Effect of combined heat and high-pressure treatments on the texture of chicken breast muscle (Pectoralis fundus)

Commercially supplied chicken breast muscle was subjected to simultaneous heat and pressure treatments. Treatment conditions ranged from ambient temperature to 70 °C and from 0.1 to 800 MPa, respectively, in various combinations. Texture profile analysis (TPA) of the treated samples was performed to determine changes in muscle hardness. At treatment temperatures up to and including 50 °C, heat and pressure acted synergistically to increase muscle hardness. However, at 60 and 70 °C, hardness decreased following treatments in excess of 200 MPa. TPA was performed on extracted myofibrillar protein gels that after treatment under similar conditions revealed similar effects of heat and pressure. Differential scanning calorimetry analysis of whole muscle samples revealed that at ambient pressure the unfolding of myosin was completed at 60 °C, unlike actin, which completely denatured only above 70 °C. With simultaneous pressure treatment at >200 MPa, myosin and actin unfolded at 20 °C. Unfolding of myosin and actin could be induced in extracted myofibrillar protein with simultaneous treatment at 200 MPa and 40 °C. Electrophoretic analysis indicated high pressure/temperature regimens induced disulfide bonding between myosin chains.

Evidence for an inhibitory role of bone morphogenetic protein(s) in the follicular-luteal transition in cattle

Bone morphogenetic proteins (BMPs) and their receptors are expressed in ovarian theca cells (TC) and granulosa cells (GC) and BMPs have been implicated in the regulation of several aspects of follicle development including thecal androgen production and granulosal oestrogen production. Their potential involvement in luteal function has received less attention. in this study, we first compared relative abundance of mRNA transcripts for BMPs, activin-beta A and BMP/activin receptors in bovine corpus luteum (CL) and follicular theca and granulosa layers before undertaking functional in vitro experiments to test the effect of selected ligands (BMP6 and activin A) on luteinizing bovine TC and GC. Relative to P-actin transcript abundance, CL tissue contained more BMP4 and -6 mRNA than granulosa, more BMP2 mRNA than theca but much less activin-beta A mRNA than both granulosa and theca. Transcripts for all seven BMP/activin receptors were readily detected in each tissue and two transcripts (BMPRII, ActRIIA) were more abundant in CL than either theca or granulosa, consistent with tissue responsiveness. In vitro luteinization of TC and GC from antral follicles (4-6 mm) was achieved by culturing with 5% serum for 6 days. Treatment with BMP6 (0, 2, 10, and 50 ng/ml) and activin A (0, 2, 10 and 50 ng/ml) under these conditions dose-dependently suppressed forskolin-induced progesterone (P-4) secretion from both cell types without affecting cell number. BMP6 reduced forskolin-stimulated upregulation of STAR mRNA and raised 'basal' CYP17A1 mRNA level in theca-lutein cells without affecting expression of CYP11A1 or hydroxy-Delta-5-steroid dehydrogenase, 3 beta- and steroid Delta-isomerase 1 (HSD3B1). In granulosa-lutein cells, STAR transcript abundance was not affected by BMP6, whereas forskolin-induced expression of CYP11A1, HSD3B1, CYP19A1 and oxytocin transcripts was reduced. In both cell types, follistatin attenuated the suppressive effect of activin A and BMP6 on forskolin-induced P4 secretion but had no effect alone. Granulosa-lutein cells secreted low levels of endogenous activin A (similar to 1 ng/ml); BMP6 reduced this, while raising follistatin secretion thus decreasing activin A:follistatin ratio. Collectively, these findings support inhibitory roles for BMP/activin signalling in luteinization and steroidogenesis in both TC and GC.

S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.

Dual-specificity phosphatases in the hypo-osmotic stress response of keratin-defective epithelial cell lines

Although mutations in intermediate filament proteins cause many human disorders, the detailed pathogenic mechanisms and the way these mutations affect cell metabolism are unclear. In this study, selected keratin mutations were analysed for their effect on the epidermal stress response. Expression profiles of two keratin-mutant cell lines from epidermolysis bullosa simplex patients (one severe and one mild) were compared to a control keratinocyte line before and after challenge with hypo-osmotic shock, a common physiological stress that transiently distorts cell shape. Fewer changes in gene expression were found in cells with the severely disruptive mutation (55 genes altered) than with the mild mutation (174 genes) or the wild type cells (261 genes) possibly due to stress response pre-activation in these cells. We identified 16 immediate-early genes contributing to a general cell response to hypo-osmotic shock, and 20 genes with an altered expression pattern in the mutant keratin lines only. A number of dual-specificity phosphatases (MKP-1, MKP-2, MKP-3, MKP-5 and hVH3) are differentially regulated in these cells, and their downstream targets p-ERK and p-p38 are significantly up-regulated in the mutant keratin lines. Our findings strengthen the case for the expression of mutant keratin proteins inducing physiological stress, and this intrinsic stress may affect the cell responses to secondary stresses in patients' skin.

Annexins: multifunctional components of growth and adaptation

Plant annexins are ubiquitous, soluble proteins capable of Ca2+-dependent and Ca2+-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.

Expression of metallothionein genes I and II in bank vole clethrionomys glareolus populations chronically exposed in situ to heavy metals

The expression of two metallothionein genes (Mt-I and Mt-II) in the liver, kidney, and gonad of bank voles collected at four metal-contaminated sites (Cd, Zn, Pb, and Fe) were measured using the quantitative real-time PCR method (QPCR). Relative Mt gene expression was calculated by applying a normalization factor (NF) using the expression of two housekeeping genes, ribosomal 18S and beta-actin. Relative Mt expression in tissues of animals from contaminated sites was up to 54.8-fold higher than those from the reference site for Mt-I and up to 91.6-fold higher for Mt-II. Mt-II gene expression in the livers of bank voles from contaminated sites was higher than Mt-I gene expression. Inversely, Mt-II expression in the kidneys of voles was lower than Mt-I expression. Positive correlations between cadmium levels in the tissues and Mt-I were obtained in all studied tissues. Zinc, which undergoes homeostatic regulation, correlated positively with both Mt-I and Mt-II gene expression only in the kidney. Results showed that animals living in chronically contaminated environments intensively activate detoxifying mechanisms such as metallothionein expression. This is the first time that QPCR techniques to measure MT gene expression have been applied to assess the impact of environmental metal pollution on field collected bank voles.

Expression of target and reference genes in Daphnia magna exposed to ibuprofen

Background: Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR). The accurate measurement of target gene expression requires normalisation to an invariant internal control e. g., total RNA or reference genes. Reference genes are preferable, as they control for variation inherent in the cDNA synthesis and PCR. However, reference gene expression can vary between tissues and experimental conditions, which makes it crucial to validate them prior to application. Results: We evaluated 10 candidate reference genes for QPCR in Daphnia magna following a 24 h exposure to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) at 0, 20, 40 and 80 mg IB l(-1). Six of the 10 candidates appeared suitable for use as reference genes. As a robust approach, we used a combination normalisation factor (NF), calculated using the geNorm application, based on the geometric mean of three selected reference genes: glyceraldehyde-3-phosphate dehydrogenase, ubiquitin conjugating enzyme and actin. The effects of normalisation are illustrated using as target gene leukotriene B4 12-hydroxydehydrogenase (Ltb4dh), which was upregulated following 24 h exposure to 63-81 mg IB l(-1). Conclusions: As anticipated, use of the NF clarified the response of Ltb4dh in daphnids exposed to sublethal levels of ibuprofen. Our findings emphasise the importance in toxicogenomics of finding and applying invariant internal QPCR control(s) relevant to the study conditions.

Metallothionein I and II gene expression as a response to metal contamination in bank vole Clethrionomys glareolus

The expression of two metallothionein genes (Mt-I and Mt-II) in the liver, kidney, and gonad of bank voles collected at four metal-contaminated sites (Cd, Zn, Pb, and Fe) were measured using the quantitative real-time PCR method (QPCR). Relative Mt gene expression was calculated by applying a normalization factor (NF) using the expression of two housekeeping genes, ribosomal 18S and beta-actin. Relative Mt expression in tissues of animals from contaminated sites was up to 54.8-fold higher than those from the reference site for Mt-I and up to 91.6-fold higher for Mt-II. Mt-II gene expression in the livers of bank voles from contaminated sites was higher than Mt-I gene expression. Inversely, Mt-II expression in the kidneys of voles was lower than Mt-I expression. Positive correlations between cadmium levels in the tissues and Mt-I were obtained in all studied tissues. Zinc, which undergoes homeostatic regulation, correlated positively with both Mt-I and Mt-II gene expression only in the kidney. Results showed that animals living in chronically contaminated environments intensively activate detoxifying mechanisms such as metallothionein expression. This is the first time that QPCR techniques to measure MT gene expression have been applied to assess the impact of environmental metal pollution on field collected bank voles.

Mechanisms of internalization and recycling of the chemokine receptor, CCR5

CCR5 is a G protein-coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV-1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV-1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand-induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine-induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP-1. CCR5 internalization and recycling were inhibited by Toxin B and C-3 exoenzyme treatment in CHO and THP-1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand-induced activation of CCR5 leads to Rho kinase-dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho-dependent manner.

Altered expression of cell cycle proteins and prolonged duration of cardiac myocyte hyperplasia in p27KIP1 knockout mice

The precise role of cell cycle-dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains to be determined. We report that loss of p27(KIP1) in the mouse results in a significant increase in heart size and in the total number of cardiac myocytes. In comparison to p27(KIP1)+/+ myocytes, the percentage of neonatal p27(KIP1)-/- myocytes in S phase was increased significantly, concomitant with a significant decrease in the percentage of G(0)/G(1) cells. The expressions of proliferating cell nuclear antigen, G(1)/S and G(2)/M phase-acting cyclins, and cyclin-dependent kinases (CDKs) were upregulated significantly in ventricular tissue obtained from early neonatal p27(KIP1)-/- mice, concomitant with a substantial decrease in the expressions of G(1) phase-acting cyclins and CDKs. Furthermore, mRNA expressions of the embryonic genes atrial natriuretic factor and alpha-skeletal actin were detectable at significant levels in neonatal and adult p27(KIP1)-/- mouse hearts but were undetectable in p27(KIP1)+/+ hearts. In addition, loss of p27(KIP1) was not compensated for by the upregulation of other CDK inhibitors. Thus, the loss of p27(KIP1) results in prolonged proliferation of the mouse cardiac myocyte and perturbation of myocyte hypertrophy.

The advection of high-resolution tracers by low-resolution winds

The usefulness of any simulation of atmospheric tracers using low-resolution winds relies on both the dominance of large spatial scales in the strain and time dependence that results in a cascade in tracer scales. Here, a quantitative study on the accuracy of such tracer studies is made using the contour advection technique. It is shown that, although contour stretching rates are very insensitive to the spatial truncation of the wind field, the displacement errors in filament position are sensitive. A knowledge of displacement characteristics is essential if Lagrangian simulations are to be used for the inference of airmass origin. A quantitative lower estimate is obtained for the tracer scale factor (TSF): the ratio of the smallest resolved scale in the advecting wind field to the smallest “trustworthy” scale in the tracer field. For a baroclinic wave life cycle the TSF = 6.1 ± 0.3 while for the Northern Hemisphere wintertime lower stratosphere the TSF = 5.5 ± 0.5, when using the most stringent definition of the trustworthy scale. The similarity in the TSF for the two flows is striking and an explanation is discussed in terms of the activity of potential vorticity (PV) filaments. Uncertainty in contour initialization is investigated for the stratospheric case. The effect of smoothing initial contours is to introduce a spinup time, after which wind field truncation errors take over from initialization errors (2–3 days). It is also shown that false detail from the proliferation of finescale filaments limits the useful lifetime of such contour advection simulations to 3σ−1 days, where σ is the filament thinning rate, unless filaments narrower than the trustworthy scale are removed by contour surgery. In addition, PV analysis error and diabatic effects are so strong that only PV filaments wider than 50 km are at all believable, even for very high-resolution winds. The minimum wind field resolution required to accurately simulate filaments down to the erosion scale in the stratosphere (given an initial contour) is estimated and the implications for the modeling of atmospheric chemistry are briefly discussed.

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.

Role of NleH, a Type III Secreted Effector from Attaching and Effacing Pathogens, in Colonization of the Bovine, Ovine, and Murine Gut

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157: H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappa B to the nucleus. In this study we investigated the role of NleH during EHEC O157: H7 infection of calves and lambs. We found that while EHEC Delta nleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157: H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappa B reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappa B response elements, we found that NleH causes an increase in NF-kappa B activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Interaction of enterohemorrhagic Escherichia coli O157 : H7 with mouse intestinal mucosa

In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10(9) and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.