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Complementation of an Escherichia coli DnaK Defect by Hsc70-DnaK Chimeric Proteins. Jean-Philippe Suppini, 2004.Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein [Hsp70] family that showstrong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones . We show here that,while the DnaK protein is, as expected, able to complement anE . coli dnaK mutant strain for growth at high temperatures and 
phage propagation, Hsc70 protein is not . However, an Hsc70in which
the peptide-binding domain has been replaced by thatof DnaK is able
to complement this strain for both phenotypes,suggesting that the
peptide-binding domain of DnaK is essentialto fulfill the specific
functions of this protein necessaryfor growth at high temperatures
and for
phage replication.The implications of these findings on the
functional specificitiesof the Hsp70s and the role of
protein-protein interactions inthe DnaK chaperone system are
discussed.
In Vitro Comparison of Topical Microbicides for Prevention of Human Immunodeficiency Virus Type 1 Transmission. Charlene S. Dezzutti, 2004.A standardized protocol was used to compare cellular toxicities and anti-human immunodeficiency virus type 1 (HIV-1) activities of candidate microbicides formulated for human use . The microbicides evaluated were cellulose acetate phthalate (CAP), Carraguard, K-Y plus nonoxynol-9 (KY-N9), PRO 2000 (0.5 and 4%), SPL7013 (5%), UC781 (0.1 and 1%), and Vena Gel, along with their accompanying placebos . Products were evaluated for toxicity on cervical and colorectal epithelial cell lines, peripheral blood mononuclear cells (PBMCs), and macrophages (M  ) by using an ATP release assay, and they were tested for their effect on transepithelial resistance (TER) of polarized epithelial monolayers . Anti-HIV-1 activity was evaluated in assays for transfer of infectious HIV-1 from epithelial cells to activated PBMCs and for PBMC and M infection . CAP, Carraguard, PRO 2000, SPL7013, and UC781 along with their placebos were 20- to 50-fold less toxic than KY-N9 and Vena Gel . None of the nontoxic product concentrations disrupted the TER . Transfer of HIV-1Ba-L from epithelial cells to PBMCs and PBMC and M infection with laboratory-adapted HIV-1Ba-L and HIV-1LAI isolates were inhibited by all products except Carraguard, KY-N9, and Vena Gel . KY-N9, Vena Gel, and Carraguard were not effective in blocking PBMC infection with primary HIV-1A, HIV-1C, and HIV-1CRF01-AE isolates . The concordance of these toxicity results with those previously reported indicates that our protocol may be useful for predicting toxicity in vivo . Moreover, our systematic anti-HIV-1 testing provides a rational basis for making better informed decisions about which products to consider for clinical trials .
Safety, Pharmacokinetics, Pharmacodynamics, and Plasma Lipoprotein Distribution of Eritoran (E5564) during Continuous Intravenous Infusion into Healthy Volunteers. Daniel P. Rossignol, 2004.Eritoran, a structural analogue of the lipid A portion of lipopolysaccharide (LPS), is an antagonist of LPS in animal and human endotoxemia models . Previous studies have shown that low doses (350 to 3,500 µg) of eritoran have demonstrated a long pharmacokinetic half-life but a short pharmacodynamic half-life . The present study describes the safety, pharmacokinetics and pharmacodynamics, and lipid distribution profile of eritoran during and after a 72-h intravenous infusion of 500, 2,000, or 3,500 µg/h into healthy volunteers . Except for the occurrence of phlebitis, eritoran administration over 72 h was safe and well tolerated . Eritoran demonstrated a slow plasma clearance (0.679 to 0.930 ml/h/kg of body weight), a small volume of distribution (45.6 to 49.8 ml/kg), and a relatively long half-life (50.4 to 62.7 h) . In plasma, the majority (  55%) of eritoran was bound to high-density lipoproteins . During infusion and for up to 72 h thereafter, ex vivo response of blood to 1- or 10-ng/ml LPS was inhibited by
 85%, even when the lowest dose of eritoran (500 µg/h) was infused . Inhibition of response was dependent on eritoran dose and the concentration of LPS used as an agonist . Finally, in vitro analysis with purified lipoprotein and protein fractions from plasma obtained from healthy volunteers indicated that eritoran is inactivated by high-density but not low-density lipoproteins, very-low-density lipoproteins, or albumin . From these results, we conclude that up to 252 mg of eritoran can be safely infused into normal volunteers over 72 h and even though it associates extensively with high-density lipoproteins, antagonistic activity is maintained, even after infusion ceases .
Variable Surface Protein Vmm of Mycoplasma mycoides subsp . mycoides Small Colony Type. Anja Persson, 2002.A variable surface protein, Vmm, of the bovine pathogen Mycoplasma mycoides subsp . mycoides small colony type (M . mycoides SC) has been identified and characterized . Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzed M . mycoides SC strains . The protein was found to undergo reversible phase variation at a frequency of 9 x 10-4 to 5 x 10-5 per cell per generation . The vmm gene was present in all of the 69 tested M . mycoides SC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified . DNA sequencing of the vmm gene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer . Vmm-like genes were also found in four closely related mycoplasmas, Mycoplasma capricolum subsp . capricolum, M . capricolum subsp . capripneumoniae, Mycoplasma sp . bovine serogroup 7, and Mycoplasma putrefaciens . However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by the vmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed . Chimeric Analysis of the Multicomponent Multidrug Efflux Transporters from Gram-Negative Bacteria. Elena B. Tikhonova, 2002.Many multidrug transporters from gram-negative bacteria belong to the resistance-nodulation-cell division (RND) superfamily of transporters . RND-type multidrug transporters have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane . They usually function as three-component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of gram-negative bacteria . The structural determinants of RND transporters responsible for multidrug recognition and complex assembly remain unknown . We constructed chimeric RND transporters composed of N-terminal residues of AcrB and C-terminal residues of MexB, the major RND-type transporters from Escherichia coli and Pseudomonas aeruginosa, respectively . The assembly of complexes and multidrug efflux activities of chimeric transporters were determined by coexpression of hybrid genes either with AcrA, the periplasmic component of the AcrAB transporter from E . coli, or with MexA and OprM, the accessory proteins of the MexAB-OprM pump from P . aeruginosa . We found that the specificity of interaction with the corresponding periplasmic component is encoded in the T60-V612 region of transporters . Our results also suggest that the large periplasmic loops of RND-type transporters are involved in multidrug recognition and efflux . Role of ctc from Listeria monocytogenes in Osmotolerance. Rozenn Gardan, 2003.Listeria monocytogenes is a food-borne pathogen with the ability to grow under conditions of high osmolarity . In a previous study, we reported the identification of 12 proteins showing high induction after salt stress . One of these proteins is highly similar to the general stress protein Ctc of Bacillus subtilis . In this study, induction of Ctc after salt stress was confirmed at the transcriptional level by using RNA slot blot experiments . To explore the role of the ctc gene product in resistance to stresses, we constructed a ctc insertional mutant . No difference in growth was observed between the wild-type strain LO28 and the ctc mutant either in rich medium after osmotic or heat stress or in minimal medium after heat stress . However, in minimal medium after osmotic stress, the growth rate of the mutant was increased by a factor of 2 . Moreover, electron microscopy analysis showed impaired morphology of the mutant grown under osmotic stress conditions in minimal medium . Addition of the osmoprotectant glycine betaine to the medium completely abolished the osmotic sensitivity phenotype of the ctc mutant . Altogether, these results suggest that the Ctc protein of L . monocytogenes is involved in osmotic stress tolerance in the absence of any osmoprotectant in the medium . Identification of an Emergent and Atypical Pseudomonas viridiflava Lineage Causing Bacteriosis in Plants of Agronomic Importance in a Spanish Region. Ana J. González, 2003.Pseudomonas strains with an atypical LOPAT profile (where LOPAT is a series of determinative tests: L, levan production; O, oxidase production; P, pectinolitic activity; A, arginine dihydrolase production; and T, tobacco hypersensibility) can be regarded as emergent pathogens in the Principality of Asturias (Spain), where they have been causing, since 1999, severe damage in at least three taxonomically unrelated orchard plants of agronomic importance: common bean (Phaseolus vulgaris), kiwifruit (Actinidia deliciosa), and lettuce (Lactuca sativa) . These strains are mainly differentiated by production of yellowish mucoid material in hypersucrose medium, used for the levan test, and by a variable pectinolytic activity on different potato varieties . The atypical organisms were identified as Pseudomonas viridiflava based on their 16S rRNA sequences . Among them a certain intraspecies genetic heterogeneity was detected by randomly amplified polymorphic DNA (RAPD) typing . To differentiate between isolates of P . viridiflava and Pseudomonas syringae pathovars, a 16S ribosomal DNA restriction fragment length polymorphism method employing the restriction endonucleases SacI and HinfI was developed . This could be used as a means of reliable species determination after the usual phenotypical characterization, which includes the LOPAT tests .
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