Effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina

The effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina were tested in laboratory bioassays. In all three species egg mortality and time needed for embryonic development to hatching increased when pH declined, but no significant effects were found on embryonic development when aluminium level was elevated. In R. arvalis and R. temporaria larval mortality was affected by both pH and aluminium. In both species the frequency of occurrence of larval deformities increased in acid water, and there was a synergistic effect of pH and aluminium. In R. arvalis swimming behaviour was disturbed by high levels of aluminium at pH 5. In all three species the frequency of stressed larvae increased when pH was depressed and aluminium concentration elevated, and there was a synergistic effect when both were combined. The three species differed significantly in egg mortality, time needed for embryonic development, larval mortality, larval deformities and larval stress al low pH and high aluminium levels. R. arvalis showed the highest acid tolerance and R. dalmatina was the most sensitive to low pH.     

Objective Assessment of Nuclear and Cortical Cataracts through Scheimpflug Images: Agreement with the LOCS III Scale

PurposeTo assess nuclear and cortical opacities through the objective analysis of Scheimpflug images, and to check the correlation with the Lens Opacity Classification System III (LOCS III).MethodsNuclear and cortical opacities were graded according to the LOCS III rules after pupil dilation. The maximum and average pixel intensity values along an elliptical mask within the lens nucleus were taken to analyse nuclear cataracts. A new metric based on the percentage of opaque pixels within a region of interest was used to analyse cortical cataracts. The percentage of opaque pixels was also calculated for half, third and quarter areas from the region of interest’s periphery.ResultsThe maximum and average intensity values along the nucleus were directly proportional to the LOCS III grade: The larger the LOCS III value, the larger maximum and average intensity ones. These metrics showed a positive and significant correlation with the LOCS grade: The larger the LOCS grade, the higher was percentage of opaque pixels along the cortex within the same mask’s size. This metric showed a significant correlation to the LOCS grade.ConclusionThe metrics used to assess nuclear opacities showed good correlation with the LOCS III. The percentage of opaque pixels showed to be a useful metric to measure objectively the severity of the cortical opacity. These metrics could be implemented in an algorithm to detect and grade lens opacities automatically and objectively.     

Characterization of Saccharomyces cerevisiae Nop17p, a novel Nop58p-interacting protein that is involved in Pre-rRNA processing

In eukaryotes, pre-rRNA processing depends on cis-acting elements and on a large number of non-ribosomal trans-acting factors, including endonucleases and exonucleases, RNA helicases, rRNA modifying enzymes and components of snoRNPs. The exosome is a conserved eukaryotic protein complex containing multiple 3'-5' exonucleases, which has been implicated in pre-rRNA, snoRNA and snRNA processing, as well as in mRNA degradation. In order to identify new proteins involved in rRNA processing, we have screened a yeast two-hybrid cDNA library, to isolate proteins interacting with the exosome subunit Rrp43p. In this screen, a novel nucleolar protein, Nop17p, was identified which also interacts with the box C/D snoRNP protein Nop58p. The NOP17 gene is not essential for cell viability but its deletion causes a temperature-sensitive phenotype. Pre-rRNA processing analyses revealed that rRNA formation is affected in the Deltanop17 strain subjected to the non-permissive temperature, although it is not blocked completely. In addition, primer extension analyses of RNA isolated from Nop17p-depleted cells subjected to the non-permissive temperature indicates that the pre-rRNA is undergoing different modification or degradation processes in these cells as compared to the parental strain. Nop17p was recently described in the same complex as Nop58p and, interestingly, its depletion leads to mislocalization of Nop1p, Nop56p, Nop58p and Snu13p, which are the core proteins of the box C/D ribonucleoprotein (snoRNP), indicating that Nop17p function is required either for nucleolar retention or for the proper assembly of the box C/D snoRNP.     

Ultrastructural studies of muscle cells and vascular endothelium immediately after freeze-thaw injury

Little is known about the ultrastructural changes which occur in vascular endothelium immediately after an in vivo freezethaw insult, although most investigators will agree that tissue viability relates directly to the degree of vascular damage. In this study an electron microscopic examination of an in vivo model for frostbite injury was initiated. The horseradish peroxidase technique was utilized to follow early alterations in capillary flow and the independent effects of hypoxia, cooling to 2 °C, supercooling, and a single freeze-thaw insult were assessed. No precipitous changes in muscle cell mitochondria or capillary endothelium were detected as a result of brief hypoxia, cooling at 2 °C, or supercooling to ?13 °C. Reducing the temperature by 1 °C/min until freezing occurred, continuing to cool for 10 min postheat of fusion, and rapidly rewarming resulted in consistent mitochondrial damage in muscle cells and marked degeneration of associated capillaries. Peroxidase injected iv prior to thawing was rarely localized in the capillaries of previously frozen muscle. Since peroxidase was found in the capillaries of unfrozen legs of the same animals, it is inferred that little or no flow occurred in most capillaries postthaw. Ultrastructural integrity of capillaries immediately after thawing may be a good index for predicting tissue loss.“In conducting the research described in this report, the investigators adhered to the ‘Guide for Laboratory Animal Facilities and Care,’ as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council.”     

The structure-function relationship of I-A molecules: correlation of serologic and functional phenotypes of four I-Ak mutant cell lines

We have isolated and characterized four mutant I-Ak-expressing cell lines derived from the B cell-B lymphoma hybrid antigen-presenting cell line TA3. The mutants were isolated by first selecting against expression of one Ak epitope by treatment with a monoclonal antibody in the presence of complement and then selecting for retention of a second Ak epitope by electronic cell-sorting of cells stained for fluorescence with a second monoclonal antibody. The serologic and functional phenotypes of the mutants were characterized by using panels of I-Ak-specific monoclonal antibodies and I-Ak-restricted T hybridomas. We obtained one Ak alpha mutant (J4) that no longer reacts with any Ak alpha-specific antibody and also is incapable of stimulating any I-Ak-restricted T hybridoma. We obtained three Ak beta mutants (LD3, K5, G1) that express a wide range of serologic and functional phenotypes. Correlation of the serologic and functional phenotypes reveals that the serologic epitope Ia.1 may overlap with a major site of T cell recognition, whereas the Ia.17 serologic epitope appears to be only a minor site for T cell recognition.     

A model including photoperiod in degree days for estimating Hevea bud growth

 The aim of this study was to verify the applicability of a heat-sum model and an alternative model that included photoperiod to describe Hevea bud growth. Considering 19° C as the base temperature, approx. 172 degree days were required for bud growth up to leaf blade expansion. The applicability of the heat-sum model for Hevea bud growth was confirmed. The inclusion of day length as an exponential term in accumulated degree days resulted in the model fitting much better for the spring than for the winter conditions. Received: 21 May 1995 / Revised: 28 October 1996 / Accepted: 27 January 1997     

The α-Subunit of the Arabidopsis Heterotrimeric G Protein,GPA1, Is a Regulator of Transpiration Efficiency

Land plants must balance CO₂ assimilation with transpiration in order to minimize drought stress and maximize their reproductive success. The ratio of assimilation to transpiration is called transpiration efficiency (TE). TE is under genetic control, although only one specific gene, ERECTA, has been shown to regulate TE. We have found that the α-subunit of the heterotrimeric G protein in Arabidopsis (Arabidopsis thaliana), GPA1, is a regulator of TE. gpa1 mutants, despite having guard cells that are hyposensitive to abscisic acid-induced inhibition of stomatal opening, have increased TE under ample water and drought stress conditions and when treated with exogenous abscisic acid. Leaf-level gas-exchange analysis shows that gpa1 mutants have wild-type assimilation versus internal CO₂ concentration responses but exhibit reduced stomatal conductance compared with ecotype Columbia at ambient and below-ambient internal CO₂ concentrations. The increased TE and reduced whole leaf stomatal conductance of gpa1 can be primarily attributed to stomatal density, which is reduced in gpa1 mutants. GPA1 regulates stomatal density via the control of epidermal cell size and stomata formation. GPA1 promoter::β-glucuronidase lines indicate that the GPA1 promoter is active in the stomatal cell lineage, further supporting a function for GPA1 in stomatal development in true leaves.Land plants, in particular plants that utilize C₃ photosynthesis, must balance CO₂ acquisition with water loss in order to maximize fitness. The water loss cost per unit of biomass acquired can be expressed as transpiration efficiency (TE; also referred to as water-use efficiency), the ratio of CO₂ assimilation (A) to transpiration. TE strongly correlates with the δ¹³C of plant tissue, the ratio of ¹³C to ¹²C relative to a standard (Farquhar et al., 1982, 1989; Dawson et al., 2002). The physiological basis of this correlation is that in plants there is diffusional and biochemical discrimination against ¹³C, the heavier and less abundant stable isotope of carbon. Discrimination against ¹³C decreases with decreasing internal CO₂ concentration (C_i), which can result from either increased A or reduced stomatal conductance (g_s; Farquhar et al., 1982). While it is known that g_s (a main factor controlling transpiration) correlates with A (Wong et al., 1979), genetic variation for TE and/or δ¹³C has been documented in a number of species (Farquhar and Richards, 1984; Virgona et al., 1990; Ehleringer et al., 1991; Comstock and Ehleringer, 1992; Hammer et al., 1997; Lambrides et al., 2004). In Arabidopsis (Arabidopsis thaliana), multiple quantitative trait loci associated with TE have been identified, indicating that TE is under genetic control (Juenger et al., 2005; Masle et al., 2005; McKay et al., 2008). However, only one gene, ERECTA, has been specifically identified as a regulator of TE (Masle et al., 2005). ERECTA encodes a Leu-rich repeat receptor-like kinase (Torii et al., 1996) and regulates TE via the control of stomatal density, g_s, mesophyll cell proliferation, and photosynthetic capacity (Masle et al., 2005).Heterotrimeric G proteins are GTP-binding proteins that function in the transduction of extracellular signals into intracellular responses. In its inactive state, the G protein classically exists as a trimer consisting of an α-subunit (Gα) bound to GDP, a β-subunit (Gβ), and a γ-subunit (Gγ). When a ligand binds to a G protein-coupled receptor (GPCR), a conformational change occurs in the G protein, resulting in the exchange of GDP for GTP and the dissociation of Gα-GTP from the Gβγ dimer. The G protein subunits remain active until the intrinsic GTPase activity of Gα results in the hydrolysis of GTP to GDP and the reassociation of the inactive trimer. The Arabidopsis genome contains canonical Gα and Gβ genes, GPA1 and AGB1, and two genes known to encode Gγs, AGG1 and AGG2 (Assmann, 2002). One likely GPCR, GCR1, has been functionally characterized (Pandey and Assmann, 2004), and additional GPCRs have been predicted using bioinformatics (Moriyama et al., 2006; Gookin et al., 2008) and interaction with GPA1 in yeast-based protein-protein interaction assays (Gookin et al., 2008). Recently, a new class of G proteins, GPCR-type G proteins (GTG1 and GTG2), have been identified in Arabidopsis that also serve as one class of abscisic acid (ABA) receptors (Pandey et al., 2009).Despite the paucity of heterotrimeric G protein subunit genes in the Arabidopsis genome as compared with mammalian systems, functional studies of heterotrimeric G protein mutants suggest that G protein function is diverse in Arabidopsis. G proteins have been shown to function in developmental processes and hormonal and environmental signaling, including stomatal aperture regulation (Perfus-Barbeoch et al., 2004; Joo et al., 2005; Chen et al., 2006; Pandey et al., 2006; Trusov et al., 2006; Warpeha et al., 2007; Fan et al., 2008; Zhang et al., 2008a, 2008b). In response to drought stress, ABA concentration increases in the leaves (Davies and Zhang, 1991; Davies et al., 2005), where it promotes stomatal closure and inhibits stomatal opening (Schroeder et al., 2001). The G protein α- and β-subunit mutants, gpa1 and agb1, respectively, are hyposensitive to ABA inhibition of stomatal opening while displaying wild-type ABA promotion of stomatal closure (Wang et al., 2001; Fan et al., 2008). ABA inhibits stomatal opening in part by inhibiting inward-rectifying K⁺ channels, reducing K⁺ influx and therefore water entry into the cell (Schroeder et al., 2001). ABA inhibition of inward K⁺ channel activity is reduced in both gpa1 and agb1 mutants (Wang et al., 2001; Fan et al., 2008). agg1 and agg2 mutants show no altered regulation of ABA-induced stomatal movements or ion channel activities, suggesting that the genome contains additional unknown Gγ(s) or that heterotrimeric G protein signaling in plants does not always operate according to the mammalian paradigm (Trusov et al., 2008). gcr1 mutants are hypersensitive to both ABA inhibition of opening and ABA promotion of stomatal closure (Pandey et al., 2006). gtg1 gtg2 double mutants show a wild-type response for ABA inhibition of stomatal opening and are hyposensitive in ABA promotion of stomatal closure (Pandey et al., 2009).While the altered stomatal sensitivities of the G protein mutants to ABA suggest that heterotrimeric G proteins may function in the regulation of whole plant water status, few experiments have been performed at the whole leaf or whole plant level. gpa1 mutants in the Wassilewskija background display increased water loss from excised leaves (Wang et al., 2001); however, there are no published reports of experiments assessing whole plant water status in gpa1 or agb1 mutants. gcr1 mutants show reduced water loss from excised leaves, drought tolerance, and improved recovery following the cessation of drought stress (Pandey and Assmann, 2004). In addition to their altered guard cell sensitivities to ABA, gpa1, agb1, and gcr1 mutants are hypersensitive to ABA inhibition of root and seedling development (Pandey et al., 2006), which could have impacts on whole plant water status. Finally, it has been recently reported that gpa1 and agb1 mutants have reduced and increased stomatal densities, respectively, in cotyledons (Zhang et al., 2008a). While stomatal density of leaves can be an important component of whole plant water status, the study by Zhang et al. (2008a) was performed on cotyledons only, whose developmental programs are often independent from those of true leaves (Chandler, 2008). Therefore, it is difficult to infer how this cotyledon phenotype will affect water relations at the whole plant level. Taken together, the stomatal aperture, electrophysiology, and tissue-specific ABA phenotypes of the G protein mutants, in addition to the possibility for altered stomatal density in the G protein mutant leaves, make it difficult to predict how G proteins contribute to the regulation of whole-plant TE. For example, the ABA-hyposensitive stomatal phenotype of gpa1 could result in increased transpiration, possibly reducing TE under certain conditions. Conversely, if gpa1 mutant leaves have reduced stomatal density, transpiration may be reduced, which could enhance TE under a range of conditions. Previous attempts to address the contributions of G proteins to whole plant transpiration, TE, and drought response using excised leaf/rosette assays to measure water loss are not sufficient, because both transpiration and A must be taken into account. Therefore, we investigated the role of GPA1 in regulating TE under ample water and drought stress conditions and in the presence of ABA. We have identified GPA1 as a negative regulator of TE in Arabidopsis via the control of g_s and stomatal proliferation.     

Ganglioside GM2-tetraspanin CD82 complex inhibits met and its cross-talk with integrins, providing a basis for control of cell motility through glycosynapse

Glycosphingolipids (GSLs) at the cell surface membrane are associated or complexed with signal transducers (Src family kinases and small G-proteins), tetraspanins, growth factor receptors, and integrins. Such organizational framework, defining GSL-modulated or -dependent cell adhesion, motility, and growth, is termed "glycosynapse" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92; Hakomori, S. (2004) Ann. Braz. Acad. Sci. 76, 553-572). We describe here the functional organization of the glycosynaptic microdomain, and the mechanisms for control of cell motility and invasiveness, in normal bladder epithelial HCV29 cells versus highly invasive bladder cancer YTS1 cells, both derived from transitional epithelia. (i) Ganglioside GM2, but not GM3 or globoside, interacted specifically with tetraspanin CD82, and such a complex inhibited hepatocyte growth factor (HGF)-induced activation of Met tyrosine kinase in a dose-dependent manner. (ii) Depletion of GM2 in HCV29 cells by treatment with D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), or reduction of CD82 expression by RNA interference, significantly enhanced HGF-induced Met tyrosine kinase and cell motility. (iii) In contrast, YTS1 cells, lacking CD82, displayed HGF-independent activation of Met tyrosine kinase and high cell motility. Transfection of the CD82 gene to YTS1 inhibited HGF dose-dependent Met tyrosine kinase activity and cell motility, due to formation of the GM2-CD82 complex. (iv) Adhesion of YTS1 or YTS1/CD82 cells to laminin-5-coated plates, as compared with noncoated plates, strongly enhanced Met activation, and the degree of activation was further increased in association with GSL depletion by P4. Laminin-5-dependent Met activation was minimal in HCV29 cells. These findings indicate that GSL, particularly GM2, forms a complex with CD82, and that such complex interacts with Met and thereby inhibits HGF-induced Met tyrosine kinase activity, as well as integrin to Met cross-talk.     

Additive Effect of Anemia and Renal Impairment on Long-Term Outcome after Percutaneous Coronary Intervention

Introduction

Anemia and renal impairment are important co-morbidities among patients with coronary artery disease undergoing Percutaneous Coronary Intervention (PCI). Disease progression to eventual death can be understood as the combined effect of baseline characteristics and intermediate outcomes.

Methods

Using data from a prospective cohort study, we investigated clinical pathways reflecting the transitions from PCI through intermediate ischemic or hemorrhagic events to all-cause mortality in a multi-state analysis as a function of anemia (hemoglobin concentration <120 g/l and <130 g/l, for women and men, respectively) and renal impairment (creatinine clearance <60 ml/min) at baseline.

Results

Among 6029 patients undergoing PCI, anemia and renal impairment were observed isolated or in combination in 990 (16.4%), 384 (6.4%), and 309 (5.1%) patients, respectively. The most frequent transition was from PCI to death (6.7%, 95% CI 6.1–7.3), followed by ischemic events (4.8%, 95 CI 4.3–5.4) and bleeding (3.4%, 95% CI 3.0–3.9). Among patients with both anemia and renal impairment, the risk of death was increased 4-fold as compared to the reference group (HR 3.9, 95% CI 2.9–5.4) and roughly doubled as compared to patients with either anemia (HR 1.7, 95% CI 1.3–2.2) or renal impairment (HR 2.1, 95% CI 1.5–2.9) alone. Hazard ratios indicated an increased risk of bleeding in all three groups compared to patients with neither anemia nor renal impairment.

Conclusions

Applying a multi-state model we found evidence for a gradient of risk for the composite of bleeding, ischemic events, or death as a function of hemoglobin value and estimated glomerular filtration rate at baseline.