Quinpramine ameliorates rat experimental autoimmune neuritis and redistributes MHC class II molecules

Activation of inflammatory cells is central to the pathogenesis of autoimmune demyelinating diseases of the peripheral nervous system. The novel chimeric compound quinpramine--generated from imipramine and quinacrine--redistributes cholesterol rich membrane domains to intracellular compartments. We studied the immunological and clinical effects of quinpramine in myelin homogenate induced Lewis rat experimental autoimmune neuritis (EAN), a model system for acute human inflammatory neuropathies, such as the Guillain-Barré syndrome. EAN animals develop paresis of all limbs due to autoimmune inflammation of peripheral nerves. Quinpramine treatment ameliorated clinical disease severity of EAN and infiltration of macrophages into peripheral nerves. It reduced expression of MHC class II molecules on antigen presenting cells and antigen specific T cell proliferation both in vitro and in vivo. Quinpramine exerted its anti-proliferatory effect on antigen presenting cells, but not on responder T cells. Our data suggest that quinpramine represents a candidate pharmaceutical for inflammatory neuropathies.     

Trade-offs between pathogen and herbivore resistance

During the past year genetic and pharmacological experiments have revealed a molecular basis for the cross-talk between signaling pathways mediating pathogen and herbivore resistance. These findings provide considerable insight into the apparently contradictory results reported for trade-offs between pathogen and herbivore resistance.     

Ascorbic acid reduction of compound I of mammalian catalases proceeds via specific binding to the NADPH binding pocket

Mammalian (Clade 3) catalases utilize NADPH as a protective cofactor to prevent one-electron reduction of the central reactive intermediate Compound I (Cpd I) to the catalytically inactive Compound II (Cpd II) species by re-reduction of Cpd I to the enzyme's resting state (ferricatalase). It has long been known that ascorbate/ascorbic acid is capable of reducing Cpd I of NADPH-binding catalases to Cpd II, but the mode of this one-electron reduction had hitherto not been explored. We here demonstrate that ascorbate-mediated reduction of Cpd I, generated by addition of peroxoacetic acid to NADPH-free bovine liver catalase (BLC), requires specific binding of the ascorbate anion to the NADPH binding pocket. Ascorbate-mediated Cpd II formation was found to be suppressed by added NADPH in a concentration-dependent manner, for the achievement of complete suppression at a stoichiometric 1:1 NADPH:heme concentration ratio. Cpd I → Cpd II reduction by ascorbate was similarly inhibited by addition of NADH, NADP(+), thio-NADP(+), or NAD(+), though with 0.5-, 0.1-, 0.1-, and 0.01-fold reduced efficiencies, respectively, in agreement with the relative binding affinities of these dinucleotides. Unexpected was the observation that although Cpd II formation is not observed in the presence of NADP(+), the decay of Cpd I is slightly accelerated by ascorbate rather than retarded, leading to direct regeneration of ferricatalase. The experimental findings are supported by molecular mechanics docking computations, which show a similar binding of NADPH, NADP(+), and NADH, but not NAD(+), as found in the X-ray structure of NADPH-loaded human erythrocyte catalase. The computations suggest that two ascorbate molecules may occupy the empty NADPH pocket, preferably binding to the adenine binding site. The biological relevance of these findings is discussed.     

A molecular switch for specific stimulation of the BKCa channel by cGMP and cAMP kinase

The cGMP and the cAMP pathways control smooth muscle tone by regulation of BK(Ca) (BK) channel activity. BK channels show considerable diversity and plasticity in their regulation by cyclic nucleotide-dependent protein kinases. The underlying molecular mechanisms are unclear but may involve expression of splice variants of the BK channel alpha subunit. Three isoforms, BK(A), BK(B), and BK(C), which were cloned from tracheal smooth muscle, differed only in their C terminus. When expressed in HEK293 cells, cGMP kinase (cGK) but not cAMP kinase (cAK) stimulated the activity of BK(A) and BK(B) by shifting the voltage dependence of the channel to more negative potentials. In contrast, BK(C) was exclusively stimulated by cAK. BK(C) lacks a C-terminal tandem phosphorylation motif for protein kinase C (PKC) with Ser(1151) and Ser(1154). Mutation of this motif in BK(A) switched channel regulation from cGK to cAK. Furthermore, inhibition of PKC in excised patches from cells expressing BK(A) abolished the stimulatory effect of cGK but allowed channel stimulation by cAK. cAK and cGK phosphorylated the channel at different sites. Thus, phosphorylation/dephosphorylation by PKC determines whether the BK channel is stimulated by cGK or cAK. The molecular mechanisms may be relevant for smooth muscle relaxation by cAMP and cGMP.     

Compliance of a cobalt chromium coronary stent alloy – the COVIS trial

Aim

Female cardiac transplant recipients' aerobic capacity is 60% lower than sex and age-predicted values. The effect of exercise training on restoring the impaired aerobic endurance and muscle strength in female cardiac transplant recipients is not known. This study examined the effect that aerobic and strength training have on improving aerobic endurance and muscle strength in female cardiac transplant recipients.

Methods

20 female cardiac transplant recipients (51 ± 11 years) participated in this investigation. The subjects performed a baseline six-minute walk test and a leg-press strength test when they were discharged following cardiac transplantation. The subjects then participated in a 12-week exercise program consisting of aerobic and lower extremity strength training. Baseline assessments were repeated following completion of the exercise intervention.

Results

At baseline, the cardiac transplant recipients' aerobic endurance was 50% lower than age-matched predicted values. The training program resulted in a significant increase in aerobic endurance (pre-training: 322 ± 104 m vs. post-training: 501 ± 99 m, p < 0.05) and leg-press strength (pre-training: 48 ± 16 kg. vs. post-training: 78 ± 27 kg, p < 0.05).

Conclusion

Aerobic and strength training are effective interventions that can partially restore the impaired aerobic endurance and strength found in female cardiac transplant recipients.     

C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects

A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK(a) of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.     

Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool

Enzymatic reduction of physiological Fe(III) complexes of the "labile iron pool" has not been studied so far. By use of spectrophotometric assays based on the oxidation of NAD(P)H and formation of [Fe(II) (1,10-phenanthroline)3]2+ as well as by utilizing electron paramagnetic resonance spectrometry, it was demonstrated that the NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase (diaphorase, EC 1.8.1.4) effectively catalyzes the one-electron reduction of Fe(III) complexes of citrate, ATP, and ADP at the expense of the co-enzymes NAD(P)H. Deactivated or inhibited lipoyl dehydrogenase did not reduce the Fe(III) complexes. Likewise, in the absence of NAD(P)H or in the presence of NAD(P)+, Fe(III) reduction could not be detected. The fact that reduction also occurred in the absence of molecular oxygen as well as in the presence of superoxide dismutase proved that the Fe(III) reduction was directly linked to the enzymatic activity of lipoyl dehydrogenase and not mediated by O2. Kinetic studies revealed different affinities of lipoyl dehydrogenase for the reduction of the low molecular weight Fe(III) complexes in the relative order Fe(III)-citrate > Fe(III)-ATP > Fe(III)-ADP (half-maximal velocities at 346-485 microm). These Fe(III) complexes were enzymatically reduced also by other flavoenzymes, namely glutathione reductase (EC 1.6.4.2), cytochrome c reductase (EC 1.6.99.3), and cytochrome P450 reductase (EC 1.6.2.4) with somewhat lower efficacy. The present data suggest a (patho)physiological role for lipoyl dehydrogenase and other flavoenzymes in intracellular iron metabolism.     

Use of a Western blotting technique in the purification of a cysteine proteinase inhibitor

In Western blotting procedures, proteins are resolved in sodium dodecyl sulfate-polyacrylamide gels with subsequent electrophoretic transfer onto nitrocellulose membranes. Although this procedure is generally employed as an analytical technique for assessing interactions of proteins with antibodies, the present report describes the use of Western blotting as a preparative procedure in the purification of a biologically active proteinase inhibitor from the cellular slime mold, Dictyostelium discoideum. The feasibility of using Western blotting for inhibitor purification depended upon the unique stability properties of the inhibitor under denaturing conditions.