Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP)

Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N-terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP). The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation. However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed. Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D-NMR to possess two helical portions separated by a three amino acid (RGP) linker. When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP. Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix. Native or linker deleted constructs were used. Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed. It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein. Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP. Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.     

Pregnancy and estradiol decrease GTPase activity in the guinea pig uterine artery

The mechanisms by which pregnancy redistributes cardiac output in an organ-specific manner are poorly understood. We propose that it is consequential to estrogen-mediated alterations in G protein-mediated signal transduction. Aortas and uterine (UAs) and mesenteric arteries (MAs) were obtained from late-pregnant, nonpregnant, or ovariectomized guinea pigs chronically treated with 17beta-estradiol. High-affinity GTPase activity was assayed enzymatically. The cGMP generated in response to the endothelium-dependent agonist ACh was measured in UAs incubated with or without cholera toxin (CTX, which inhibits G(s)alpha). Pregnancy significantly decreased UA but not aorta or MA GTPase activity. 17beta-Estradiol decreased UA GTPase activity compared with untreated ovariectomized animals. ACh increased cGMP in pregnant but not nonpregnant UAs. Pretreatment of nonpregnant UAs with CTX increased ACh-induced cGMP levels similar to pregnancy. Thus pregnancy and estradiol decrease the GTPase activity of a CTX-sensitive G protein in UAs, increasing receptor-dependent cGMP release. This alteration in receptor-mediated G protein coupling in UAs may contribute to the characteristic cardiovascular adaptation to pregnancy.     

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.     

Cytotoxic Peptides: Naphthoquinonyl Derivatives of Luteinizing Hormone-Releasing Hormone

In an attempt to produce efficient cytotoxic derivatives of luteinizing hormone-releasing hormone (LH-RH), two novel 1,4-naphthoquinone derivatives of [D-Lys⁶]-LH-RH were synthesized primarily by solid-phase peptide synthesis, in good yield and high purity. The ability of each analog to produce reactive oxygen species using enzymatic reduction, i.e. NADPH-cytochrome P-450 reductase, was evaluated employing electron spin resonance (ESR) spectroscopy and spin-trapping techniques. The ESR results suggest that the novel cytotoxic analogs are extremely effective in generating oxygen radicals.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Molecular mechanical studies of proflavine and acridine orange intercalation.

Previous workers have reported that proflavine and acridine orange form various structurally different complexes with the dinucleoside phosphates rCpG and dCpG, with uniform C3'-endo and mixed C3'-endo (3'-5') C2'-endo sugar puckers being observed. We present theoretical calculations, based on the method of molecular mechanics, which support the experimental observations. The results suggest that the mixed C3'-edo (3'-5') C2'-endo pucker conformation isi intrinsically more stable than the uniform C3'-endo conformation, but that the additional stabilisation gained from specific, hydrogen bonding, interactions between nucleic acid and solvent, or intramolecularly within the nucleic acid, can lead to the adoption of the latter conformation, or of variants between the two. The role played by hydrogen bonding between amino-groups and nucleic acid phosphate appears more subtle than previously supposed.     

Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli

The cDNA coding for the precursor (p-ALDH) or mature (m-ALDH) rat liver mitochondrial aldehyde dehydrogenase was cloned in an expression vector pT7-7 and expressed in Escherichia coli strain BL21 (DE3)/plysS. The p-ALDH expressed in E. coli was a soluble tetrameric protein. It exhibited virtually the same specific activity and KmS for substrates as m-ALDH. N-terminal sequencing of isolated p-ALDH provided the evidence that the catalytic activity was not derived from a partially processed mature-like enzyme. The assembly states of both p-ALDH and m-ALDH synthesized in a rabbit reticulocyte lysate were also determined. Both of them were monomers and could not bind to a 5'-AMP-Sepharose column, showing that the monomeric form of the enzyme is inactive. The stabilities in vivo and in vitro were compared between p-ALDH and m-ALDH expressed in E. coli. p-ALDH was less stable than was m-ALDH both in vivo and in vitro. Thus, although the conformations of p-ALDH and m-ALDH are similar, the presence of signal peptide is a destabilizing factor to the p-ALDH. p-ALDH expressed in E. coli could bind to and be translocated into rat liver mitochondria, however, with lower efficiency when compared to the import of p-ALDH synthesized in reticulocyte lysate.