Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.     

A dynamic analysis of the shade-induced plasticity in Arabidopsis thaliana rosette leaf development reveals new components of the shade-adaptative response

BACKGROUND AND AIMS: It is well known that plant aerial development is affected by light intensity in terms of the date of flowering, the length of stems and petioles, and the final individual leaf area. The aim of the work presented here was to analyse how shade-induced changes in leaf development occur on a dynamic basis from the whole rosette level to that of the cells. METHODS: Care was taken to ensure that light intensity was the only source of micro-meteorological variation in the study. The dynamics of leaf production, rosette expansion, individual leaf area expansion and epidermal cell expansion were analysed in Arabidopsis thaliana plants grown under two light intensities in three independent experiments. KEY RESULTS: The total area of rosette leaves was reduced by the shading treatment. Both the number of leaves produced and their individual leaf areas were reduced. The reduction in leaf number was associated with a reduction in leaf initiation rate and the duration of the phase of leaf production. The reduction in individual leaf area was associated with a reduction in leaf expansion rate and an increase in the duration of leaf expansion. The changes in leaf expansion dynamics were accompanied by a decrease in epidermal cell number which was partly compensated for by an increase in epidermal cell area. Overall, the whole rosette leaf expansion rate was reduced by shading, whereas the total duration of rosette leaf expansion was unaffected. This was mainly due to the accumulation of the increases in the durations of expansion of each individual leaf which was associated with an increase in cell expansion. CONCLUSIONS: The dynamic analysis presented here reveals a new shade-adaptative response mediated via the control of area expansion at the cell, organ and whole plant levels.     

Sexual dimorphism in the renin-angiotensin system in aging spontaneously hypertensive rats

In young adult spontaneously hypertensive rats (SHR), mean arterial pressure (MAP) is higher in males than in females and inhibition of the renin-angiotensin system (RAS) eliminates this sex difference. After cessation of estrous cycling in female SHR, MAP is similar to that in male SHR. The purpose of this study was to determine the role of the RAS in maintenance of hypertension in aging male and female SHR. At 16 mo of age, MAP was similar in male and female SHR (183+/-5 vs. 193+/-8 mmHg), and chronic losartan (40 mg.kg-1.day-1 po for 3 wk) reduced MAP by 52% (to 90+/-8 mmHg, P<0.05 vs. control) in males and 37% (to 123+/-11 mmHg, P<0.05 vs. control) in females (P<0.05, females vs. males). The effect of losartan on angiotensin type 1 (AT1) receptor blockade was similar: MAP responses to acute doses of ANG II (62.5-250 ng/kg) were blocked to a similar extent in losartan-treated males and females. F2-isoprostane excretion was reduced with losartan more in males than in females. There were no sex differences in plasma renin activity, plasma angiotensinogen or ANG II, or renal expression of AT1 receptors, angiotensin-converting enzyme, or renin. However, renal angiotensinogen mRNA and protein expression was higher in old males than females, whereas renal ANG II was higher in old females than males. The data show that, in aging SHR, when blood pressures are similar, there remains a sexual dimorphism in the response to AT1 receptor antagonism, and the differences may involve sex differences in mechanisms responsible for oxidative stress with aging.     

Effects of acute hyperosmolality on blood-brain barrier function in ovine fetuses and lambs

We examined the effects of hyperosmolality on blood-brain barrier (BBB) permeability during development to test the vulnerability of the immature barrier to stress. The BBB response to hyperosmolality was quantified using the blood-to-brain transfer constant (Ki) with alpha-aminoisobutyric acid in fetuses at 60% and 90% gestation, premature, newborn, and older lambs. Ki plotted against osmolality increased as a function of increases in osmolality in all groups and brain regions. The relationship was described (P < 0.05) by a segmented regression model. At lower osmolalities, changes in Ki were minimal, but after a break point (threshold) was reached, the increase (P < 0.05) was linear. We examined the responses of Ki to hyperosmolality within each brain region by comparing the thresholds and slopes of the second regression segment. Lower thresholds and higher slopes imply greater vulnerability to hyperosmolality in the younger groups. Thresholds increased (P < 0.05) with development in the thalamus, superior colliculus, pons, and spinal cord, and slopes of the second regression segment decreased (P < 0.05) in the cerebellum, hippocampus, inferior colliculus, medulla, and spinal cord. BBB resistance to hyperosmolality increased (P < 0.05) with development in most brain regions. The pattern of the Ki plotted against osmolality was (P < 0.05) heterogenous among brain regions in fetuses and premature and newborn lambs, but not in older lambs. We conclude that 1) BBB permeability increased as a function of changes in osmolality, 2) the barrier becomes more resistant to hyperosmolality during development, and 3) the permeability response to hyperosmolality is heterogenous among brain regions in fetuses and premature and newborn lambs.     

PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit

The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit.     

Hematology, serum chemistry, and serology of Galápagos penguins (Spheniscus mendiculus) in the Galápagos Islands, Ecuador

The Galápagos penguin (Spheniscus mendiculus) is an endangered species endemic to the Galápagos Islands, Ecuador. In 2003 and 2004, 195 penguins from 13 colonies on the islands of Isabela and Fernandina in the Galápagos archipelago were examined. Genetic sexing of 157 penguins revealed 62 females and 95 males. Hematology consisted of packed cell volume (n = 134), white blood cell differentials (n = 83), and hemoparasite blood smear evaluation (n = 114). Microfilariae were detected in 22% (25/114) of the blood smears. Female penguins had significantly higher eosinophil counts than males. Serum chemistry on 83 penguins revealed no significant differences between males and females. Birds were seronegative to avian paramyxovirus type 1-3, avian influenza virus, infectious bursal disease virus, Marek's disease virus (herpes), reovirus, avian encephalomyelitis virus, and avian adenovirus type 1 and 2 (n = 75), as well as to West Nile virus (n = 87), and Venezuelan, western and eastern equine encephalitis viruses (n = 26). Seventy-five of 84 (89%) penguins had antibodies to Chlamydophila psittaci but chlamydial DNA was not detected via polymerase chain reaction in samples from 30 birds.     

The anatomy of organogenesis: novel solutions to old problems

Understanding anatomical aspects of mammalian organ development, in both normal and mutant animals, is important for basic biology and also for regenerative medicine and tissue engineering. The size and complexity of developing organs, together with variations in their detailed anatomy, has made the obtaining of high-resolution time-courses of anatomical change difficult to obtain. The fact that organ development tends to use the same genes as early embryogenesis also makes genetic manipulation difficult, as so many mutant embryos die before organogenesis begins. These problems have seriously hampered the study of organogenesis. Here, we describe three significant advances that promise solutions: (1) the production of GFP-reporter mice that can be used for high-resolution live-imaging of small tissues as they grow, (2) RNA interference, which allows the manipulation of specific genes at any stage of organ development, and (3) optical projection tomography, which allows medium-resolution three-dimensional images of complete embryos to be obtained easily. We finish by looking ahead to the prospects of uniting these three technologies to allow accurate, high-throughput screening of mutants and automated comparison of biological data with computer predictions.     

Plasticity to soil water deficit in Arabidopsis thaliana: dissection of leaf development into underlying growth dynamic and cellular variables reveals invisible phenotypes

Genetic variability in the plasticity of leaf area expansion in response to water deficit has been reported in Arabidopsis thaliana. Here, the objective was to identify the underlying dynamic and cellular processes involved in this variability. Twenty-five accessions were subjected to identical soil water deficit treatments. In all accessions, the plasticity of leaf production was low compared with that of individual leaf expansion. A subset of accessions was selected for further dissection of individual leaf expansion into its underlying variables: the rate and duration of leaf expansion and epidermal cell number and area. In all accessions, water deficit had opposite effects on the rate and duration of leaf expansion. The accumulation of these effects was reflected in changes in final leaf area. At the cellular level, moderate water deficits had opposite effects on cell number and cell size, but more severe ones reduced both variables. The importance of these opposing effects is highlighted by the behaviour of the accession An-1, for which the compensation between the decrease in leaf expansion rate and the increase in the duration of expansion is total. This dynamic plasticity in response to water deficit is not detectable when only final measurements are done.     

Temporal and Spatial Distribution Patterns of Larval Trichoptera in Madeiran Streams

Temporal and spatial distribution patterns of lotic larval trichopteran assemblages in relation to environmental variables were investigated in Madeiran streams using multivariate analyses. TWINSPAN classification detected distinct faunal assemblages related to spatial factors between non-polluted high altitude sites and lower lying enriched sites where tolerant taxa were predominant but showed strong seasonal shifts in species composition and abundance. The 15 TWINSPAN end groups were grouped into five arbitrary clusters based upon the seasonal and spatial changes in the trichopteran assemblages detected by the analysis. Significant differences between environmental variables (distance from source, altitude, temperature, conductivity, alkalinity and nitrate) and the trichopteran assemblages (using trichopteran based metrics) of these clusters were confirmed by the Kruskal-Wallis test (H) and Dunn’s test. Chemical classification of samples within the clusters revealed a strong association between trichopteran assemblages and water quality. Canonical Correspondence Analysis and Monte Carlo global permutation tests also identified significant associations between the larval assemblages and physicochemical variables such as temperature and conductivity along a strong physical gradient (altitude, slope) and nitrate along a weaker seasonal gradient. Analysis of functional feeding group distribution patterns clearly showed that mid to high altitude indigenous woodland sites were trophically diverse whilst the lower reaches of the islands streams are trophically impoverished with strong seasonal shifts between two feeding groups of enrichment tolerant taxa. Trichopteran shredders are exclusive to indigenous woodland sites, indicating a limited distribution associated with land use, allochthonous input and habitat destruction. The results indicate that several ‘environmental filters’ operate at different levels upon the islands trichopteran fauna, producing temporally and spatially distinct ‘subsets’ of species best able to exploit conditions and resources at a given site or time, confounding the direct comparison of these insular systems with the findings of the River Continuum Concept, traditionally associated with unaffected continental lotic systems.