Opacification of SC60B-OUV lens implant following routine phacoemulsification surgery: case report and EM study.
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Aged↗
Biomedical subjects
G J Phillips
Publications and source records attributed to G J Phillips.
Chromosomal integration and expression of the Escherichia coli K88 gene cluster in Salmonella enterica ser. Choleraesuis strain 54 (SC54).
Attempts to develop live vaccines to protect against enterotoxigenic Escherichia coli (ETEC) infection by induction of both cell-mediated and mucosal immunity, and serum antibody responses have included use of recombinant Salmonella strains that produce K88 fimbrial antigens (Hone et al., 1988; Attridge et al., 1988; Morona et al., 1994). However, none of the recombinant Salmonella vectors has been licensed by the United States Department of Agriculture (USDA) for use as a live vaccine in pigs in the United States. A variant of Salmonella enterica ser. Choleraesuis strain 54 (SC54) is currently used as a safe and effective intranasal attenuated live vaccine in pigs. In order to expand the efficacy of this live vaccine strain, we sought to modify strain SC54 to express the K88 antigens of ETEC. To accomplish this, a plasmid-based system was used to integrate the K88 gene cluster into the chromosome of strain SC54 by site-specific recombination. The K88 antigens were expressed by strain SC54, and the gene cluster was stably maintained in the host.
Administration, Intranasal↗
Green fluorescent protein--a bright idea for the study of bacterial protein localization.
Use of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for protein and DNA localization has provided sensitive, new approaches for studying the organization of the bacterial cell, leading to new insights into diverse cellular processes. GFP has many characteristics that make it useful for localization studies in bacteria, primarily its ability to fluoresce when fused to target polypeptides without the addition of exogenously added substrates. As an alternative to immunofluorescence microscopy, the expression of gfp gene fusions has been used to probe the function of cellular components fundamental for DNA replication, translation, protein export, and signal transduction, that heretofore have been difficult to study in living cells. Moreover, protein and DNA localization can now be monitored in real time, revealing that several proteins important for cell division, development and sporulation are dynamically localized throughout the cell cycle. The use of additional GFP variants that permit the labeling of multiple components within the same cell, and the use of GFP for genetic screens, should continue to make this a valuable tool for addressing complex questions about the bacterial cell.
Bacteria↗
Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY.
Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.
Bacterial Proteins↗
YidC mediates membrane protein insertion in bacteria.
The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.
ATP-Binding Cassette Transporters↗
Genetic system for reversible integration of DNA constructs and lacZ gene fusions into the Escherichia coli chromosome.
A plasmid system for site-specific integration into and excision and recovery of gene constructs and lacZ gene fusions from the Escherichia coli chromosome was developed. Plasmid suicide vectors utilizing the origin of replication of R6K plasmids and containing the attP sequence of bacteriophage lambda, multiple cloning site, and antibiotic resistance markers facilitate reversible integration into the E. coli chromosome by site-specific recombination. Additional vectors permit construction of lacZ gene fusions in three possible reading frames for recombination with the bacterial chromosome. These suicide vectors can be propagated in newly constructed E. coli strains that harbor different pir alleles. Two helper plasmids that encode the necessary gene products for integration (Int) and excision (Int and Xis) were also constructed. This plasmid system was shown to be a reliable and efficient means to integrate and subsequently recover plasmids from the E. coli attB site.
Artificial Gene Fusion↗
Green fluorescent protein functions as a reporter for protein localization in Escherichia coli.
The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.
ATP-Binding Cassette Transporters↗
High copy number plasmids compatible with commonly used cloning vectors.
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Cloning, Molecular↗
Comparison of the quality of protection elicited by toxoid and peptide liposomal vaccine formulations against ricin as assessed by markers of inflammation.
Ricin is a very toxic substance which inhibits protein synthesis and produces severe tissue damage and inflammation. It is very potent when inhaled as an aerosol and protection has been examined in a series of studies using vaccine candidates including a formaldehyde inactivated ricin toxoid and the A chain of ricin, a polypeptide equivalent to half of the toxin molecule. Initially, subcutaneous injections of both compounds were found to protect against inhaled ricin but not without some subsequent adverse signs. Intra-pulmonary vaccination using liposomal formulations of these compounds was investigated with a view to improving lung condition following challenge. Using the humoral and local pulmonary immune responses as indices of vaccine performance, no significant difference between toxoid or peptide vaccines was found. In the third and current study, the quality of lung protection by vaccines was assessed using markers of inflammation. Thus, the profiles of inflammatory cell and protein influx into the lung were determined following intratracheal (i.t.) challenge with ricin of rats treated with liposomal vaccine formulations. Results showed that liposomal ricin toxoid offered a better quality of protection with a significantly lower influx of polymorphonuclear leucocytes (neutrophils) and little pulmonary oedema compared with the A chain/liposome formulation. Further, there was no significant difference between the quality of protection offered by the A chain when administered subcutaneously or locally into the lung by i.t. instillation. Liposomal ricin toxoid is a good candidate vaccine and optimised pulmonary delivery by inhalation should be further examined.
Animals↗
Alteration of open reading frames by use of new gene cassettes.
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Base Sequence↗
New cloning vectors with temperature-sensitive replication.
A series of cloning vectors with conditional, temperature-sensitive replication that are selectable with ampicillin, chloramphenicol, and kanamycin has been constructed. These vectors are derivatives of a pSC101 mutant that can replicate only at low temperatures. The cloning vectors carry a number of unique restriction sites and provide for screening of recombinant plasmids by alpha complementation. These vectors have proven useful for a variety of applications where conditional replication of a recombinant plasmid is desired.
Cloning, Molecular↗
Molecular characterization of Escherichia coli FtsE and FtsX.
The genes ftsE and ftsX are organized in one operon together with ftsY. FtsY codes for the receptor of the signal recognition particle (SRP) that functions in targeting a subset of inner membrane proteins. We have found no indications for a structural relationship between FtsE/X and FtsY. Evidence is presented that FtsE and FtsX form a complex in the inner membrane that bears the characteristics of an ATP-binding cassette (ABC)-type transporter. FtsE is a hydrophilic nucleotide-binding protein that has a tendency to dimerize and associates with the inner membrane through an interaction with the integral membrane protein FtsX. An FtsE null mutant showed filamentous growth and appeared viable on high salt medium only, indicating a role for FtsE in cell division and/or salt transport.
ATP-Binding Cassette Transporters↗
Use of in vitro models of haemofiltration and haemodiafiltration to estimate dosage regimens for critically ill patients prescribed cefpirome.
BACKGROUND: The physico-chemical properties of cefpirome (low protein binding, high water solubility and low molecular weight) suggest that it may be lost readily from the extracorporeal circulation of intensive care unit patients during continuous renal replacement therapy. METHOD: In order to make informed dosage recommendations for patients receiving artificial renal support, cefpirome loss from human blood has been quantified using in vitro models of continuous haemofiltration and haemodiafiltration. Cefpirome clearance was measured using three membrane types at varying ultrafiltrate (UFR) and dialysis flow rates (Qd). RESULTS: During haemofiltration cefpirome was found to cross hollow fibre polyamide (PA) and polyacrylonitrile (PAN) membranes with equal efficiency. The mean sieving coefficients (S) of both PA and PAN membranes were consistently high (> 0.7) when two different ultrafiltration rates were used. Changing the ultrafiltration rate or membrane type had no significant effect on the sieving coefficient of cefpirome but did result in an increase in cefpirome filter clearance (Fcl). Using the haemodiafiltration model, cefpirome penetrated PAN membranes (flat plate AN69S) more efficiently than hollow fibre PA membranes (FH66D). In each case, increasing the dialysis flow rate reduced the S-value. However, although increasing Qd was associated with a greater Fcl of cefpirome when PAN membranes were employed, no such relationship was found for the PA hollow fibre membrane. CONCLUSION: The information generated can be used to estimate a dosing regimen for intensive care patients prescribed cefpirome and receiving continuous renal replacement therapy.
Cephalosporins↗
Recovery of YAC-end sequences through complementation of an Escherichia coli pyrF mutation.
We have developed a genetic means to recover sequences from YAC-ends near the yeast selectable marker URA3. This strategy is based on the ability of URA3 to complement mutations in pyrF, an Escherichia coli gene required for pyrimidine biosynthesis. We have developed an E.coli strain with a non-reverting allele of pyrF that is also suitable for cloning (recA-, hsdR-). We demonstrate the utility of this complementation strategy to obtain right-end clones from three YACs containing Arabidopsis thaliana DNA.
Chromosomes, Artificial, Yeast↗
Fetal exposure to low protein maternal diet alters the susceptibility of young adult rats to sulfur dioxide-induced lung injury.
The maternal diet is an important determinant of glutathione-related metabolism in rats. Glutathione (GSH) may play a major role in the detoxification of sulfur dioxide (SO2) within the lungs. The effects of fetal exposure to a low protein maternal diet upon later susceptibility to pulmonary injury induced by chronic SO2 exposure were evaluated in young adult rats. Pregnant rats were fed purified diets containing 180 g casein/kg (control diet) or 120, 90 or 60 g casein/kg (experimental diets). After parturition, all dams were fed a standard non-purified diet (189 g protein/kg diet). The pups thus differed only in terms of protein nutrition during gestation. At seven wk of age the male pups were housed in either room air or 286 microg SO2/m3 for 5 h/d during a 28-d period. At the end of the final SO2 treatment period, the rats exposed to 90 or 60 g casein/kg diets in utero exhibited significantly greater pulmonary injury, as assessed by bronchoalveolar lavage, than did those exposed to control diet in utero. Significant maternal diet-induced differences in activities of enzymes of the gamma-glutamyl cycle were noted in the lungs and livers of rats which had not undergone SO2 treatment. Furthermore, the response of these enzyme activities to SO2 treatment was determined by prior exposure to the maternal diet. SO2-treated rats exposed to control diet (180 g casein/kg) and low protein diet (60 g casein/kg), but not those exposed to 120 or 90 g casein/kg diets, tended to augment the activities, relative to rats not treated with SO2, of enzymes which maintain tissue GSH status either through synthesis or recycling. Differences in susceptibility to SO2-induced tissue injury may be related to programming of GSH metabolism by the maternal diet. Alternatively, impaired immune and acute phase responses to an inflammatory insult may account for a failure to resolve initial SO2-induced injury in rats exposed to low protein maternal diets.
Air Pollutants↗
Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle.
Targeting of the cytoplasmic membrane protein leader peptidase (Lep) and a Lep mutant (Lep-inv) that inserts with an inverted topology compared to the wild-type protein was studied in Escherichia coli strains that are conditional for the expression of either Ffh or 4.5S RNA, the two components of the E. coli SRP. Depletion of either component strongly affected the insertion of both Lep and Lep-inv into the cytoplasmic membrane. This indicates that SRP is required for the assembly of cytoplasmic membrane proteins in E. coli.
Escherichia coli↗
Sulphur dioxide: a potent glutathione depleting agent.
Sulphur dioxide (SO2) is an air pollutant implicated in the initiation of asthmatic symptoms. Glutathione (GSH) has been proposed to play a role in detoxification of SO2 through the sulfitolysis of glutathione disulphide (GSSG) to S-sulphoglutathione (GSSO3-). Rats were exposed to concentrations of SO2 between 5 and 100 ppm for 5 hr a day between 7 and 28 days. Lung injury as assessed by bronchoalveolar lavage and tissue GSH status were evaluated. SO2 5 ppm failed to elicit any lung injury or inflammatory response but did deplete GSH pools in lung, liver, heart and kidney. Activities of gamma-glutamylcysteine synthetase (GCS), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GRed) in lung were lowered relative to those in control animals. In liver, GRed activity was decreased. SO2 50 ppm exposure also failed to elicit injury or inflammation but did lower inflammatory cell numbers in the circulation. Rats exposed to 50 ppm SO2 maintained tissue GSH status, but activities of GCS, GPx, GRed and gamma-glutamyltranspeptidase in lung and hepatic GRed and GPx were significantly lower than in control rats. Unaltered GST activity in lung and liver was suggestive of an impairment of the sulfitolysis reaction in these animals, perhaps through lower substrate flux through the GPx reaction, as GSSO3- is a known inhibitor of GST in the rat. Rats exposed to 100 ppm SO2 exhibited evidence of inflammation (120-fold increase in neutrophil numbers recovered in lavage fluid) and like the 5 ppm exposed rats had lower tissue GSH concentrations and GSH-related enzyme activities in lung. We conclude that sulfitolysis of GSSG does occur in vivo during SO2 exposure and that SO2, even in the absence of pulmonary injury, is a potent glutathione depleting agent.
Air Pollutants↗
New plasmids carrying antibiotic-resistance cassettes.
A series of new plasmid vectors is described that carry gene cassettes imparting resistance to the antibiotics chloramphenicol (CmR), kanamycin (KmR), tetracycline (TcR) and spectinomycin/streptomycin (Sp/SmR). The gene cassettes are symmetrically flanked by several restriction sites. In addition, several restriction sites that are normally found internal to the gene cassettes have been eliminated, thereby expanding the number of restriction enzymes available to excise an intact antibiotic-resistance gene. The gene cassettes are carried by high-copy-number plasmids that confer ampicillin resistance (ApR).
DNA Transposable Elements↗