DcuB of Escherichia coli catalyzes C 4 -dicarboxylate/succinate antiport during growth by fumarate respiration. The expression of genes of fumarate respiration, including the genes for DcuB (dcuB) and fumarate reductase (frdABCD) is transcriptionally activated by C 4 -dicarboxylates via the DcuS-DcuR two-component system, comprising the sensor kinase DcuS, which contains a periplasmic sensing domain for C 4 -dicarboxylates. Deletion or inactivation of dcuB caused constitutive expression of DcuSregulated genes in the absence of C 4 -dicarboxylates. The effect was specific for DcuB and not observed after inactivation of the homologous DcuA or the more distantly related DcuC transporter. Random and site-directed mutation identified three point mutations (T394I, D398N, and K353A) in DcuB that caused a similar derepression as dcuB deletion, whereas the transport activity of the DcuB mutants was retained. Constitutive expression in the dcuB mutants depended on the presence of a functional DcuS-DcuR two-component system. Mutation of residues E79A, R83A, and R127A of DcuB, on the other hand, inactivated growth by fumarate respiration and transport of [ 14 C]succinate, whereas the expression of dcuBlacZ was not affected. Therefore, the antiporter DcuB is a bifunctional protein and has a regulatory function that is independent from transport, and sites for transport and regulation can be differentiated.The fumarate/succinate antiporter DcuB (dicarboxylate uptake) of Escherichia coli catalyzes the uptake of external C 4 -dicarboxylates like fumarate, L-malate, or aspartate (1-3). Fumarate is used as an electron acceptor in fumarate respiration by fumarate reductase, which carries the active site at the cytoplasmic side of the membrane (for reviews, see Refs. 4 -6). L-Malate and aspartate are converted to fumarate by fumarase and aspartase, respectively, and are then metabolized in the same way as fumarate. Fumarate respiration results in the generation of a proton potential and drives ATP synthesis and growth of the bacteria. The product of fumarate respiration, succinate, is not further catabolized in anaerobic growth and is excreted by DcuB. DcuB, therefore, is responsible for the substrate/product antiport of fumarate, L-malate, or aspartate against succinate. DcuB and fumarase B are encoded by the dcuB fumB gene cluster (1, 7).

Abbreviations used: BBB, blood-brain barrier; CCI, controlled cortical impact; ICP, intracranial pressure; MLCK, myosin light chain kinase; PBS, phosphate buffered saline; pMLC, phosphorylated myosin light chain. AbstractThe role of the endothelial contractile apparatus in the process of brain edema formation after brain trauma is not characterized. Phosphorylation of myosin light chains by myosin light chain kinases (MLCK) activates endothelial contractile elements and results in a rearrangement of the cytoskeleton. This may enhance post-traumatic blood-brain barrier dysfunction. In order to investigate the role of the MLCK on brain edema formation and blood-brain barrier permeability after brain injury, mice were anesthetized and subjected to a controlled cortical impact (CCI). MLCK expression is significantly up-regulated after CCI with a maximum 12 h post-injury. Specific inhibition of MLCK by ML-7 resulted in a reduction of phosphorylation of myosin light chains and improved bloodbrain-barrier integrity. Accordingly, ML-7 attenuated posttraumatic brain edema formation and intracranial hypertension 24 h after CCI. Prevention of brain edema formation did not translate into improved neurological outcome or reduced brain lesion. In conclusion, the results confirm that the endothelial contractile apparatus is activated by CCI and opens the endothelial barrier leading to vasogenic brain edema formation. Lack of neurological and histological improvement suggests that specific targeting of vasogenic brain edema at the endothelial level is not sufficient to limit secondary brain damage and has, therefore, to be combined with other potential neuroprotective strategies.

Protein stability under changing conditions is of vital importance for the cell and under the control of a fine-tuned network of molecular chaperones. Aging and age-related neurodegenerative diseases are directly associated with enhanced protein instability. Employing C. elegans expressing GFP-tagged luciferase as a reporter for evaluation of protein stability we show that the chaperoning strategy of body wall muscle cells and neurons is significantly different and that both are differently affected by aging. Muscle cells of young worms are largely resistant to heat stress, which is directly mediated by the stress response controlled through Heat Shock Transcription Factor 1. During recovery following heat stress the ability to refold misfolded proteins is missing. Young neurons are highly susceptible to chronic heat stress, but show a high potency to refold or disaggregate proteins during subsequent recovery. The particular proteome instability in neurons results from a delayed induction of the heat shock response. In aged neurons protein stability is increased during heat stress, whereas muscle cells show enhanced protein instability due to a deteriorated heat shock response. An efficient refolding activity is absent in both aged tissues. These results provide molecular insights into the differential protein stabilization capacity in different tissues and during aging.