Immunochemical subfractionation of a microsomal fraction of rat liver with antibody-coated Staphylococcus aureus cells

The procedure for immunochemical adsorption of vesicles with specific antigen on their outer surfaces was improved. When microsomal vesicles were mixed with Staphylococcus aureus cells coated with the antibody against NADPH-cytochrome c reductase, more than 90% of the enzyme activity was adsorbed on the cell, whereas, only about 10% of the activity was adsorbed on cells coated with the same amount of anti-ovalbumin antibody. NADH-cytochrome c reductase and aldehyde dehydrogenase activities were adsorbed on the cell to the same extent as was NADPH-cytochrome c reductase activity. Under this condition, there was no adsorption of the activities of the marker enzymes of lysosomes and Golgi apparatus, whereas large amounts of the activities of the plasma membrane enzymes were adsorbed. The specific activity of NADPH-cytochrome c reductase in the adsorbed vesicles from the microsomal fractions increased considerably. In contrast, marker enzymes of the Golgi or of the plasma membranes could be enriched in unadsorbed vesicles from the Golgi fractions.     

Metabolism of 3-phosphoglyceroyl phosphate in phosphoenolpyruvate-enriched human erythrocytes

The concentration of 3-phosphoglyceroyl phosphate in erythrocytes was increased by more than 100-fold when red cells were incubated with extracellular phosphoenolpyruvate at 37 degrees C. Since these elevated levels were maintained for 60 min, the metabolism of 3-phosphoglyceroyl phosphate and related compounds could be investigated in phosphoenolpyruvate-treated erythrocytes. 2,3-Bisphosphoglycerate synthesis was not affected by intracellular pH when the 3-phosphoglyceroyl phosphate level was constant but did vary with 3-phosphoglyceroyl phosphate concentration. On the other hand, the relationship between the rate of 2,3-bisphosphoglycerate synthesis and 3-phosphoglyceroyl phosphate concentration was not straightforward. At relatively low concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis agreed with a rate calculated from a formula incorporating kinetic parameters of purified 2,3-bisphosphoglycerate synthase (Rose, Z.B. (1973) Arch. Biochem. Biophys. 158, 903-910). However, at high concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis was lower than the calculated value. The concentration of glucose 1,6-bisphosphate did not increase even when 3-phosphoglyceroyl phosphate was elevated to 200 microM. Elevated levels of intracellular 2,3-bisphosphoglycerate did not inhibit glycolytic activity in these erythrocytes. These results suggest that incubation of erythrocytes with phosphoenolpyruvate is a useful technique to investigate the effect of metabolic perturbations at the intermediate stages of glycolysis.     

Calcium-activated protease in the giant algaChara australis

Summary There is a protease, which is activated by Ca²⁺ (about 100 M), works at neutral pH and exists in the cytoplasm inChara australis. This protease may correspond to calpain, the calcium-activated neutral protease, which has been studied in animal cells. This is the first report showing the existence of a calcium-activated protease in plant cells.     

Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 m in diameter) and less electron-dense than the D-granules (less than 0.6 m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Isolation of a new cDNA clone encoding an Rh polypeptide associated with the Rh blood group system

We searched for a human chromosome that would restore the cholesterol metabolism in 3T3 cell lines (SPM-3T3) derived from homozygous sphingomyelinosis mice (spm/spm). Mouse A9 cells containing a single copy of pSV2neo-tagged chromosomes 9, 11, or 18 derived from normal human fibroblasts served as donor cells for transfer of human chromosomes. Purified A9 microcells were fused with SPM-3T3 cells, and the microcell hybrids were selected in medium containing G418 antibiotics. The microcell hybrids that contained human chromosomes 9, 11, or 18 in a majority of cells were examined. The accumulation of intracellular cholesterol in the microcell hybrids containing a chromosome 18 decreased markedly, whereas in the microcell hybrids containing either chromosomes 9 or 11 it was similar to that in SPM3T3 cells. The SPM-3T3 cells with an intact chromosome 18 were further passaged and subcloned. Clones which again accumulated intracellular cholesterol had concurrently lost the introduced chromosome 18. The abnormal accumulation was associated with a decrement in the esterification of exogenous cholesterol. These findings suggest that the gene responsible for the abnormal cholesterol metabolism in the spm/spm mice can be restored by a hu man chromosome 18. The gene was tentatively mapped on 18pter18p11.3 or 18q21.3qter that was lost during subcloning, thereby resulting in reaccumulation of the intracellular cholesterol.     

N-(Carboxyacyl)chitosans

Hemicelluloses were isolated from pineapple-leaf fibers under different conditions. Study of the properties of these hemicelluloses gave direct evidence of some ester linkages between the hemicellulose and the lignin in this fiber. An aldobiouronic acid was isolated from this fiber hemicellulose, and characterized as 2-O-(4-O-methyl-α-d-glucopyranosyluronic acid)-d-xylose. This indicates that the general nature of the hemicellulose is similar to those of jute and other fiber hemicelluloses.     

Identification and Characterization of a Novel aac(6′)-Iag Associated with the bla IMP-1–Integron in a Multidrug-Resistant Pseudomonas aeruginosa

In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6′)-Iag, bla _IMP-1, a truncated form of bla _IMP-1, and a truncated form of aac(6′)-Iag. The aac(6′)-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6′)-Iz. Recombinant AAC(6′)-Iag protein showed aminoglycoside 6′-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6′)-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for bla _IMP-1 and aac(6′)-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.