Perturbed regulation of ZAP-70 and sustained tyrosine phosphorylation of LAT and SLP-76 in c-Cbl-deficient thymocytes.

Recent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protein tyrosine kinases. This finding is supported by studies showing that c-Cbl interacts directly with a negative regulatory tyrosine in ZAP-70, and that the levels of tyrosine-phosphorylated ZAP-70 and numerous other proteins are increased in TCR-stimulated thymocytes from c-Cbl-deficient mice. Here, we demonstrate that this enhanced phosphorylation of ZAP-70 and that of two substrates, LAT and SLP-76, is not due to altered protein levels but is the consequence of two separate events. First, we find increased expression of tyrosine-phosphorylated TCRzeta chain in c-Cbl-deficient thymocytes, which results in a higher level of zeta-chain-associated ZAP-70 that is initially accessible for activation. Thus, more ZAP-70 is activated and more of its substrates (LAT and SLP-76) become tyrosine-phosphorylated after TCR stimulation. However, an additional mechanism of ZAP-70 regulation is evident at a later time poststimulation. At this time, ZAP-70 from both normal and c-Cbl-/- thymocytes becomes hyperphosphorylated; however, only in normal thymocytes does this correlate with ZAP-70 down-regulation and a diminished ability to phosphorylate LAT and SLP-76. In contrast, c-Cbl-deficient thymocytes display altered phosphorylation kinetics, for which LAT phosphorylation is increased and SLP-76 phosphorylation is sustained. Thus, the ability to down-regulate the phosphorylation of two ZAP-70 substrates is impaired in c-Cbl-/- thymocytes. These findings provide evidence that c-Cbl is involved in the negative regulation of the phosphorylation of LAT and SLP-76 by ZAP-70.     

Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein.

The c-Cbl protooncogene is a negative regulator for several receptor tyrosine kinases (RTKs) through its ability to promote their polyubiquitination. Hence, uncoupling c-Cbl from RTKs may lead to their deregulation. In testing this, we show that c-Cbl promotes ubiquitination of the Met RTK. This requires the c-Cbl tyrosine kinase binding (TKB) domain and a juxtamembrane tyrosine residue on Met. This tyrosine provides a direct binding site for the c-Cbl TKB domain, and is absent in the rearranged oncogenic Tpr-Met variant. A Met receptor, where the juxtamembrane tyrosine is replaced by phenylalanine, is not ubiquitinated and has transforming activity in fibroblast and epithelial cells. We propose the uncoupling of c-Cbl from RTKs as a mechanism contributing to their oncogenic activation.     

ENDOLITHIC MICROFLORA ARE MAJOR PRIMARY PRODUCERS IN DEAD CARBONATE SUBSTRATES OF HAWAIIAN CORAL REEFS1

To quantify the contribution of endolithic phototrophs to primary production of dead carbonate substrates, experimental blocks of cleaned Porites lobata Dana skeleton were placed at three different sites in Kaneohe Bay, Hawaii: inshore, lagoonal, and oceanic. After 6 months of exposure, experimental blocks were colonized by communities characteristic of their estuarine (inshore, lagoonal) and oceanic (ocean) environments. Blocks were sub-sampled; net photosynthesis (NP) and chl a concentrations of the whole blocks (epi- and endoliths) and scrapped blocks (only endoliths) were quantified. Green turf algae colonized predominantly inshore and lagoonal blocks, while encrusting corallines were the dominant epiliths colonizing oceanic blocks. Four main species of endolithic phototrophs were identified in all blocks: Mastigocoleus testarum Lagerheim, Plectonema terebrans Bornet and Flahault (cyanobacteria), Phaeophila dendroides Crouan and Crouan, and Ostreobium quekettii Bornet and Flahault (Chlorophytes). While epiliths were very different between sites, NP rates and chl a concentration of endoliths did not vary significantly and were positively correlated (191±25 mmol C·m⁻²·day⁻¹ and 590±150 mg chl a·m⁻² of reef, respectively). Assimilation numbers for whole communities, including both epilithic and endolithic communities, were similar to those measured for endolithic communities alone (average of 0.3 g C·g chl a·h⁻¹). Under experimental conditions, the contribution of endolithic phototrophs to community NP rates of blocks ranged from 56% to 81%, and under natural conditions, we estimated that this contribution ranged between 32% and 46%. Thus, we showed that the endolithic phototrophs are one of the major primary producers in dead coral substrates in a wide range of coral reef environments.     

The influence of locust bean gum and dextran on the gelation of κ-carrageenan

The interaction of κ-carrageenan with locust bean gum and dextran has been studied by rheology, differential scanning calorimetry (DSC), and electron spin resonance spectroscopy (ESR). Rheological measurements show that the carrageenan gel characteristics are greatly enhanced in the presence of locust bean gum but not in the presence of dextran. Carrageenan/locust bean gum mixtures show two peaks in the dsc cooling curves. The higher temperature peak corresponds to the temperature of gelation and its intensity increases at the expense of the lower temperature peak as the proportion of locust bean gum in the mixture increases. Furthermore, the DSC heating curves show enhanced broadening when locust bean gum is present, indicating increased aggregation. These results are taken as evidence of carrageenan/locust bean gum association. The gelation process has also been followed by ESR using spin-labeled carrageenan. On cooling carrageenan solutions, an immobile component appears in the ESR spectra signifying a loss of segmental mobility consistent with chain stiffening due to the coil → helix conformational transition and helix aggregation. For carrageenan/locust bean gum mixtures, carrageenan ordering occurs at temperatures corresponding to the higher temperature DSC setting peak and the temperature of gelation. Similar studies using spin-labeled locust bean gum show that its mobility remains virtually unaffected during the gelation process. It is evident, therefore, that carrageenan and locust bean gum interact only weakly. It is proposed that at low carrageenan concentrations the gel network consists of carrageenan helices cross-linked by locust bean gum chains. At high carrageenan concentrations the network is enhanced by the additional self-aggregation of the “excess” carrageenan molecules. For carrageenan/dextran mixtures, only one peak is observed in the dsc cooling curves. The onset of gelation shifts to higher temperatures only at very high (20%) dextran concentrations and this is attributed to volume exclusion effects. Furthermore, there is no enhanced broadening of the peaks in the DSC heating curves as for the carrageenan/locust bean gum systems. It is therefore concluded that carrageenan/dextran association does not occur. The difference in behavior between locust bean gum and dextran is attributed to the greater flexibility of the dextran chains. © 1996 John Wiley & Sons, Inc.     

Prediction of high airway pressure using a non-linear autoregressive model of pulmonary mechanics

Background

For mechanically ventilated patients with acute respiratory distress syndrome (ARDS), suboptimal PEEP levels can cause ventilator induced lung injury (VILI). In particular, high PEEP and high peak inspiratory pressures (PIP) can cause over distension of alveoli that is associated with VILI. However, PEEP must also be sufficient to maintain recruitment in ARDS lungs. A lung model that accurately and precisely predicts the outcome of an increase in PEEP may allow dangerous high PIP to be avoided, and reduce the incidence of VILI.

Methods and results

Sixteen pressure-flow data sets were collected from nine mechanically ventilated ARDs patients that underwent one or more recruitment manoeuvres. A nonlinear autoregressive (NARX) model was identified on one or more adjacent PEEP steps, and extrapolated to predict PIP at 2, 4, and 6 cmH₂O PEEP horizons. The analysis considered whether the predicted and measured PIP exceeded a threshold of 40 cmH₂O. A direct comparison of the method was made using the first order model of pulmonary mechanics (FOM(I)). Additionally, a further, more clinically appropriate method for the FOM was tested, in which the FOM was trained on a single PEEP prior to prediction (FOM(II)). The NARX model exhibited very high sensitivity (> 0.96) in all cases, and a high specificity (> 0.88). While both FOM methods had a high specificity (> 0.96), the sensitivity was much lower, with a mean of 0.68 for FOM(I), and 0.82 for FOM(II).

Conclusions

Clinically, false negatives are more harmful than false positives, as a high PIP may result in distension and VILI. Thus, the NARX model may be more effective than the FOM in allowing clinicians to reduce the risk of applying a PEEP that results in dangerously high airway pressures.

     

Salmonid biomass and habitat relationships for small lakes

Lake morphometry and water chemistry were analyzed as predictors of brook trout and total salmonid biomass (brook trout, Atlantic salmon and Arctic char) in water bodies of Newfoundland. Lake morphometric variables included surface area, depth, perimeter and catchment area while water chemistry variables included conductivity/TDS and total Phosphorus. The broadly used Morpho-Edaphic Index was also assessed. Fish biomass/ha varied by an order of magnitude in the study lakes, despite similar water chemistry. Interactive stepwise multiple regression analysis suggested that Basin Permanence Index best explains brook trout biomass while surface area best explains the variance of total salmonid biomass in the relatively small, unproductive water bodies of Insular Newfoundland. In contrast, the Morphoedaphic Index performed poorly. The results suggest that biomass relationships with easily measured habitat variables such as surface area provide useful information, especially in management of water bodies under similar climate and nutrient regimes.     

Essential role of E3 ubiquitin ligase activity in Cbl-b-regulated T cell functions

E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T cells in vivo. Thus, modulation of the E3 ligase activity of Cbl-b might be a novel modality to control T cell immunity in vaccination, cancer biology, or autoimmunity.     

Multiple driving factors explain spatial and temporal variability in coral calcification rates on the Bermuda platform

Experimental studies have shown that coral calcification rates are dependent on light, nutrients, food availability, temperature, and seawater aragonite saturation (Ω _arag), but the relative importance of each parameter in natural settings remains uncertain. In this study, we applied Calcein fluorescent dyes as time indicators within the skeleton of coral colonies (n = 3) of Porites astreoides and Diploria strigosa at three study sites distributed across the northern Bermuda coral reef platform. We evaluated the correlation between seasonal average growth rates based on coral density and extension rates with average temperature, light, and seawater Ω _arag in an effort to decipher the relative importance of each parameter. The results show significant seasonal differences among coral calcification rates ranging from summer maximums of 243 ± 58 and 274 ± 57 mmol CaCO₃ m^?2 d^?1 to winter minimums of 135 ± 39 and 101 ± 34 mmol CaCO₃ m^?2 d^?1 for P. astreoides and D. strigosa, respectively. We also placed small coral colonies (n = 10) in transparent chambers and measured the instantaneous rate of calcification under light and dark treatments at the same study sites. The results showed that the skeletal growth of D. strigosa and P. astreoides, whether hourly or seasonal, was highly sensitive to Ω _arag. We believe this high sensitivity, however, is misleading, due to covariance between light and Ω _arag, with the former being the strongest driver of calcification variability. For the seasonal data, we assessed the impact that the observed seasonal differences in temperature (4.0 °C), light (5.1 mol photons m^?2 d^?1), and Ω _arag (0.16 units) would have on coral growth rates based on established relationships derived from laboratory studies and found that they could account for approximately 44, 52, and 5 %, respectively, of the observed seasonal change of 81 ± 14 mmol CaCO₃ m^?2 d^?1. Using short-term light and dark incubations, we show how the covariance of light and Ω _arag can lead to the false conclusion that calcification is more sensitive to Ω _arag than it really is.