Salmonella enterica serovar Typhimurium DT104 displays a rugose phenotype

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25 degrees C after 3 days of incubation. Incubation at 37 degrees C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25 degrees C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.     

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B + C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.     

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.     

Tom34 unlike Tom20 does not interact with the leader sequences of mitochondrial precursor proteins

Tom20 and Tom34 are mammalian liver proteins previously identified by others to be components of the mitochondrial import translocation apparatus. It has been shown that Tom20 interacts with the leader sequence of nuclear coded matrix space precursor proteins. Here we show with recombinantly expressed Tom proteins that Tom34 binds the mature portion of the precursor and not the leader. Both these Tom proteins inhibited the import of newly translated precursor of aldehyde dehydrogenase in an in vitro assay. Only Tom20 inhibited the import of a fusion protein of the leader of aldehyde dehydrogenase attached to dihydrofolate reductase. Antibodies against Tom20 coprecipitated both the precursor of aldehyde dehydrogenase (pALDH) and of dihydrofolate reductase (pA-DHFR). Antibodies against Tom34 interacted only when the mature portion of aldehyde dehydrogenase was present. Similar import inhibition patterns were found when other precursor and chimeric constructs we investigated. When Tom34-green fluorescence protein was transfected to HeLa cells it was observed that Tom34 was found through out the cell. It is concluded from our observation that Tom34 is a cytosolic protein, whose role appeared to be to interact with mature portion of some preproteins and may keep them in an unfolded, import compatible state.     

Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes

CCA-adding enzymes catalyze the addition of CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences. We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP. Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous. Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes. Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA. The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA. The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.     

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.     

Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance

The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K(2)TeO(3) at 128 microg/ml, compared to the MIC of 2 microg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a approximately 20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.     

Molecular mechanism of cGMP-mediated smooth muscle relaxation

Contraction and relaxation of smooth muscle is a tightly regulated process involving numerous endogenous substances and their intracellular second messengers. We examine the key role of cyclic guanosine monophosphate (cGMP) in mediating smooth muscle relaxation. We briefly review the current art regarding cGMP generation and degradation, while focusing on the recent identification of the molecular mechanisms underlying cGMP-mediated smooth muscle relaxation. cGMP-induced SM relaxation is mediated mainly by cGMP-dependent protein kinase activation. It involves several molecular events culminating in a reduction in intracellular Ca(2+) concentration and a decrease in the sensitivity of the contractile system to Ca(2+). We propose that the cGMP-induced decrease in Ca(2+) sensitivity is a strategic way to achieve "active relaxation" of the smooth muscle. In summary, we present compelling evidence supporting a key role for cGMP as a mediator of smooth muscle relaxation in physiological and pharmacological settings.     

Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis

Immunity to tumors as well as to viral and bacterial pathogens is often mediated by cytotoxic T lymphocytes (CTLs). Thus, the ability to induce a strong cell-mediated immune response is an important requirement of novel immunotherapies. Antigen-presenting cells (APCs), including dendritic cells (DCs), are specialized in initiating T-cell immunity. Harnessing this innate ability of these cells to acquire and present antigens, we sought to improve antigen presentation by targeting antigens directly to DCs in vivo through apoptosis. We engineered Fas-mediated apoptotic death of antigen-bearing cells in vivo by co-expressing the immunogen and Fas in the same cell. We then observed that the death of antigen-bearing cells results in increased antigen acquisition by APCs including DCs. This in vivo strategy led to enhanced antigen-specific CTLs, and the elaboration of T helper-1 (Th1) type cytokines and chemokines. This adjuvant approach has important implications for viral and nonviral delivery strategies for vaccines or gene therapies.     

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.