Biomedical subjects

A Brooun

Publications and source records attributed to A Brooun.

9 recordsLinked to original sources

PubMed2001

HIV-1 preintegration complexes preferentially integrate into longer target DNA molecules in solution as detected by a sensitive, polymerase chain reaction-based integration assay.

After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using lambda exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.

Binding, Competitive↗

PubMed2000

Expression and fast-flow purification of a polyhistidine-tagged myoglobin-like aerotaxis transducer.

A Co(2+)-affinity, fast-flow perfusion chromatography method to purify a polyhistidine-tagged myoglobin-like aerotaxis transducer HemAT-Hs has been developed. The method relies upon a six-histidine affinity tag fused to the C-terminus and N-terminus of HemAT-Hs for expression in the native host, an extremely halophilic Archaeon Halobacterium salinarum, and in the heterologous host Escherichia coli, respectively. The His-tagged HemAT-Hs can be purified rapidly using either low or high ionic strength buffers. Purified His-tagged HemAT-Hs in high or low salt buffers demonstrated no difference in spectral characteristics and retained reversible oxygen binding capacity. This fast-flow Co(2+)-affinity perfusion chromatography provides a simple method for preparation of halophilic heme containing soluble proteins for biophysical and structural studies.

Archaeal Proteins↗

PubMed2000

A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms.

Bacterial biofilms show enormous levels of antibiotic resistance, but little is known about the underlying molecular mechanisms. Multidrug resistance pumps (MDRs) are responsible for the extrusion of chemically unrelated antimicrobials from the bacterial cell. Contribution of the MDR-mediated efflux to antibiotic resistance of Pseudomonas aeruginosa biofilms was examined by using strains overexpressing and lacking the MexAB-OprM pump. Resistance of P. aeruginosa biofilms to ofloxacin was dependent on the expression of MexAB-OprM but only in the low concentration range. Unexpectedly, biofilm resistance to ciprofloxacin, another substrate of MexAB-OprM, did not depend on the presence of this pump. Dose-dependent killing indicated the presence of a small "superresistant" cell fraction. This fraction was primarily responsible for very high resistance of P. aeruginosa biofilms to quinolones. Bacterial cells recovered from a biofilm and tested under nongrowing conditions with tobramycin exhibited higher resistance levels than planktonic cells but lower levels than cells of an intact biofilm.

Anti-Bacterial Agents↗

PubMed1999

Purification and ligand binding of EmrR, a regulator of a multidrug transporter.

EmrR, the repressor of the emrRAB operon of Escherichia coli, was purified to 95% homogeneity. EmrR was found to bind putative ligands of the EmrAB pump-2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone-with affinities in the micromolar range. Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR.

2,4-Dinitrophenol↗

PubMed1998

Sensory rhodopsin II transducer HtrII is also responsible for serine chemotaxis in the archaeon Halobacterium salinarum.

Previously, we demonstrated that the methyl-accepting protein HtrII is the transducer for photoreceptor sensory rhodopsin II. Here, we provide experimental evidence that HtrII is also a chemotransducer. Using an agarose-in-plug bridge method, we show that an HtrII overexpression strain has a quicker response to serine than does an HtrII deletion strain. Furthermore, an in vivo flow assay demonstrates that the deletion strain is unable to modulate methylesterase activity after serine addition or photostimulation, while the overexpression strain shows distinct methanol peaks following both types of stimuli.

Archaeal Proteins↗

PubMed1998

An archaeal aerotaxis transducer combines subunit I core structures of eukaryotic cytochrome c oxidase and eubacterial methyl-accepting chemotaxis proteins.

Signal transduction in the archaeon Halobacterium salinarum is mediated by three distinct subfamilies of transducer proteins. Here we report the complete htrVIII gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome c oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the htrVIII gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.

Amino Acid Sequence↗

PubMed1997

Primary structure and functional analysis of the soluble transducer protein HtrXI in the archaeon Halobacterium salinarium.

Signal transduction in the archaeon Halobacterium salinarium is mediated by a family of 13 soluble and membrane-bound transducers. Here, we report the primary structure and functional analysis of one of the smallest halobacterial putative transducers, HtrXI. Hydropathy plot analysis of the primary structure predicts no membrane-spanning segments in HtrXI. The fractionation of the H. salinarium proteins confirmed that HtrXI is a soluble protein. Capillary assay with an HtrXI deletion mutant and a complemented strain revealed that this soluble transducer is involved in Asp and Glu taxis. In vivo analysis of the methylesterase activity of the htrXI-1 deletion mutant suggests that HtrXI plays an important role in the adaptation of the chemotactic responses to His, Asp, and Glu, which are attractants for halobacteria. Stimulation by Asp and Glu causes demethylation of HtrXI and of another putative transducer, HtrVII. But addition of His to halobacterial cells increases HtrXI methylation together with that of other putative transducers. In the absence of HtrXI, stimulation by either Glu or His does not decrease or increase the methylation of any putative transducers. Therefire, the HtrXI transducer appears to have a complex role in chemotaxis signal transduction.

Amino Acid Sequence↗

PubMed1996

The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein.

Recently, a large family of transducer proteins in the Archaeon Halobacterium salinarium was identified. On the basis of the comparison of the predicted structural domains of these transducers, three distinct subfamilies of transducers were proposed. Here we report isolation, complete gene sequences, and analysis of the encoded primary structures of transducer gene htrII, a member of family B, and its blue light receptor gene (sopII) of sensory rhodopsin II (SRII). The start codon ATG of the 714-bp sopII gene is one nucleotide beyond the termination codon TGA of the 2298-bp htrII gene. The deduced protein sequence of HtrII predicts a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm. HtrII has a common feature with HtrI, the sensory rhodopsin I transducer; like HtrI, HtrII possesses a hydrophilic loop structure just after the second transmembrane segment. The C-terminal 299 residues (765 amino acid residues total) of HtrII show strong homology to the signaling and methylation domain of eubacterial transducer Tsr. The hydropathy plot of the primary structure of SRII indicates seven membrane-spanning alpha-helical segments, a characteristic feature of retinylidene proteins ("rhodopsins") from a widespread family of photoactive pigments. SRII shows high identity with SRI (42%), bacteriorhodopsin (BR) (32%), and halorhodopsin (24%). The crucial positions for retinal binding sites in these proteins are nearly identical, with the exception of Met-118 (numbering according to the mature BR sequence), which is replaced by Val in SRII. In BR, residues Asp-85 and Asp-96 are crucial in proton pumping. In SRII, the position corresponding to Asp-85 in BR is conserved, but the corresponding position of Asp-96 is replaced by an aromatic Tyr. Coexpression of the htrII and sopII genes restores SRII phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI and insertion in htrII/sopII regions. This paper describes the first example that both HtrI and HtrII exist in the same halobacterial cell, confirming that different sensory rhodopsins SRI and SRII in the same organism have their own distinct transducers.

Amino Acid Sequence↗

PubMed1996

Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins.

Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments. We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.

Amino Acid Sequence↗