Molecular genetic differentiation in earthworms inhabiting a heterogeneous Pb-polluted landscape

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages ('A' and 'B'), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central 'ecological island' site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies.

Differentiation status of limbal epithelial cells cultured on intact and denuded amniotic membrane before and after air-lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under ‘air-lifting’ conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting and quantitative PCR. Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane. On denuded amniotic membrane the proportion of differentiated cells remained unaltered following airlifting. Within cells grown on intact amniotic membrane, however, the number of differentiated cells increased significantly following air-lifting. These results have important implications for both basic and clinical research. Firstly, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Secondly, these findings serve as a warning to clinicians that the affect of amniotic membrane on transplantable cells is not fully understood; the use of intact or denuded amniotic membrane can produce different results in terms of the amount of differentiation, once cells are exposed to the air.

CD27 mediates interleukin-2-independent clonal expansion of the CD8+ T cell without effector differentiation

The clonal expansion of antigen-specific CD8+ T cells in response to microbial infections is essential for adaptive immunity. Although IL-2 has been considered to be primarily responsible for this process, quantitatively normal expansion occurs in the absence of IL-2 receptor signaling. Here, we show that ligating CD27 on CD8+ T cells that have been stimulated through the T cell receptor causes their expansion in the absence of IL-2 by mediating two distinct cellular processes: enhancing cell cycling and promoting cell survival by maintaining the expression of IL-7 receptor alpha. This pathway for clonal expansion of the CD8+ T cell is not associated with the development of a capacity either for production of IFN-gamma or for cytotoxic T lymphocyte function and, therefore, is uncoupled from differentiation. Furthermore, ligating CD27 increases the threshold concentration at which IL-2 induces IFN-gamma-producing capability by the CD8+ T cell, suggesting that CD27 signaling may suppress effector differentiation. Finally, CD8+ T cells that have been stimulated by the TCR/CD27 pathway maintain their capacity for subsequent expansion and effector differentiation in response to a viral challenge in vivo. Thus, the TCR/CD27 pathway enables the CD8+ T cell to replicate by a process of self-renewal, which may contribute to the continuous generation of new effector CD8+ T cells in persistent viral infections.

Glucosinolate-accumulating S-cells in Arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation

Plant secondary metabolites glucosinolates (GSL) have important functions in plant resistance to herbivores and pathogens. We identified all major GSL that are accumulated in S-cells in Arabidopsis by MALDI-TOF MS, and estimated by LC-MS that the total GSL concentration in these cells is above 130 mM. The precise locations of the S-cells outside phloem bundles in rosette and cauline leaves and in flower stalks were visualised using sulphur mapping by cryo-SEM/EDX. S-cells contain up to 40% of total sulphur in flower stalk tissues. S-cells in emerging flower stalks and developing leaf tissues show typical signs of Programmed Cell Death (PCD) or apoptosis, such as chromatin condensation in the nucleus and blebbing of the membranes. TUNEL staining for DNA double strand breaks confirmed PCD in S-cells in postmeristematic tissues in the flower stalk as well as in the leaf. Our results show that S-cells in postmeristematic tissues proceed to an extreme degree of metabolic specialisation besides PCD. Accumulation and maintenance of a high concentration of GSL in these cells are accompanied by degradation of a number of cell organelles. The substantial changes in the cell composition during S-cell differentiation indicate the importance of this particular GSL-based phloem defence system. The specific anatomy of the S-cells and ability to accumulate specialised secondary metabolites is similar to that of the non-articulated laticifer cells in latex plants and thus indicates a common evolutionary origin.

Variation in the ovarian reserve Is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

Objectives: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. Methods: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We. determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. Results: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. Conclusions: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

A contribution to the differentiation between nectar honey and honeydew honey

Thirty genuine honey samples were analyzed for pH, acidity, water, ash, net absorbance, total polyphenols (Folin-Ciocalteau method) and glucose, fructose, melezitose and erlose (as their trimethylsilyl oximes and trimethylsilyl ethers) by capillary gas chromatography. The resulting data were used, along with palynological analysis, to characterize the samples in relation to their possible source (nectar, honeydew and mixture honeys). Some minor components (carboxylic acids and cyclitols), eluting before monosaccharides, were also determined. One of these compounds was quercitol (1,3,4/2,5-cyclohexane-pentol), a deoxyinositol which has been previously determined in Quercus sp. samples. Quercitol was present in a broad concentration range (0.01-1.50 g/100 g) in honeys whose major source was honeydew but it was never higher than 0.01 g/100 g in samples characterized as nectar honeys. Quercitol concentrations appear to be related to the presence and amount of Quercus sp. honeydew as honey source, although further research is required to confirm this. (C) 2004 Elsevier Ltd. All rights reserved.

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g gluconodelta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

Myostatin regulates cardiomyocyte growth through modulation of Akt signaling

Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine - threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

A role for Notch signaling in human corneal epithelial cell differentiation and proliferation

PURPOSE. To identify the role of Notch signaling in the human corneal epithelium. METHODS. Localization of Notch1, Notch2, Delta1, and Jagged1 in the human corneal epithelium was observed with the use of indirect immunofluorescence microscopy. Gene and protein expression of Notch receptors and ligands in human corneal epithelial cells was determined by RT-PCR and Western blot analysis, respectively. The effects of Notch inhibition (by {gamma}-secretase inhibition) and activation (by recombinant Jagged1) on epithelial cell proliferation (Ki67) and differentiation (CK3) were analyzed after Western blotting and immunocytochemistry. RESULTS. Immunofluorescent labeling localized Notch1 and Notch2 to suprabasal epithelial cell layers, whereas Delta1 and Jagged1 were observed throughout the corneal epithelium. Notch1, Notch2, Delta1, and Jagged1 genes and proteins were expressed in human corneal epithelial cells. {gamma}-Secretase inhibition resulted in decreased Notch1 and Notch2 expression, with an accompanying decrease in Ki67 and increased CK3 expression. The activation of Notch by Jagged1 resulted in the upregulation of active forms of Notch1 and 2 proteins (P < 0.05), with a concurrent increase in Ki67 (P < 0.05) and a decrease in CK3 (P < 0.05) expression. Interestingly, {gamma}-secretase inhibition in a three-dimensional, stratified corneal epithelium equivalent had no effect on Ki67 or CK3 expression. In contrast, Jagged1 activation resulted in decreased CK3 expression (P < 0.05), though neither Notch activation nor inhibition affected cell proliferation in the 3D tissue equivalent. CONCLUSIONS. Notch family members and ligands are expressed in the human corneal epithelium and appear to play pivotal roles in corneal epithelial cell differentiation.

Can the cardiomyocyte cell cycle be reprogrammed?

Cardiac repair following myocardial injury is restricted due to the limited proliferative potential of adult cardiomyocytes. The ability of mammalian cardiomyocytes to proliferate is lost shortly after birth as cardiomyocytes withdraw from the cell cycle and differentiate. We do not fully understand the molecular and cellular mechanisms that regulate this cell cycle withdrawal, although if we could it might lead to the discovery of novel therapeutic targets for improving cardiac repair following myocardial injury. For the last decade, researchers have investigated cardiomyocyte cell cycle control, commonly using transgenic mouse models or recombinant adenoviruses to manipulate cell cycle regulators in vivo or in vitro. This review discusses cardiomyocyte cell cycle regulation and summarises recent data from studies manipulating the expressions and activities of cell cycle regulators in cardiomyocytes. The validity of therapeutic strategies that aim to reinstate the proliferative potential of cardiomyocytes to improve myocardial repair following injury will be discussed. (c) 2007 Elsevier Inc. All rights reserved.

Genetic differentiation between hosts and locations in populations of latent Botrytis cinerea in southern England

This study tested the hypothesis that aggressive, localized infections and asymptomatic systemic infections were caused by distinct specialized groups of Botrytis cinerea, using microsatellite genotypes at nine loci of 243 isolates of B. cinerea obtained from four hosts (strawberry (Fragaria ´ananassa), blackberry (Rubus fruticosus agg.), dandelion, (Taraxacum of®- cinale agg.) and primrose (Primula vulgaris)) in three regions in southern England (in the vicinities of Brighton, Reading and Bath). The populations were extremely variable, with up to 20 alleles per locus and high genic diversity. Each host in each region had a population of B. cinerea with distinctive genetic features, and there were also consistent host and regional distinctions. The B. cinerea population from strawberry was distinguished from that on other hosts, including blackberry, most notably by a common 154-bp amplicon at locus 5 (present in 35 of 77 samples) that was rare in isolates from other hosts (9¤166), and by the rarity (3¤77) of a 112-bp allele at locus 7 that was common (58¤166) in isolates from other hosts. There was signi®cant linkage disequilibrium overall within the B. cinerea populations on blackberry and strawberry, but with quite different patterns of association among isolates from the two hosts. No evidence was found for differentiation between populations of B. cinerea from systemically infected hosts and those from locally infected fruits.

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.