Dansylated analogues of the opioid peptide [Dmt1]DALDA: in vitro activity profiles and fluorescence parameters

Dansylated analogues of the potent and selective micro opioid peptide agonist [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) were prepared either by substitution of N(beta)-dansyl-alpha,beta-diaminopropionic acid or N(epsilon)-dansyllysine for Lys(4), or by attachment of a dansyl group to the C-terminal carboxamide function via a linker. All three analogues displayed high micro agonist potency in vitro and the C-terminally dansylated one retained significant micro receptor selectivity. The three analogues showed interesting differences in their fluorescence emission maxima and quantum yields, indicating that the dansyl group in two of them was engaged in intramolecular hydrophobic interactions. These dansylated [Dmt(1)]DALDA analogues represent valuable tools for binding studies, cellular uptake and intracellular distribution studies, and tissue distribution studies.     

Design and synthesis of potent, highly selective vasopressin hypotensive agonists.

We report here the solid-phase synthesis and vasodepressor potencies of a new lead vasopressin (VP) hypotensive peptide [1(beta-mercapto-beta,beta-pentamethylenepropionic acid)-2-0-ethyl-D-tyrosine, 3-arginine, 4-valine, 7-lysine, 9-ethylenediamine] lysine vasopressin, d(CH(2))(5)[D-Tyr(Et)(2), Arg(3), Val(4), Lys(7), Eda(9)]LVP (C) and 21 analogues of C with single modifications at positions 9 (1-13), 6 (14), 2 (16-20) and combined modifications at positions 6 and 10 (15) and 2 and 10 (21). Peptides 1-13 have the following replacements for the Eda residue at position 9 in C: (1) Gly-NH(2); (2) Gly-NH-CH(3); (3) Ala-NH(2); (4) Ala-NH-CH(3), (5) Val-NH(2); (6) Cha-NH(2); (7) Thr-NH(2); (8) Phe-NH(2); (9) Tyr-NH(2); (10) Orn-NH(2); (11) Lys-NH(2); (12) D-Lys-NH(2); (13) Arg-NH(2). Peptide 14 has the Cys residue at position 6 replaced by Pen. Peptide 15 is the retro-Tyr(10) analogue of peptide 14. Peptides 16-20 have the D-Tyr(Et) residue at position 2 in C replaced by the following substituents: D-Trp (16); D-2-Nal (17); D-Tyr(Bu(t))(18); D-Tyr(Pr(n)) (19); D-Tyr(Pr(i)) (20). Peptide 21 is the retro-Tyr(10) analogue of peptide 20. C and peptides 1-21 were evaluated for agonistic and antagonistic activities in in vivo vasopressor (V(1a)-receptor), antidiuretic (V(2)-receptor), and in in vitro (no Mg(2+)) oxytocic (OT-receptor) assays in the rat, and, like the original hypotensive peptide, d(CH(2))(5)[D-Tyr(Et)(2), Arg(3), Val(4)]AVP (A) (Manning et al., J. Peptide Science 1999, 5:472-490), were found to exhibit no or negligible activities in these assays. Vasodepressor potencies were determined in anesthetized male rats with baseline mean arterial blood pressure (BP) maintained at 100-120 mmHg. The effective dose (ED), in microg/100 g i.v., the dose required to produce a vasodepressor response of 5 cm(2) area under the vasodepressor response curve (AUC) during the 5-min period following the injection of the test peptide, was determined. The EDs measure the vasodepressor potencies of the hypotensive peptides C and 1-21 relative to that of A (ED = 4.66 microg/100 g) and to each other. The following ED values in microg/100 g were obtained for C and for peptides 1-21; C 0.53; (1) 2.41; (2) 1.13; (3) 1.62; (4) 0.80; (5) 1.83; (6) 1.56; (7) 2.12, (8) 2.58; (9) 1.40; (10) 0.88; (11) 0.90; (12) 0.85; (13) 0.68; (14) 0.99; (15) 1.05; (16) 0.66; (17) 0.54; (18) 0.33; (19) 0.18; (20) 0.15; (21) 0.14. All of the hypotensive peptides reported here are more potent than A. Peptides 20 and 21 exhibit a striking 30-fold enhancement in vasodepressor potencies relative to A. With a vasodepressor ED = 0.14, peptide 21 is the most potent VP vasodepressor agonist reported to date. Because it contains a retro-Tyr(10) residue, it is a promising new radioiodinatable ligand for the putative VP vasodilating receptor. Some of these new hypotensive peptides may be of value as research tools for studies on the complex cardiovascular actions of VP and may lead to the development of a new class of antihypertensive agents.     

Constituents of the leaves of Pseuderanthemum carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb.

A new iridoid, 5β,6β-dihydroxyantirrhide (1) was isolated from the dried leaves of Pseuderanthemum carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb. (Acanthaceae), together with 13 known compounds, including two iridoids, linarioside and antirrhinoside; five phenylethanoids, echipuroside A, verbascoside, isoverbascoside, isomartynoside and osmanthuside B; and six flavonoids, luteolin 7-O-β-d-glucopyranoside, luteolin 7-O-rutinoside, apigenin 7-O-rutinoside, apigenin 6-C-α-l-arabinopyranosyl–8-C-β-l-arabinopyranoside, apigenin 6,8-di-C-α-l-arabinopyranoside and apigenin 6-C-β-d-xylopyranosyl–8-C-α-l-arabinopyranoside. Their chemical structures were elucidated by 1D and 2D NMR as well as HR-ESI-MS spectroscopic analysis. Some purified compounds were evaluated the acetylcholinesterase inhibition and cytotoxic activities against the HeLa cervical cancer cell line and the MCF-7 breast cancer cell line at the concentration of 100 μg/mL. Luteolin 7-O-β-d-glucopyranoside exhibited cytotoxic activities against both the HeLa cervical cancer cell line and the MCF-7 breast cancer cell line. Verbascoside and isoverbascoside showed strong cytotoxic activity against the MCF-7 breast cancer cell line. The tested compounds showed the AChE inhibitory activity fairly weak.     

Rhizobacterially induced protection of watermelon against Didymella bryoniae

Aims: To identify rhizobacteria from the Mekong Delta of Vietnam, which can systemically protect watermelon against Didymella bryoniae and elucidate the mechanisms involved in the protection conferred by isolate Pseudomonas aeruginosa 23_1‐1. Methods and Results: Bacteria were isolated from watermelon roots and their antagonistic ability tested in vitro. Of 190 strains, 68 were able to inhibit D. bryoniae by production of antibiotics. Four strains were able to reduce foliar infection by D. bryoniae when applied to watermelon seeds before sowing. Strain Ps. aeruginosa 23_1‐1 was chosen for investigations of the mechanisms involved in protection and ability to control disease under field conditions. In the field, the bacterium was able to significantly reduce disease in two consecutive seasons and increase yield. Furthermore, it colonized watermelon plants endophytically, with higher numbers in plants infected by D. bryoniae than in noninoculated plants. To elucidate the mechanisms involved in protection, the infection biology of the pathogen was studied in bacterially treated and control plants. Pseudomonas aeruginosa 23_1‐1 treatment inhibited pathogen penetration and this was associated with hydrogen peroxide accumulation, increased peroxidase activity and occurrence of new peroxidase isoforms, thus indicating that resistance was induced. Conclusions: The endophytic bacterium Ps. aeruginosa 23_1‐1 can control D. bryoniae in watermelon by antibiosis and induced resistance under greenhouse and field conditions. Significance and Impact of the Study: These findings suggest that rhizobacteria from native soils in Vietnam can be used to control gummy stem blight of watermelon through various mechanisms including induction of resistance.     

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10² colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.     

The Sudden Dominance of bla CTX–M Harbouring Plasmids in Shigella spp. Circulating in Southern Vietnam

Background

Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.

Methodology

We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla _CTX–M encoding plasmid.

Principal Findings

We show that two different bla _CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla _CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the bla _CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.

Significance

The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.     

Arabidopsis GIGANTEA protein is post-transcriptionally regulated by light and dark

GIGANTEA (GI) is a key regulator of photoperiodic flowering in Arabidopsis and encodes a protein with no domains of known biochemical function. Expression of GI mRNA is controlled by the circadian clock, but GI protein accumulation has not been previously investigated. We generated plants that produced functional epitope-tagged GI to enable us to track the protein through the daily cycle. Here we show that GI protein levels oscillate when either constitutively overexpressed or driven by its promoter and that its accumulation is modulated by day length as well as by phase-specific factors. Also, we demonstrate that one of the mechanisms underlying GI protein oscillation occurs post-translationally via dark-induced proteolysis by the 26S proteasome.