Critical role for an acidic amino acid region in platelet signaling by the HemITAM (hemi-immunoreceptor tyrosine-based activation motif) containing receptor CLEC-2 (C-type lectin receptor-2)

CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.

Bmp4 regulates chick Ebf2 and Ebf3 gene expression in somite development

The chick Early B-cell Factor-2 and 3 (cEbf2 and cEbf3) genes are members of EBF family of helix loop helix transcription factors. The expression, regulation and importance of these genes have been extensively studied in lymphatic, nervous and muscular tissues. Recently, a new role for some members of EBF in bone development has been investigated. However, the expression profile and regulation in the axial skeleton precursor, the somite, have yet to be elucidated. Therefore, this study was aimed to investigate the expression and regulation of cEbf2 and cEbf3 genes in the developing chick embryo somite from HH4 to HH28. The spatiotemporal expression study revealed predominant localization of cEbf2 and cEbf3 in the lateral sclerotomal domains and later around vertebral cartilage anlagen of the arch and the proximal rib. Subsequently, microsurgeries, ectopic gene expression experiments were performed to analyze which tissues and factors regulate cEbf2 and cEbf3 expression. Lateral barriers experiments indicated the necessity for lateral signal(s) in the regulation of cEbf2 and cEbf3 genes. Results from tissue manipulations and ectopic gene expression experiments indicate that lateral plate-derived Bmp4 signals are necessary for the initiation and maintenance of cEbf2 and cEbf3 genes in somites. In conclusion, cEbf2 and cEbf3 genes are considered as lateral sclerotome markers which their expression is regulated by Bmp4 signals from the lateral plate mesoderm.

Muscular weakness in the mdx mouse.

mdx mice are believed to be virtually free from neuromuscular symptoms, despite the presence of a degenerative/regenerative process that involves all skeletal muscles. We analyzed both the spontaneous motility and treadmill motor activity of mdx mice aged 15 days to 6 months. Our results indicate that there is an early period, between the end of the second and up to the fifth week of life, when mdx mice experience extreme weakness. After this critical period, both spontaneous motility and endurance of mdx mice, although lower than those of controls, do not show statistically significant differences up to 6 months of age. We also carried out a detailed histological analysis of proximal and distal muscle groups in mdx mice during this early critical motility period. The occurrence of extensive necrosis followed by regeneration and involving proximal muscles before distal ones was documented in mice as young as 16-17 days of age and reached a peak at day 18. We conclude that dystrophin deficiency induces muscle degeneration and significant weakness in mdx mice, but only in an early period. Later on, during development, mdx mice adapt to the lack of this protein and do not show detectable in vivo functional muscle impairment up to 6 months of age.

Regulation of Bcl-xL expression by H2O2 in cardiac myocytes.

Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.

Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c-jun expression.

Cardiac myocyte hypertrophy is associated with an increase in expression of immediate early genes (e.g. c-jun) via activation of pre-existing transcription factors. The activity of CREB transcription factor is regulated through phosphorylation of Ser-133 by one of several protein kinases (e.g. protein kinase A (PKA), p90 ribosomal S6 kinases (RSKs) and the related kinase, MSK1). A cell-permeable form of cAMP, hypertrophic agonists (endothelin-1 (ET-1), phenylephrine (PE)) and hyperosmotic shock all promoted phosphorylation of CREB(Ser-133) in rat neonatal cardiac myocytes. The response to endothelin-1 required the extracellular signal-regulated kinase cascade which stimulates both RSKs and MSK1. Phosphorylation of CREB(Ser-133) in response to ET-1 was not associated with any increase in DNA binding to a consensus cAMP-response element (CRE). The rat c-jun promoter contains elements which may bind either c-Jun/ATF2 or CREB/ATF1 dimers. Using extracts from rat cardiac myocytes, we identified at least two complexes which bind to the most proximal of these elements, one of which contained CREB and the other c-Jun. Thus, phosphorylation and activation of CREB in cardiac myocytes may be effected by a range of different stimuli to influence the expression of immediate early genes such as c-jun.

Influence of ground surface characteristics on the mean radiant temperature in urban areas

The effect of variations in land cover on mean radiant surface temperature (Tmrt) is explored through a simple scheme developed within the radiation model SOLWEIG. Outgoing longwave radiation is parameterised using surface temperature observations on a grass and an asphalt surface, whereas outgoing shortwave radiation is modelled through variations in albedo for the different surfaces. The influence of surface materials on Tmrt is small compared to the effects of shadowing. Nevertheless, altering ground surface materials could contribute to a reduction on Tmrt to reduce the radiant load during heat-wave episodes in locations where shadowing is not an option. Evaluation of the new scheme suggests that despite its simplicity it can simulate the outgoing fluxes well, especially during sunny conditions. However, it underestimates at night and in shadowed locations. One grass surface used to develop the parameterisation, with very different characteristics compared to an evaluation grass site, caused Tmrt to be underestimated. The implications of using high resolution (e.g. 15 minutes) temporal forcing data under partly cloudy conditions are demonstrated even for fairly proximal sites.

Characterisation of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2),which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Convergence accommodation in orthoptic practice

Aims. Orthoptists are familiar with AC/A ratios and the concept that accommodation drives convergence, but the reverse relationship, that of the accommodation associated with convergence, is rarely considered. Methods. This article reviews published evidence from our laboratory which has investigated the drives to both vergence and accommodation. All studies involved a method by which accommodation and vergence were measured concurrently and objectively to a range of visual stimuli which manipulate blur, disparity and proximal/looming cues in different combinations. Results Results are summarised for both typical and atypical participants, and over development between birth and adulthood. Conclusions For the majority of typical children and adults, as well as patients with most heterophorias and intermittent exotropia, disparity is the main cue to both vergence and accommodation. Thus the convergence→accommodation relationship is more influential than that of accommodative vergence. Differences in “style” of near cue use may be a more useful way to think about responses to stimuli moving in depth, and their consequences for orthoptic patients, than either AC/A or CA/C ratios. The implications of a strong role for vergence accommodation in orthoptic practice are considered.

Digital image correlation analysis of the load transfer by implant-supported restorations

This study compared splinted and non-splinted implant-supported prosthesis with and without a distal proximal contact using a digital image correlation method. An epoxy resin model was made with acrylic resin replicas of a mandibular first premolar and second molar and with threaded implants replacing the second premolar and first molar. Splinted and non-splinted metal-ceramic screw-retained crowns were fabricated and loaded with and without the presence of the second molar. A single-camera measuring system was used to record the in-plane deformation on the model surface at a frequency of 1.0 Hz under a load from 0 to 250 N. The images were then analyzed with specialist software to determine the direct (horizontal) and shear strains along the model. Not splinting the crowns resulted in higher stress transfer to the supporting implants when the second molar replica was absent. The presence of a second molar and an effective interproximal contact contributed to lower stress transfer to the supporting structures even for non-splinted restorations. Shear strains were higher in the region between the molars when the second molar was absent, regardless of splinting. The opposite was found for the region between the implants, which had higher shear strain values when the second molar was present. When an effective distal contact is absent, non-splinted implant-supported restorations introduce higher direct strains to the supporting structures under loading. Shear strains appear to be dependent also on the region within the model, with different regions showing different trends in strain changes in the absence of an effective distal contact. (C) 2011 Elsevier Ltd. All rights reserved.

Influence of thermoplastic extrusion on the nutritive value of bovine rumen protein

This Study investigated the impact of thermoplastic extrusion on the nutritive quality of bovine rumen protein. Proximal composition, amino acid profile and in vivo true protein digestibility among rats were determined in raw (RBR) and extruded (EBR) rumen. Raw and extruded bovine rumen presented high percentages of protein (more than 95% on dry basis). Neither raw nor extruded proteins had any limiting amino acid, and the RBR and EBR amino acid scores were, respectively, 1.28 (leucine) and 1.25 (methionine plus cystine). Extrusion reduced significantly true protein digestibility from 97.7% to 93.1% (p < 0.001), but protein digestibility-corrected amino acid scores for both proteins (RBR and EBR) were 100%. Animal growth presented comparable profiles using raw and extruded rumen. In conclusion, thermoplastic extrusion did not affect the protein quality of bovine rumen, and this does not hinder the use of this material as a food ingredient. (C) 2009 Elsevier Ltd. Ail rights reserved.

Adding Value to the Meat of Spent Laying Hens Manufacturing Sausages with a Healthy Appeal

The aim of this study was to evaluate the viability of the use of spent laying hens` meat in the manufacturing of mortadella-type sausages with healthy appeal by using vegetable oil instead of animal fat. 120 Hy-line (R) layer hens were distributed in a completely randomized design into two treatments of six replicates with ten birds each. The treatments were birds from light Hy-line (R) W36 and semi-heavy Hy-line (R) Brown lines. Cold carcass, wing, breast and leg fillets yields were determined. Dry matter, protein, and lipid contents were determined in breast and leg fillets. The breast and legg fillets of three replicates per treatment were used to manufacture mortadella. After processing, sausages were evaluated for proximal composition, objective color, microbiological parameters, fatty acid profile and sensory acceptance. The meat of light and semi-heavy spent hens presented good yield and composition, allowing it to be used as raw material for the manufacture of processed products. Mortadellas were safe from microbiological point of view, and those made with semi-heavy hens fillets were redder and better accepted by consumers. Values for all sensory attributes were evaluated over score 5 (neither liked nor disliked). Both products presented high polyunsaturated fatty acid contents and good polyunsaturated to saturated fatty acid ratio. The excellent potential for the use of meat from spent layer hens of both varieties in the manufacturing of healthier mortadella-type sausage was demonstrated.

Rosiglitazone reverses tenofovir-induced nephrotoxicity

Tenofovir disoproxil fumarate (TDF) is a first-line drug used in patients with highly active retroviral disease; however, it can cause renal failure associated with many tubular anomalies that may be due to down regulation of a variety of ion transporters. Because rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist induces the expression of many of these same transporters, we tested if the nephrotoxicity can be ameliorated by its use. High doses of TDF caused severe renal failure in rats accompanied by a reduction in endothelial nitric-oxide synthase and intense renal vasoconstriction; all of which were significantly improved by rosiglitazone treatment. Low-dose TDF did not alter glomerular filtration rate but produced significant phosphaturia, proximal tubular acidosis, polyuria and a reduced urinary concentrating ability. These alterations were caused by specific downregulation of the sodium-phosphorus cotransporter, sodium/hydrogen exchanger 3 and aquaporin 2. A Fanconi`s-like syndrome was ruled out as there was no proteinuria or glycosuria. Rosiglitazone reversed TDF-induced tubular nephrotoxicity, normalized urinary biochemical parameters and membrane transporter protein expression. These studies suggest that rosiglitazone treatment might be useful in patients presenting with TFV-induced nephrotoxicity especially in those with hypophosphatemia or reduced glomerular filtration rate.

PKC stimulated by glucagon decreases UT-A1 urea transporter expression in rat IMCD

It is well-known that glucagon increases fractional excretion of urea in rats after a protein intravenous infusion. This effect was investigated by using: (a) in vitro microperfusion technique to measure [(14)C]-urea permeability (Pu x 10(-5) cm/s) in inner medullary collecting ducts (IMCD) from normal rats in the presence of 10(-7) M of glucagon and in the absence of vasopressin and (b) immunoblot techniques to determine urea transporter expression in tubule suspension incubated with the same glucagon concentration. Seven groups of IMCDs (n = 47) were studied. Our results revealed that: (a) glucagon decreased urea reabsorption dose-dependently; (b) the glucagon antagonist des-His(1)-[Glu(9)], blocked the glucagon action but not vasopressin action; (c) the phorbol myristate acetate, decreased urea reabsorption but (d) staurosporin, restored its effect; e) staurosporin decreased glucagon action, and finally, (f) glucagon decreased UT-A1 expression. We can conclude that glucagon reduces UT-A1 expression via a glucagon receptor by stimulating PKC.