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Biomedical subjects

M Geiser

Publications and source records attributed to M Geiser.

At least 19 recordsLinked to original sources
PubMed2001

Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase.

Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement is that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multiparallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.

Cloning, Molecular↗
PubMed2000

Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology.

The selective infective phage (SIP) technology allows a rapid positive selection of interacting pairs of biological molecules that restore to non-infectious phages their ability to infect the bacterial host. After a successful infection, the phage is amplified and the DNA encoding the interacting ligand is isolated from the phage genome and sequenced. In our studies we have evaluated the usefulness of SIP for the identification and cloning of proteins interacting with a biotinylated target binding to a newly designed adapter molecule consisting of streptavidin fused to the C-terminus of the extracellular domain of the phage minor coat protein III. The new adapter was expressed in Escherichia coli and refolded from inclusion bodies. The two different domains joined within the chimaera were found to be biologically functional. We also demonstrated that non-covalent interactions between a non-infectious phage displaying a short peptide, which specifically binds the streptavidin, and the adapter molecule restore phage infectivity. To evaluate the potential of SIP as a general and generic tool for the screening of cDNA libraries that encode the ligands displayed at the surface of the phage and binding to biotinylated targets, we have increased both the size of the displayed ligand on the phage and the size of the biotinylated target bound to the streptavidin domain of the adapter molecule. In our model systems we show that the size of either the ligand or the target is a limiting factor for the technology.

Antibodies, Anti-Idiotypic↗
PubMed2000

[The mucociliary system of the lung--role of surfactants].

Many pollution particles enter the organism via the lung. In the lung, on a surface of 140 m2, the blood is separated from the air by a tissue barrier of only 1/1000 mm. The conducting airways (trachea, bronchi, bronchioli) are a very effective aerodynamic filter for inhaled particles. The mucociliary transport system functions like a self-cleaning mechanism within the filter. Inhaled particles and particles deposited in the lungs play a crucial aetiological and therapeutic role. The discussion in health policy on the relationship between the increase in air pollution and lung damage is of great importance at the present time. Epidemiological studies of recent years have shown very clearly that there is a correlation between morbidity and mortality as a consequence of respiratory and cardiogenic problems and the concentration of PM10 particles in ambient air. So far, however, this correlation has not been explained. The intrathoracic airways are coated by a respiratory epithelium. This has an irregular coating of viscous liquid, consisting of a low viscous sol phase and a high viscous gel phase. It seems, however, that those phases are not clearly distinguishable. The gel phase is moved towards the pharynx by the metachronal ciliary beat transporting the particles out of the lungs. Furthermore, at the air-liquid interface, there exists a continuous surfactant film which reduces the surface tension as is the case in the alveoli. When particles are deposited on the airway wall, that is, on the surfactant film, they are wetted by surface forces and displaced into the liquid phases. Thus, the surfaces of the particles are probably changed by the surfactant or by surfactant components. Many of these particles are transported in the liquid (gel phase) towards the pharynx (mucociliary transport), whereas some of them remain in close association with the epithelium (sol phase). Such particles remain in the airways for days or even weeks. They are either phagocytised by macrophages and carried off via the airways or taken up by dendritic cells and transported into the tissue from where they reach the lymph nodes via lymph drainage and are presented to the T-lymphocytes. The displacement of particles into the liquid phases, caused by the surfactant, can be considered as the initial step in a complex cascade of defence processes in the lungs. The surface of the particles is probably modified by surfactant or surfactant components. These modified particles may be directed to that clearance pathway which is most beneficial for our health, that is, out of the lungs or into the lymphatic glands, where an immune reaction can be triggered. We therefore consider surfactant to be a primary immune barrier.

Animals↗
PubMed2000

[Effect of light on choroidal blood flow in the fovea centralis].

PURPOSE: To investigate whether light and dark exposures induce a response of choroidal blood flow (ChBF) in the foveal region in humans. METHODS: In a group of healthy volunteers (age 25-60 years) ChBF was measured using a new confocal laser Doppler flowmeter (probing laser at 785 nm, power at the cornea = 90 microW). ChBF was recorded at room light, in darkness, at room light following dark adaptation, and during strong light exposure following room light. RESULTS: While ChBF was stable during room light condition, it decreased significantly by 15% (p < 0.01) during darkness. After 6 min of room light following darkness, ChBF was back to baseline. Strong diffuse, green light exposure over a field of 45 degrees had no detectable effect on ChBF. In all the experiments, no significant change of blood pressure was detected. CONCLUSIONS: Our findings did not confirm the presence of an active process of ChBF regulation in response to strong light exposure in humans. They demonstrate, however, a reversible decrease in ChBF occurring after a transition from room light to darkness.

Adaptation, Ocular↗
PubMed2000

Interaction of fungal spores with the lungs: distribution and retention of inhaled puffball (Calvatia excipuliformis) spores.

BACKGROUND: The biologic responses to inhaled airborne fungal spores, which are well-known allergen carriers, would be better understood if we had an insight into their pattern of distribution and interaction with lung structures. OBJECTIVES: To investigate the retention characteristics of inhaled basidiospores, which often represent the major portion of the spore load in air-sampling surveys and to analyze their regional distribution within and interaction with the lungs. METHODS: Intubated and anesthetized Syrian Golden hamsters inhaled aerosols of puffball (Calvatia excipuliformis) spores, with an aerodynamic diameter of 3.1 micrometer, either by spontaneous breathing (group A, n = 3) or by continuous negative-pressure ventilation (group B, n = 4). Lungs were fixed by intravascular perfusion of fixative solution within 29 minutes of the initial inhalation, and tissue samples were then processed for light and electron microscopy. RESULTS: Stereological (fractionator) analysis of lung tissue revealed that the greatest number of spores was deposited within the alveoli (67.2% in group A and 89.8% in group B). The intrapulmonary conducting airways retained an intermediate proportion (32.3% in group A and 10.0% in group B), whereas the extrapulmonary mainstem bronchi and trachea held the lowest proportion (0.5% or less). Deposited spores were lodged within the aqueous lining layer and in close proximity to the epithelial cells. Within the intrapulmonary conducting airways, 22. 3% of the spores in group A and 9.0% of those in group B had been engulfed by macrophages. CONCLUSION: This study demonstrates that inhaled 3-micrometer-diameter basidiospores become distributed over a large surface area. It also reveals that such particles are displaced by surfactant (surface forces) into the aqueous lining layer of airways and alveoli, thereby facilitating subsequent phagocytosis by macrophages. This interaction of spores with lung structures may be important for the development of respiratory allergies induced by airborne fungal allergens.

Animals↗
PubMed2000

Retention of Teflon particles in hamster lungs: a stereological study.

The significance of aerosols in medicine is increased when the distribution of inhaled aerosols in the different respiratory tract compartments and their interaction with lung structures are known. The aim of this study was to investigate the retention of the hydrophobic Teflon spheres used in human beings so as to analyze their regional distribution and to study their interaction with lung structures at the deposition site. Six intubated and anesthetized Syrian Golden hamsters inhaled aerosols of Teflon particles with an aerodynamic diameter of 5.5 microns by continuous negative-pressure ventilation adjusted to slow breathing. Lungs were fixed by intravascular perfusion within 21 minutes after inhalation was started, and tissue samples were taken and processed for light and electron microscopy. The stereological (fractionator) analysis revealed that particle retention was the greatest in alveoli (72.4%), less in intrapulmonary conducting airways (22.9%), and the least in extrapulmonary mainstem bronchi (0.3%) and trachea (4.4%). Particles were found submerged in the aqueous lining layer and in close vicinity to epithelial cells. In intrapulmonary conducting airways, 21.5% of Teflon particles had been phagocytized by macrophages. This study with highly hydrophobic Teflon particles clearly demonstrates that for spheres of this size, surface tension and line tension forces rather than the particles' surface free energy are decisive for the displacement of particles into the aqueous phase by surfactant. It was this displacement that enabled subsequent interaction with macrophages. Refined knowledge of particle retention may help us to better understand the biological response to inhaled particles.

Administration, Inhalation↗
PubMed2000

Subfoveal choroidal blood flow in response to light-dark exposure.

PURPOSE: To document the response of subfoveal choroidal blood flow (ChBF) in the human eye induced by light and dark exposures and provide some insight into the mechanism underlying this response. METHODS: In a group of 12 volunteers (age, 25-60 years), ChBF was measured with a confocal laser Doppler flowmeter. Wavelength of the probing laser beam was 785 nm (90 microW at the cornea). ChBF was recorded in room light, in darkness, in room light after dark adaptation, and during strong green light exposure after exposure to room light. After dark adaptation of both eyes, ChBF was also measured in one eye while only the fellow eye was exposed to strong visible light. RESULTS: Although ChBF was stable during room light condition, it decreased significantly by 15% (P < 0.01) during dark adaptation. After 6 minutes of room light following 20 minutes of darkness, ChBF was back to baseline. Strong, diffuse, green light exposure over a field of 40 degrees, as well as the probing laser beam, had no detectable effect on ChBF. No change in ChBF was detected when the fellow eye was illuminated after both eyes had been dark adapted. CONCLUSIONS: The findings did not confirm the presence of an active process of ChBF regulation in response to light exposure in humans. They demonstrate, however, a reversible decrease in ChBF that occurs after a transition from room light to darkness, which could involve a neural mechanism.

Adult↗
PubMed1999

Identification of the human melanoma-associated chondroitin sulfate proteoglycan antigen epitope recognized by the antitumor monoclonal antibody 763.74 from a peptide phage library.

To identify the epitope of the melanoma-associated chondroitin sulfate proteoglycan (MCSP) recognized by the monoclonal antibody (mAb) 763.74, we first expressed random DNA fragments obtained from the complete coding sequence of the MCSP core glycoproteins in phages and selected without success for binders to the murine mAb 763.74. We then used a library of random heptapeptides displayed at the surface of the filamentous M13 phage as fusion protein to the NH2-terminal portion of the minor coat protein III. After three rounds of selection on the bound mAb, several phages displaying related binding peptides were identified, yielding the consensus sequence Val-His-Leu-Asn-Tyr-Glu-His. Competitive ELISA experiments showed that this peptide can be specifically prevented from binding to mAb 763.74 by an anti-idiotypic MK2-23 mouse:human chimeric mAb and by A375 melanoma cells expressing the antigen MCSP. We screened the amino acid sequence of the MCSP molecule for a region of homology to the consensus sequence and found that the amino acid sequence Val-His-Ile-Asn-Ala-His spanning positions 289 and 294 has high homology. Synthetic linear peptides corresponding to the consensus sequence as well as to the MCSP-derived epitope inhibit the binding of mAb 763.74 to the phages displaying the consensus amino acid sequence. Finally, the biotinylated consensus peptide absorbed to streptavidin-microtiter plates can be used for the detection of mAb 763.74 in human serum. These results show clearly that the MCSP epitope defined by mAb 763.74 has been identified.

Amino Acid Sequence↗
PubMed1999

A new methodology for controlled particle inhalation by small rodents.

In order to investigate the deposition, retention, and clearance mechanisms implicated in particle inhalation under standardized conditions, we developed a continuous negative-pressure ventilation system, whereby the breathing pattern in small rodents could be controlled during exposure to aerosols. Using an on-line open-flow set-up, 19 anesthetized, intubated, and paralyzed Syrian golden hamsters, individually contained within a whole-body box, were artificially ventilated under the said continuous negative-pressure conditions, 1 of 5 different combinations of breathing frequency and tidal volume being established. The animals were then exposed to aerosols containing 6-micron diameter polystyrene spheres, and the deposition of particles in the conducting airways was monitored photometrically. During exposure, the level of respiration (mean lung inflation) was stabilized by means of a negative-pressure vent. Breathing frequency and tidal volume, as well as the compliance of the system, remained virtually unchanged during the course of a single experiment, and in each case, a reproducible deposition of particles was achieved. Our findings indicate that tidal volume, but not breathing frequency, has a marked influence on the particle deposition ratio. Breathing frequency exerts opposing and counterbalancing effects on this latter parameter by enhancing the impaction of particles on the one hand, and by decreasing sedimentation on the other.

Administration, Inhalation↗
PubMed1998

Neuropathology of ablation of rat gliosarcomas and contiguous brain tissues using a microplanar beam of synchrotron-wiggler-generated X rays.

Adult-rat-brain tissues display an unusually high resistance to necrosis when serially irradiated with parallel, thin slices of a microplanar (i.e., microscopically thin and macroscopically broad) beam of synchrotron-wiggler-generated, approx. 35-120 keV (median approx. 50 keV) Gd-filtered X rays at skin-entrance absorbed doses of 312 to 5000 Gy per slice. Such microplanar beams were used to irradiate young adult rats bearing right frontocerebral 9L gliosarcomas (approx. 4 mm diameter), through a volume of tissue containing the tumor and contiguous brain tissue, either in a single array or in 2 orthogonally crossed arrays of tissue slices. Each array included 101 parallel microplanar slices, 100 microm center-to-center distance, each slice being approx. 25 microm wide and 12 mm high, with skin-entrance absorbed doses of 312.5 Gy or 625 Gy per slice. Compared with unirradiated controls with a median survival time of 20 days after tumor initiation, the median survival time was extended in irradiated rats by 139 days (625 Gy, crossed arrays), 96 days (312 Gy, crossed arrays) or 24 days (625 Gy, single array). The tumors disappeared in 22 of the 36 irradiated rats, 4/11 even after unidirectional microbeam irradiation. The extent and severity of radiation damage to the normal brain in rats with or without tumor was graded histopathologically. Correlation of those grades with radiation doses shows that loss of tissue structure was confined to beam-crossing regions and that only minor damage was done to zones of the brain irradiated unidirectionally.

Animals↗
PubMed1998

Bioassay for hamster macrophage chemotaxis: application to study particle-lung interactions.

Attraction of lung macrophages to particle deposition sites has been demonstrated in different animal species. We reported a threefold increase of the number of macrophages to occur within 40 min after polystyrene particle deposition in hamster airways [Geiser et al. (1994) Am. J. Respir. Cell Mol. Biol. 160: 594-603]. Complement-derived chemotactic activity is one of the mechanisms postulated for macrophage recruitment. It was the aim of this study to test whether complement-derived chemotactic activity is involved in the rapid recruitment of macrophages to the site of deposited polystyrene particles in hamster airways. We first developed an in vitro cell migration assay for hamster macrophages to assess complement-derived chemotaxis. Second, the bronchoalveolar lavage fluids (BALF) of four hamsters that had inhaled aerosols of polystyrene microspheres were tested for chemotactic activity by this bioassay and compared with BALF of four sham-exposed hamsters. Chemotactic response of macrophages was found toward complement-activated hamster serum, whereas macrophage migration was not increased toward BALF of particle and sham-exposed hamsters. In contrast, macrophage migration to BALF of both groups was reduced by 1.6-fold. Thus, the stimulus for macrophage recruitment to the site of deposited polystyrene particles in hamster airways could not be demonstrated using this bioassay.

Animals↗
PubMed1997

Ultrastructure of the aqueous lining layer in hamster airways: is there a two-phase system?

For particle retention and clearance, the structure and surface properties of the airway lining layer are important. Due to difficulties of its preservation, structural analysis has been hampered, and, hence, the existence of two distinct and continuous phases and how much osmiophilic material is available are unclear. It was the objective of this study to investigate the ultrastructure of the aqueous lining layer in the intrathoracic conducting airways of hamsters. By means of transmission electron microscopy, we investigated the ultrastructure of the airway lining layer in hamsters whose lungs have been fixed by the application of fixative dissolved in nonpolar fluorocarbon, either by instillation via the trachea or injection into the gas exchange parenchyma, together with intravascular perfusion of aqueous fixatives. The results were compared to lungs fixed by intravascular perfusion only. In twelve hamsters, the airway lining layer was found to consist of an aqueous phase and was coated by an osmiophilic film that follows fairly closely the upper-extending contours of cilia protruding from epithelial cells. Substantially less osmiophilic material was preserved in extrapulmonary airways and when nonaqueous fixative was injected. We found that the aqueous lining layer of the intrathoracic airways in hamsters essentially surrounds and covers the cilia, the microvilli, and any other structures like macrophages or deposited particles contained in it and is coated by an osmiophilic film of variable thickness. In healthy animals, a gel phase is expected to be very thin, not clearly separated from the periciliary fluid, and located just beneath the osmiophilic film.

Animals↗
PubMed1997

Diffuse luminance flicker increases retinal vessel diameter in humans.

PURPOSE: To determine retinal vessel diameter variations in response to neuronal activity induced by diffuse luminance flicker. METHODS: The diameter of retinal arteries and veins was measured in 9 normal subjects by computer analysis of fundus pictures taken in monochromatic light under normal conditions of illumination and after 1 min of sinusoidally varying diffuse luminance flicker at 10 Hz. RESULTS: The diameter immediately after flicker was significantly larger than the pre-stimulus diameter by 4.2 +/- 2.2% for the retinal arteries and 2.7 +/- 1.7% for the retinal veins (mean +/- SD). Six is after cessation of the flicker, arterial diameter was not significantly different from that of pre-flicker value. CONCLUSIONS: Diffuse luminance flicker induces an increase in retinal vessel diameter. This increase most probably reflects an increase in retinal blood flow previously evidenced in humans by the blue field simulation technique. The technique needs to be optimized in terms of flicker parameters, to determine whether flicker-evoked retinal diameter changes could represent a useful clinical measure of the capability of the retinal vascular system to vasodilate.

Adolescent↗
PubMed1997

Prediction of the biologically active sites in eclosion hormone from the silkworm, Bombyx mori.

The structure-activity relationship of eclosion hormone from the silkworm, Bombyx mori, was analyzed. First, the probable active residues in silkworm eclosion hormone and also tobacco hornworm eclosion hormone were predicted by the average distance map method. To examine the contributions of those residues to the activity of silkworm eclosion hormone, Gly-substituted mutants for those predicted residues were produced by site-directed mutagenesis and their activities were evaluated by a bioassay. Finally, Glu12, Met24 and Phe25 were estimated to be the crucial residues for the eclosion hormone activity. The possibility of the development of a blocker of an eclosion hormone receptor on the basis of the present work is also discussed.

Amino Acid Sequence↗
PubMed1996

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

Animals↗