantages to using cell surface-engineered yeast as an immobilized biocatalyst, e.g., the enzyme was anchored covalently on the cell surface without enzyme loss or additional treatments for immobilization, and the mass transfer resistance between the enzyme and the substrate was sharply reduced in contrast to conventional immobilization methods (17). Several studies have successfully used engineered yeast with immobilized target enzymes as a biocatalyst for a single use, such as the cell surface engineering of a β-glucosidase from Aspergillus oryzae for isoflavone aglycone production and a chitosanase from Paenibacillus fukuinensis for chitooligosaccharide production (7, 19). However, there have been no reports of the use of engineered yeast for consecutive batch production without loss of enzyme activity during the reaction process.The objective of this work was to present a novel approach for GOS synthesis by anchoring BGase from Penicillium expansum F3 on the cell surface of S. cerevisiae as an immobilized enzyme. Figure ​Figure11 shows the main principle of this strategy. The BGase that was cell surface engineered and anchored to yeast (BGase-anchored yeast) could directly utilize lactose for GOS synthesis in batches without loss of enzyme activity. The carbon source (glucose) for cell growth and the inducer (galactose) for enzyme production were the by-products of lactose. The yield of GOS was greatly increased because of the removal of glucose and the continuous expression of BGase. The results showed that this method was especially suitable for GOS synthesis, and it has great promise for industrial oligosaccharide production in the future.Open in a separate windowFIG. 1.Schematic of GOS synthesis by BGase-anchored yeast. The BGase-anchored yeast is represented by modified ovals. The surface BGase converted lactose into GOS, glucose, and a small quantity of galactose. The undesirable glucose was consumed by the yeast for cell growth, and the galactose induced the continuous expression of BGase. Thus, BGase-anchored yeast cells were grown and successively utilized lactose to produce GOS. After a batch reaction, BGase-anchored yeast with higher BGase activity could be harvested and recycled for another batch of GOS synthesis under the same cultivation conditions as the first batch.     

Characteristics of a nickel–albumin binding assay for assessment of myocardial ischaemia

Background: The aim of this study was to describe a method to measure ischaemia-induced alterations of the binding capacity of serum albumin to exogenous nickel.

Methods: We measured the levels of cardiac troponin I (cTnI), serum albumin, ischaemia-modified albumin (IMA) measured by a cobalt–albumin binding assay (CABA), and a nickel–albumin binding assay (NABA) in the following groups: myocardial infarction (n?=?32) and non-ischaemic chest pain (n?=?64).

Results: IMA, cTnI and NABA levels were higher in the myocardial infarction group. NABA presented a higher ability to discriminate myocardial ischaemia than CABA.

Conclusions: Patients with myocardial infarction have reduced nickel binding to human serum albumin, and NABA may have an important role as an early marker of myocardial ischaemia.