Comparative Proteomics of Inner Membrane Fraction from Carbapenem-Resistant Acinetobacter baumannii with a Reference Strain

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A. baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A. baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A. baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A. baumannii are: i) β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A. baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A. baumannii to carbapenem

Ionic liquids at electrified interfaces

Until recently, “room-temperature” (<100–150 °C) liquid-state electrochemistry was mostly electrochemistry of diluted electrolytes(1)–(4) where dissolved salt ions were surrounded by a considerable amount of solvent molecules. Highly concentrated liquid electrolytes were mostly considered in the narrow (albeit important) niche of high-temperature electrochemistry of molten inorganic salts(5-9) and in the even narrower niche of “first-generation” room temperature ionic liquids, RTILs (such as chloro-aluminates and alkylammonium nitrates).(10-14) The situation has changed dramatically in the 2000s after the discovery of new moisture- and temperature-stable RTILs.(15, 16) These days, the “later generation” RTILs attracted wide attention within the electrochemical community.(17-31) Indeed, RTILs, as a class of compounds, possess a unique combination of properties (high charge density, electrochemical stability, low/negligible volatility, tunable polarity, etc.) that make them very attractive substances from fundamental and application points of view.(32-38) Most importantly, they can mix with each other in “cocktails” of one’s choice to acquire the desired properties (e.g., wider temperature range of the liquid phase(39, 40)) and can serve as almost “universal” solvents.(37, 41, 42) It is worth noting here one of the advantages of RTILs as compared to their high-temperature molten salt (HTMS)(43) “sister-systems”.(44) In RTILs the dissolved molecules are not imbedded in a harsh high temperature environment which could be destructive for many classes of fragile (organic) molecules

Hairpin and duplex forms of a self-complementary dodecamer, d-AGATCTAGATCT, and interaction of the duplex form with the peptide KGWGK: can a pentapeptide destabilize DNA?

Ordered forms of a synthetic dodecamer, d-AGATCTAGATCT, a direct repeat of the BglII recognition sequence, have been investigated using UV, CD, and fluorescence spectroscopy. Complex hairpin-duplex equilibria are manifest in UV thermal transitions, which are monophasic in the presence of very low or high NaCl concentrations but distinctly biphasic at intermediate ionic strengths. In 100 mM NaCl, the 1/Tm vs 1n C curve has a reasonable positive slope, which yields &#916;H and &#916;S for duplex formation as -66.2 kcal/mol and -190 cal/mol, respectively. Interaction of the dodecamer in duplex form with a tryptophan-containing peptide, KGWGK, has also been investigated to test the "bookmark" hypothesis (Gabbay et al., 1976) under the uniform structural constraint of the oligonucleotide of defined sequence. CD spectra of the peptide (P), the oligonucleotide (N), and their mixtures at different P/N ratios show a dramatic change in peptide spectrum but little change in nucleic acid dichroism with peptide binding. The Tm of P-N complexes decreases with an increase in peptide binding and levels off at saturation binding of P/N = 2.0. The data are interpreted in terms of a groove-cum-intercalation mode of binding, where intercalation to the tryptophan side chain destabilizes the double helix. A Scatchard plot of the binding data is nonlinear, with best-fit values for an overall association constant K = 4.33 x 105 M-1, and the number of binding sites n = 3.23 when fitted to the site-exclusion model of binding

Binding of oligopeptides to d-AGATCTAGATCT and d-AAGCTTAAGCTT: can tryptophan intercalate in DNA hairpins?

The interactions of three tryptophan-containing peptides, KWK, KGWK tert-butyl ester, and KGWGK, with two self-complementary dodecamers of the same base composition but different sequence were studied by UV, CD, and fluorescence spectroscopy. The oligonucleotides, d-AGATCTAGATCT and d-AAGCTTAAGCTT, contain tandem repeats of the recognition site for the restriction enzyme BglII in the former and HindIII in the latter. Thermal transition data in dilute solutions and in 0.01 M NaCl indicate these dodecamers to be present in hairpin forms. Binding of peptides to these hairpins was followed by tryptophan fluorescence quenching titrations at 10 mM Na+; the data suggest intercalation of the indole ring. The association constants for the peptide-oligonucleotide (PN) complexes are an order of magnitude higher (105 M) than those reported with polynucleotides [104 M; Rajeswari et al. (1987) Biochemistry 26, 6825]. The pentapeptide, KGWGK, discriminates between BglII and HindIII sequences with higher affinity for the HindIII dodecamer. The CD maximum of KGWGK, at 220 nm, is drastically diminished upon interaction with oligonucleotides. The ellipticity at 220 nm is halved at 10 times less P/N ratio with the HindIII dodecamer than the BglII dodecamer, suggesting stronger binding to the HindIII dodecamer. The results are discussed in terms of two different modes of binding of oligopeptides to the DNA hairpins