Centrifuge microscope as a tool in the study of cell motility.

The centrifuge microscope (CM) is composed of a centrifuge and a microscope optical system designed to observe minute objects, especially living cells, during the application of centrifugal acceleration. Structures and characteristics of various types of CM designed and constructed up to the present and studies done with the CM on cell biology, especially cell motility, are reviewed. These studies include observations of the behavior of cells and cell components in a centrifugal field, determination of the mechanical properties of the cell surface and cytoplasm, microsurgical operations on cells with centrifugal force, and determination of the magnitude and the site of generation of motive force for cell motility.

Effect of systemic dexamethasone on extracellular amino acids, GABA and acetylcholine release in rat hippocampus using in vivo microdialysis.

In order to elucidate the mechanism of the cytotoxic effect of glucocorticoid on the rat hippocampus, we studied the effects of dexamethasone, a synthetic glucocorticoid, on neurotransmitter release from the hippocampus by in vivo microdialysis. Dexamethasone (10 mg/kg) was administered i.p.

Sperm-egg fusion in the sea urchin is blocked in Mg(2+)-free seawater.

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev.

Fertilization Reaction without Changes in Intracellular Ca in Medaka Eggs-An Experiment with Acetone-Treated Eggs.

To investigate whether or not causal relationship exists between the increase in intracellular Ca and other cortical reactions at fertilization in the medaka, Oryzias latipes, intracellular Ca was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca . Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e.

[Secondary autonomic diseases--diabetes mellitus].

Diabetic autonomic neuropathy is an established disease entity. Nevertheless, it was not until 1945 that diabetic autonomic neuropathy became a target for extensive systemic research works. This is partly because of insidious progress of this disorder and lack of precise techniques of examinations.

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes: (aequorin/calcium/oocyte maturation/1-methyladenine/starfish).

A transient increase in intracellular Ca upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca transient followed by GVBD.

Time Sequence of Early Events in Fertilization in the Medaka Egg: (micropyle/exocytosis/sperm movement/calcium release/medaka egg).

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10 /ml at 23°-25°C).

Identification of the site important for the actin-activated MgATPase activity of myosin subfragment-1.

The lysine-rich sequence (-KKGGKKK-) located at the 50,000/20,000 Mr junction of myosin subfragment-1 (S-1) was cleaved by endoprotease Arg-C or by trypsin in the presence of ATP and an equimolar amount of actin. Under these conditions, cleavage by Arg-C was between the first and second lysine residues, whereas cleavage by trypsin was between the third and fourth lysine residues. The actin-activated MgATPase activity of the S-1 cleaved by Arg-C was almost the same as native S-1, but S-1 cleaved by trypsin showed markedly reduced ATPase activity.

Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography.

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine.

Shift of binding site at the interface between actin and myosin.

The molar ratio dependent change in the binding manner between actin and the lysine-rich sequence at the junction between 50K and 20K domains of subfragment 1 was studied by both protease digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The tryptic cleavage site at the function between 50K and 20K was found to be located between the third and fourth lysine residues in the lysine-rich sequence -KKGGKKK-. This site was not protected by actin when the molar ratio of actin to subfragment 1 was 1:1 but was protected at 2:1 and 3:1.

Mechanical properties of the cortex before and during cleavage.

The changes in stiffness of the cell before and during cleavage reported previously for various kinds of echinoderm eggs are classified into three types. There is no general rule that cleavage starts when the stiffness attains a maximum or a minimum. Because the stiffness of the cell surface is much greater than that of the mitotic apparatus, the change in stiffness of the cell represents mainly the change in stiffness of the cell surface.

ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure.

To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved.

Binding manner of actin to the lysine-rich sequence of myosin subfragment 1 in the presence and absence of ATP.

Actin was cross-linked to myosin subfragment 1 with a water-soluble carbodiimide both in the presence and in the absence of ATP, and the cross-linking of the N-terminal acidic sequence of actin to the lysine-rich sequence (--KKGGKKK--) at the junction between the 50K and the 20K fragments of lysines in the lysine-rich sequence were compared between the resulting acto-22K fragment and the uncross-linked 22K fragment by using a protein sequencer. It was found that, in the presence of ATP, a very small amount of cross-linked product was produced and, in the product, only one lysine residue which lies closest to the 50K fragment mainly decreased in its amount as compared to the corresponding lysine residue in un-cross-linked 22K. In the absence of ATP, on the other hand, the amounts of all five lysine residues in acto-22K were about 60% those of the corresponding residues in 22K.

Nuclear Involvement in Localization of the Initiation Site of Surface Contraction Waves in Xenopus Eggs: (surface contraction waves/Xenopus laevis/nucleus/colchicine/cleavage).

The initiation site of surface contraction waves (SCWs) was examined in fertilized, parthenogenetically activated and enucleated Xenopus eggs after either rotation through 90° off the vertical axis or injection of colchicine. In enucleated eggs, SCWs always started from a top site of the egg under all conditions examined. In fertilized or activated eggs, SCWs started, depending on the experimental conditions, from either the sperm entry point, the animal pole region located sideward or the top site of the egg.

Analysis of Cellular Mosaicism in a Transgenic Mouse by Histological In Situ Hybridization: (Transgenic mice/mosaics/cell lineage/in situ hybridization).

One of the transgenic mice carrying a chicken δ-crystallin gene was found to be mosaic with regard to the distribution of the exogenous gene. Taking advantage of the exogenous DNA sequences as a cell lineage marker detectable by histological in situ hybridization technique, we studied cellular mosaicism in mouse 7-5. This mouse carried the exogenous gene in 20-40% of its cells, probably reflecting chromosomal integration of the exogenous DNA which occurred in a blastomere of around the 4-cell stage.

The sites on calmodulin cross-linked to myosin light chain kinase and troponin I with water-soluble carbodiimide.

To elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis.