IBRDC2, an IBR‐type E3 ubiquitin ligase,is a regulatory factor for Bax and apoptosis activation

Bax, a pro‐apoptotic protein from the Bcl‐2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax conformation and expression levels. We report that IBRDC2, an IBR‐type RING‐finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination‐dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl‐xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax^?/? cells, combined with the use of active Bax‐specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.     

Apoptosis in the monkey small intestinal epithelium: structural and functional alterations in the mitochondria

Our earlier studies have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. Because mitochondria have been implicated in the apoptotic process, this study looked at the function and lipid composition of mitochondria isolated from apoptotic villus tip cells and compared it with middle and crypt cells. Decreased MTT reduction and respiratory control ratio, increased swelling and altered mitochondrial enzyme activities were seen in the villus tip cell mitochondria when compared to other cells. The lipid composition of the villus tip mitochondria were different from the other mitochondria. A decrease in phosphatidylethanolamine and phosphatidyl-inositol and an increase in phosphatidic acid was seen in these mitochondria. Fatty acid composition analysis showed more unsaturated fatty acids in the free fatty acid and phospholipid fraction in villus tip cell mitochondria as compared to other cells. These studies suggest that in the monkey small intestinal epithelium, apoptotic process is associated with functional and structural alterations in the mitochondria.     

Transport of nitrogen and zinc to rhodes grass by arbuscular mycorrhiza and roots as affected by different nitrogen sources (NH<Subscript>4</Subscript><Superscript>+</Superscript>-N and NO<Subscript>3</Subscript><Superscript>−</Superscript>-N)

We investigated the effect of mineral nitrogen forms on transfer of nitrogen (N) and zinc (Zn) from attached compartments to rhodes grass (Chloris gayana) colonised with arbuscular mycorrhizal fungi (AMF). After being pre-cultivated in substrates with adequate nutrient supply and either AMF inoculated (+AM) or left non-inoculated (?AM), rhodes grass was positioned adjacent to an outer compartment holding a similar substrate but applied with labelled nitrogen (¹⁵N) either as ammonium (NH₄ ⁺) or nitrate (NO₃ ^?), and a high supply of Zn (150 mg kg^?1 DS). Plant roots together with fungal mycelium were either allowed to explore the outer compartment (with root access) or only mycorrhizal hyphae were allowed (without root access). Within each access treatment, biomasses of rhodes grass were not significantly affected by AMF inoculation or N form. AMF contribution to plant ¹⁵N uptake was about double in NH₄ ⁺ compared with NO₃ ^?-supplied treatments while the mycorrhizal influence on plant Zn uptake was insignificant. Without root access, the shoot ¹⁵N/Zn concentration ratio was up to ten-fold higher in +AM than –AM treatments and this ratio increase was clearly more pronounced in NH₄ ⁺ than NO₃ ^?-supplied treatments. In conclusion, rhodes grass in symbiosis with the tested AMF acquired more N when supplied with ammonium. Moreover, there is clear indication that although the AMF have transported both nutrients (N and Zn), N was preferentially transferred as compared to Zn. We confirmed that, while rhodes grass is not able to prevent excessive Zn uptake via roots under conditions of high Zn, mycorrhiza is able to avoid excessive Zn supply to the host plant when the fungus alone has access to contaminated patches.     

Lactisole interacts with the transmembrane domains of human T1R3 to inhibit sweet taste

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisole's inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisole's action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.     

Molecular mechanism of maternal rescue in the clk-1 mutants of Caenorhabditis elegans

The clk-1 mutants of Caenorhabditis elegans display an average slowing down of physiological rates, including those of development, various behaviors, and aging. clk-1 encodes a hydroxylase involved in the biosynthesis of the redox-active lipid ubiquinone (co-enzyme Q), and in clk-1 mutants, ubiquinone is replaced by its biosynthetic precursor demethoxyubiquinone. Surprisingly, homozygous clk-1 mutants display a wild-type phenotype when issued from a heterozygous mother. Here, we show that this maternal effect is the result of the persistence of small amounts of maternally derived CLK-1 protein and that maternal CLK-1 is sufficient for the synthesis of considerable amounts of ubiquinone during development. However, gradual depletion of CLK-1 and ubiquinone, and expression of the mutant phenotype, can be produced experimentally by developmental arrest. We also show that the very long lifespan observed in daf-2 clk-1 double mutants is not abolished by the maternal effect. This suggests that, like developmental arrest, the increased lifespan conferred by daf-2 allows for depletion of maternal CLK-1, resulting in the expression of the synergism between clk-1 and daf-2. Thus, increased adult longevity can be uncoupled from the early mutant phenotypes, indicating that it is possible to obtain an increased adult lifespan from the late inactivation of processes required for normal development and reproduction.     

Rapid allopatric speciation in logperch darters (Percidae: Percina)

Theory predicts that clades diversifying via sympatric speciation will exhibit high diversification rates. However, the expected rate of diversification in clades characterized by allopatric speciation is less clear. Previous studies have documented significantly higher speciation rates in freshwater fish clades diversifying via sympatric versus allopatric modes, leading to suggestions that the geographic pattern of speciation can be inferred solely from knowledge of the diversification rate. We tested this prediction using an example from darters, a clade of approximately 200 species of freshwater fishes endemic to eastern North America. A resolved phylogeny was generated using mitochondrial DNA gene sequences for logperches, a monophyletic group of darters composed of 10 recognized species. Divergence times among logperch species were estimated using a fossil calibrated molecular clock in centrarchid fishes, and diversification rates in logperches were estimated using several methods. Speciation events in logperches are recent, extending from 4.20 +/- 1.06 million years ago (mya) to 0.42 +/- 0.22 mya, with most speciation events occurring in the Pleistocene. Diversification rates are high in logperches, at some nodes exceeding rates reported for well-studied adaptive radiations such as Hawaiian silverswords. The geographic pattern of speciation in logperches was investigated by examining the relationship between degree of sympatry and the absolute age of the contrast, with the result that diversification in logperches appears allopatric. The very high diversification rate observed in the logperch phylogeny is more similar to freshwater fish clades thought to represent examples of sympatric speciation than to clades representing allopatric speciation. These results demonstrate that the geographic mode of speciation for a clade cannot be inferred from the diversification rate. The empirical observation of high diversification rates in logperches demonstrates that allopatric speciation can occur rapidly.     

Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbetagamma, and beta-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.     

Chronopharmacokinetics of imipenem in the rat

There are no studies indicating a possible modification of imipenem pharmacokinetics related to the hour (i.e., circadian time) of its administration. The aim of this study was to evaluate the influence of different times of intramuscular imipenem administration on its disposition in Wistar AF EOPS rats. Four groups of eight animals were given a single intramuscular injection of 140 mg/kg of imipenem either at 10:00, 16:00, 22:00, or 04:00 h. Blood samples were collected 0.5, 1, 2, 3, 4, 6, and 8 h after drug injection, and the main pharmacokinetic parameters determined were C_max, T_max, elimination half-life (t_1/2), area under the concentration-versus-time curve (AUC), total serum clearance (CL/F), and volume of distribution (V/F). Circadian variation of C_max (49%), T_max (92%), and AUC (19%) was observed leading to variability of imipenem exposure. Clearance and volume of distribution were modified according to the circadian time of drug injection but did not reach statistical significance. The results suggest that varying the time of administration induces intra-individual variability.     

Development of Standard Fuel Models in Boreal Forests of Northeast China through Calibration and Validation

Understanding the fire prediction capabilities of fuel models is vital to forest fire management. Various fuel models have been developed in the Great Xing''an Mountains in Northeast China. However, the performances of these fuel models have not been tested for historical occurrences of wildfires. Consequently, the applicability of these models requires further investigation. Thus, this paper aims to develop standard fuel models. Seven vegetation types were combined into three fuel models according to potential fire behaviors which were clustered using Euclidean distance algorithms. Fuel model parameter sensitivity was analyzed by the Morris screening method. Results showed that the fuel model parameters 1-hour time-lag loading, dead heat content, live heat content, 1-hour time-lag SAV(Surface Area-to-Volume), live shrub SAV, and fuel bed depth have high sensitivity. Two main sensitive fuel parameters: 1-hour time-lag loading and fuel bed depth, were determined as adjustment parameters because of their high spatio-temporal variability. The FARSITE model was then used to test the fire prediction capabilities of the combined fuel models (uncalibrated fuel models). FARSITE was shown to yield an unrealistic prediction of the historical fire. However, the calibrated fuel models significantly improved the capabilities of the fuel models to predict the actual fire with an accuracy of 89%. Validation results also showed that the model can estimate the actual fires with an accuracy exceeding 56% by using the calibrated fuel models. Therefore, these fuel models can be efficiently used to calculate fire behaviors, which can be helpful in forest fire management.     

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECM components.