Behavior of free-swimming cells under various accelerations

The different steps of the gravity signal-transduction chain on the cellular level are not identified. In our experiments performed up to now we mainly stressed our attention on the last step, the response of the cells. Swimming behavior is a suitable indicator for the physiological status of a Paramecium cell. Depending on membrane potential and/or concentrations of Ca++, cGMP and cAMP the beating direction and the beating velocity of the cilia are influenced in a characteristical way leading to a changed swimming activity of the cell. The behavior of Paramecium is influenced by various stimuli from their environment. Previous studies have demonstrated that under controlled conditions Paramecium shows a clear gravity-dependent behavior resulting in negative gravitaxis and gravikinesis (speed regulation in dependence of gravity). By changing the orienting stimulus (gravity) we expected changes of the swimming behavior. Additional experiments were performed using pawn mutant d4-500r. Due to defective Ca(2+)-channels the membrane of this mutant cannot depolarize. As a consequence d4-500r cannot perform phobic responses and swim backwards. Comparative experiments are also performed with the ciliate Loxodes striatus. In contrast to Paramecium this ciliate possesses statocyst-like organelles--the Müller Organelles.     

Real time NMR monitoring of local unfolding of HIV-1 protease tethered dimer driven by autolysis

Structural studies in proteases have been hampered because of their inherent autolytic function. However, since autolysis is known to be mediated via protein unfolding, careful monitoring of the autolytic reaction has the potential to throw light on the folding-unfolding equilibria. In this paper we describe real time nuclear magnetic resonance investigations on the tethered dimer construct of the human immunodeficiency virus-1 protease, which have yielded insights into the relative stabilities of several residues in the protein. The residues lying along the active site (bottom, side and top of the active site) and those in helix have lower unfolding free energy values than the other parts of the protein. The residue level stability differences suggest that the protein is well suited to adjust itself in almost all the regions of its structure, as and when perturbations occur, either due to ligand binding or due to mutations.     

Functional roles of the hook in a rotating tethered cell

A bacterial cell tethered through a flagellum on a glass slide rotates its cell body in either counter-clockwise or clockwise at around 10 Hz. To analyze the detailed manner of rotation, we have constructed and expressed yellow fluorescent protein (YFP)-FliN fusion protein in a fliN deletion mutant, resulting in the recovery of motility of the Fla^- mutant cells. The tethered cells that incorporated the fusion protein in the flagellar motor rotate around one of the fluorescent spots. Tracing the center spot of a rotating cell, we have found that the rotating circles of the tethered cells were often distorted, and that the cell has seldom rotated smoothly but gyrated around the center point. The radii of the gyrating circles were 100-200 nm for the wild-type cells, and 50 nm for the cells carrying short hooks, suggesting that the flexibility of the hook is responsible for asymmetrical rotation. These observations indicate that tethered cells almost always interact with the glass surface in one cycle of rotation, where the length and flexibility of the hook have an important role.     

Real time visualization of protein kinase activity in living cells.

A library of fluorescently labeled protein kinase C (PKC) peptide substrates was prepared to identify a phosphorylation-induced reporter of protein kinase activity. The lead PKC substrate displays a 2.5-fold change in fluorescence intensity upon phosphorylation. PKC activity is readily sampled in cell lysates containing the activated PKCs. Immunodepletion of conventional PKCs from the cell lysate eliminates the fluorescence response, suggesting that this peptide substrate is selectively phosphorylated by PKCalpha, beta, and gamma. Finally, living cells microinjected with the peptide substrate exhibit a 2-fold increase in fluorescence intensity upon exposure to a PKC activator. These results suggest that peptide-based protein kinase biosensors may be useful in monitoring the temporal and spatial dynamics of PKC activity in living cells.     

Real time imaging of human progenitor neurogenesis

Human neural progenitors are increasingly being employed in drug screens and emerging cell therapies targeted towards neurological disorders where neurogenesis is thought to play a key role including developmental disorders, Alzheimer's disease, and depression. Key to the success of these applications is understanding the mechanisms by which neurons arise. Our understanding of development can provide some guidance but since little is known about the specifics of human neural development and the requirement that cultures be expanded in vitro prior to use, it is unclear whether neural progenitors obey the same developmental mechanisms that exist in vivo. In previous studies we have shown that progenitors derived from fetal cortex can be cultured for many weeks in vitro as undifferentiated neurospheres and then induced to undergo neurogenesis by removing mitogens and exposing them to supportive substrates. Here we use live time lapse imaging and immunocytochemical analysis to show that neural progenitors use developmental mechanisms to generate neurons. Cells with morphologies and marker profiles consistent with radial glia and recently described outer radial glia divide asymmetrically and symmetrically to generate multipolar intermediate progenitors, a portion of which express ASCL1. These multipolar intermediate progenitors subsequently divide symmetrically to produce CTIP2(+) neurons. This 3-cell neurogenic scheme echoes observations in rodents in vivo and in human fetal slice cultures in vitro, providing evidence that hNPCs represent a renewable and robust in vitro assay system to explore mechanisms of human neurogenesis without the continual need for fresh primary human fetal tissue. Knowledge provided by this and future explorations of human neural progenitor neurogenesis will help maximize the safety and efficacy of new stem cell therapies by providing an understanding of how to generate physiologically-relevant cell types that maintain their identities when placed in diagnostic or transplantation environments.     

Real time ultrasonic monitoring of hepatic cryosurgery

Cryosurgery has a number of advantages that make it particularly appealing in the treatment of liver cancer. However, a major problem in the clinical application of hepatic cryosurgery is the lack of a precise means of monitoring the freezing process in situ. Preliminary investigations on simulated tissue have shown that standard ultrasonography is capable of accurately determining the amount of frozen material during a cryosurgical procedure. To extend these results to living tissue, cryosurgery was performed, in vivo, on the livers of four mongrel dogs. An ultrasound imaging device using a new intraoperative ultrasound transducer monitored the entire process in real time. The results indicate that the entire freezing and thawing cycle can be monitored easily using real time ultrasound. During freezing, the solidification interface can be seen to move through the tissue allowing clear imaging of the cryolesion. After complete thawing, the cryolesion became less echogenic than before freezing and was therefore distinguishable under ultrasound. Postsurgical pathologic examination showed excellent correlation between the lesion size and its ultrasonic image.     

Ecology and behaviour of a free-swimming tube-dwelling rotifer Cephalodellaforficula

SUMMARY. 1. Cephalodella for ficula (Ploima, Rotifera) lives in tubes it constructs itself. These tubes are built of mueus in detritus-rich environments. The tubes are often closed at both ends, arc not used as sieves, and are not eaten directly.
2. The rotifer swims back and forth in its tube and apparently lives on bacteria which are shed from the inner walls of the tubes. Because of surface-to-volume considerations, this feeding strategy is probably only possible for animals smaller than roughly 1 mm. Under low food conditions, rotifers inside a tube have a distinctly higher fitness than rotifers removed from their tube.
3. Given high food conditions, rotifers removed from a tube immediately build another. Grazing on particles outside the tube appears to take place when a tube is being lengthened. Rotifers do not leave the tube for routine feeding, but under conditions of starvation or very low oxygen concentration they will leave the tube and swim about.     

Computer-based tracking of living cells

A computer-based tracing technique has been developed to follow the movement of living cells and keep them centered in the field of view of an optical microscope. With the use of an image-processing system, the video image of a cell can be sufficiently processed to allow computer-recognition of the cell boundaries. Determination of the location of the center of the cell enables comparison of successive cell positions and correction for any cell movement. In order to illustrate the versatility of this technique, patterns of movement were obtained of cancerous and non-cancerous cells in an effort to determine the difference in motility between the two cell types. After examination of the data gathered, it was found that there is no difference in the motility between the two cell types over 1-h periods.     

Real time, high resolution video imaging of apoptosis in single cells with a polymeric nanoprobe

We report a new apoptosis nanoprobe (Apo-NP) designed on the basis of a polymer nanoparticle platform. This simple one-step technique is capable of boosting fluorescence signals upon apoptosis in living cells, enabling real-time imaging of apoptosis in single cells and in vivo. The Apo-NP efficiently delivers chemically labeled, dual-quenched caspase-3-sensitive fluorogenic peptides into cells, allowing caspase-3-dependent strong fluorescence amplification to be imaged in apoptotic cells in real-time and at high resolution. The design platform of the Apo-NP is flexible and can be fine-tuned for a wide array of applications such as identification of caspase-related apoptosis in pathologies and for monitoring therapeutic efficacy of apoptotic drugs in cancer treatment.     

Real time PCR profiling of 330 human micro-RNAs

The small size of miRNAs makes profiling of all the 462 known human miRNAs difficult using single cell samples. Recently, we showed that judicious sequence partitioning between RT primers and second strand synthesis primers permitted multiplexed RT-PCR amplification of miRNA in very small samples to allow individual real time PCR quantification. Here, we show that zip coding the primers and TaqMan probes with sequences specific to each miRNA greatly improves reaction specificity, which permits the profiling of all miRNAs in a single multiplexed RT-PCR reaction.     

Residence time distributions of gas flowing through rotating drum bioreactors

Residence time distribution studies of gas through a rotating drum bioreactor for solid-state fermentation were performed using carbon monoxide as a tracer gas. The exit concentration as a function of time differed considerably from profiles expected for plug flow, plug flow with axial dispersion, and continuous stirred tank reactor (CSTR) models. The data were then fitted by least-squares analysis to mathematical models describing a central plug flow region surrounded by either one dead region (a three-parameter model) or two dead regions (a five-parameter model). Model parameters were the dispersion coefficient in the central plug flow region, the volumes of the dead regions, and the exchange rates between the different regions. The superficial velocity of the gas through the reactor has a large effect on parameter values. Increased superficial velocity tends to decrease dead region volumes, interregion transfer rates, and axial dispersion. The significant deviation from CSTR, plug flow, and plug flow with axial dispersion of the residence time distribution of gas within small-scale reactors can lead to underestimation of the calculation of mass and heat transfer coefficients and hence has implications for reactor design and scale-up.     

Real time spectral analysis during phytochrome chromophore and chromoprotein purification

The plant photoreceptor phytochrome senses light quality and quantity in the red region of the spectrum, directing adaptation and development. The functional holo-protein is a dimeric chromoprotein which is formed by an autoassembly reaction between the translation product and the open chain tetrapyrroles phytochromobilin (PPhiB) or phycocyanobilin (PCB). We are interested in structure/function relationships within the phytochrome molecule, in particular chromophore/protein interaction during the assembly and photoactivation, using IR and NMR spectroscopy. For this we use an automated F/HPLC system running in a darkroom to purify large amounts of protein and chromophore separately. To obtain highly pure PCB chromophore we developed improved extraction and purification methods in which the final step is RPC-18 HPLC. As there are many spectrally only slightly different tetrapyrroles in the extract, the triple-wavelength monitoring offered by the F/HPLC detector was inadequate for distinguishing between PCB and impurities. Furthermore, lambda(max) for the phytochrome Pfr signalling state lies between 705 and 730 nm, beyond the range of the detector. Also, as both holo-protein and chromophore are photoactive, we wished to minimize light exposure of the eluate. We therefore implemented a miniature CCD-based flow UV-vis spectrophotometer using a xenon flash and quartz fiber optics enabling us monitor the entire 250-800 nm spectrum of the eluate to an accuracy of +/-3 x 10(-3)A in real time. The instrumentation described can be added to any chromatographic system, thereby allowing the purification of any molecule to be monitored easily and efficiently.     

Real time evaluation of overranging in helical computed tomography

Overranging or overscanning increases the dose delivered to patients undergoing helical Computed Tomography examinations. In order to reduce it, nowadays most of the multidetector tomographs close the X-ray beam aperture at the scan extremes. This technical innovation, usually referred to as dynamic or adaptive collimation, also influences the overranging assessment methods. In particular, the film free approach proposed in previous studies is not suitable for these modern tomographs. The present study aims to introduce a new method of estimating overranging with real time dosimetry, even suitable for tomographs equipped with adaptive collimation. The approach proposed is very easy to implement and time saving because only a pencil chamber is required. It is also equivalent in precision and in accuracy to the film based one, considered an absolute benchmark.     

Real time non-invasive imaging of receptor-ligand interactions in vivo

Non-invasive longitudinal detection and evaluation of gene expression in living animals can provide investigators with an understanding of the ontogeny of a gene's biological function(s). Currently, mouse model systems are used to optimize magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging modalities to detect gene expression and protein function. These molecular imaging strategies are being developed to assess tumor growth and the tumor microenvironment. In addition, pre-labeling of progenitor cells can provide invaluable information about the developmental lineage of stem cells both in organogenesis and tumorigenesis. The feasibility of this approach has been extensively tested by targeting of endogenous tumor cell receptors with labeled ligand (or ligand analog) reporters and targeting enzymes with labeled substrate (or substrate analog). We will primarily discuss MRI, PET, and SPECT imaging of cell surface receptors and the feasibility of non-invasive imaging of gene expression using the tumor microenvironment (e.g., hypoxia) as a conditional regulator of gene expression.