Mode of inhibitory action of Deltalac-acetogenins, a new class of inhibitors of bovine heart mitochondrial complex I

We have revealed that Deltalac-acetogenins, a new class of inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), act differently from ordinary inhibitors such as rotenone and piericidin A [Ichimaru et al. (2005) Biochemistry 44, 816-825]. Since a detailed study of these unique inhibitors might provide new insight into the terminal electron transfer step of the enzyme, we further characterized their inhibitory action using the most potent Deltalac-acetogenin derivative (compound 1). Unlike ordinary complex I inhibitors, 1 had a dose-response curve for inhibition of the reduction of exogenous short-chain ubiquinones that was difficult to explain with a simple bimolecular association model. The inhibitory effect of 1 on ubiquinol-NAD(+) oxidoreductase activity (reverse electron transfer) was much weaker than that on NADH oxidase activity (forward electron transfer), indicating a direction-specific effect. These results suggest that the binding site of 1 is not identical to that of ubiquinone and the binding of 1 to the enzyme secondarily (or indirectly) disturbs the redox reaction of ubiquinone. Using endogenous and exogenous ubiquinone as an electron acceptor of complex I, we investigated the effect of 1 in combination with different ordinary inhibitors on the superoxide production from the enzyme. The results indicated that the level of superoxide production induced by 1 is significantly lower than that induced by ordinary inhibitors probably because of fewer electron leaks from the ubisemiquinone radical to molecular oxygen and that the site of inhibition by 1 is downstream of that by ordinary inhibitors. The unique inhibitory action of hydrophobic Deltalac-acetogenins may be closely associated with the dynamic function of the membrane domain of complex I.     

FKBP133: a novel mouse FK506-binding protein homolog alters growth cone morphology

FK506-binding proteins are the peptidyl prolyl cis-trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.     

Dissecting the role of Rho-mediated signaling in contractile ring formation

In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.     

Identification of Valine- or Leucine-Containing Glycopeptidolipids from Mycobacterium avium–intracellulare Complex

Mycobacterium avium-intracellulare complex is a species of acid-fast microorganisms that cause opportunistic infections in immuno-compromised hosts. The cell wall of this microbe is rich in glycopeptidolipids (GPLs), which are composed of a fatty acyl moiety, several sugar moieties and a tripeptide-amino alcohol, D-Phe-D-alloThr-D-Ala-L-Alaninol. GPLs have molecular diversity in the hydrocarbon chain variety of the acyl moiety, and methyl and acetyl modifications of the sugar moiety, but there has been no report of any variety in the tripeptide-amino alcohol component. In this study, we showed two atypical GPL ions of 34 or 48 Da less than the dominant ions of GPLs by mass spectrometry. These ions could not be explained as resulting from conventional molecular diversity. To investigate the reasons why these ions appeared, we made a preparation of the lipopeptide component from intact GPLs and structurally analyzed the molecules. The results suggested that these atypical ions differed from the typical ions in amino acid composition. We further determined its composition by amino acid analysis, and the results showed that the tripeptide portion of the two atypical ions is composed of the Val-alloThr-Ala or the Leu-alloThr-Ala amino acid sequence. In this study, we present novel variations in the tripeptide portion of GPL molecules.     

Role of Somatodendritic and Postsynaptic 5-HT1A Receptors on Learning and Memory Functions in Rats

Memory impairment is a major problem afflicting mankind. The association between memory functions and neurotransmitter functions is of great interest for understanding brain function. Serotonergic pathways play an important role in the modulation of memory functions but the importance of its receptor types and subtypes on memory functions is still unclear. Activation and blockade of various serotonin (5-HT) receptors has been reported to alter cognitive processes and 5-HT receptor antagonism could be beneficial in the treatment of cognitive diseases. The role of 5-HT on memory functions is complicated. Among the 5-HT receptors subtypes, 5-HT(1A) receptors are of special interest because these receptors are present in the brain areas involved in learning and memory functions such as hippocampus and cortex. The present study was therefore designed to investigate the effect of activation and blockade of somatodendritic and/or postsynaptic 5-HT(1A) receptor on learning and memory functions in rats using modified version of water maze. In this study, 8-OH-DPAT (8-hydroxy-2-(di-N-propylamino) tetralin) at 0.3?mg/kg significantly enhanced learning acquisition (LA), short-term memory (STM) and long term memory (LTM) of rats pre-injected with saline suggesting that the activation of pre-synaptic 5-HT(1A) receptors by its agonist enhanced the memory functions of rats. Conversely, rats injected with 8-OH-DPAT at 1.0?mg/kg exhibited impaired LA and STM and had no effect on LTM. It was also shown in this study that blockade of 5-HT(1A) receptors by spiperone enhanced LA, had no effect on STM but impaired the LTM, which showed that the blockade of 5-HT(1A) receptors by its antagonist exerts different effect on different types of memory. This study suggests that 5-HT(1A) receptor could be used as a significant pharmacological target for the treatment of CNS diseases. Unraveling the role of serotonin in cognition and memory disorders could provide better therapy and it may lead to new insights in our understandings of learning and memory.     

Toxicoproteomic analysis of a mouse model of nonsteroidal anti-inflammatory drug-induced gastric ulcers

Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents; however, their use has been limited by their association with mucosal damage in the upper gastrointestinal tract. NSAIDs inhibit cyclooxygenase and consequently block the synthesis of prostaglandins, which have cytoprotective effects in gastric mucosa; these effects on prostaglandins have been thought to be major cause of NSAID-induced ulceration. However, studies indicate that additional NSAID-related mechanisms are involved in formation of gastric lesions. Here, we used a toxicoproteomic approach to understand cellular processes that are affected by NSAIDs in mouse stomach tissue during ulcer formation. We used fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS)-which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry-in this proteomic analysis of pyrolic stomach from control and diclofenac (Dic)-treated mice. FD-LC-MS/MS results were highly sensitive; 10 differentially expressed proteins were identified, and all 10 were more highly expressed in Dic-treated mice than in control mice. Specifically, expression levels of 78 kDa glucose-regulated protein (GRP78), heat shock protein beta-1 (HSP27), and gastrin were more than 3-fold higher in Dic-treated mice than in control mice. This study represents a first step to ascertain the precise actors of early NSAID-induced ulceration.     

Symmetry distortion in the human hemoglobin tetramer induced by asymmetric ligation

To investigate the conformational changes in human tetrameric (αβ)(2) hemoglobin upon binding of the first two ligands, we have measured the kinetics of reactions between 4,4'-dithiodipyridine and β93Cys sulfhydryl groups of four diliganded hemoglobins by using CO-bound Fe(II)-Ni(II) hybrids with and without β-β cross-linking. The data show that all the diliganded intermediates have high sulfhydryl reactivities, which are greater than or equal to that for the fully-liganded end state, especially when containing liganded α subunit(s). The results also reveal that both the asymmetrically (α1β1 and α1β2) diliganded species show similar high rates of sulfhydryl reactivity and biphasic kinetics, suggesting a new conformation but only slight functional distortion caused by asymmetric ligation.     

Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor

Background

The aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx).

Methods

Post-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer''s (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2).

Results

Using the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion.

Conclusions

Normothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.     

Construction and application of an Escherichia coli bioreporter for aniline and chloroaniline detection

Aniline and chlorinated anilines (CAs) are classified as priority pollutants; therefore, an effective method for detection and monitoring is required. In this study, a green-fluorescence protein-based bioreporter for the detection of aniline and CAs was constructed in Escherichia coli DH5α, characterized and tested with soil and wastewater. The sensing capability relied on the regulatory control between a two-component regulatory protein, TodS/TodT, and the P _todX promoter of Pseudomonas putida T-57 (PpT57), since the gene expression of todS, todT, and todC2 are positively induced with 4-chloroaniline. The bioreporter system (DH5α/pPXGFP–pTODST) is markedly unique with the two co-existing plasmids. The inducibility of the fluorescence response was culture-medium- and time-dependent. Cells grown in M9G medium exhibited a low background fluorescence level and were readily induced by 4CA after 3-h exposure, reaching the maximum induction level at 9?h. When tested with benzene, toluene, ethyl-benzene and xylene, aniline and CAs, the response data were best fit by a sigmoidal dose–response relationship, from which the K _1/2 value was determined for the positive effectors. 3CA and 4CA were relatively powerful inducers, while some poly-chlorinated anilines could also induce green fluorescence protein expression. The results indicated a broader recognition range of PpT57’sTodST than previously reported for P. putida. The test results with environmental samples were reliable, indicating the potential application of this bioreporter in the ecotoxicology assessment and bioremediation of areas contaminated with aniline- and/or CAs.