Overexpression of a human and a fungal ABC transporter similarly suppresses the differentiation defects of a fungal peroxisomal mutant but introduces pleiotropic cellular effects

Among the peroxisome membrane proteins, some are required for peroxisome biogenesis (e.g. PEX2) while others are not, e.g. ABC (ATP-binding cassette) transporters. Unexpectedly, overproduction of the peroxisomal ABC transporter PMP70 was found to be able to restore peroxisome biogenesis in mammalian pex2 mutant cell lines. In the filamentous fungus Podospora anserina, pex2 mutations not only impair peroxisome biogenesis but also cause a precise cell differentiation defect. Here, we show that both defects are partially suppressed by expression of the human cDNA encoding PMP70. In addition, PMP70 expression causes new developmental defects, different from those induced by pex2 mutations. We also show that overexpression of the P. anserina pABC1 gene, which encodes a peroxisomal ABC transporter, leads to similar effects. Taken together, our results demonstrate that: (i) the genetic relationship between PEX2 and PMP70, initially observed in mammals, has been conserved through evolution; (ii) the cell differentiation defect observed in the P. anserina pex2 mutants is indeed linked to impairment in peroxisome biogenesis; and (iii) unexpected detrimental cellular defects result from overproduction of peroxisomal ABC transporters.     

The intracellular granzyme B inhibitor,proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation,and its overexpression enhances CTL potency

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells, CD8(+) T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-), CD4(+), CD8(-), CD45(+)), tonsillar DCs, and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.     

A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release

Lysophosphatidylcholines(lyso-PCs), generated during blood storage, are etiologic in atwo-insult, sepsis-based model of transfusion-related acute lung injury(TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do notactivate neutrophils (PMNs). We hypothesized that priming of PMNsalters their reactivity such that a second priming agent causes PMNactivation and endothelial cell damage. PMNs were primed or not withLPS and then treated with lyso-PCs, and oxidase activation and elastaserelease were measured. For coculture experiments, activation of humanpulmonary microvascular endothelial cells (HMVECs) was assessed byICAM-1 expression and chemokine release. HMVECs were stimulated ornot with LPS, PMNs were added, cells were incubated with lyso-PCs, andthe number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 andrelease of ENA-78, GRO, and IL-8. PMN-mediated HMVEC damage wasdependent on LPS activation of HMVECs, chemokine release, PMNadhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates themicrobicidal arsenal, and PMN-mediated HMVEC damage was the resultof two insults: HMVEC activation and PMN oxidase assembly.

     

Comparative trajectories of active and S195A inactive trypsin upon binding to serpins

Serpins inhibit proteinases through a complicated multistep mechanism. The precise nature of these steps and the order by which they occur are still debated. We compared the fate of active and S195A inactive rat trypsin upon binding to alpha(1)-antitrypsin and P(1)-Arg-antichymotrypsin using stopped-flow kinetics with fluorescence resonance energy transfer detection and time-resolved fluorescence resonance energy transfer. We show that inhibition of active trypsin by these serpins leads to two irreversible complexes, one being compatible with the full insertion of the serpin-reactive site loop but not the other one. Binding of inactive trypsin to serpins triggers a large multistep reversible rearrangement leading to the migration of the proteinase to an intermediate position. Binding of inactive trypsin, unlike that of active trypsin, does not perturb the rhodamine fluorescence at position 150 on the helix F of the serpin. Thus, inactive proteinases do not migrate past helix F and do not trigger full serpin loop insertion.     

Xanthan–galactomannan interactions as related to xanthan conformations

The influence of xanthan conformation on the physicochemical behaviour of their mixtures with galactomannan from Schizolobium parahybae mannose:galactose ratio (M/G=3), was studied by viscoelastic measurements, differential scanning calorimetry (DSC) and chiroptical (circular dichroism) methods. The results suggested a more effective interaction of the galactomannan with disordered xanthan segments, which are more abundant in low salt concentrations but are still present in lower proportion at temperatures lower than the temperature of xanthan conformational transition (T_m). The dependence of ellipticity with temperature in a circular dichroism (CD) spectra suggested an ordering of the xanthan chains induced by galactomannan at the temperature of gel formation (T_g≈25°C), under conditions where xanthan alone exhibits a disordered conformation. The lower T_g value found (≈25°C) compared with that (60°C) usually described in the literature is certainly related to the M/G ratio and the galactosyl unit distribution along the mannan main chain.     

Transpiration and stomatal conductance of two wetland macrophytes (Cladium jamaicense and Typha domingensis) in the subtropical Everglades

In the subtropical Everglades, greater than 85% of water input through atmospheric precipitation, the primary source of water to the ecosystem, is lost through evapotranspiration (ET). Two dominant macrophytes that could influence ET rates through transpiration and stomatal control were investigated in a field study: Cladium jamaicense, presently the species with the greatest distribution (65%–70%); and Typha domingensis, a species increasing in distribution due to eutrophication and hydrological modifications to the ecosystem. Transpiration rates and stomatal conductance of the two species were compared at eutrophic, mesotrophic, and oligotrophic sites throughout an annual wet and dry season of subtropical Florida. Typha domingensis possessed higher transpiration and conductance rates (>; 11 mmol m^-2 sec^-1) than C. jamaicense (<;7 mmol m^-2 sec^-1; P <; 0.01), during the winter and spring months when ambient temperatures and vapor pressure were at a minimum. However, rates for the two species converged and were not significantly different during the summer and fall months. Stomatal conductance of C. jamaicense remained constant over the range of ambient vapor pressures, but significantly increased in T. domingensis with decreasing vapor pressure. Vapor pressure and light were the best predictors of seasonal and diurnal transpiration rates of T. domingensis, whereas temperature explained the most variability in C. jamaicense. Annual transpiration rates for both species were 1 to 2 mmol m^-2 sec^-1 greater at the eutrophic site than at the transitional and oligotrophic sites. Leaf area increased six times in C. jamaicense and twofold in T. domingensis from the control to nutrient-enriched sites. Results at the leaf scale suggest vegetation shifts and nutrient enrichment have the potential to alter water balances in the Everglades. However, canopy level studies may be necessary to support these conclusions when applied to the larger ecosystem or regional scale.     

Abrupt permafrost thaw drives spatially heterogeneous soil moisture and carbon dioxide fluxes in upland tundra

Permafrost thaw causes the seasonally thawed active layer to deepen, causing the Arctic to shift toward carbon release as soil organic matter becomes susceptible to decomposition. Ground subsidence initiated by ice loss can cause these soils to collapse abruptly, rapidly shifting soil moisture as microtopography changes and also accelerating carbon and nutrient mobilization. The uncertainty of soil moisture trajectories during thaw makes it difficult to predict the role of abrupt thaw in suppressing or exacerbating carbon losses. In this study, we investigated the role of shifting soil moisture conditions on carbon dioxide fluxes during a 13-year permafrost warming experiment that exhibited abrupt thaw. Warming deepened the active layer differentially across treatments, leading to variable rates of subsidence and formation of thermokarst depressions. In turn, differential subsidence caused a gradient of moisture conditions, with some plots becoming consistently inundated with water within thermokarst depressions and others exhibiting generally dry, but more variable soil moisture conditions outside of thermokarst depressions. Experimentally induced permafrost thaw initially drove increasing rates of growing season gross primary productivity (GPP), ecosystem respiration (R_eco), and net ecosystem exchange (NEE) (higher carbon uptake), but the formation of thermokarst depressions began to reverse this trend with a high level of spatial heterogeneity. Plots that subsided at the slowest rate stayed relatively dry and supported higher CO₂ fluxes throughout the 13-year experiment, while plots that subsided very rapidly into the center of a thermokarst feature became consistently wet and experienced a rapid decline in growing season GPP, R_eco, and NEE (lower carbon uptake or carbon release). These findings indicate that Earth system models, which do not simulate subsidence and often predict drier active layer conditions, likely overestimate net growing season carbon uptake in abruptly thawing landscapes.     

A comment on the earliest spalacinae (rodentia,Muroidea)

According to F. M. Catzefliset al. (Trends Ecol. Evol. 7, 122–126, 1992), comparative dating of the oldest dichotomy in the muroid group (Spalacinae-other Muridae) by molecular and paleontological time scales produces conflicting results. For the earliest Spalacinae they refer to a date of 25–37 Ma; however, it has been demonstrated thatRhizospalax is not a muroid rodent, and a review of the record of fossil Spalacinae suggests that paleontological and molecular dating produces a comparable age (±19 Ma).     

Genetic evidence for ribosomal antisuppressors inPodospora anserina

Summary Antisuppressors were screened for with the help of informational suppressors inPodospora anserina. Four mutations in the AS₁ locus and two in the AS₂ locus were isolated, using allele non specific suppressors supposed to be ribosomal ambiguity mutations. Four mutations in the AS₃ locus and 45 in the AS₄ locus were obtained, using a nonsense (tRNA like) suppressor. All antisuppressors are partially dominant. Most mutations in the AS₄ locus are lethal. The four mutants at the AS₃ locus and 6 out of the 8 viable mutants at the AS₄ locus are cold sensitive. Phenotypic properties and action spectra of the antisuppressors suggest that they are restrictive ribosomal mutations.