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Biomedical subjects

G Georgiou

Publications and source records attributed to G Georgiou.

90 records · Page 5Linked to original sources
PubMed1992

Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide.

beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.

Ampicillin↗
PubMed1991

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli.

The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A-beta-lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5 less than or equal to pH less than or equal to 6.0) and a low temperatures. These conditions were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn2+ ions.

Bacterial Proteins↗
PubMed1991

Structure and morphology of protein inclusion bodies in Escherichia coli.

We have studied the structure and characteristics of inclusion bodies formed by the enzyme beta-lactamase in the periplasmic space of Escherichia coli or in the cytoplasm, following expression of the protein without its signal sequence. Electron microscopy of highly purified protein aggregates using a novel sucrose gradient centrifugation procedure revealed striking morphological differences. Periplasmic inclusion bodies were essentially amorphous whereas the protein particles in the cytoplasm were highly regular. Depending on the cellular location, the inclusion bodies exhibited differences in protein composition even though they were formed by the expression of the same polypeptide chain. It was shown that the chaperonins GroEL and SecB are not incorporated into the inclusion bodies. Furthermore, the degree of solubilization of the inclusion bodies in the presence of denaturants and the sensitivity of the aggregated proteins to protease digestion indicated that the differences between cytoplasmic and periplasmic inclusion bodies extend to the conformation of the associated polypeptide chains.

Cell Compartmentation↗
PubMed1991

Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo.

Protease III, the product of the ptr gene, is a 110-kDa periplasmic protease with specificity towards insulin and other low-molecular-weight substrates (less than 7,000 molecular weight) in vitro (Y.-S.E. Cheng and D. Zipser, J. Biol. Chem. 254:4698-4706, 1979). Escherichia coli strains deficient in protease III were constructed by insertional inactivation of the ptr gene. This mutation did not appear to affect the function of the adjoining recB and recC genes. Expression of protein A-beta-lactamase, a protease-sensitive secreted polypeptide, was increased approximately twofold in ptr cells. A comparable increase in the half-life of protein A-beta-lactamase was observed by pulse-chase experiments, suggesting that protease III is involved in the catabolism of high-molecular-weight substrates in vivo, ptr mutants exhibited no detectable phenotypic alterations except for a slight reduction in growth rate. When the ptr mutation was transferred to a strain deficient in the secreted protease DegP, a further decrease in growth rate, as well as an additive increase in the expression of the fusion protein, was observed. A ptr degP ompT mutant strain resulted in a further increase in expression in minimal medium but not in rich medium.

Blotting, Southern↗
PubMed1990

Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli.

High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli. The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm. With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate. Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein. Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis. These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway. Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G. (1988) Biotechnol. Prog. 4, 97-101). We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing. It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro.

Cytosol↗
PubMed1990

Affinity immobilization of a genetically engineered bifunctional hybrid protein.

Protein A from Staphylococcus aureus (SpA) is a receptor for the Fc domain of several classes of antibodies including immunoglobin G (IgG). A hybrid protein consisting of protein A and the enzyme beta-lactamase has been constructed using recombinant DNA techniques. The functional characteristics of the hybrid protein adsorbed on IgG-coated Sepharose matrices were studied in detail and compared to those of (i) the hybrid protein in solution and (ii) beta-lactamase covalently immobilized on CNBr-activated Sepharose. Protein A--beta-lactamase bound tightly and specifically to IgG-Sepharose and could be stored for at least 4 weeks without dissociation. The rate of penicillin G hydrolysis by the beta-lactamase domain of the immobilized hybrid protein was found to depend on the amount of IgG covalently coupled to the support. For all IgG loads, higher specific activities and lower Km values relative to covalently immobilized beta-lactamase were obtained. Adsorption of the hybrid protein on the support resulted in increased stability to thermal deactivation. These results indicate that bifunctional hybrid proteins can be useful for the affinity immobilization of enzymes.

Chromatography, Affinity↗
PubMed1990

In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT.

The Escherichia coli outer membrane protease OmpT (protease VII) has been shown to degrade several proteins in vitro, but its function in vivo is uncertain. We demonstrate that OmpT participates in the degradation of a fusion protein secreted into the periplasmic space. A strain with mutations in degP (K.L. Strauch and J. Beckwith, Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988) and ompT exhibits a cumulative decrease in protein degradation and should be useful for the expression of proteolytically sensitive secreted proteins.

Bacterial Proteins↗
PubMed1990

A study of serum free medium culture on the cloning efficiency of human bone marrow myeloid and erythroid progenitors.

Thirteen different combinations of serum-free media were tested to assess their suitability to replace serum containing medium for in vitro culture of human hemopoietic progenitors (CFU-GM and BFU-E). Bone marrow samples from patients with and without hematological diseases were tested. All tested media supported the growth of CFU-GM and BFU-E colonies, however our results have shown that the cloning efficiency of all commercially available serum-free media tested was lower (mean 18% and 12% of controls for CFU-GM and BFU-E respectively) and the colony size was smaller than those in serum-containing medium. In the serum-free cultures, there was no linear relationship between the colony numbers and cell concentration plated. Depletion of T-lymphocytes and monocytes did not improve the cloning efficiency of the serum-free medium culture. Furthermore, the addition of high concentration of insulin, transferrin and other supplements to the serum-free media did not improve the cloning efficiency. These results have indicated that the currently available commercial serum-free media do not provide optimal requirements for hemopoietic progenitor cell cultures and that other factors contained in serum are essential for their optimal growth.

Blood Physiological Phenomena↗
PubMed1988

A human complement-fixing monoclonal anti-human lymphocyte antibody of rat origin.

MARCH 1E11 is an IgM monoclonal anti-human lymphocyte antibody of rat origin with the capacity to utilise both human and rabbit complement. The antibody reacts with all thymocytes and with all peripheral blood T and B lymphocytes. The treatment of human bone marrow or human peripheral blood mononuclear cells (PBMC) with MARCH 1E11 and either pooled human serum or autologous serum as a complement source resulted in cytolysis of greater than 99% of OKT3-positive lymphocytes. Under these conditions, progenitor cell recovery (colony-forming unit (CFU-c), burst forming unit--erythroid (BFU-e) and colony-forming unit--mixed (CFU-mix)) was greater than 90% of that of untreated cells. The response of treated marrow or PBMC to phytohaemagglutinin stimulation and in mixed leucocyte reactions demonstrated a reduction in thymidine incorporation to values similar to those obtained for unstimulated cells. The antibody does not cause modulation of the cell surface antigen and does not react significantly with non-lymphoid tissues. This monoclonal antibody may be useful for in vitro elimination of T lymphocytes from allogeneic bone marrow used for transplantation. The antibody may also be useful for treatment protocols requiring lymphoid depletion or immunosuppression as in organ transplantation.

Animals↗
PubMed1987

Penetration of albendazole sulphoxide into hydatid cysts.

The penetration of albendazole sulphoxide, the principal metabolite of albendazole into hydatid cysts (E granulosus) was measured by means of in vitro animal and clinical studies. The drug freely diffuses across the parasitic membranes. Cyst/serum concentrations of 22% were achieved in patients, longer pre-operative therapy produced higher concentrations.

Albendazole↗
PubMed1986

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.

Alkaline Phosphatase↗
PubMedDate not supplied

Selective attentional biases for physical threat in physical anxiety sensitivity.

Anxiety sensitivity is a trait susceptibility associated with the fear of anxiety-related sensations. One reason why such fears exist may be because those high in anxiety sensitivity selectively attend towards such sensations. However, few studies have actually investigated these cognitive biases in high anxiety sensitive individuals. The current study, therefore, sought to investigate selective attentional biases using the visual dot-probe paradigm. Since recent research suggests that at least one component of anxiety sensitivity is linked to the fear of physical sensations, individual were selected on the basis as to whether they were high or low in their anxious concern for physical sensations. In order to determine whether a general or specific attentional bias exists, the emotionality of material presented to participants was varied in terms of whether it was physically threat-related, socially threat-related, or positive. Consistent with predictions, those high in physical anxiety sensitivity were found to exhibit a selective attentional bias in favour of the location of physically threatening material. Furthermore, those low in anxiety sensitivity were found to avoid such material. Interestingly, a similar attentional bias was not found for either socially threatening or positive material. If anything, those high in physical anxiety sensitivity avoided positive material. These findings are discussed in light of current theories of anxiety sensitivity and future research.

Adolescent↗
PubMedDate not supplied

Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli. 1. Composition.

We have determined the macromolecular composition of inclusion bodies formed by overexpressing beta-lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition. Specifically, contaminating polypeptides ranged from less than 5% to over 50% of the total protein content. Phospholipids composed 0.5-13% of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature beta-lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown at basic pH. Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.

Bacterial Proteins↗
PubMedDate not supplied

A quantitative immunoassay utilizing Escherichia coli cells possessing surface-expressed single chain Fv molecules.

A facile, quantitative immunoassay is described that utilizes Escherichia coli (E. coli) bacteria expressing single chain Fv (scFv) antibody fragments attached to the cell surface. A Scatchard analysis demonstrated that the antibodies on the surface of the cells retained full binding activity (Kd = 2.2 x 10(-9) M) and that there are 60,000 scFv molecules per cell. The cells are used as the antibody reagent in the assay, and, following incubation with analyte, simple centrifugation is used to separate the antibody-bound from unbound analyte. The immunoassay is rapid and accurate down to the nanomolar level. In addition, a variety of detection strategies can be used, and the immunoassay is not adversely affected by the presence of animal serum. A key advantage of the new immunoassay is that the antibody reagent can be inexpensively produced in a "ready to use" form by simply growing cultures of the bacteria.

Antigens, Surface↗
PubMedDate not supplied

Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain.

Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.

Endopeptidases↗
PubMedDate not supplied

Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase.

Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.

Animals↗
PubMedDate not supplied

Frontal lobe changes in alcoholism: a review of the literature.

Alcohol can induce a wide spectrum of effects on the central nervous system. These effects can be recognized at the neurophysiological, morphological and neuropsychological levels. Several studies of the effect of alcohol on the frontal lobes were identified for review from MedLine, PsychLIT databases and by manual searching. In this review article, the different changes are examined in detail. Computed tomography studies have reported changes of frontal lobe in alcoholism, while magnetic resonance imaging studies supported these findings. Neurophysiological studies with positron emission tomography and single photon emission computed tomography have reported a decreased frontal lobe glucose utilization and reduced cerebral blood flow. There is also evidence from neuropsychological studies that there are specific deficits in alcoholism that suggest frontal lobe dysfunction. Considered together, these studies lend a strong credence to the concept of frontal lobe pathology in alcoholism. However, frontal lobe is not an isolated part of the brain and should be considered with its heavy connections to different cortical and subcortical areas of the brain.

Alcoholism↗