High-throughput FACS method for directed evolution of substrate specificity.
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Catalysis↗
Biomedical subjects
George Georgiou
Publications and source records attributed to George Georgiou.
How to flip the (redox) switch.
Redox-sensitive transcription factors such as the E. coli OxyR and S. cerevisiae Yap1, are activated by conformational changes that stem from the post-translational modification of reactive protein thiols. Recent studies provide new insights on how different agents that cause redox stress mediate the activation of transcription through distinct and specific pathways.
Bacterial Proteins↗
Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer.
Reagents for proteome research must of necessity be generated by high throughput methods. Aptamers are potentially useful as reagents to identify and quantitate individual proteins, yet are currently produced for the most part by manual selection procedures. We have developed automated selection methods, but must still individually purify protein targets. Therefore, we have attempted to select aptamers against protein targets generated by in vitro transcription and translation of individual genes. In order to specifically immobilize the protein targets for selection, they are also biotinylated in vitro. As a proof of this method, we have selected aptamers against translated human U1A, a component of the nuclear spliceosome. Selected sequences demonstrated exquisite mimicry of natural binding sequences and structures. These results not only reveal a potential path to the high throughput generation of aptamers, but also yield insights into the incredible specificity of the U1A protein for its natural RNA ligands.
Automation↗
Cell-Surface display of heterologous proteins: From high-throughput screening to environmental applications.
A variety of expression systems for the display of either short peptides or fully folded proteins on E.coli and, to a lesser extent, on Gram-positive bacteria have been developed. The expression of proteins on the surface of microbial cells has proved extremely important for numerous applications ranging from combinatorial library screening and protein engineering, to whole cell biocatalysts and adsorbants for bioremediation purposes.
Bacterial Proteins↗
Genetic analysis of the twin arginine translocator secretion pathway in bacteria.
The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tat-specific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wild-type TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.
Amino Acid Sequence↗
Measurement of the lateral diffusion of human MHC class I molecules on HeLa cells by fluorescence recovery after photobleaching using a phycoerythrin probe.
The mobility of cell surface MHC class I molecules on HeLa cells was measured by fluorescence recovery after photobleaching (FRAP). The probe used for these studies was the phycobiliprotein R-phycoerythrin coupled to Fab fragments of a monoclonal antibody specific for human monomorphic MHC class I molecules. It was found that the recovery curves could be equally well fitted by either a random diffusion model with an immobile component or by an anomalous diffusion model. In the latter case, the anomalous diffusion exponent was consistent with that previously determined by single-particle tracking (SPT) experiments using the same probe (P. R. Smith, I. E. G. Morrison, K. M. Wilson, N. Fernandez, and R. J. Cherry. 1999. Biophys. J. 76:3331-3344). The FRAP experiments, however, yielded a considerably higher value of D(0), the diffusion coefficient for a time interval of 1 s. To determine whether the results were probe dependent, FRAP measurements were also performed with the same monoclonal antibody labeled with Oregon Green. These experiments gave similar results to those obtained with the phycoerythrin probe. FRAP experiments with the lipid probe 5-N-(octadecanoyl) aminofluoroscein (ODAF) bound to HeLa cells gave typical results for lipid diffusion. Overall, our observations and analysis are consistent with anomalous diffusion of MHC class I diffusion on HeLa cells, but quantitative differences between FRAP and SPT data remain to be explained.
Antibodies, Monoclonal↗
The prognostic significance of CA 125 in patients with non-Hodgkin's lymphoma.
OBJECTIVE: To assess the association of serum CA 125 in patients with non-Hodgkin's lymphoma (NHL) with prognostic parameters of the disease, response to treatment, and survival. PATIENTS AND METHODS: Sixty-eight patients [38 males, median age 56 (range 17-82) yr] with NHL were evaluated. CA 125 was measured by an enzyme immunoradiometric assay at diagnosis and at the end of first-line treatment. RESULTS: Median overall CA 125 was 49 (1-963) U mL-1, whereas 49 patients had initially abnormal (>35 U mL) CA 125 levels. High CA 125 was found to correlate with failure of treatment (P = 0.001) and relapse (P = 0.01), and to be independently associated with bulky disease, effusions, LDH, and the International Prognostic Index (IPI) score (P<0.01 for each of these four variables). An initially abnormal CA 125 value was associated with poorer 5-yr survival [median survival of patients with CA 125>35 U mL-1 33 (18-72) months compared to 58 (20-77) months for those with CA 125 = 35 U mL-1, P = 0.012]. Moreover, CA 125>35 U mL-1 (among stage III/IV and LDH>460 mU mL-1) emerged as an independent predictor of death within 5 yr from diagnosis (Relative Risk (RR) 3.1, 95% CI 1.5-12.8, P = 0.02). CONCLUSION: Measurement of serum CA 125 is useful for staging, monitoring, and estimating prognosis in patients with NHL.
Adolescent↗
Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity.
The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. We have engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 nM. The entire antibody panel showed high serum, thermal, and denaturant stability. In vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the K(d) of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies may be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.
Amino Acid Sequence↗
In vitro scanning-saturation mutagenesis.
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Amino Acid Substitution↗
Increased activity of lysosomal enzymes in the peritoneal fluid of bacterial peritonitis.
OBJECTIVE: The activity of lysosomal enzymes is increased in the cerebrospinal fluid of bacterial meningitis, suggesting that inflammation may cause leakage of lysosomal enzymes into the extracellular fluid. Our objective was to study the activity of 3 lysosomal enzymes in cell-free peritoneal fluid of patients with peritoneal inflammation. METHODS: The beta-galactosidase, beta-glucuronidase, and alpha-mannosidase activity (nmol 4-methylumbelliferone/mL/h); the total, polymorphonuclear, and mononuclear cell number; and chemical parameters were determined in the peritoneal fluid of 26 patients with culture-positive acute bacterial peritonitis, 13 patients (under antibiotic treatment) with culture-negative bacterial peritonitis, 6 patients with acute mesenteric lymphadenitis, and 26 control subjects who were operated on for surgical conditions without peritoneal inflammation. RESULTS: The median beta-galactosidase activity in the culture-positive bacterial peritonitis, mesenteric lymphadenitis, and controls was 175 (range: 63--2210), 50 (range: 37--56), and 16 (range: 8--32), respectively. The beta-glucuronidase was 488 (range: 79--998), 53 (range: 27--98), and 15 (range: 3--22), respectively. The alpha-mannosidase was 801 (range: 100-3172), 78 (range: 33--157), and 41 (range: 16--63), respectively. The differences of the enzyme activities among the groups of the subjects studied were significant, with the exception of the alpha-mannosidase activity between mesenteric lymphadenitis and controls. There was no significant correlation between the enzyme activities and the cytologic or chemical parameters studied. CONCLUSIONS: The elevation of the lysosomal enzymes' activity in the peritoneal fluid of patients with bacterial peritonitis seems to be a reliable index of peritoneal infection. Of the enzymes studied, the beta-glucuronidase and beta-galactosidase activities provide the best means for diagnosing bacterial inflammation of the peritoneal cavity.
Adolescent↗
The many faces of glutathione in bacteria.
Glutathione is one of the most abundant thiols present in cyanobacteria and proteobacteria, and in all mitochondria or chloroplast-bearing eukaryotes. In bacteria, in addition to its key role in maintaining the proper oxidation state of protein thiols, glutathione also serves a key function in protecting the cell from the action of low pH, chlorine compounds, and oxidative and osmotic stresses. Moreover, glutathione has emerged as a posttranslational regulator of protein function under conditions of oxidative stress, by the direct modification of proteins via glutathionylation. This review summarizes the biosynthesis and function of glutathione in bacteria from physiological and biotechnological standpoints.
Antioxidants↗
Scale-up considerations for a hollow-fiber-membrane bioreactor treating trichloroethylene-contaminated water.
Scale-up of a hollow-fiber-membrane (HFM) bioreactor treating trichloroethylene- (TCE-) contaminated water via co-metabolism with the methanotroph Methylosinus trichosporium OB3b PP358 was investigated through cost comparisons, bioreactor experiments, and mathematical modeling. Cost comparisons, based on a hypothetical treatment scenario of 568-L/min (150-gpm) flowrate with an influent TCE concentration of 100 microg/L, resulted in a configuration of treatment trains with two HFM modules in series and an overall annual cost of US dollar 0.36/m3 treated. Biological experiments were conducted with short lumen and shell residence times, 0.16 and 0.40 min, respectively, as a result of the cost comparisons. A new variable, specific transformation, was defined for characterizing the cometabolic transformation in continuous-flow systems, and values as large as 38.5 microg TCE/mg total suspended solids were sustainable for TCE treatment. Using mathematical modeling, HFM bioreactor system design was investigated, resulting in a five-step system design strategy to facilitate sizing of the unit processes.
Biodegradation, Environmental↗