A cationic lumen in the Wzx flippase mediates anionic O-antigen subunit translocation in Pseudomonas aeruginosa PAO1.

Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Through experimentally based and unbiased topological mapping, our group previously observed that Wzx possesses many charged and aromatic amino acid residues within its 12 transmembrane segments (TMS).

ProQ is an RNA chaperone that controls ProP levels in Escherichia coli.

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E.

Transmembrane helix I and periplasmic loop 1 of Escherichia coli ProP are involved in osmosensing and osmoprotectant transport.

Osmoregulatory transporters stimulate bacterial growth by mediating osmoprotectant uptake in response to increasing osmotic pressure. The ProP protein of Escherichia coli transports proline and other osmoprotectants. Like LacY, ProP is a member of the major facilitator superfamily and a H(+)-solute symporter.

Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae.

Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain.

Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality.

ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV.

Core residue replacements cause coiled-coil orientation switching in vitro and in vivo: structure-function correlations for osmosensory transporter ProP.

Protein ProP acts as an osmosensory transporter in diverse bacteria. C-Terminal residues 468-497 of Escherichia coli ProP (ProPEc) form a four-heptad homodimeric alpha-helical coiled coil. Arg 488, at a core heptad a position, causes it to assume an antiparallel orientation.

The structure and function of Saccharomyces cerevisiae proteinase A.

Saccharomyces cerevisiae proteinase A (saccharopepsin; EC 3.4.23.

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli.

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally.

Evidence that WbpD is an N-acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O-antigen biosynthesis in Pseudomonas aeruginosa PAO1.

Di-N-acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3-N-acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid [UDP-d-Man(2NAc3NAc)A], a precursor for the d-Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium.

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template.

Overexpression, purification, and characterization of ProQ, a posttranslational regulator for osmoregulatory transporter ProP of Escherichia coli.

ProP is an osmosensor and osmoregulatory transporter in Escherichia coli. Osmotic activation of ProP is attenuated 5-fold in the absence of soluble protein ProQ, but proQ lesions do not influence proP transcription or ProP levels. The mechanism by which ProQ amplifies ProP activity is unknown.

The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa.

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P.

Assessment of three variations of the 1,9-dimethylmethylene blue assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid.

Objective: To determine whether 3 variations of the 1,9-dimethylmethylene blue (DMMB) assay yield comparable results when measuring sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF).

Sample Population: 25 samples of SF collected from affected joints of 13 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses.

Procedure: Sulfated glycosaminoglycan concentrations were measured by the direct spectrophotometric (ie, Farndale), microplate, and indirect DMMB assays in samples of SF collected from normal and affected joints and in samples digested with nucleases, papain, and hyaluronidase.

Development of a solid-phase assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid.

Objective: To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays.

Sample Population: 25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses.

Procedure: A solid-phase DMMB assay was developed to measure sGAG concentrations in SE Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Famdale method) and microplate DMMB assay.

Facial trauma and ocular/orbital injury.

Background And Objectives: Ocular injuries occur commonly in patients with facial trauma. Patients with significant eye injuries may present with grossly normal eyes and good visual acuity; however, subsequent ocular disorders may become apparent. The estimates of incidence vary considerably.

Small-diameter corneal inlay in presbyopic or pseudophakic patients.

We report the results of using the small-diameter corneal inlay to create a bifocal cornea. The inlay was implanted in five eyes in January 1993. At 12 months postoperatively, uncorrected near vision had improved from J4 to better than J2 in four of the five.

Comparison of three keratometry instruments.

We compared the reliability of measurements of three keratometers and keratometry readings from a corneal topography system to determine if they were as accurate as the "gold standard" Javal-Schiotz keratometer. Same-day measurement of the steepest and flattest powers of the central 3 mm of the corneas of 200 eyes (100 patients) revealed no statistically significant difference in reliability between the newer keratometers and the Javal-Schiotz. In addition, the newer machines are more convenient and efficient in some clinical settings.

The use of collagen shields for drug delivery in cataract and intraocular lens surgery.

Collagen shields are an appealing route of drug administration because they avoid complications associated with periocular injections and frequent topical drug application. A review of past studies establishes the capability of collagen shields to reach therapeutic aqueous drug levels in animal models. More recent studies on human subjects reaffirm the capability of collagen shields to provide adequate prophylaxis against the most common pathogens of postoperative endophthalmitis.

Ab-interno neodymium:YAG versus erbium:YAG laser sclerostomies in a rabbit model.

This study was undertaken to determine whether thermally-induced tissue necrosis was a factor in ab-interno contact-laser sclerostomy failure. A rabbit model was used to compare the continuous-wave Neodymium (Nd):YAG with the pulsed Erbium (Er):YAG laser with respect to such failure. Laser energy was focused into a fused-silica fiber optic (400 microns) for the Nd:YAG laser (12 W; 3 to 5 seconds), and into a single-crystal, uncladded sapphire fiber optic (250 microns) for the Er:YAG laser (7 to 8 mJ; 250 microseconds; 6 to 8 pulses).

Combined microwave heating and surface cooling of the cornea.

We investigated a nonsurgical means of reshaping the cornea to correct hyperopia, keratoconus, or myopia. The object was to heat the central stroma of the cornea to the shrinkage temperature of collagen, 55-58 degrees C. The heating device was an open-ended, coaxial, near-field applicator driven at 2450 MHz; it incorporates cooling of the cornea surface by flow of saline.

Absorption of 308-nm excimer laser radiation by balanced salt solution, sodium hyaluronate, and human cadaver eyes.

Absorption of the excimer laser radiations of 193-nm argon fluorine and 308-nm xenon chloride in balanced salt solution, sodium hyaluronate, and human cadaver eyes was measured. The absorption of these materials as considerably different for the two wavelengths; we found that 308-nm light experienced much less absorption than the 193-nm light. The extinction coefficient (k) for 308 nm was k = 0.