Proteasome-mediated degradation of tau proteins occurs independently of the chymotrypsin-like activity by a nonprocessive pathway

Mitochondria can regenerate ascorbic acid from its oxidized forms, which may help to maintain the vitamin both in mitochondria and in the cytoplasm. In this work, we sought to determine the site and mechanism of mitochondrial ascorbate recycling from dehydroascorbic acid. Rat skeletal muscle mitochondria incubated for 3 h at 37 degrees C with 500 microM dehydroascorbic acid and energy substrates maintained ascorbate concentrations more than twice those observed in the absence of substrate. Succinate-dependent mitochondrial reduction of dehydroascorbic acid was blocked by inhibitors of mitochondrial Complexes II and III. Neither cytochrome c nor the outer mitochondrial membrane were necessary for the effect. The ascorbate radical was generated by mitochondria during treatment with dehydroascorbic acid and was abolished by ferricyanide, which does not penetrate the mitochondrial inner membrane. Together, these results show that energy substrate-dependent ascorbate recycling from dehydroascorbic acid involves an externally exposed portion of mitochondrial complex III.     

Evidence that enzymatic conversion of N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, a specific inhibitor of endopeptidase 24.15, to N-[1 (R,S)-carboxy-3-phenylpropyl]-Ala-Ala is necessary for inhibition of angiotensin converting enzyme

N-[1 (R,S)-Carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB) is a potent, substrate-related, specific inhibitor of endopeptidase 24.15, an enzyme involved in the metabolism of bioactive peptides including bradykinin, neurotensin, and proenkephalin, and prodynorphin-derived enkephalin precursors. The observation that this inhibitor causes a pronounced decrease in blood pressure after intravenous infusion into normotensive rats posed the question of the mechanism of this hypotensive response. It was suggested previously that cFP-AAF-pAB is an inhibitor of angiotensin converting enzyme (ACE) and that this function can account for the hypotensive response to the inhibitor. We present here evidence that cFP-AAF-pAB has no intrinsic ACE-inhibitory activity. The previously observed inhibition is shown to be dependent on cleavage of the Ala-Phe bond in the inhibitor by endopeptidase 24.11 (enkephalinase, EC 3.4.24.11), a contaminant of some ACE preparations.     

Field studies of ammonia excretion in Aphanius iberus (Pisces; Cyprinodontidae): body size and habitat effects

The effects of body size and habitat variability on ammonia excretion rates (R_AMs) of Aphanius iberus were analyzed in situ for the first time. At hourly intervals during a 5‐h field experiment, ammonia excretion was measured in 75 mature specimens from three sampling sites (small creek, marine salt‐mine, and salt‐marsh) established in a gradient of water salinity (0–5; 35–40; 65–70‰). Our results showed a specific size dependence pattern of R_AMs in the reproduction period, which might reflect an effect of the reproductive effort. In addition, the results point to a significant decrease in mean R_AM values of each population from freshwater aquatic systems (3.81 ± 0.58 μmol g⁻¹ h⁻¹ in fish of 2.8 ± 0.3 mm total length, TL) to salt aquatic systems with significantly higher alkalinity (2.52 ± 0.35 μmol g⁻¹ h⁻¹ in fish of 3.1 ± 0.5 mm TL in marine salt‐mine; 1.98 ± 0.55 μmol g⁻¹ h⁻¹ in fish of 3.1 ± 0.4 mm TL in salt‐marsh). Due to the size‐dependent pattern, R_AM in different habitats cannot be compared directly; ancova , followed by residual compared analysis (regression‐related techniques), is seen as a valid method for this purpose. This work presents the first field data on ammonia excretion in the Aphanius genus and the flexible physiologic response characteristic of Cyprinodontids has been demonstrated.     

Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8⁺ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim^+/−, Bim^−/− mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim^−/− mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim^+/− mice. At the peak of parasitemia, peritoneal macrophages of Bim^−/− mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim^−/− splenocytes. Moreover, an impaired anti-T. cruzi CD8⁺ T-cell response was found in Bim^−/− mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim^−/− mice and place Bim as an important protein in the control of T. cruzi infections.Subject terms: Cell death and immune response, Infectious diseases     

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH₂) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH₂-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.     

Gα<Subscript>16</Subscript> interacts with tetratricopeptide repeat 1 (TPR1) through its β3 region to activate Ras independently of phospholipase Cβ signaling

Background  

G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα₁₆ as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cβ (PLCβ).