Asparaginyl endopeptidase mapping of proteins with subsequent matrix-assisted laser desorption/ionization mass spectrometry.

A matrix-assisted laser desorption/ionization (MALDI) mass spectrometric mapping strategy for the identification and characterization of isolated and purified proteins is described. The method, which employs the combined usage of a new site-specific enzyme Asparaginyl endopeptidase (Asn-EP) for proteolysis, and MALDI for subsequent mass analysis, is capable of rapidly and sensitively examining the components of complex mixtures without any chromatographic or electrophoretic separation steps. Subpicomole sample quantities typically suffice to permit the confirmation of deduced primary structures and/or the identification of possible post-translational modifications.

[Stability studies on L-ascorbic acid dl-alpha-tocopherol phosphoric acid diester potassium salt (EPC-K)].

A compound having structures of both vitamins E and C, L-ascorbic acid dl-alpha-tocopherol phosphoric acid diester potassium salt (EPC-K), which was proven to have both anti-oxidative3%!d moisturizing effects, has been formulated in the quasi drug hair growing products to mainly prevent dandruff and itching which may be one of the causes for the hair loss. Stabilities of EPC-K in 75% ethanolic solutions with various pHs (2-10) were examined extensively by storing them at 50 degrees C for 30 d and by sunlight exposure for 30 d. Decomposition of EPC-K was only observed under pH 2 at a level of 50% and under sunlight exposure at 25% level.

"Varicoid change" of bile canaliculi in rat liver at an early phase of ischaemia-reperfusion injury.

To elucidate early changes and the mechanism of ischaemia-reperfusion liver injury, we investigated three-dimensional microstructural changes of cellular actin filaments in rat livers using confocal laser scanning microscopy. The liver tissues of a reperfusion group were examined 12 h after removal of a vascular clamp. Fixed tissues were stained with fluorescein-labelled phalloidin to obtain stereoscopic images of the actin filaments and these were compared with histological findings.

Secretagogue-induced calcium wave shows higher and prolonged transients of nuclear calcium concentration in mast cells.

To clarify the mechanism of secretagogue (compound 48/80)-induced calcium signaling in rat peritoneal mast cells, we analyzed serial confocal calcium images with high spatial and temporal resolution using different Ca(2+)-probes. The Ca(2+)-wave began at the periphery of the cytoplasm, and then spread to the center of the nucleus. Nuclear [Ca2+]i was clearly higher than cytoplasmic [Ca2+]i.

Cytotoxic activity of rev protein of human immunodeficiency virus type 1 by nucleolar dysfunction.

The rev protein (Rev) of the human immunodeficiency virus type 1 (HIV-1) is known as a post-transcriptional regulator of viral gene expression. It is located in the cell nucleolus. Transiently expressed Rev caused nucleolar ballooning and deformity with aberrant accumulation of rRNAs, and de novo synthesis of rRNAs decreased dramatically in these cells.

[Stability of allantoin and identification of its degradation compounds].

The stability of allantoin was studied in pH 3.0, 6.0, and 8.

3-D observation of N-cadherin expression during cardiac myofibrillogenesis of the chick embryo using a confocal laser scanning microscope.

It is not yet understood whether cell adhesion molecules play an active role in early cardiac morphogenesis or not. We present here the spatial and temporal expressions of N-cadherin and its relationships to actin filaments during looping (7- to 13-somite stages) of the chick embryonic heart tube observed by means of a confocal laser scanning microscope. Serial optical tomograms were obtained from the whole-mounted heart tubes stained with antibody to N-cadherin (fluorescein-conjugated) and phalloidin (rhodamine-conjugated).

Determination of eperisone in human plasma by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method was developed to determine eperisone hydrochloride, 4'-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride, in human plasma over the concentration range 0.2-40 ng/ml. Excellent sensitivity was achieved by selection of a favorable fragment ion, m/z 98, of eperisone and reduction of heat decomposition of eperisone by using a splitless injector and a shortened capillary column.

[Confocal laser scanning microscopy for image cytometry].

By using a focused laser beam as the light source of the microscope, to minimized flare, together with a pinhole in front of a photo detector to eliminate out-of-focus data, the confocal laser scanning microscope (CLSM) provides a depth-discriminated fluorescence image with high spatial resolution. The CLSM, therefore, has been widely used as a tool for accurate morphometric and densitometric analyses. We discussed here the biological applications of CLSM for demonstrating the three-dimensional features of the embryonic heart tube and chromosome and for imaging fast dynamic changes of [Ca2+]i in the living heart muscle cell pairs.

[Elucidation on abnormal behavior of glycyrrhizin salts in high performance liquid chromatography determination for quasi-drug products].

Glycyrrhizinates such as monoammonium and dipotassium glycyrrhizinates which are extracted from licorice, converted to easily water-soluble salts and refined have been formulated in many quasi-drug products as an anti-inflammatory agent. A quantitative analysis of glycyrrhizinates in quasi-drug products has usually and successfully been carried out by high performance liquid chromatography (HPLC). However, a slightly higher value than the actual content has sometimes been observed in daily analysis.

Intranuclear topological distribution of HIV-1 trans-activators.

Subcellular localization of human immunodeficiency virus type I (HIV-1) Tat and Rev was examined using a confocal laser scanning microscope (CLSM). In transfected COS-7 cells, Tat resided exclusively in the perinocleolar region, while Rev infiltrated fully into the nucleoli. The chimeric Tat in which the nucleolar targeting signal was replaced by that of Rev, which retains trans-acting activity of Tat, remained still in the perinucleolar region as wild-type Tat.

Quantitative histological analysis of the human sinoatrial node during growth and aging.

Background: Fibrosis or fatty infiltration of the human sinoatrial (SA) node is generally believed to represent replacement of the SA nodal cells by connective tissue. Quantitative analysis, however, has not been performed precisely to validate the interpretation of such histological changes.

Methods And Results: The actual volume of the SA node and its components were calculated according to the sum of the pixel number representing the colors of SA nodal cells and connective tissue in serial sections using a digital color image analyzer.

[Cold agglutinin hemolytic anemia complicating mycoplasma pneumonia].

A case of Mycoplasma pneumonia complicated with severe hemolytic anemia, which occurred as a result of a high titer of cold agglutinin is presented. A 49 year-old male was admitted because of fever, jaundice and dyspnea. Chest x-ray showed diffuse small nodular infiltrates throughout both lung fields.

3-D observation of actin filaments during cardiac myofibrinogenesis in chick embryo using a confocal laser scanning microscope.

Using a confocal laser scanning microscope (CLSM), we observed subcellular three-dimensional (3-D) arrangements of actin filaments stained with fluorescein-labeled phalloidin during myofibrinogenesis of chick embryonic heart (7- to 13-somite stages). Serial optical tomograms were obtained from whole-mounted heart tubes and reconstructed into stereoscopic images. Development of myofibrils in myocardial differentiation considerably differed in inner and outer myocardial cell layers.

Comparison of protein tyrosine phosphorylation and morphological changes induced by IL-2 and IL-3.

We constructed cell lines which can proliferate in response to IL-2 or IL-3 by introducing a wild-type and mutant forms of cDNAs encoding the human IL-2R p75 chain into an IL-3 dependent hematopoietic cell line which expresses the p55 chain of the IL-2R. We compared early events that were induced in these cells by IL-2 and IL-3. Analysis of protein tyrosine phosphorylation showed that two common protein bands, pp95 and pp90, were phosphorylated by stimulation of either IL-2 or IL-3, suggesting the possible sharing of part of a signal transduction pathway between IL-2R and IL-3R.

Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy.

We describe here the use of a confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca2+]i) in isolated ventricular cell pairs. The scanning apparatus of our system, paired galvanometer mirrors, can perform narrow band scanning of an area of interest at a high temporal resolution of less than 70 msec per image. The actual [Ca2+]i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of [Ca2+]i in optically sectioned unit volumes of the cell.

[Effect of bite force on parotid secretion in human].

To determine the physiological roles of periodontal mechanoreceptors in parotid salivary secretion, we studied the effects of strength and frequency of unilateral bites on the salivary secretion from both sides of human parotid glands. Parotid saliva was collected bilaterally via modified Lashley cups, and the flow rate of parotid saliva was measured with a device using a strain gauge. The bite force was monitored with a pressure transducer.

Calcium waves in mammalian heart: quantification of origin, magnitude, waveform, and velocity.

A dual, digital, indo-1 fluorescence imaging system was used to obtain high-speed ratiometric images of [Ca2+]i waves in single voltage-clamped mammalian cardiac cells. The spatiotemporal origin of [Ca2+]i waves in depolarized cells was detected as the spontaneous appearance, over 100-300 ms, of domelike regions of elevated [Ca2+]i, approximately 20 microns in diameter and 300 nM at the center. Images of [Ca2+]i taken at 67-ms intervals during propagation of [Ca2+]i waves revealed that the [Ca2+]i wave front was 1) constant in shape, 2) spatially steep, typically rising from 500 to 1200 nM in about 10 microns, and 3) propagating at constant velocity, typically 100 microns/s at 22 degrees C.

[A case of autoimmune hemolytic anemia and bullous pemphigoid-like skin lesion combined with idiopathic thrombocytopenic purpura].

A case of autoimmune hemolytic anemia (AIHA) and bullous pemphigoid (BP)-like skin lesion combined with Idiopathic thrombocytopenic purpura (ITP) is reported. A 25-years-old male, who had been diagnosed as ITP and treated at another hospital, was admitted in this hospital recently complaining of disseminated bullous-vesicular eruptions on the whole body and autoimmune hemolytic anemia. Examinations, disclosed that RBC was 364 X 10(4)/microliters, reticulocyte 40, platelet 3000/microliters, direct and indirect Coombs test positive, and platelet Coombs consumption test was positive leading to the diagnosis of AIHA and ITP, known as "Evans syndrome.

High temporal resolution video imaging of intracellular calcium.

We have developed a system for imaging intracellular free calcium ion concentration ([Ca2+]i) at the highest rate possible with conventional video equipment. The system is intended to facilitate quantitative study of rapid changes in [Ca2+]i in cells that move. It utilizes intensified video cameras with nearly ideal properties and digital image processing to produce two images that can be ratioed without artifacts.

MR imaging of spastic diplegia. Comparative study between preterm and term infants.

Eighteen children (eleven preterm birth and seven term birth) with clinical evidence of spastic diplegia (SD) were studied to clarify the differences of the lesions between preterm and term SD on MR imaging. All with preterm birth showed similar abnormalities of the periventricular white matter represented by high intensity in T2-weighted imaging and low intensity in T1 imaging. It seemed that the lesions were periventricular leukomalacia (PVL) and dysmyelination pathologically and correlated well clinically with spastic diplegia.