A potential link between Alzheimer's disease and hypersensitive pupil dilation has been reported. Patients diagnosed with probable Alzheimer's disease by standard clinical criteria exhibited a marked hypersensitivity in the pupil dilation response to a cholinergic antagonist, tropicamide, placed in their eyes. We have devised a system to measure pupil area stably and intermittently for 60 minutes. This system could potentially be used for Alzheimer's disease diagnosis using this neurobiological test. The system consists of a probe with a high resolution infrared CCD camera and a real time pupil area analysis system using computer imaging processing. Averaging the pupil area during times of eye open, pupillary waves and blinking, and using the constant pupil+iris area to compensate for eye image size variation allows accurate measurement.
Spikes detected on EEG (electroencephalogram) records give important information for clinical diagnosis, especially that of epileptic disorders, and therefore automatization of spike detection will be a powerful aid for electroencephalographer in the visual inspection of spikes. A new method for detecting spikes in EEG records was developed by taking account of properties of the spikes which had been employed by electroencephalographers for the visual inspection. The proposed method has two key features as follows: 1) adjustment of the threshold values for the spike detection depending upon the characteristics of spikes in each EEG record and 2) renovation of the templates for the spike waveform according to the detected spikes. The discrimination of the artifacts such as eye blinks and EMG (electromyogram) from the spikes was also taken into account. The proposed method was applied to 71 segments (1 segment=5sec) of EEGs recorded from 10 patients. A detection accuracy of 85.3% (64/75) was obtained as the result of the automatic detection, which was in good agreement with the electroencephalographer's visual inspection. Since the proposed method was based on adaptive decision criteria, it is automatically adaptable to the spike of various waveforms derived from respective subjects. The proposed spike detection method can be effectively applicable for clinical use.
In this paper, the difference of light reflex between the normal and the senile dementia is studied by using light-stimuli. The senile dementia patients show us smaller changes (p<0.01) and longer delay times (p<0.05) than the normal elders when their pupils are lightened by pocket-light. The results may be applied to the new objective method to evaluate the senile dementia.
A new technique for imaging single fluorescent dye molecules in aqueous solution has been developed by refining epifluorescence and total internal reflection fluorescence microscopies. This approach enabled us to directly image the processive movement of individual fluorescently labeled kinesin molecules along a microtubules. This method was also used to visualize individual ATPase reactions of single myosin molecules. Single molecule imaging using green fluorescent protein as a fluorescent tag allowed us to assay functions of single protein molecules expressed in vitro. This method can be applied to examining other enzymatic reactions, interaction of polymerases and helicases with DNA, and a variety of protein-protein or protein-ligand associations at a single molecular level.
Numerical chromosome aberrations of cancer cells can be detected by counting the number of FITC-labeled spots per nucleus. Each spot has bright green fluorescence emitted from the FITC-labeled DNA probe that hybridizes with the peri-centromere of the chromosome. The nuclei are stained with a red fluorescent dye. We have devised a system for automatic detection of numerical chromosome abnormalities. The hardware of the system includes a fluorescence microscope with a computer-controllable stage, a CCD camera, and a personal computer. The software for automatic counting of FITC-labeled fluorescent spots in nuclei consists of the following parts. The focal plane of the microscope is changed in order to determine the nuclear region at each focus, and to detect the FITC-labeled spots. If in a red region, which is considered a candidate of nuclear region, the following three values each lie within a specified range, then the red region is regarded as a nucleus: (1) the area of the red region, (2) the ratio between the height and width of a rectangle which circumscribes the red region, and (3) the ratio between the area of the red region and the area of the rectangle circumscribing it. If the region does not satisfy these values, it is regarded as an undesirable object such as clustered or injured nuclei, or a noise spot, and removed from further processing. The number of FITC-labeled spots per nucleus is counted. The time required for the automatic measurement is similar to that achieved by the human eye. The histogram of the nuclear population assorted according to the number of FITC-labeled spots resulting from hybridization to female X chromosomes showed that the result obtained by automated counting corresponded well to that achieved by the human eye.
We have been developing a 3-dimensional internal structure microscope for the observation of internal structures of samples. The internal structure is obtained by processing over 400 stored images of sliced faces of the sample, sliced continuously by the precise rotary slicer proceeding under observation with a microscope. 3D-ISM has been utilized to observe food, farm products and biological organs. Under white light, it is difficult to get clear images of expressed gene under high magnification. In order to overcome the problem, we installed a fluorescence microscope function into the 3D-ISM for detection of fluorescent materials. The performance of the apparatus are as follows. The resolution of the sample sending is 0.5μm. The maximum observation speed is five sections per second. The lowest sample cooling temperature is -50°C. The object lens magnification are from 2 to 100. U and B and G excitations are available by the fluorescent wave length. Each sliced face image is recorded on a postscript type laser disk after sliced, followed the next slicing. With the recorded section's image a 3-dimensional image is constructed on a workstation by volume rendering method, and hence the internal of the sample is observable. A cancer field gene (c-kit) in a embryo of ICR strain mice which is 11.5 day olds was observed. The embryo was dyed with a FITC labeled DNA probe (c-kit) using whole mount fluorescence in situ hybridization method. The existence of fluorescence inside the embryo's vertebra and internal organs were confirmed, and the topography of c-kit was observable. A device which is possible in fluorescent observation was constructed on trial, and succeeded in observation of topography of the gene in an embryo, which was difficult so far.
Osteocytes, dendritic resident cells in bone, transduce signals of mechanical loading that results in anabolic responses of bone. In our previous report with primary osteocytes derived from newborn rat calvaria, we showed that cAMP levels were elevated 15min after the initiation of streching, and that the protein levels of both IGF-I and osteocalcin were elevated 36h later. Here we report that stretching induces the expression of an immediate early gene, cfos, at 15min and of osteocalcin in several hours in these cells. Furthermore, the blockers of stretch activated channels and epithelial-like sodium channels, in combination, abolish the effects of stretching; elevated expression of osteocalcin and calcium influx into the stretched cells. Independent experiment with a blocker of L type channels also inhibited the osteocalcin expression caused by stretching. Therefore, it is conceivable that the calcium influxes, both primary and secondary, are the essential responses of stretched osteocytes that result in the anabolic reaction of bone.
Proteins located within the lipid bilayer, surrounding the intracellular bacterial magnetic particles from Magnetospirillum sp. AMB-1 were purified and characterized using two dimensional polyacrylamide gel-electrophoresis. Three major proteins of approximate molecular weight 55.9, 36.4 and 28.4kDa were identified. The N-terminal amino acid sequence of one of these proteins, designated MpsA, was determined and used to design a pair of PCR primers which amplified a 105bp DNA fragment from AMB-1 genomic DNA. Gene-walking, using anchored PCR, was used to determine the complete nucleotide sequence (951bp) of the mpsA gene. mpsA encodes a 317 amino acid protein which does not have an N-terminal cytoplasmic transport signal sequence. Intracellular localization studies were carried out using a mpsA-luc gene fusion expressed in AMB-1 following gene transfer by conjugation. The gene fusion was constructed by cloning a 1.6kb mpsA fragment upstream of luc in the conjugal plasmid pKLC. The MpsA-Luc fusion protein was preferentially located on the magnetic particle membrane. Although the function of MpsA remains unknown, homologies indicate a similarity with biotin dependent carboxylase/decarboxylases.
Mouse endothelial cells (ECs) were exposed to controlled levels of shear stress in flow-loading device, and we measured changes in the expression of vascular cell adhesion molecule-1 (VCAM-1) on both protein and mRNA levels. The surface expression of VCAM-1 was decreased by flow in mouse endothelial cells (ECs). Shear stress decreased VCAM-1 mRNA levels time-dependently. The decrease in VCAM-1 gene expression by flow depended on the intensity of shear stress applied. To determine the signal transduction for the downregulation of VCAM-1 gene expression, we applied laminar flow to ECs in the presence of various inhibitors and examined the changes in the gene expression of VCAM-1 by Northern blot analysis. H-89, Chelerhythrine Chloride and TEA did not affect the shear stress-induced downregulation of VCAM-1 gene expression. Herbimycin A and BAPTA decreased the basal mRNA levels of VCAM-1 in static conditioned cells. We performed a luciferase assay to evaluate the effect of shear stress on VCAM-1 gene transcription. The luciferase activity of shear-stressed cells was markedly lower than that of static control cells. These findings suggest that shear stress downregulates the VCAM-1 gene expression transcriptionally in mouse endothelial cells.
It has been known that myosin heavy chain isoform changes from embryonic to adult type during development of chicken skeletal muscles. However, the reg ulatory mechanism of the transition is not understood. We hypothesized that gravitational stress may play an important role in the isoform transition. To test this idea, we kept neonatal chickens for 2 weeks at various centrifugal forces. Myosin heavy chain expression was studied by immunofluorescence, SDS-PAGE and northern blot analyses. In anterior latissimus dorsi (ALD) muscle, an antigravity muscle which holds wing weight, it was found to that hyper gravity made the muscle hypertrophied and the transition of myosin heavy chains from embryonic type SM 1 to adult type SM 2 occured faster than ground controls. We concluded that gravitational stress is a key environmental factor which influences differential expression of myosin heavy chain gene in antigravity muscle during postnatal development.
Endothelial cells in vivo are subjected to hemodynamic forces such as shear stresses by blood flow, and stretch by vessel expansion due to blood pressure change. These forces are known to elicit many physiological responses to maintain vascular tonus. To further investigate the stretch-induced responses and the intracellular signaling pathway in vascular endothelial cells, an application of in vitro mechanical stimulation is required. Using these apparatus, we obtained following data; 1) Stretch induced a transient increase in [Ca2+]i. 2) Cyclic uni-axial stretch aligned endothelial cells perpendicular to the stretch direction. 3) Cyclic uni-axial stretch induced tyrosine phosphorylation of focal adhesion proteins and the stretch-induced morphological change was inhibited by tyrosine kinase inhibitors. 4) Cyclic uni-axial stretch induced re-organization of cytoskeleton and focal adhesion. 5) Antisense experiments revealed that focal adhesion kinase (FAK) plays an important role in the stretch-induced morphological change. These data indicate that we could apply mechanical stretch to endothelial cells reproducibly and quantitatively and could investigate the stretch-induced intracellular signaling pathway using our stretch apparatus.
Morphological responses of endothelial cells were studied using excised rabbit aortic segments in culture. The specimen with a new longitudinal axis 90° from the original vessel axis was carefully fixed on a silicone rubber with holder, which is set to the flow chamber. In this study shear stress of 1Pa was applied for 24, 48, 60 and 72 hours. After flow exposure specimens were stained with silver nitrate and the endothelial cell shape and orientation were measured. Further, the cytoskeletal structure was observed by staining actin filaments with rhodamine-phalloidin. As a control, aortic segments were kept in an incubator under no flow condition. Time course of changes in cell shape and orientation was quite similar until 48 hours in both cases of static culture conditions and shear stress exposure conditions. At 60 hours after exposure to shear stress, cell with elongated shape at first to vessel axis became to nearly round shape. After then, endothelial cells started to elongate and align to flow direction at around 72 hours. However the actin filaments seemed to already respond to shear stress and had different structure from static culture conditions at around 24 hours. It was found that our new experimental apparatus would be very useful to study the morphology and functions of endothelial cells under flow conditions using excised aortic segments. Behavior of actin filaments in endothelial cells on the excised segment under shear flow was quite different from those cultured on glass or plastics. The results suggest that the substrates under endothelial cells would be very important for their morphology and functions.
The effects of the change of flow direction on the morphology of cultured bovine aortic endothelial cells were studied. Fully confluent endothelial cells cultured on glass plates were subjected to fluid-imposed shear stress of 2Pa for 24 hours. Experiments on shear flow exposure were performed for alternating orthogonal flow with a 3, 30min and 1 hour interval. (Data of 30min interval were previously reported.) After flow exposure, endothelial cells were fixed and F-actin filaments were stained with rhodamine phalloidin and shape index (SI) and angle of cell orientation were measured. After application of 2Pa shear stress for 24 hours in the alternating orthogonal flow with 30min interval the endothelial cells elongated (SI=0.63±0.14) and slightly aligned with the center of two flow directions. With 1 hour interval, cells were more elongated (0.55±0.16) than the cells with 30min and aligned with the center of two flow directions. In the both flow conditions, thick and clear stress fibers which aligned with cell axes were observed in central portion of the cells. In the flow experiments with 3min interval, many cells detatched from the glass plates. Moreover, in the remained cells on the glass plates after 24 hours, stress fiber was not observed. The results of this study indicate that morphology of endothelial cells are affected with the change of flow direction. In the atherosclerotic regions, such as arterial bends and branches, the blood flow is not simple but very complex with reverse and secondary flows. From these points of view, the change of the flow direction may affect the morphology and function of endothelial cells in in vivo flow condition.