PubMed Health⌕ Search

Biomedical subjects

P Fuchs

Publications and source records attributed to P Fuchs.

At least 37 records · Page 2Linked to original sources
PubMed1996

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS.

BACKGROUND: The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). OBJECTIVES: pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. STUDY DESIGN AND RESULTS: Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. CONCLUSIONS: Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.

Amino Acid Sequence↗
PubMed1996

Mechanical ventilation in stroke patients--is it worthwhile?

We assessed the therapeutic efficacy and outcome of mechanical ventilation (MV) in patients with acute respiratory failure (ARF) following ischemic stroke (IS) or intracerebral hemorrhage (ICH), retrospectively graded by patients with IS (n = 881) and ICH (n = 108) admitted to our service during 11 years, according to the severity of their clinical state and to whether we employed MV. Outcome was recorded in terms of survival and duration of MV and compared with patients with neuromuscular (NM) diseases. We found a very high in-hospital mortality in stroke patients who were treated with ventilation (90.5% for IS and 87.5% for ICH) compared with NM patients (29%). We conclude that MV in stroke patients with ARF is not life-saving, and its use should be considered only after considering other potentially important factors.

Aged↗
PubMed1996

Cell surface display of a single-chain antibody for attaching polypeptides.

To provide an efficient means of coupling proteins, peptides and other suitable moieties to cells, we have constructed a retroviral expression vector for cell surface display of a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-oxazo-line-5-one (phOx). The hapten phOx can be easily conjugated to primary amino and sulfhydryl groups, thus providing points of attachment for the cell surface-bound anti-phOx scFv. This universal cell coupling system could prove to be particularly useful for anchoring monoclonal antibodies for tumor targeting and to present co-stimulatory molecules and other ligands (even mixtures) at the cell surface for gene therapy.

Animals↗
PubMed1996

Epithelial cell polarity and embryo implantation in mammals.

At embryo implantation we are confronted with the fact that uterine and trophoblast epithelium make contact via their apical cell membranes. This epithelium-epithelium adhesion leading to definitive attachment of the embryo to the uterine wall, however, is far from being trivial and has been called a cell biological paradox. It has been proposed that some of the molecular events involved in epithelium-to-mesenchyme transformation might play a role in the interaction between uterine cells and trophoblast. As a mechanism to achieve uterine epithelium adhesiveness for trophoblast it is postulated that uterine cells partially modulate their epithelial phenotype. Data from recent in vitro experiments give support to this hypothesis and suggest that loss of apical-basal cell polarity might prepare the apical cell pole of uterine epithelium for cell-to-cell contact with trophoblast in vivo.

Animals↗
PubMed1996

Adhesiveness of the apical surface of uterine epithelial cells: the role of junctional complex integrity.

Embryo implantation necessitates that the apical plasma membrane of uterine epithelial cells acquires adhesiveness. Recent studies have indicated that modulation of a major element of the epithelial phenotype, i.e. apical-basal cell polarity, might be critical in this respect. Here, we analyze polar characteristics of nonadhesive vs. adhesive uterine epithelial cell lines focusing on cytoskeletal-junctional interactions that may play a role in regulating adhesiveness of the apical plasma membrane. HEC-1-A is a human uterine epithelial cell line exhibiting nonadhesive properties of its apical surface for trophoblast, whereas RL95-2 represent another such cell line exhibiting adhesive properties enabling trophoblast attachment. Homotypic intercellular contacts and functionally related proteins, i.e. ZO-1, E-cadherin, alpha-catenin, beta-catenin, plakoglobin, and desmoplakin 1, were examined by transmission electron microscopy, immunocytochemistry, confocal laser scanning microscopy, and immunoprecipitation techniques. In addition, details of actin filament architecture were studied after phalloidin labeling. While nonadhesive HEC-1-A exhibited the well-known pattern of cell-to-cell contacts of polarized epithelial cells, adhesive RL95-2 showed a lack of ZO-1 expression, tracer leakiness of the paracellular pathway, and atypical features in adherens junctions: E-cadherin, alpha-catenin and plakoglobin were colocalized in all plasma membrane domains and beta-catenin was localized in lateral membrane domains. Immunoprecipitations showed in both cell lines the presence of two different E-cadherin-catenin complexes, one composed of E-cadherin, alpha-catenin and beta-catenin, and the other of E-cadherin, alpha-catenin and plakoglobin. Concerning RL95-2 these data indicate that E-cadherin/plakoglobin complexes are randomly distributed, whereas E-cadherin/beta-catenin complexes are laterally localized in these cells. Additionally, the actin-based cytoskeleton of RL95-2 lacked a polar organization. With respect to the intermediate filament-desmosome system, both cell types expressed desmoplakin I, but the vast majority of RL95-2 lacked well-formed desmosomes as demonstrated by electron microscopy. It is concluded that modulation of tight junctions and/or remodelling of adherens junctions, e.g. differential distribution of E-cadherin/plakoglobin complexes and E-cadherin/beta-catenin complexes, are correlated with the development of apical adhesiveness of human uterine epithelial cells. This model system should allow to test experimentally whether this correlation is due to any causal function in the development of epithelial cell polarity.

Actins↗
PubMed1995

Human antibody libraries in Escherichia coli.

A potentially vast pool of human antibodies with novel specificities for diagnostic and therapeutic purposes can be generated in Escherichia coli. Antibodies to infectious agents have already been isolated by amplifying the heavy and light chain repertoires of donor lymphocytes and they have even been rescued many years after the initial infection from memory cells cultivated in SCID mice. Eventually, however, the creation of extremely large and diverse libraries from the naive antibody repertoire of unactivated B lymphocytes or by gene synthesis using random oligonucleotides for the hypervariable regions could provide a rapid means of obtaining human antibodies to any particular antigen. An important breakthrough for exploiting the potential size and diversity of these libraries has been the development of systems for the surface display of antibodies that are physically linked to their own genes. This allows large numbers of clones to be screened simultaneously and antibodies with affinities of up to 10(8) M-1 have already been obtained using these vectors. It seems quite feasible, therefore, that antibodies with affinities approaching those obtained in the secondary immune response can be obtained by systematically optimizing the strategies for making antibody libraries. Furthermore, it might be possible to establish extremely large antibody repertoires in E. coli by the in vivo recombination of phage and plasmid antibody libraries. The affinity of the selected antibodies could be increased by chain shuffling or random mutagenesis followed by several rounds of selection under increasingly stringent conditions.

Animals↗
PubMed1995

DNA measurements reveal differences in the life cycles of Babesia bigemina and B. canis, two typical members of the genus Babesia.

The relative DNA levels of different developmental stages of Babesia bigemina and B. canis were measured by cytophotometry using the fluorochrome Hoechst 33258 as a staining dye. The DNA measurements provided direct proof of sexual reproduction, i.e., fusion of gametes resulting in the formation of zygotes followed by a meiotic division. Both Babesia species are considered to be typical members of the genus Babesia; however, the DNA measurements revealed important differences in the life cycle of these parasites, indicating that Babesia species are not characterized by a life cycle, which is specific for this genus.

Animals↗
PubMed1995

Comparative antimicrobial activities of the penem WY-49605 (SUN5555) against recent clinical isolates from five U.S. medical centers.

The in vitro activity of WY-49605 (SUN5555) (WY) was compared with those of cefaclor, cefixime, and amoxicillin-clavulanic acid against 2,958 consecutive clinical isolates from five medical centers and 402 respiratory pathogens from 18 other facilities. Most members of the family Enterobacteriaceae were inhibited by WY (MIC at which 50% of the isolates are inhibited [MIC50], < or = 2.0 micrograms/ml). MIC90s of > or = 8.0 micrograms/ml were observed for Enterobacter cloacae, Serratia spp., and Proteus mirabilis. WY was the most active drug against methicillin-susceptible Staphylococcus aureus (MIC90, 0.12 microgram/ml) and other coagulase-negative staphylococci (MIC90, 4.0 micrograms/ml). The four drugs were not active against nonenteric gram-negative bacilli, methicillin-resistant Staphylococcus aureus, and Staphylococcus haemolyticus. At 2.0 micrograms/ml, WY inhibited 82% of Enterococcus faecalis strains and was equal to or superior to the other drugs against streptococci, Haemophilus influenzae, and Moraxella catarrhalis.

Amoxicillin↗
PubMed1995

Cell adhesion to the apical pole of epithelium: a function of cell polarity.

Human uterine epithelium displays a distinct polarized organization with apical, lateral, and basal plasma membrane domains. Although non-adhesive throughout most of the menstrual cycle, epithelial cells allow attachment of trophoblast cells to their apical pole during embryo implantation. A recent hypothesis postulates that epithelial cells turn off genes for apical-basal polarity and turn on genes for a more mesenchyme-like phenotype allowing cell-cell interaction with trophoblast. Using an in vitro assay human uterine cell lines (RL95-2, HEC-1-A, AN3-CA) were selected on the basis of adhesiveness for trophoblast-type cells (JAR). Subsequently, uterine cells were examined for epithelium-specific ultrastructure using transmission electron microscopy, and for the expression of E-cadherin, alpha 6-, beta 1-, beta 4-integrin subunits and cytokeratin using immunocytochemistry, confocal laser scanning microscopy, and surface replication technique. HEC-1-A monolayers are non-adhesive for JAR cells and appear highly polarized expressing E-cadherin, alpha 6-, beta 1-, beta 4-integrin subunits, and cytokeratin. Both, integrins and E-cadherin, are present at the lateral membrane. RL95-2 monolayers which are adhesive for JAR cells appear non-polarized. Like HEC-1-A cells, RL95-2 cells express E-cadherin, alpha 6-, beta 1-, and beta 4-integrin subunits, and cytokeratin. In contrast to HEC-1-A cells, integrins and E-cadherin are distributed at the entire cell surface. AN3-CA monolayers are non-adhesive for JAR cells and appear non-polarized. Cells lack epithelial-specific markers such as keratin and E-cadherin. They show only low expression of alpha 6-, beta 1-integrin subunits and lack beta 4-integrin subunit. Conversely, they express vimentin. Thus, modulation of the epithelial phenotype of uterine cells, i.e. loss of apical-basal polarity, might prepare the apical cell pole for cell-cell interaction with trophoblast. However, loss of cell polarity would not lead to enhancement of adhesiveness for trophoblast if accompanied by a loss of epithelium-specific adhesion molecules.

Cadherins↗
PubMed1994

Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers.

To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.

Amino Acid Sequence↗
PubMed1994

Defective transformation of chromosomal markers in DNA polymerase I mutants of the radioresistant bacterium Deinococcus radiodurans.

The transformation efficiency of six independently selected chromosomal markers (four for rifampicin resistance and two for acriflavine resistance) was found to be reduced by about 3 logs in a Deinococcus radiodurans strain that was isogenic with wild type except for an insertional mutation in the pol gene that eliminated DNA polymerase I activity (strain 6R1A). D. radiodurans strains UV17 and 303, previously obtained by chemical mutagenesis, were determined to be partially deficient in DNA Pol I activity as assessed in a permeabilized cell system. Both UV17 and 303 demonstrated intermediate transforming efficiencies that correlated with their levels of residual polymerase activity. The transformation efficiency of strain 6R1A could be greatly restored by expression of cloned E. coli DNA Pol I, but not to wild-type levels. Plasmid transfer and chromosomal duplication insertion were not substantially affected by lack of DNA Pol I activity. D. radiodurans is known to possess extraordinarily efficient repair pathways for DNA damage, and is refractory to DNA damage-induced mutagenesis caused by numerous agents, including several that cause base mispairing. We suggest that D. radiodurans may differ from other naturally transformable bacteria in that DNA Pol I is needed to efficiently convert most drug-resistance markers. This unusual mechanism may be required to accomplish chromosomal conversion prior to correction of donor DNA by this organism's efficient repair pathways.

Acriflavine↗
PubMed1994

Interpretive criteria and quality control for antimicrobial susceptibility tests of levofloxacin.

To confirm preliminary interpretive breakpoints for prototype 5 micrograms levofloxacin disks, 490 strains were tested in vitro using commercially manufactured disks. For in vitro susceptibility testing, 5 micrograms levofloxacin disks can be used with interpretive criteria of < or = 12 mm for resistant (MIC > or = 8.0 micrograms/ml) and > or = 16 mm for susceptible (MIC < or = 2.0 micrograms/ml). Proposed quality control limits for tests of levofloxacin are as follows: Escherichia coli ATCC 25922, zones 29-37 mm or MIC 0.008-0.03 microgram/ml; Pseudomonas aeruginosa ATCC 27853, zones 19-26 mm or MIC 0.5-2.0 micrograms/ml; Staphylococcus aureus ATCC 25923, zones 24-31 mm; Staphylococcus aureus ATCC 29213, MIC 0.06-0.25 microgram/ml and Enterococcus faecalis ATCC 29212, MIC 0.25-2.0 micrograms/ml.

Bacteriological Techniques↗
PubMed1994

Restoration of the DNA damage resistance of Deinococcus radiodurans DNA polymerase mutants by Escherichia coli DNA polymerase I and Klenow fragment.

Deinococcus radiodurans and other species of this genus share extreme resistance to ionizing radiation and many other agents that damage DNA. D. radiodurans mutant strains defective in a deinococcal DNA polymerase that is homologous with E. coli DNA polymerase I are highly sensitive to DNA damage. In the current work we have inquired whether E. coli DNA Pol I can substitute for D. radiodurans Pol in partially or fully restoring to pol- D. radiodurans mutants the extreme DNA damage-resistance typical of this organism. The E. coli polA gene or a 5'-truncated polA gene that encodes the Klenow fragment were introduced and expressed in two different D. radiodurans pol- mutants: Strain 303, which is a chemically mutagenized derivative, and strain 6R1A, which is isogenic with wild-type D. radiodurans except for an insertional mutation within the pol gene. Expression of E. coli polA in both of these mutants fully restored wild-type resistance to ionizing- and UV254-radiation and mitomycin-C exposure. Expression of the Klenow fragment-encoding gene restored wild-type resistance to D. radiodurans strain 303, but only partial resistance to strain 6R1A. The observation that E. coli DNA Pol I is as effective as D. radiodurans Pol in restoring damage resistance, indicates that D. radiodurans DNA Pol per se does not have special properties that are essential or prerequisite for expression of the extreme resistance of D. radiodurans.

Bacterial Proteins↗
PubMed1994

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.

Chromosomes, Bacterial↗