Insights into the arginine paradox: evidence against the importance of subcellular location of arginase and eNOS.

Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role.

A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

[Establishment and identification of stable transfection of CHO and COS7 cells with TIF3cDNA].

OBJECTIVE: To establish and identify CHO and COS7 cell lines transfected with TIF3cDNA. METHODS: CHO and COS7 cell lines transfected with TIF3cDNA were established with plasmid (pcDNA3.1/V5-His-TOPO) as the vector, using calcium phosphate inductive transfection techniques and G418 cell selection protocols. Expression of the transfected gene was analyzed and identified using western blotting analysis. The non-transfected group and vector-transfected group were designed to compare with the target gene-transfected group. RESULTS: 3 out of 7 CHO cell lines transfected with TIF3cDNA expressed stably high levels of TIF3 encoded protein. Similarly, 2 out of 4 COS7 cell lines had significant over-expression of TIF3 encoded protein. Conclusion Three CHO and two COS7 cell lines transfected with TIF3cDNA have been successfully established. The cell lines are of great value for the future exploration of Cd compounds related oncogenens and for the studies of the other biological functions of the compounds.

CAR1, a TNFR-related protein, is a cellular receptor for cytopathic avian leukosis-sarcoma viruses and mediates apoptosis.

Viral envelope (Env)-receptor interactions have been implicated in the cell death associated with infection by subgroups B and D avian leukosis-sarcoma viruses (ALVs). A chicken protein, CAR1, was identified that permitted infection of mammalian cells by these viral subgroups. CAR1 bound to a viral Env fusion protein, comprising an ALV-B surface Env protein and the Fc region of an immunoglobulin, indicating that it is a specific viral receptor. CAR1 contains two extracellular cysteine-rich domains characteristic of the TNFR family and a cytoplasmic region strikingly similar to the death domain of TNFR1 and Fas, implicating this receptor in cell killing. Chicken embryo fibroblasts susceptible to ALV-B infection and transfected quail QT6 cells expressing CAR1 underwent apoptosis in response to the Env-Ig fusion protein, demonstrating that this cytopathic ALV receptor can mediate cell death.