Plant-wide names for partial cDNA sequences of rice usingBlast and4th dimension

A total of 687 DNA sequence accessions from the Mendel database (release 1.04, 3 November 1994) assigned standardized designations for plant genes and gene products were used in aBLAST similarity search of 7557 rice partial cDNA sequences and 287 other rice sequences from the Japanese Rice Genome Research Program. We describe procedures for data manipulation, import and export from and to Macintosh and Unix, and the use of 4th Dimension relational database management system (RDBMS) in data processing. Altogether 275 sequences showed strong similarity hits. Using the CPGN nomenclature, we assign putative designations for genes and gene products. Assignments include representatives of 26 gene products, including 58 cDNA sequences similar to α-tubulins (TubA), 23 similar to β-tubulins (TubB) and 51 similar to cytosolic subunit C of glyceraldehyde-3-phosphate dehydrogenase (NAD) (GapC). The results of the similarity searches are listed and are also available electronically. The assignments have been submitted to the CPGN working groups for verification and for later inclusion in the GenBank/EMBL/DDBJ sequence databases, which will include the standardized designations in the accession data fields. Member of the ISPMB Commission on Plant Gene Nomenclature, representing the Rice Genome Research Program of Japan. Reprint requests to T. Sasaki.     

Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in trachco-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heatlabile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.     

Endogenous activating factor for guanylate cyclase in synaptosomal-soluble fraction of rat brain.

When the crude mitochondrial fraction of rat brain was homogenized with distilled water and centrifuged, most of guanylate cyclase activity was detected in the soluble fraction. The total guanylate cyclase activity recovered in the soluble fraction was 5- to 8-fold higher than that of the crude mitochondrial fraction. The greater recovery of guanylate cyclase activity was found to be due to a release of an endogenous activating factor for guanylate cyclase. The activating factor was partially purified by acid extraction followed by a gel filtration and ion exchange resin columns. The factor was a dialyzable small molecule. The molecular weight was estimated to be between 300 and 600 by a Sephadex G-15 column and Diaflo ultrafilter membranes. It was stable in dilute acids, but labile in alkaline solution. It was readily soluble in water, but insoluble in organic solvents. Treatment with various enzymes, so far as tested, failed to abolish the activity. The activating factor stimulated the initial velocity of the reaction. It altered neither the Km value for GTP nor the dependency of the enzyme on divalent metals. The activation by the factor was due to an increase in the Vmax of the reaction. The activation was prevented by lysolecithin, Lubrol PX, hydroxylamine, methylhydroxylamine, or hemoglobin.     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 × 4 mm I.D. column packed with 7 μm NH₂-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion—molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]⁻, with little fragmentation, and it is particularly useful for the molecular weight determination of sugars.