A digital readout attachment for the radiometer syringe burette unit.
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Automation↗
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Differential epithelial outgrowth of plucked and microdissected human hair follicles in explant culture.
In the present study we prepared explant cultures of plucked total hair follicles and of fragments microdissected from the following regions: B1 (bulb region), B2 (intermediate region), B3-1 (lower central outer root sheath, ORS), B3-2 (upper central ORS) and B4 (area of fracture). The growth capacities, the start of epithelial outgrowth, the stages of differentiation and apoptosis were studied immunohistochemically in early and late explant cultures using a battery of antibodies against cytokeratins, growth factor receptors and cell adhesion molecules and proliferation markers. Whole plucked hair follicles showed epithelial outgrowths exclusively in the upper central ORS (B3-2) starting early, mostly by day 3. In microdissected fragments, in contrast, outgrowths were more widespread, mostly in B3-2 and B3-1, and started early, but were also of late onset in some cases of B2 and B4. Epithelial outgrowths exhibited a basal layer of small cuboidal cells in a low stage of differentiation and one to two suprabasal layers of large prickle-like cells expressing late differentiation markers. The former expressed the receptor of nerve growth factor (NGF) heterogeneously whereas epidermal growth factor (EGF) receptor was not detectable. This is similar to ORS cells of this area in vivo. The proliferative activity of the outgrowths was always restricted to peripheral cells. Thus no essential differences in differentiation of outgrowing cells were detected. These results suggest that keratinocytes with the highest growth capacities in plucked human hair follicles are localized in the lower central ORS (corresponding to B3-2) and some with a lower capacity in the upper central ORS (corresponding to B3-1) as established after microdissection. This is in agreement with the bulge activation theory. NGF may also play a role in hair growth.
Adult↗
Mechanical manipulation of polymorphonuclear leukocyte plasma membranes with optical tweezers causes influx of extracellular calcium through membrane channels.
Optical tweezers are used mechanically to manipulate the plasma membrane of polymophonuclear leukocytes attached to the bottom of a glass manipulation chamber. The laser trapping beam is dragged across the membrane of cells in calcium-containing and calcium-depleted extracellular medium. This treatment causes a significant rise in the intracellular calcium concentration compared with controls, in cells in calcium-containing medium (239.8 +/- 49.0% against 75.4 +/- 16.4%, respectively), but not in cells in calcium-depleted medium (69.1 +/- 9.6% against 83.4 +/- 18.5%, respectively), indicating that the calcium rise is caused by an influx of calcium from the environment. The rise in calcium concentration is blocked (23.5 +/- 7.1% against 17.1 +/- 4.1%, respectively) by the addition of lansoprazole, indicating that the influx is not due to unspecific membrane damage caused by the mechanical manipulation of the cell. It can therefore be concluded that mechanical manipulation of the neutrophil membrane, in the piconewton force range exerted by the optical tweezer, does not damage the plasma membrane but stimulates a mechanically inducible, membrane channel-mediated influx of extracellular calcium.
2-Pyridinylmethylsulfinylbenzimidazoles↗
A model for cochlear outer hair cell deformations in micropipette aspiration experiments: an analytical solution.
We propose a mathematical model to describe the deformations of the cochlear outer hair cell (OHC) in the micropipette aspiration experiments. The bending effect is considered, and the OHC is treated as a cylindrical shell. The pipette effect is modeled by two-dimensional normal loading. Considering the OHC wall as an infinitely long cylinder, we obtain solution in terms of Fourier series with respect to the circumferential coordinate, where coefficients are expressed by closed formulae. We keep leading terms in fourier series and derive a closed formula for the length of tongue of the aspirated cell surface in terms of pipette pressure, cell geometry, and elastic moduli. To demonstrate application of the theory, we use data recently reported from the micropipette aspiration experiments and obtain an estimate of the elastic shear modulus for the OHC lateral wall.
Biomechanical Phenomena↗
A piconewton force transducer and its application to measurement of the bending stiffness of phospholipid membranes.
The bending stiffness of a phospholipid bilayer (Kc) was measured by forming thin bilayer cylinders (tethers) from giant phospholipid vesicles. Based on the balance of forces, the tether force was expected to be proportional to the square root of the membrane tension, with a constant of proportionality containing Kc. The membrane tension was controlled via the aspiration pressure in a micropipette used to hold the vesicle. The force on the tether was generated by an electromagnet acting on a paramagnetic bead attached to the vesicle surface. The magnitude of the force was determined from measurements of the magnet current, which was adjusted to maintain the position of the bead. Measurements were performed on vesicles composed of stearoyl-oleoyl-phosphatidylcholine plus 5% (by mole) biotinylated phosphatidylethanolamine to mediate adhesion to streptavidin-coated beads. From each vesicle, tethers were formed repeatedly at different values of the membrane tension. The expected relationship between membrane tension and tether force was observed. The mean value of Kc for 10 different vesicles was 1.17 x 10(-19) J (SD = 0.08 x 10(-19) J). The precision of these data demonstrates the reliability of this approach, which avoids uncertainties of interpretation and measurement that may be associated with other methods for determining Kc.
Calibration↗
Assisted hatching does not enhance IVF success in good-prognosis patients.
PURPOSE: The role of assisted hatching in good-prognosis IVF patients was evaluated in a prospective, randomized, controlled pilot study, which was followed by a retrospective observational series. METHODS: After assisted hatching was proved successful in a mouse embryo study, 20 good-prognosis IVF patients were randomly assigned to either assisted hatching (13) or no assisted hatching (7; the controls). Following this series, 27 good-prognosis IVF patients were retrospectively evaluated to determine the outcome with assisted hatching. RESULTS: In the prospective study, clinical pregnancies resulted from 3 (23%) of 13 patients in the hatching group, compared to 3 (43%) of 7 in the control group. Implantation rates were similar: 9.6% in the hatching group and 10.7% in the controls. In the retrospective series, the 11.1% implantation rate with assisted hatching was significantly less than the 42.9% implantation rate seen with traditional IVF. CONCLUSIONS: Implantation and pregnancy rates are high in young women undergoing traditional IVF. Assisted hatching is not beneficial in these patients.
Adult↗
Interaction between rough phenotype and amino acid auxotrophy in Saccharomyces cerevisiae.
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Adenine↗
Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli.
Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanically-induced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.
Actins↗
Current applications of single-cell PCR.
The advent of the polymerase chain reaction (PCR) has revolutionised the way in which molecular biologists view their task at hand, for it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell. It is especially in the field of prenatal diagnostics that this ability has been readily seized upon, as it has opened up the prospect of preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. However, apart from diagnostic applications, single-cell PCR has proven to be of enormous use to basic scientists, addressing diverse immunological, neurological and developmental questions, where both the genome but also messenger RNA expression patterns were examined. Furthermore, recent advances, such as optimised whole genome amplification (WGA) procedures, single-cell complementary DNA arrays and perhaps even single-cell comparative genomic hybridisation will ensure that the genetic analysis of single cells will become common practice, thereby opening up new possibilities for diagnosis and research.
Animals↗
["One shot" CO2 laser stapedotomy].
BACKGROUND: In order to further optimize the surgical technique with CO(2) laser in stapes surgery, a scanner system was used to obtain a footplate perforation of 0.5-0.6 mm with only one laser application ("one-shot" stapedotomy). PATIENTS AND METHODS: A total of 255 patients with otosclerosis were submitted to primary CO(2) laser stapedotomy with a SurgiTouch scanner. This study discusses the surgical technique and clinical results. RESULTS: An adequately large perforation diameter could be achieved with a single shot in 68% of the patients treated. In 14% of the patients, a second laser application at the same site was necessary. In 18% the perforation had to be enlarged by several slightly overlapping laser applications without using the scanner. There was no evidence of laser dependent inner ear affections. CONCLUSION: CO(2) laser, combined with modern scanner systems, is well suited for application in stapes surgery.
Adolescent↗
Analysis of single-cell differences by use of an on-chip microculture system and optical trapping.
A method is described for continuous observation of isolated single cells that enables genetically identical cells to be compared; it uses an on-chip microculture system and optical tweezers. Photolithography is used to construct microchambers with 5-microm-high walls made of thick photoresist (SU-8) on the surface of a glass slide. These microchambers are connected by a channel through which cells are transported, by means of optical tweezers, from a cultivation microchamber to an analysis microchamber, or from the analysis microchamber to a waste microchamber. The microchambers are covered with a semi-permeable membrane to separate them from nutrient medium circulating through a "cover chamber" above. Differential analysis of isolated direct descendants of single cells showed that this system could be used to compare genetically identical cells under contamination-free conditions. It should thus help in the clarification of heterogeneous phenomena, for example unequal cell division and cell differentiation.
Bacteria↗
Double microcatheter technique for endovascular coiling of wide-neck aneurysms using a new guiding device for the transcarotid approach: technical note.
In the endovascular treatment of cerebrovascular diseases, positioning the guiding catheter is of great importance. In cases where vessel tortuosity prohibits positioning the guiding catheter in the carotid artery via the femoral approach, we use a direct carotid approach via common carotid artery puncture. For direct puncture of the common carotid artery we devised a 6-Fr sheath with double insertion points. This new device facilitates the use of double microcatheters and provides safe and effective vascular access.
Aged↗
Combined nanomanipulation by atomic force microscopy and UV-laser ablation for chromosomal dissection.
Nanomanipulation and nanoextraction on a scale close to and beyond the resolution limit of light microscopy is needed for many modern applications in biological research. For the manipulation of biological specimens a combined microscope allowing for ultraviolet (UV) microbeam laser manipulation together with manipulation by an atomic force microscope (AFM) was used. In a one-step procedure, human metaphase chromosomes were dissected optically by the UV-laser ablation and mechanically by AFM manipulation. With both methods, sub-400-nm cuts could be achieved routinely. Thus, the AFM is an indispensable tool for in situ quality control of nanomanipulation. However, already on this scale the dilation of the topographic AFM image due to the tip geometry can become significant. Therefore the AFM images were restored using a tip geometry obtained by a blind tip-reconstruction algorithm. Cross-sectional analysis of the restored image reveals a 380-nm-wide UV-laser cut and AFM cuts between 70 nm and 280 nm.
Chromosomes↗
Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes.
A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.
Cell Separation↗
Deformation of intracellular endosomes under a magnetic field.
We present a non-invasive method to monitor the membrane tension of intracellular organelles using a magnetic field as an external control parameter. By exploiting the spontaneous endocytosis of anionic colloidal ferromagnetic nanoparticles, we obtain endosomes possessing a superparamagnetic lumen in eukaryotic cells. Initially flaccid, the endosomal membrane undulates because of thermal fluctuations, restricted in zero field by the resting tension and the curvature energy of the membrane. When submitted to a uniform magnetic field, the magnetized endosomes elongate along the field, resulting in the flattening of the entropic membrane undulations. The quantification of the endosome deformation for different magnetic fields allows in situ measurement of the resting tension and the bending stiffness of the membrane enclosing the intracellular organelle.
Cell Membrane↗
Force generation by recombinant myosin heads trapped between two functionalized surfaces.
Fluorescence resonance energy transfer measurements have revealed that the lever-arm domain of myosin swings when it hydrolyzes Mg-ATP. It is generally accepted that this swing of the lever arm of myosin is the molecular basis of force generation. On the other hand, the possibility that the force might be generated at the interface between actin and myosin cannot be ignored. However, there is a third possibility, namely, that myosin itself generates force without actin. Thus, using recombinant subfragment 1 molecules of Dictyostelium myosin II that were trapped between two functionalized surfaces of a surface-force apparatus, we determined whether myosin itself could actually generate force. Here, we report that, despite the absence of actin, myosin heads themselves have a capacity to generate a force (at least approximately 0.2 pN/molecule) that is coupled to the structural changes. Although the role of actin should not be neglected because muscle physiologically shortens as a result of the interaction between actin and myosin, in this work the focus is on the question of whether the catalytic domain of myosin has the capacity to generate force.
Adenosine Triphosphate↗
Probing molecular interaction between concanavalin A and mannose ligands by means of SFM.
Recently, the scanning force microscope (SFM) has been widely used for direct monitoring of specific interactions between biologically active molecules. Such studies have employed the SFM liquid-cell setup, which allows measurements to be made in the native environment with force resolution down to a tenth of a picoNewton. In this study, the ligand-receptor strength of monoclonal anti-human prostatic acid phosphatase and prostatic acid phosphatase, representing an antigen-antibody system with a single type of interaction, was determined. Then, the interaction force occurring between concanavalin A and the carbohydrate component of the glycoproteins arylsulfatase A and carboxypeptidase Y was measured. High mannose-type glycans were sought on the human prostate carcinoma cell surface. Application of an analysis based on the Poisson distribution of the number of bonds formed in all these measured systems allowed the strength of the molecular interaction to be calculated. The values of the force acting between two single molecules were 530+/-25, 790+/-32, and 940+/-39 pN between prostatic acid phosphatase and monoclonal anti-human prostatic acid phosphatase, between concanavalin A and arylsulfatase A, and between concanavalin A and carboxypeptidase Y, respectively. The value calculated from data collected for the force between concanavalin A and mannose-containing ligands present on the surface of human prostate carcinoma cells was smaller, 116+/-17 pN. The different values of the binding force between concanavalin A and mannose-containing ligands were attributed to the structural changes of the carbohydrate components.
Binding Sites↗
Permeability and the hidden area of lipid bilayers.
The passive water permeability of a lipid vesicle membrane was studied, related to the hydrostatic (not osmotic) pressure difference between the inner and the outer side of the vesicle in a water environment without additives. Each pressure difference was created by sucking a vesicle into a micropipette at a given sucking pressure. The part of the membrane sucked into the micropipette (the projection length) was measured as a function of time. The time dependence can be divided into two intervals. We put forward the idea that smoothing of membrane defects, accompanied by an increase of the membrane area, takes place during the initial time interval, which results in a faster increase of the projection length. In the second time interval the volume of the vesicle decreases due to the permeability of its membrane and the increase of the projection length is slower. The hidden area and the water permeability of a typical lipid bilayer were estimated. The measured permeability, conjugated to the hydrostatic pressure difference, is an order of magnitude higher than the known value of the permeability, conjugated to the osmotic pressure difference. A hypothesis, based on pore formation, is proposed as an explanation of this experimental result.
Computer Simulation↗