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Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

The formation, maturation, and dissolution of focal adhesions are basic prerequisites of cell migration and rely on the recruitment, signalling, and endocytosis of integrins. In many instances, extracellular matrix molecules are recognised by a number of integrins, and it is the sequential involvement of different integrins that allows establishment of cell polarity and migration towards a matrix stimulus. In this review, we consider both the similarities and differences between two key fibronectin receptors, alpha(v)beta(3) and alpha(5)beta(1) integrin. By considering the GTPase and kinase signalling and trafficking of two such closely-related receptors, we begin to understand how cell migration is coordinated.

Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin-ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin alpha(5)beta(1)-Fc fusion protein or membrane alpha(5)beta(1). Force prolonged bond lifetimes in the 10-30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca(2+)/Mg(2+) to Mg(2+)/EGTA and to Mn(2+) caused longer lifetime in the same 10-30-pN catch bond region. A truncated alpha(5)beta(1) construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

Aim Areas of endemism are the fundamental units of cladistic biogeographical analysis but there is no consensus on the most appropriate method for their delimitation. In this paper, the relative performance of a number of algorithmic approaches for the delimitation of areas of endemism is investigated within the context of the Canary Islands flora, and areas of endemism within the Canary Islands archipelago are defined. Location The Canary Islands. Methods A data matrix comprising the distributions of 609 endemic spermatophyte taxa (c. 90% of the endemic flora) scored on a 10 × 10 km UTM grid was analysed using: (1) UPGMA (unweighted pair group method with arithmetic mean) clustering of Jaccard and Kulczynski similarity coefficient matrices, (2) parsimony analysis of endemicity (PAE), and (3) the program ndm (eNDeMism). The performance of each method was then determined by the extent to which the resulting areas of endemism met three criteria: (1) possession of two or more strict endemic taxa, (2) diagnosability, and (3) geographical contiguity. Results Each of the four methods resulted in substantially different sets of areas. ndm analysis resolved 17 areas of endemism consistent with all three criteria, and collectively these accounted for 59% of all cells. In the hierarchical analyses none of the methods recovered more than eight areas of endemism, and the total coverage of cells ranged from 13% to 33% when the results were confined to intra-island areas of endemism. Main conclusions ndm outperforms hierarchical clustering methods in terms of both the number of intra-island areas of endemism delimited that meet the three evaluation criteria and the total coverage of those areas. ndm may also be considered preferable because it is non-hierarchical, incorporates spatial information into the delimitation of areas, and permits overlap between areas of endemism where there is evidence to support it. The results support the use of ndm as the most appropriate method currently available for the delimitation of areas of endemism. The areas of endemism identified by the ndm analysis are discussed.

For two rooted phylogenetic trees T and T ′ , the rooted subtree prune and regraft distance between T and T ′ has often been used as a replacement for the hybridization number of T and T ′ . However, Baroni et al. [M. Baroni, S. Grunewald, V. Moulton, C. Semple, Bounding the number of hybridisation events for a consistent evolutionary history, J. Math. Biol. 51 (2005) 171–182] constructed particular instances that showed that both the difference and the ratio between this number and the distance can be arbitrarily large. In this note, we show that the difference and ratio values obtained in the above reference of Baroni et al. are the best possible, thus answering a problem posed in [C. Semple, Hybridization networks, in: O. Gascuel, M. Steel (Eds.), Reconstructing Evolution: New Mathematical and Computational Advances, Oxford University Press, 2007, pp. 277–314].

A total of 133 samples (53 fermented unprocessed, 19 fermented processed. 62 urea-treated processed) of whole crop wheat (WCW) and 16 samples (five fermented unprocessed, six fermented processed, five urea-treated processed) of whole crop barley (WCB) were collected from commercial farms over two consecutive years (2003/2004 and 2004/2005). Disruption of the maize grains to increase starch availability was achieved at the point of harvest by processors fitted to the forage harvesters. All samples were subjected to laboratory analysis whilst 50 of the samples (24 front Year 1, 26 front Year 2 all WCW except four WCB in Year 2) were subjected to in vivo digestibility and energy value measurements using mature wether sheep. Urea-treated WCW had higher (P

Samples of whole crop wheat (WCW, n = 134) and whole crop barley (WCB, n = 16) were collected from commercial farms in the UK over a 2-year period (2003/2004 and 2004/2005). Near infrared reflectance spectroscopy (NIRS) was compared with laboratory and in vitro digestibility measures to predict digestible organic matter in the dry matter (DOMD) and metabolisable energy (ME) contents measured in vivo using sheep. Spectral models using the mean spectra of two scans were compared with those using individual spectra (duplicate spectra). Overall NIRS accurately predicted the concentration of chemical components in whole crop cereals apart from crude protein. ammonia-nitrogen, water-soluble carbohydrates, fermentation acids and solubility values. In addition. the spectral models had higher prediction power for in vivo DOMD and ME than chemical components or in vitro digestion methods. Overall there Was a benefit from the use of duplicate spectra rather than mean spectra and this was especially so for predicting in vivo DOMD and ME where the sample population size was smaller. The spectral models derived deal equally well with WCW and WCB and Would he of considerable practical value allowing rapid determination of nutritive value of these forages before their use in diets of productive animals. (C) 2008 Elsevier B.V. All rights reserved.

Inclusion of rapeseed feeds in dairy cow diets has the potential to reduce milk fat saturated fatty acid (SFA) and increase cis-monounsaturated fatty acid (cis-MUFA) content, but effectiveness may depend on the form in which the rapeseed is presented. Four mid-lactation Holstein dairy cows were allocated to four maize silage-based dietary treatments according to a 4 × 4 Latin Square design, with 28-day experimental periods. Treatments consisted of a control diet (C) containing 49 g/kg dry matter (DM) of calcium salts of palm oil distillate (CPO), or 49 g/kg DM of oil supplied as whole rapeseeds (WR), rapeseeds milled with wheat (MR) or rapeseed oil (RO). Replacing CPO with rapeseed feeds had no effect (P > 0.05) on milk fat and protein content, while milk yields were higher (P < 0.05) for RO and MR compared with WR (37.1, 38.1 and 34.3 kg/day, respectively). Substituting CPO with RO or MR reduced (P < 0.05) milk fat total SFA content (69.6, 55.6, 71.7 and 61.5 g/100 g fatty acids for C, RO, WR and MR, respectively) and enhanced (P < 0.05) milk cis-9 18:1 MUFA concentrations (corresponding values 18.6, 24.3, 17.0 and 23.0 g/100 g fatty acids) compared with C and WR. Treatments RO and MR also increased (P < 0.05) milk trans-MUFA content (4.4, 6.8, 10.5 g/100 g fatty acids, C, MR and RO, respectively). A lack of significant changes in milk fat composition when replacing CPO with WR suggests limited bioavailability of fatty acids in intact rapeseeds. In conclusion, replacing a commercial palm oil-based fat supplement in the diet with milled rapeseeds or rapeseed oil represented an effective strategy to alter milk fatty acid composition with the potential to improve human health. Inclusion of processed rapeseeds offered a good compromise for reducing milk SFA and increasing cis-MUFA, whilst minimising milk trans-MUFA and negative effects on animal performance.

Clinical and biomedical studies have provided evidence for the critical role of n-3 fatty acids on the reduction of chronic disease risk in humans, including cardiovascular disease. In the current experiment, the potential to enhance milk n-3 content in two breeds with inherent genetic differences in mammary lipogenesis and de novo fatty acid synthesis was examined using extruded linseeds. Six lactating cows (three Holstein and three Jersey) were used in a two-treatment switchback design with 3 × 21-day experimental periods to evaluate the effect of iso-energetic replacement of calcium salts of palm oil distillate (CPO) in the diet (34 g/kg dry matter (DM)) with 100 g/kg DM extruded linseeds (LIN). For both breeds, replacing CPO with LIN had no effect (P > 0.05) on DM intake or milk yield, but reduced (P < 0.05) milk fat and protein yield (on average, from 760 to 706 and 573 to 552 g/day, respectively). Relative to CPO, the LIN treatment reduced (P < 0.01) total saturated fatty acid content and enhanced (P < 0.001) 18:3n-3 in milk, whereas breed by diet interactions were significant for milk fat 16:0, total trans fatty acid and conjugated linoleic acid concentrations. Increases in 18:3n-3 intake derived from LIN in the diet were transferred into milk with a mean marginal transfer efficiency of 1.8%. Proportionate changes in milk fatty acid composition were greater in the Jersey, highlighting the importance of diet–genotype interactions on mammary lipogenesis. More extensive studies are required to determine the role of genotype on milk fat composition responses to oilseeds in the diet.

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

The endemic vascular flora of the Canary Islands comprises over 680, taxa collectively accounting for more than 50% of the total native flora. To investigate geographical patterns of diversity within the endemic flora, distribution data from published sources together with other field observation and herbarium data were used to compile a data matrix comprising the distributions of ca. 90% of endemic taxa scored on a 10 × 10km UTM grid. WORLDMAP was then used to investigate patterns of endemic diversity, range size rarity (a measure of endemicity), phylogenetic diversity and threatened taxon richness. Endemic taxon richness was found to be highly heterogeneous across the archipelago, with cells containing between one and 139 taxa each (0.05-22.82% of endemic diversity). Patterns of variation in range size rarity and phylogenetic diversity were found to be largely congruent with endemic diversity, although some cells exhibited markedly higher range size rarity scores than would be predicted by their endemic diversity scores. In contrast, the pattern of endangered taxon richness across the archipelago differed markedly from endemic taxon richness. Many cells in Lanzarote, Fuerteventura and Gran Canaria exhibit higher endangered taxon richness scores than would be predicted from their endemic richness scores whereas in Tenerife, El Hierro, La Palma and La Gomera, the converse is generally true. The implications of the results both for understanding the evolution of Canary Island endemic diversity and for the conservation of the region’s unique and vulnerable flora are considered.La flora vascular endémica de las Islas Canarias comprende unos 680 táxones, lo que viene a representar más del 50% de la flora nativa. Con objeto de investigar patrones geográficos de diversidad en la flora endémica, se recopilaron los datos publicados que, junto con otras observaciones de campo y datos de herbario, sirvieron para completar una matriz de datos que abarca la distribución de cerca del 90% de los táxones endémicos usando cuadrículas UTM de10 × 10 km. A continuación, se utilizó el programa WORLDMAP para investigar los patrones de diversidad de los endemismos, el rango del grado de rareza (una medida de endemicidad), la diversidad filogenética y la riqueza en táxones amenazados. Se observó que la riqueza en endemismos es muy heterogénea a lo largo del archipiélago, con unos valores por cuadrícula que oscilan entre 1 y 139 táxones (0,05-22,82% de la diversidad de táxones endémicos). Los patrones de variación del rango del grado de la rareza y la diversidad filogenética resultaron ser en gran parte congruentes con la diversidad en endemismos, aunque algunas cuadrículas mostraron valores mucho más altos de rareza de los que podían ser predichos dada su diversidad de endemismos. En contraste, los patrones de riqueza en especies amenazadas en el archipiélago difirieron marcadamente de la riqueza en táxones endémicos. Muchas cuadrículas de Lanzarote, Fuerteventura y Gran Canaria mostraron valores más altos de riqueza en especies amenazadas que las que pudieran ser predichas sobre la base de su riqueza en táxones endémicos, mientras que en Tenerife, El Hierro y La Gomera la regla fue generalmente lo contrario. Se consideran las implicaciones que estos resultados suponen para la comprensión de la evolución de la diversidad de endemismos canaria y para la conservación de su singular y vulnerable flora.

Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Association of talin with β1 integrin and paxillin with α4 integrin was dependent on both the ligand and receptor activation state, and was sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules, thus demonstrating that these compounds may induce secondary effects in vivo via integrin activation. This study provides insight into the dependence of the activity of small molecule anti-integrin compounds upon receptor conformation, and provides a novel quantitative assay for the validation of potential integrin antagonists.

Even though extensive research has examined the role of nutrition on milk fat composition, there is less information on the impact of forages on milk fatty acid (FA) composition. In the current study, the effect of replacing grass silage (GS) with maize silage (MS) as part of a total mixed ration on animal performance and milk FA composition was examined using eight multiparous mid-lactation cows in a replicated 4 × 4 Latin square with 28-day experimental periods. Four treatments comprised the stepwise replacement of GS with MS (0, 160, 334 and 500 g/kg dry matter (DM)) in diets containing a 54 : 46 forage : concentrate ratio on a DM basis. Replacing GS with MS increased (P < 0.001) the DM intake, milk yield and milk protein content. Incremental replacement of GS with MS in the diet enhanced linearly (P < 0.001) the proportions of 6:0–14:0, decreased (P < 0.01) the 16:0 concentrations, but had no effect on the total milk fat saturated fatty acid content. Inclusion of MS altered the distribution of trans-18:1 isomers and enhanced (P < 0.05) total trans monounsaturated fatty acid and total conjugated linoleic acid content. Milk total n-3 polyunsaturated fatty acid (PUFA) content decreased with higher amounts of MS in the diet and n-6 PUFA concentration increased, leading to an elevated n-6 : n-3 PUFA ratio. Despite some beneficial changes associated with the replacement of GS with MS, the overall effects on milk FA composition would not be expected to substantially improve long-term human health. However, the role of forages on milk fat composition must also be balanced against the increases in total milk and protein yield on diets containing higher proportions of MS.

PKCdelta is essential for apoptosis, but regulation of the proapoptotic function of this ubiquitous kinase is not well understood. Nuclear translocation of PKCdelta is necessary and sufficient to induce apoptosis and is mediated via a C-terminal bipartite nuclear localization sequence. However, PKCdelta is found predominantly in the cytoplasm of nonapoptotic cells, and the apoptotic signal that activates its nuclear translocation is not known. We show that in salivary epithelial cells, phosphorylation at specific tyrosine residues in the N-terminal regulatory domain directs PKCdelta to the nucleus where it induces apoptosis. Analysis of each tyrosine residue in PKCdelta by site-directed mutagenesis identified two residues, Y64 and Y155, as essential for nuclear translocation. Suppression of apoptosis correlated with suppressed nuclear localization of the Y --> F mutant proteins. Moreover, a phosphomimetic PKCdelta Y64D/Y155D mutant accumulated in the nucleus in the absence of an apoptotic signal. Forced nuclear accumulation of PKCdelta-Y64F and Y155F mutant proteins, by attachment of an SV40 nuclear localization sequence, fully reconstituted their ability to induce apoptosis, indicating that tyrosine phosphorylation per se is not required for apoptosis, but for targeting PKCdelta to the nucleus. We propose that phosphorylation/dephosphorylation of PKCdelta in the regulatory domain functions as a switch to promote cell survival or cell death.

The ability of cells to adhere to each other and to their surrounding extracellular matrices is essential for a multicellular existence. Adhesion provides physical support for cells, regulates cell positioning and enables microenvironmental sensing. The integrins and the syndecans are two adhesion receptor families that mediate adhesion, but their relative and functional contributions to cell-extracellular matrix interactions remain obscure. Recent advances have highlighted connections between the signalling networks that are controlled by these families of receptors. Here we survey the evidence that synergistic signalling is involved in controlling adhesive function and the regulation of cell behaviour in response to the external environment.

Marcelo R. de Carvalho AE Flavio A. Bockmann AE Dalton S. Amorim AE Carlos Roberto F. Brandao AE Mario de Vivo AE Jose L. de Figueiredo AE Heraldo A. Britski AE Mario C. C. de Pinna AE Naercio A. Menezes AE Fernando P. L. Marques AE Nelson Papavero AE Eliana M. Cancello AE Jorge V. Crisci AE John D. McEachran AE Robert C. Schelly AE John G. Lundberg AE Anthony C. Gill AE Ralf Britz AE Quentin D. Wheeler AE Melanie L. J. Stiassny AE Lynne R. Parenti AE Larry M. Page AE Ward C. Wheeler AE Julian Faivovich AE Richard P. Vari AE Lance Grande AE Chris J. Humphries AE Rob DeSalle AE Malte C. Ebach AE Gareth J. Nelson

Protein kinase C delta (PKC delta) mediates apoptosis downstream of many apoptotic stimuli. Because of its ubiquitous expression, tight regulation of the proapoptotic function of PKC delta is critical for cell survival. Full-length PKC delta is found in all cells, whereas the catalytic fragment of PKC delta, generated by caspase cleavage, is only present in cells undergoing apoptosis. Here we show that full-length PKC delta transiently accumulates in the nucleus in response to etoposide and that nuclear translocation precedes caspase cleavage of PKC delta. Nuclear PKC delta is either cleaved by caspase 3, resulting in accumulation of the catalytic fragment in the nucleus, or rapidly exported by a Crm1-sensitive pathway, thereby assuring that sustained nuclear accumulation of PKC delta is coupled to caspase activation. Nuclear accumulation of PKC delta is necessary for caspase cleavage, as mutants of PKC delta that do not translocate to the nucleus are not cleaved. However, caspase cleavage of PKC delta per se is not required for apoptosis, as an uncleavable form of PKC delta induces apoptosis when retained in the nucleus by the addition of an SV-40 nuclear localization signal. Finally, we show that kinase negative full-length PKC delta does not translocate to the nucleus in apoptotic cells but instead inhibits apoptosis by blocking nuclear import of endogenous PKC delta. These studies demonstrate that generation of the PKC delta catalytic fragment is a critical step for commitment to apoptosis and that nuclear import and export of PKC delta plays a key role in regulating the survival/death pathway.

We consider infinite nested radicals in which the arguments are positive polynomial sequences. It is shown that the evaluation of such a nesting is always finite, and we prove necessary and sufficient conditions for the evaluation to be a finite polynomial.

Previous clinical trials have evaluated a large number of symptomatic individuals with heartburn. Most studies have documented the need for multiple daily dosing with H2-antagonists to achieve clinical and statistical efficacy for symptom relief. The purpose of this study was to compare the safety profile and efficacy of famotidine oral dosing regimens, 40 mg nocte and 20 mg b.d. with placebo in the relief of symptoms in patients suffering from frequent heartburn found to have endoscopically normal oesophageal mucosa or mild non-erosive oesophagitis. Famotidine (20 mg) b.d. reduced and eventually completely relieved gastro-oesophageal reflux disease symptoms in most patients during the 6-week trial. Global assessment of improvement at 2 and 6 weeks indicated significantly greater improvement with a b.d. treatment regimen than with either a 40 mg nocte or placebo treatment. No statistically significant differences between famotidine and placebo in the number of patients who experienced clinical adverse experiences were noted and no serious adverse events attributable to famotidine occurred. Based upon these findings, patients with gastro-oesophageal reflux symptoms experience good relief of their complaints with twice daily famotidine in standard doses.

To define the optimum doses of omeprazole appropriate for acute and long-term therapy of patients with gastro-oesophageal reflux disease, 24-h oesophageal pH was measured in 12 patients with symptomatic reflux and an abnormal 24-h oesophageal acid exposure time (greater than 6%) in a randomized, double-blind, four-way crossover study comparing the effects of omeprazole 10, 20, or 40 mg/day and placebo. Total reflux time over 24 hours, number of reflux episodes per hour, and the number of reflux episodes lasting greater than 5 minutes were measured by ambulatory 24-h oesophageal pH monitoring. All doses of omeprazole were superior to placebo in decreasing gastro-oesophageal reflux as measured by each index. With placebo, oesophageal acid exposure was 16.3% of the 24 hours, 10 mg omeprazole/day reduced that to 6.3%, 20 mg/day lowered acid exposure to 0.9%, and 40 mg/day to 0.6%. Thus only the 20 and 40 mg doses reduced acid exposure to within the normal range. Similar results were obtained with the other indices of reflux. These data suggest that a rational dose regimen for reflux oesophagitis is 20 mg/day, a regimen that has proved effective in clinical trials. The present study indicates that 24-hour oesophageal pH monitoring is a practical approach to the determination of drug dosage in patients with gastro-oesophageal reflux.

Integrins mediate cell adhesion to the extracellular matrix and initiate intracellular signaling. They play key roles in the central nervous system (CNS), participating in synaptogenesis, synaptic transmission and memory formation, but their precise mechanism of action remains unknown. Here we show that the integrin ligand-mimetic peptide GRGDSP induced NMDA receptor-dependent increases in intracellular calcium levels within seconds of presentation to primary cortical neurons. These were followed by transient activation and nuclear translocation of the ERK1/2 mitogen-activated protein kinase. RGD-induced effects were reduced by the NMDA receptor antagonist MK801, and ERK1/2 signaling was specifically inhibited by ifenprodil and PP2, indicating a functional connection between integrins, Src and NR2B-containing NMDA receptors. GRGDSP peptides were not significantly neuroprotective against excitotoxic insults. These results demonstrate a previously undescribed, extremely rapid effect of RGD peptide binding to integrins on cortical neurons that implies a close, functionally relevant connection between adhesion receptors and synaptic transmission.

For territorial group-living species, opportunities to reproduce on the natal territory can be limited by a number of factors including the availability of resources within a territory, access to unrelated individuals, and monopolies on reproduction by dominant group members. Individuals looking to reproduce are therefore faced with the options of either waiting for a breeding opportunity to arise in the natal territory, or searching for reproductive opportunities in non-natal groups. In the cooperatively breeding Southern pied babbler, Turdoides bicolor, most individuals who achieve reproductive success do so through taking up dominant breeding positions within non-natal groups. For subordinate pied babblers therefore, searching for breeding opportunities in non-natal groups is of primary importance as this represents the major route to reproductive success. However, prospecting (where individuals leave the group to search for reproductive opportunities within other groups) is costly and individuals rapidly lose weight when not part of a group. Here we demonstrate that subordinate pied babblers adopt an alternative strategy for mate attraction by vocal advertisement from within their natal territories. We show that subordinates focus their calling efforts on the edges of their territory, and specifically near boundaries with neighbouring groups that have potential breeding partners (unrelated individuals of the opposite sex). In contrast to prospecting, calling individuals showed no body mass loss associated with this behaviour, suggesting that calling from within the group may provide a 'cheap' advertisement strategy. Additionally, we show that subordinates use information regarding the composition of neighbouring groups to target the greatest number of potential mating partners.

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK–PINCH–kindlin, FAK–paxillin, talin–vinculin and α-actinin–zyxin–VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community. Humphries and colleagues analyse proteomic data of integrin adhesion complexes to derive a consensus integrin adhesome and characterize the temporal dynamics of adhesome component recruitment during adhesion complex assembly and disassembly.

Changes in diet carbohydrate amount and type (i.e., starch vs. fiber) and dietary oil supplements can affect ruminant methane emissions. Our objectives were to measure methane emissions, whole-tract digestibility, and energy and nitrogen utilization from growing dairy cattle at 2 body weight (BW) ranges, fed diets containing either high maize silage (MS) or high grass silage (GS), without or with supplemental oil from extruded linseed (ELS). Four Holstein-Friesian heifers aged 13 mo (BW range from start to finish of 382 to 526 kg) were used in experiment 1, whereas 4 lighter heifers aged 12 mo (BW range from start to finish of 292 to 419 kg) were used in experiment 2. Diets were fed as total mixed rations with forage dry matter (DM) containing high MS or high GS and concentrates in proportions (forage:concentrate, DM basis) of either 75:25 (experiment 1) or 60:40 (experiment 2), respectively. Diets were supplemented without or with ELS (Lintec, BOCM Pauls Ltd., Wherstead, UK; 260 g of oil/kg of DM) at 6% of ration DM. Each experiment was a 4 × 4 Latin square design with 33-d periods, with measurements during d 29 to 33 while animals were housed in respiration chambers. Heifers fed MS at a heavier BW (experiment 1) emitted 20% less methane per unit of DM intake (yield) compared with GS (21.4 vs. 26.6, respectively). However, when repeated with heifers of a lower BW (experiment 2), methane yield did not differ between the 2 diets (26.6g/kg of DM intake). Differences in heifer BW had no overall effect on methane emissions, except when expressed as grams per kilogram of digestible organic matter (OMD) intake (32.4 vs. 36.6, heavy vs. light heifers). Heavier heifers fed MS in experiment 1 had a greater DM intake (9.4kg/d) and lower OMD (755 g/kg), but no difference in N utilization (31% of N intake) compared with heifers fed GS (7.9 kg/d and 799 g/kg, respectively). Tissue energy retention was nearly double for heifers fed MS compared with GS in experiment 1 (15 vs. 8% of energy intake, respectively). Heifers fed MS in experiment 2 had similar DM intake (7.2 kg/d) and retention of energy (5% of intake energy) and N (28% of N intake), compared with GS-fed heifers, but OMD was lower (741 vs. 765 g/kg, respectively). No effect of ELS was noted on any of the variables measured, irrespective of animal BW, and this was likely due to the relatively low amount of supplemental oil provided. Differences in heifer BW did not markedly influence dietary effects on methane emissions. Differences in methane yield were attributable to differences in dietary starch and fiber composition associated with forage type and source.

Purpose Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision‐making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrin‐associated adhesion complex composition. Experimental design Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literature‐based reference set of cell adhesion‐related components and using ontological and protein–protein interaction network analyses. Results Adhesion complex‐specific proteins in MSCs were identified that comprised predominantly cell adhesion‐related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain‐containing proteins in MSC adhesion complexes were highlighted, which may act as force‐sensing components. Conclusion and clinical relevance These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention.

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry.

IntroductionReproducibility is a defining feature of science, but the extent to which it characterizes current research is unknown. Scientific claims should not gain credence because of the status or authority of their originator but by the replicability of their supporting evidence. Even research of exemplary quality may have irreproducible empirical findings because of random or systematic error.RationaleThere is concern about the rate and predictors of reproducibility, but limited evidence. Potentially problematic practices include selective reporting, selective analysis, and insufficient specification of the conditions necessary or sufficient to obtain the results. Direct replication is the attempt to recreate the conditions believed sufficient for obtaining a previously observed finding and is the means of establishing reproducibility of a finding with new data. We conducted a large-scale, collaborative effort to obtain an initial estimate of the reproducibility of psychological science.ResultsWe conducted replications of 100 experimental and correlational studies published in three psychology journals using high-powered designs and original materials when available. There is no single standard for evaluating replication success. Here, we evaluated reproducibility using significance and P values, effect sizes, subjective assessments of replication teams, and meta-analysis of effect sizes. The mean effect size (r) of the replication effects (Mr = 0.197, SD = 0.257) was half the magnitude of the mean effect size of the original effects (Mr = 0.403, SD = 0.188), representing a substantial decline. Ninety-seven percent of original studies had significant results (P < .05). Thirty-six percent of replications had significant results; 47% of original effect sizes were in the 95% confidence interval of the replication effect size; 39% of effects were subjectively rated to have replicated the original result; and if no bias in original results is assumed, combining original and replication results left 68% with statistically significant effects. Correlational tests suggest that replication success was better predicted by the strength of original evidence than by characteristics of the original and replication teams.ConclusionNo single indicator sufficiently describes replication success, and the five indicators examined here are not the only ways to evaluate reproducibility. Nonetheless, collectively these results offer a clear conclusion: A large portion of replications produced weaker evidence for the original findings despite using materials provided by the original authors, review in advance for methodological fidelity, and high statistical power to detect the original effect sizes. Moreover, correlational evidence is consistent with the conclusion that variation in the strength of initial evidence (such as original P value) was more predictive of replication success than variation in the characteristics of the teams conducting the research (such as experience and expertise). The latter factors certainly can influence replication success, but they did not appear to do so here. Reproducibility is not well understood because the incentives for individual scientists prioritize novelty over replication. Innovation is the engine of discovery and is vital for a productive, effective scientific enterprise. However, innovative ideas become old news fast. Journal reviewers and editors may dismiss a new test of a published idea as unoriginal. The claim that “we already know this” belies the uncertainty of scientific evidence. Innovation points out paths that are possible; replication points out paths that are likely; progress relies on both. Replication can increase certainty when findings are reproduced and promote innovation when they are not. This project provides accumulating evidence for many findings in psychological research and suggests that there is still more work to do to verify whether we know what we think we know.

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the “linker domain” (amino acids 38-47), with inhibitory activity toward α5β1-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward α5β1-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO α5 cell adhesion on recombinant Fn III6-10 fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III9 synergy site within the Fn III6-10 substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting α5β1 integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic α5β1 inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.

The study was designed to determine the excretion balance of radiolabeled rabeprazole in urine and feces and to examine the metabolite profile in plasma, urine and feces after a single oral dose of [14C] rabeprazole, preceded by once daily dose of rabeprazole for 7 days. Six healthy subjects were enrolled in this study. The study was a single-center, open-label, multiple-dose, mass-balance study. Each subject received a single 20 mg dose of rabeprazole tablet for 7 days followed by the administration of 20 mg of [14C] rabeprazole as an oral solution after an overnight fast on Day 8. After oral dosing of [14C] rabeprazole, the mean Cmax of total radioactivity was 1,080 +/- 215 ng equivalent/ml with 0.33 +/- 0.13 hours of the mean tmax. The apparent elimination half-life of total [14C] radioactivity was 12.6 +/- 3.4 hours. The total [14C] recovery in urine and feces was 99.8 +/-0.7% by 168 hours after oral administration of [14C] rabeprazole, and mean cumulative [14C] radioactivity excreted in urine was 90.0 +/- 1.7% by 168 hours and 79.8 +/- 2.5% of the radioactivity was excreted in urine within 24 hours. Excretion via feces added to the total by 9.8%. The major radioactive component in the early plasma samples was rabeprazole, however the thioether and thioether carboxylic acid metabolites were the main radioactive components in the later plasma sample. These results support the previous finding that the substantial contribution of the non-enzymatic thioether pathway minimizes the effect of CYP2C19 polymorphism on the inter-individual variation ofplasma clearance of rabeprazole compared with other PPIs. Low levels of the sulfone metabolite were detected only in early plasma samples. No rabeprazole was detected in any urine and feces samples. The main radioactive components in urine were thioether carboxylic acid and mercapturic acid conjugate metabolites, and in the feces, the thioether carboxylic acid metabolite. The administration of [14C] rabeprazole was safe as evidenced by the lack of serious adverse events and the fact that all observed events were mild in intensity. [14C] rabeprazole was rapidly absorbed after oral administration and mostly excreted in urine.

SUMMARY Background Omeprazole and lansoprazole are both of proven efficacy in the treatment of Zollinger‐Ellison syndrome and idiopathic gastric acid hypersecretion. Rabeprazole, which has a similar mechanism of action, has not previously been studied in these diseases. Aim To determine the dose of rabeprazole that decreased basal acid output to safe levels in patients with Zollinger‐Ellison syndrome or idiopathic gastric acid hypersecretion. Methods Patients with Zollinger‐Ellison syndrome or idiopathic gastric acid hypersecretion were given rabeprazole 60 mg once daily for uncomplicated disease or 40 mg twice daily for complicated disease. Doses were titrated according to response and continued for 2 years. Efficacy was assessed primarily by measuring basal acid output. Results All patients had basal acid output before the next dose controlled to

Eight Jersey cows were used in two balanced 4 × 4 Latin Squares to investigate the effects of replacement of dietary starch with non-forage fibre on productivity, diet digestibility and feeding behaviour. Total-mixed rations consisted of maize silage, grass silage and a soyabean meal-based concentrate mixture, each at 250 g/kg DM, with the remaining 250 g consisting of cracked wheat/soya hulls (SH) in the ratios of 250 : 0, 167 : 83; 83 : 167 and 0 : 250 g, respectively, for treatments SH0, SH83, SH167 and SH250. Starch concentrations were 302, 248, 193 and 140 g/kg DM, and NDF concentrations were 316, 355, 394 and 434 g/kg DM, for treatments SH0, SH83, SH167 and SH250, respectively. Total eating time increased (p

Background— Essential to tissue-engineered vascular grafts is the formation of a functional endothelial monolayer capable of resisting the forces of blood flow. This study targeted α 2 (VIII) collagen, a major component of the subendothelial matrix, and examined the ability of and mechanisms by which endothelial cells attach to this collagen under static and dynamic conditions both in vitro and in vivo. Methods and Results— Attachment of human endothelial cells to recombinant α 2 (VIII) collagen was assessed in vitro under static and shear conditions of up to 100 dyne/cm 2 . α 2 (VIII) collagen supported endothelial cell attachment in a dose-dependent manner, with an 18-fold higher affinity for endothelial cells compared with fibronectin. Cell attachment was significantly inhibited by function-blocking anti-α 2 (56%) and -β 1 (98%) integrin antibodies but was not RGD dependent. Under flow, endothelial cells were retained at significantly higher levels on α 2 (VIII) collagen (53% and 51%) than either fibronectin (23% and 16%) or glass substrata (7% and 1%) at shear rates of 30 and 60 dyne/cm 2 , respectively. In vivo studies, using endothelialized polyurethane grafts, demonstrated significantly higher cell retention rates to α 2 (VIII) collagen-coated than to fibronectin-coated prostheses in the midgraft area ( P Conclusions— These studies demonstrate that α 2 (VIII) collagen has the potential to improve both initial cell attachment and retention of endothelial cells on vascular grafts in vivo, which opens new avenues of research into the development of single-stage endothelialized prostheses and the next generation of tissue-engineered vascular grafts.

Protein kinase C (PKC) delta is an essential regulator of mitochondrial dependent apoptosis in epithelial cells. We have used the PKCdelta(-/-) mouse to ask if loss of PKCdelta protects salivary glands against gamma-irradiation-induced apoptosis in vivo and to explore the mechanism underlying protection from apoptosis. We show that gamma-irradiation in vivo results in a robust induction of apoptosis in the parotid glands of wild type mice, whereas apoptosis is suppressed by greater than 60% in the parotid glands of PKCdelta(-/-) mice. Primary parotid cells from PKCdelta(-/-) mice are defective in mitochondrial dependent apoptosis as indicated by suppression of etoposide-induced cytochrome c release, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. Notably, apoptotic responsiveness can be restored by re-introduction of PKCdelta by adenoviral transduction. Etoposide and gamma-irradiation-induced activation of p53 is similar in primary parotid cells and parotid glands from PKCdelta(+/+) and PKCdelta(-/-) mice, indicating that PKCdelta functions downstream of the DNA damage response. In contrast, activation of the c-Jun amino-terminal kinase is reduced in primary parotid cells from PKCdelta(-/-) cells and in parotid C5 cells, which express a dominant inhibitory mutant of PKCdelta. Similarly, c-Jun amino-terminal kinase activation is suppressed in vivo in gamma-irradiated parotid glands from PKCdelta(-/-) mice. These studies indicate an essential role for PKCdelta downstream of the p53 response and upstream of the c-Jun amino-terminal kinase activation in DNA damage-induced apoptosis in vivo and in vitro.

MIA (melanoma inhibitory activity) has been identified as a small protein secreted from malignant melanoma cells, which interacts with extracellular matrix proteins including fibronectin. Here, we show that MIA negatively regulates the activity of the mitogen-activated protein kinase pathway in malignant melanoma. Using far Western blotting and co-immunoprecipitation we searched for MIA-binding cell surface proteins. We found that MIA interacts with integrin alpha4beta1 and alpha5beta1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings also suggest that MIA may play a role in tumor progression and the spread of malignant melanomas via mediating detachment of cells from extracellular matrix molecules by modulating integrin activity. Inhibiting MIA functions in vivo may therefore provide a novel therapeutic strategy for metastatic melanoma disease.

Rota conjectured that, given $n$ disjoint bases of a rank-n matroid M, there are n disjoint transversals of these bases that are all bases of M. We prove a stronger statement for the class of paving matroids.

Aim To discover the pattern of relationships of areas of endemism for Australian genera in the plant family Rhamnaceae tribe Pomaderreae for comparison with other taxa and interpretation of biogeographical history. Location Australian mainland, Tasmania and New Zealand. Methods A molecular phylogeny and geographic distribution of species within four clades of Pomaderreae are used as a basis for recognition of areas of endemism and analysis of area relationships using paralogy-free subtrees. The taxon phylogeny is the strict consensus tree from a parsimony analysis of 54 taxa, in four clades, and sequence data for the internal transcribed spacer regions of ribosomal DNA (ITS1-5.8S-ITS2) and the plastid DNA region trnL-F. Results The biogeographical analysis identified five subtrees, which, after parsimony analysis, resulted in a minimal tree with 100% consistency and seven resolved nodes. Three sets of area relationships were identified: the areas of Arnhem and Kimberley in tropical north Australia are related based on the phylogeny of taxa within Cryptandra; the moister South-west of Western Australia, its sister area the coastal Geraldton Sandplains, the semi-arid Interzone region and arid Western Desert are related, based on taxa within Cryptandra, Spyridium, Trymalium and Pomaderris; and the eastern regions of Queensland, McPherson-Macleay, south-eastern New South Wales (NSW), Victoria, southern Australia, Tasmania and New Zealand are related based on Cryptandra, Pomaderris and Spyridium. Tasmania and NSW are related based entirely on Cryptandra, but the position of New Zealand relative to the other south-eastern Australian regions is unresolved. Main conclusions The method of paralogy-free subtrees identified a general pattern of geographic area relationships based on Australian Pomaderreae. The widespread distribution of clades, the high level of endemicity and the age of fossils for the family, suggest that the Pomaderreae are an old group among the Australian flora. Their biogeographical history may date to the early Palaeogene with subsequent changes through to the Pleistocene.

Although the cell line K562 reportedly expresses a single species of beta1 integrin, alpha5beta1, surface staining with monoclonal antibodies JB1A, 12G10 and B3B11 to the beta1 chain clearly demonstrated differences in the expression levels of the epitopes detected by these antibodies. The present studies were initiated to determine the basis for this molecular heterogeneity in the integrins. Cross-linking of surface integrins with B3B11 caused their selective aggregation. This distribution was similar to that observed for the alpha5 chain. In contrast, cross-linking the beta1 chains with 12G10 did not cause codistribution of alpha5, suggesting that these two species were not associated on the cell surface. Immunoprecipitates of the surface integrins of K562 cells indicated the presence of 120 and 140 kDa forms of the beta1 chain which were detected by 12G10 and B3B11, respectively. Immunological, biochemical and mass spectrometric analysis of K562 surface integrins also failed to demonstrate the presence of any alpha chain in association with the 120 kDa species of beta1 of K562 cells. Treatment of the two forms of beta1 with PGNase reduced their masses to approximately 90 kDa, suggesting that N-glycosylation was responsible for the mass differences. Collectively, these results provide evidence for a novel species of beta1 on the cell surface, which does not appear to be associated with any alpha chain. The data also suggest that differences in glycosylation may be involved in defining the association between the integrin alpha and beta chains and the functional properties of these integrins.

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGF beta in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-beta (TGF beta) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGFbeta into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full-length fibronectin and superfibronectin, which are capable of assembly into fibronectin fibrils, but not by other fibronectin fragments, including a 160-kDa RGD-containing fragment that activates alpha5beta1 integrins. This suggests that the critical event for LTBP1 assembly is the formation of a fibronectin fibrillar network and that integrin ligation by fibronectin molecules alone is not sufficient. Not only was fibronectin essential for the initial incorporation of LTBP1 into the ECM, but the continued presence of fibronectin was required for the continued assembly of LTBP1. These studies highlight a nonredundant role for fibronectin in LTBP1 assembly into the ECM and suggest a novel role for fibronectin in regulation of TGF beta via LTBP1 interactions.

Aim The aim of this paper was to revise the historical biogeographical method for resolving complicated distribution patterns through a technique that has come to be called assumption 2. Assumption 2 was used to resolve multiple areas on a single terminal branch (masts) as well as paralogous and missing areas in two or more areagrams. Recent examples, however, have shown that assumption 2 may be using rather than resolving paralogy. The paper attempts to resolve this problem by formulating a separate procedure to avoid using paralogous (redundant) area data in area cladistic analyses. Method The revision results in a new derivative method, the transparent method, to replace assumptions 1 and 2. It separates the procedures for resolving paralogy and for solving distribution patterns that occur in more than one area (masts). Results Several hypothetical examples show how the transparent method reduces paralogy and masts. The results show that paralogy can be reduced if the paralogy subtree method is applied after uncovering all possible relationships as single components on the terminals of areagrams. Conclusion The transparent method is a significant step forward in cladistic biogeography as it utilizes area relationships rather than generating general areagrams based on paralogous data.

The activation of bis(imino)pyridine cobalt(II) precatalysts by MAO leads initially to a bis(imino)pyridine cobalt(I) cationic species with no cobalt−C(alkyl) bond into which insertion can occur. M...

Integrins are dynamic membrane proteins that mediate adhesion of cells to the extracellular matrix. Integrins initiate signal transduction, alone and cooperatively with growth factor receptors, and regulate many aspects of cell behavior. We report here that alpha5beta1-mediated adhesion of Ntera2 neuronal cells to fibronectin decreased apoptosis in response to serum withdrawal. Adhesion induced phosphorylation of FAK, and strongly increased the AKT phosphorylation induced by growth factors, demonstrating for the first time in neuronal cells that integrin-mediated adhesion and growth factors cooperate to regulate AKT activity. Integrins exist on cells in different activation states, and cell survival on fibronectin was enhanced by the antibody 12G10, that modulates the conformation of beta1 in favor of its active form. The antibody 12G10 specifically delayed loss of phosphorylation of AKT on serine 473, and GSK-3beta on serine 9, induced by serum withdrawal, suggesting that these kinases are critical sensors of integrin activation on neuronal cells.

Integrin adhesion receptors are structurally dynamic proteins that adopt a number of functionally relevant conformations. We have produced a conformation-dependent anti-alpha5 monoclonal antibody (SNAKA51) that converts alpha5beta1 integrin into a ligand-competent form and promotes fibronectin binding. In adherent fibroblasts, SNAKA51 preferentially bound to integrins in fibrillar adhesions. Clustering of integrins expressing this activation epitope induced directional translocation of alpha5beta1, mimicking fibrillar adhesion formation. Priming of alpha5beta1 integrin by SNAKA51 increased the accumulation of detergent-resistant fibronectin in the extracellular matrix, thus identifying an integrin conformation that promotes matrix assembly. The SNAKA51 epitope was mapped to the calf-1/calf-2 domains. We propose that the action of the antibody causes the legs of the integrin to change conformation and thereby primes the integrin to bind ligand. These findings identify SNAKA51 as the first anti-integrin antibody to selectively recognize a subset of adhesion contacts, and they identify an integrin conformation associated with integrin translocation and fibronectin matrix formation.

A more complete understanding of amino acid (AA) metabolism by the various tissues of the body is required to improve upon current systems for predicting the use of absorbed AA. The objective of this work was to construct and parameterize a model of net removal of AA by the portal-drained viscera (PDV). Six cows were prepared with arterial, portal, and hepatic catheters and infused abomasally with 0, 200, 400, or 600 g of casein daily. Casein infusion increased milk yield quadratically and tended to increase milk protein yield quadratically. Arterial concentrations of a number of essential AA increased linearly with respect to infusion amount. When infused casein was assumed to have a true digestion coefficient of 0.95, the minimum likely true digestion coefficient for noninfused duodenal protein was found to be 0.80. Net PDV use of AA appeared to be linearly related to total supply (arterial plus absorption), and extraction percentages ranged from 0.5 to 7.25% for essential AA. Prediction errors for portal vein AA concentrations ranged from 4 to 9% of the observed mean concentrations. Removal of AA by PDV represented approximately 33% of total postabsorptive catabolic use, including use during absorption but excluding use for milk protein synthesis, and was apparently adequate to support endogenous N losses in feces of 18.4 g/d. As 69% of this use was from arterial blood, increased PDV catabolism of AA in part represents increased absorption of AA in excess of amounts required by other body tissues. Based on the present model, increased anabolic use of AA in the mammary and other tissues would reduce the catabolic use of AA by the PDV.

Intercellular adhesion mediated by integrin alpha4beta1 and vascular cell adhesion molecule-1 (VCAM-1) plays a crucial role in both the rolling and firm attachment of leukocytes onto the vascular endothelium. Essential to the alpha4beta1/VCAM-1 interaction is its mechanical strength that allows the complex to resist the large shear forces imposed by the bloodstream. Herein we employed single-molecule dynamic force spectroscopy to investigate the dynamic strength of the alpha4beta1/VCAM-1 complex. Our force measurements revealed that the dissociation of the alpha4beta1/VCAM-1 complex involves overcoming at least two activation potential barriers: a steep inner barrier and a more elevated outer barrier. The inner barrier grants the complex the tensile strength to withstand large pulling forces (>50 pN) and was attributed to the ionic interaction between the chelated Mg2+ ion at the N-terminal A-domain of the beta1 subunit of alpha4beta1 and the carboxyl group of Asp-40 of VCAM-1 through the use of site-directed mutations. In general, additional mutations within the C-D loop of domain 1 of VCAM-1 suppressed both inner and outer barriers of the alpha4beta1/VCAM-1 complex, while a mutation at Asp-143 of domain 2 of VCAM-1 resulted in the suppression of the outer barrier, but not the inner barrier. In contrast, the outer barrier of alpha4beta1/VCAM-1 complex was stabilized by integrin activation. Together, these findings provide a molecular explanation for the functionally relevant kinetic properties of the alpha4beta1/VCAM-1 interaction.

While it is self-evident that all extracellular molecules are an integral part of a multicellular organism, it is paradoxical that they are often considered to be dissociated from cells. The reality is that a continuum of dynamic, bi-directional interactions links the intracellular environment through cell-surface receptors to multimolecular extracellular assemblies. These interactions not only control the behaviour of individual cells, but also determine tissue architecture. Adhesion receptor function is partly determined by an ability to tether the contractile cytoskeleton to the plasma membrane, but there is also evidence that integrin receptors modulate signalling events that are essential for cellular differentiation. A major challenge is now to integrate work at the atomic, molecular and cellular levels, and obtain holistic insights into the mechanisms controlling cell adhesion. In the present study, we review current knowledge of the molecular mechanisms employed by cells to integrate with the extracellular matrix. Two main topics are covered: the adaptation of integrin structure for bi-directional signalling and the integration of integrin signalling with other receptors.