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George Georgiou

Publications and source records attributed to George Georgiou.

At least 19 recordsLinked to original sources
PubMed2007

Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides.

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.

Amino Acid Sequence↗
PubMed2006

Substrate specificity of the Escherichia coli outer membrane protease OmpP.

Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.

Amino Acid Sequence↗
PubMed2006

Assembly of multimeric phage nanostructures through leucine zipper interactions.

One barrier to the construction of nanoscale devices is the ability to place materials into 2D- and 3D-ordered arrays by controlling the assembly and ordering of connections between nanomaterials. Ordered assembly of nanoscale materials may potentially be achieved using biological tools that direct specific connections between individual components. Recently, viruses were successfully employed as scaffolds for the nucleation of nanoparticles and nanowires (Mao et al., 2004); however, there is a paucity of methods for the higher order assembly of phage-templated materials. Here we describe a general strategy for the assembly of filamentous bacteriophages into long, wire-like or into tripod-like structures. To prepare the linear phage assemblies, dimeric leucine zipper protein domains, fused to the p3 and p9 proteins of M13 bacteriophage, were employed to direct the specific end-to-end self-association of the bacteriophage particles. Electron microscopy revealed that up to 90% of the phage displaying complementary leucine zipper domains formed linear multi-phage assemblies, composed of up to 30 phage in length. To prepare tripod-like assemblies, phage were engineered to express trimeric leucine zippers as p3 fusion proteins. This resulted in 3D assembly with three individual phages attached at a single point. These ordered phage structures should provide a foundation for self-assembly of virally templated nanomaterials into useful devices.

Bacteriophage M13↗
PubMed2006

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase.

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.

Catalysis↗
PubMed2006

Differential modulation of E. coli mRNA abundance by inhibitory proteins that alter the composition of the degradosome.

In Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA-catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitor-specific changes in degradosome composition--and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.

Endoribonucleases↗
PubMed2006

A potentially insect-implantable trehalose electrooxidizing anode.

The dominant sugar in the body fluids of many insects is not glucose, the sugar of the vertebrates, but trehalose. In a step toward a cell that would operate in insects, we describe here a trehalose electrooxidizing anode. The novel component of the anode is its engineered, trehalose oxidation catalyzing, FAD-glucose-3-dehydrogenase (G3DH). Screening for gene-sources of G3DH pointed to the G3DH of Agrobacterium tumefaciens. Sequencing of the A. tumefaciens genome revealed a 1.7 kb fragment which contained the G3DH coding gene. The fragment was isolated, cloned and expressed in E. coli strain BL-21, to yield the approximately 65 kDa his-tagged flavoenzyme, with a specific activity of approximately 2.5U/mg protein. Electrical wiring of its reaction center to a carbon electrode through a high apparent electron diffusion coefficient (5.8 x 10(-6)cm(2)/s) redox hydrogel with a -0.2V versus Ag/AgCl redox potential resulted in the trehalose electrooxidizing anode. Trehalose was electrooxidized at pH 7.2 already at -0.36 V versus Ag/AgCl. At 0 V versus Ag/AgCl the trehalose electrooxidation current density was 0.1 mA/cm(2).

Animals↗
PubMed2006

Dissociation of bipolar hemiarthroplasty of the hip after dislocation. A report of five different cases and review of literature.

Little information is available about the rare but serious disadvantage of dissociation of modular components during dislocation or after close reduction in the bipolar hemiarthroplasty of the hip. In most cases, simple dislocation after primary bipolar hemiarthroplasty can safely be reduced by close methods. Dissociation leads almost always to reoperation and possible revision of the prosthesis. To avoid this complication, strict adherence to the surgical technique during the initial procedure and extra precaution during close reduction are recommended, in order to provide enhanced security over component disassembly. In the five cases presented in this study, dissociation is reported at different circumstances, along with the different methods of treatment required in each patient.

Aged↗
PubMed2006

Mood-congruent free recall bias in anxious individuals is not a consequence of response bias.

The status of mood-congruent free recall bias in anxious individuals was evaluated following incidental encoding of target words. Individuals with high and low levels of trait anxiety completed a modified Stroop task, which revealed an attentional bias for threat-related stimuli in anxious individuals. This group was significantly slower in naming the colour in which threat-related words were displayed compared to neutral words. In a subsequent free recall test for the words used in the modified Stroop task, anxious individuals recalled more threat-related words compared to low-anxious people. This difference was significant even when controlling for the false recall of items that had not been presented during study. These results support the view put forward by Russo, Fox, Bellinger, and Nguyen-Van-Tam (2001) that mood-congruent free recall bias in anxious individuals can be observed if the target material is encoded at a relatively shallow level. Moreover, contrary to Dowens and Calvo (2003), the current results show that the memory advantage for threat-related information in anxious individuals is not a consequence of response bias.

Adult↗
PubMed2006

Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays.

Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

Animals↗
PubMed2006

Regulation of RraA, a protein inhibitor of RNase E-mediated RNA decay.

The recently discovered RraA protein acts as an inhibitor of the essential endoribonuclease RNase E, and we demonstrated that ectopic expression of RraA affects the abundance of more than 700 transcripts in Escherichia coli (K. Lee, X. Zhan, J. Gao, J. Qiu, Y. Feng, R. Meganathan, S. N. Cohen, and G. Georgiou, Cell 114:623-634, 2003). We show that rraA is expressed from its own promoter, P(rraA), located in the menA-rraA intergenic region. Primer extension and lacZ fusion analysis revealed that transcription from P(rraA) is elevated upon entry into stationary phase in a sigma(s)-dependent manner. In addition, the stability of the rraA transcript is dependent on RNase E activity, suggesting the involvement of a feedback circuit in the regulation of the RraA level in E. coli.

Alkyl and Aryl Transferases↗
PubMed2006

The bacterial twin-arginine translocation pathway.

The twin-arginine translocation (Tat) pathway is responsible for the export of folded proteins across the cytoplasmic membrane of bacteria. Substrates for the Tat pathway include redox enzymes requiring cofactor insertion in the cytoplasm, multimeric proteins that have to assemble into a complex prior to export, certain membrane proteins, and proteins whose folding is incompatible with Sec export. These proteins are involved in a diverse range of cellular activities including anaerobic metabolism, cell envelope biogenesis, metal acquisition and detoxification, and virulence. The Escherichia coli translocase consists of the TatA, TatB, and TatC proteins, but little is known about the precise sequence of events that leads to protein translocation, the energetic requirements, or the mechanism that prevents the export of misfolded proteins. Owing to the unique characteristics of the pathway, it holds promise for biotechnological applications.

Amino Acid Sequence↗
PubMed2006

Pharmacological manipulation of the vasoconstrictive effects of amyloid-beta peptides by donepezil and rivastigmine.

The amyloid-beta (Abeta) peptide has been linked to the pathology of Alzheimer's disease (AD). There is now evidence to support a vasoconstrictive effect of Abeta protein that could be detected in peripheral skin microvasculature. In this study we investigated the ability of acetylcholinesterase (AChE) inhibitors, Donepezil and Rivastigmine, to modulate the vasoconstrictor activity of Abeta25-35 and Abeta1-40. The ability of these drugs to improve endothelial mediated vascular responses to acetylcholine and bradykinin subsequent to perfusion of Abeta peptides was also investigated. The vascular responses to Abeta peptides, acetylcholine, bradykinin and sodium nitroprusside and their modulation by acetylcholinesterase inhibitors were examined in the base of a vacuum induced blister raised on the rat hind footpad using laser Doppler flowmetry. Abeta25-35 (1 microM) and Abeta1-40 (0.1 microM) induced a vasoconstrictor effect and significantly reduced the vasodilator response to acetylcholine (100 microM) and bradykinin (1 microM). Donepezil (100 microM) and Rivastigmine (100 microM) both reduced the vasoconstrictor effect of Abeta peptides, and significantly restored the endothelial vascular response to acetylcholine. Similarly, Donepezil significantly restored the endothelial vascular response to bradykinin. The results also showed that the actions of acetylcholinesterase inhibitors are independent of a direct action on smooth muscle cell reactivity or on endothelial cell function in the absence of Abeta. The current study provides the first evidence in vivo to suggest that acetylcholinesterase inhibitors modulate the vasoconstrictive effects of Abeta peptides at the level of skin microvasculature. We raise the notion that Donepezil and Rivastigmine mediate these vascular modulatory effects via an action on Abeta-mediated vasoconstrictor mechanisms rather than an independent action on endothelial or smooth muscle cell mediated responses.

Amyloid beta-Peptides↗
PubMed2006

Pure infradiaphragmatic Hodgkin's lymphoma. Clinical features, prognostic factor and comparison with supradiaphragmatic disease.

BACKGROUND AND OBJECTIVES: Pure infradiaphragmatic Hodgkin's lymphoma (HL) is a rare disease. The prognostic impact of a purely infradiaphragmatic localization of this lymphoma is controversial. We aimed to evaluate the baseline clinicopathologic features, prognostic factors and outcome of a large series of consecutive patients with pure infradiaphragmatic HL. DESIGN AND METHODS: We analyzed 131 patients with clinical stage I/II infradiaphragmatic HL treated with ABVD or equivalent regimens with or without radiotherapy, and compared 54 of them with 444 patients with pure supradiaphragmatic disease, who were treated at the same center. RESULTS: Older age, clinical stage II (borderline), involvement of > or =3 sites, lymphocyte predominant histology, elevated serum beta2-microglobulin and higher International Prognostic Score were more frequent in patients with infradiaphragmatic disease than in those with supradiaphragmatic disease, while nodular sclerosis was less frequent. The complete remission rate was 100%, 97% and 82% for stages I, IIA and IIB, respectively. Only B-symptoms independently predicted for inferior failure-free survival, while inferior overall survival was independently associated with the involvement of > or =3 sites. At 10 years failure-free survival was 82+/-6% (vs. 85+/-2% for patients with supradiaphragmatic disease, p=0.45), overall survival was 74+/-8% (vs. 91+/-2%, p=0.0006), and disease-specific survival 87+/-5% (vs. 94+/-1%, p=0.04). In multivariate analysis the differences between infradiaphragmatic and supradiaphragmatic disease were obscured by older age and B-symptoms. INTERPRETATION AND CONCLUSIONS: Pure infradiaphragmatic HL presents with distinct clinicopathologic characteristics. The previously reported poorer outcome may be explained by the unfavorable profile of the patients rather than the infradiaphragmatic presentation per se. Patients with stage IIB disease should probably be classified as having advanced HL because of the unacceptable rate of primary refractory disease.

Hodgkin Disease↗
PubMed2006

Time to rest.

Explore the source record for details and available documents.

Burnout, Professional↗
PubMed2005

Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression.

The detection of methamphetamine and other chemically related illicit drugs relies extensively on immunoassays. Here we report the cloning and affinity maturation of an anti-methamphetamine antibody which is being employed in the current commercial assays. An anti-methamphetamine scFv was cloned from hybridoma cells, expressed in bacteria and its affinity towards methamphetamine and N-ethylamphetamine (ethamphetamine) was determined by Surface Plasmon Resonance (SPR). The anti-methamphetamine scFv gene was subjected to random mutagenesis by error prone PCR and variants with improved affinity were isolated from the resulting library by a novel screening methodology termed Anchored Periplasmic Expression (APEx) [Harvey, B.R., Georgiou, G., Hayhurst, A., Jeong, K.J., Iverson, B.L., Rogers, G.K. (2004). Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc. Natl. Acad. Sci. U. S. A. 101, 9193.]. The isolated clones exhibited improved affinity to these illicit drugs, yet maintained low cross-reactivity to over-the-counter drugs. In addition, all clones displayed improved expression characteristics in Escherichia coli. The affinity improved scFv antibodies are thus likely to be useful in methamphetamine class immunodiagnostics.

Amino Acid Sequence↗