Solvent-stable Pseudomonas aeruginosa PseA protease gene: identification, molecular characterization, phylogenetic and bioinformatic analysis to study reasons for solvent stability

We have previously isolated a solvent-stable protease from a novel solvent-tolerant strain of Pseudomonas aeruginosa (PseA). Here we report cloning and characterization of the gene coding for this solvent-tolerant protease. A homology search of the N-terminal amino acid sequence of the purified PseA protease revealed an exact match to a P. aeruginosa PST-01 protease gene, lasB. The c-DNA sequence of the PST-01 protease was used to design primers for the amplification of a 1,494-bp open reading frame encoding a 53.6-kDa, 498-amino-acid PseA LasB polypeptide. The deduced PseA LasB protein contained a 23-residue signal peptide (2.6 kDa) followed by a propeptide of 174 residues and a 33-kDa mature product of 301 residues. A phylogenetic analysis placed PseA lasB closest to the known zinc metalloproteases from P. aeruginosa. This gene was also found to contain a conserved HEXXH zinc-binding motif, characteristic of all zinc metallopeptidases. The 3D structure analysis of PseA protease revealed the presence of 7 alpha-helices (36% of the sequence). The molecule was found to have two disulfide bonds (between Cys-227 and Cys-255 and between Cys-467 and Cys-494) and had a number of hydrophobic clusters at the protein surface. These hydrophobic patches (21% of the sequence) and disulfide bonds may possibly be responsible for the solvent-stable nature of the enzyme.     

Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

Bigendothelin-1 via p38-MAPK-dependent mechanism regulates adult rat ventricular myocyte contractility in sepsis

We tested the hypothesis that exogenous administration of the ET-1 precursor, bigET-1, would regulate adult rat ventricular myocyte (ARVM) contractility in a p38-mitogen activated protein kinase (p38-MAPK)-dependent mechanism during sepsis. Ventricular myocytes from adult rat hearts (both sham and septic) were stimulated to contract at 0.5 Hz and mechanical properties were evaluated using an IonOptix Myocam system. Immunoblot analysis was used to determine the phosphorylation of p38-MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2). ARVMs were treated with vehicle, bigET-1 and inhibitors for 24 h and then subjected to functional and biochemical estimations. Septic ARVM displayed a distorted cell membrane and irregular network within the cells along with increased cell contractility as evidenced by elevated peak shortening (PS), maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt) in comparison to sham ARVM. BigET-1 treatment caused ARVM enlargement in both sham and sepsis groups. BigET-1 (100 nM) produced an increase in ARVM contractility in sham group as compared to vehicle treatment. However, septic ARVM treated with bigET-1 exhibited unaltered ARVM contractility, and upregulated ET(B) receptors as compared to respective sham group. BigET-1 increased the concentration of ET-1 and upregulated phosphorylation of p38-MAPK but not of ERK1/2 in sham and septic ARVM. Furthermore, inhibition of p38-MAPK by SB203580 (10 microM) increased ARVM contractility in sham but not in sepsis group. BigET-1 reversed SB203580-induced increase in PS in sham group but accentuated it in sepsis group. BigET-1 also reversed SB203580-induced inhibition of p38-MAPK phosphorylation in sham but not in septic ARVM. SB203580 pretreatment followed by bigET-1 administration significantly decreased p38-MAPK phosphorylation and downregulated ET(B) receptor expression as compared to bigET-1 treatment per se in sepsis group but not in sham. We concluded that a bigET-1-induced non-responsive effect on septic ARVM contractile function could be due to upregulation of p38-MAPK phosphorylation and ET(B) receptor expression.     

Benzothiophene carboxamide derivatives as novel antimalarials

Bromo-benzothiophene carboxamide derivatives have been shown in the preceding article to inhibit Plasmodium falciparum Enoyl-ACP reductase. Here, we report bromo-benzothiophene carboxamide derivatives as potent inhibitors of Plasmodium asexual blood-stages in vitro as well as in vivo in the mouse model. These compounds specifically impair the development of metabolically active trophozoite stage of intraerythrocytic cycle and the intravenous administration of 3-bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) enhances the longevity of P. berghei infected mice by 2 weeks compared to disease control animals thereby preventing the onset of ataxia and convulsions in treated mice. These compounds thus hold promise for the development of potent antimalarials.     

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.     

Alanine scanning of all cysteines and construction of a functional cysteine-less Cdr1p, a multidrug ABC transporter of Candida albicans

Herein, we discuss the role of the native cysteines present in a major multidrug ABC transporter of Candida albicans, Cdr1p, and describe the construction of this transporter's functional cysteine-less (cysless) protein version for cross-linking studies. In the experiments in which all 23 cysteines were replaced individually, we observed that most of the cysteine replacements were tolerated by the protein, but the replacement of C1056, C1091, C1106, C1294 or C1336 resulted in an enhanced drug susceptibility together with an abrogated drug efflux. Notably, the ATPase activity was uncoupled, which largely remained unaffected in these variants. The substitution of the critical cysteines with serines restored the normal expression and functionality of Cdr1p because serine can effectively mimic the hydrogen bonding properties of cysteine. Finally, we constructed a functional cysless His-tagged Cdr1p in which all the cysteines of the native protein were replaced with alanines and the critical cysteines were replaced with serines. Notably, cysless GFP-tagged variant of Cdr1p was non-functional. The cysless His-tagged variant of Cdr1p is the first example of a cysless ABC transporter in yeast, and it will lead to a greater understanding of the architecture of this important protein and provide insight into the nature of drug binding and interdomain communication.     

Cloning and Expression of Small cDNA Fragment Encoding Strong Antiviral Peptide from <Emphasis Type="Italic">Celosia cristata</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.