Distinct genotype and antigenicity among genogroup II sapoviruses

SaV, a pathogen of acute gastroenteritis, is divided into five genogroups, GI to GV. However, the relation between SaV antigenicity and genetic clusters is not fully understood. We have recently identified two GII SaV strains, Mc10 and C12, which are grouped into the same cluster based on the polymerase but are grouped into distinct clusters based on the capsid. To evaluate the difference in antigenicity between these two strains, VLP were expressed in mammalian cells. An antigen ELISA demonstrated for the first time that strains in the same GII SaV genogroup, but within different clusters, have distinct antigenicities.     

Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

A simple procedure for the isolation of highly purified lysosomes from normal rat liver

A procedure for the isolation of highly purified lysosomes from normal rat liver is described. The method depends on the swelling of mitochondria when the postnuclear supernatant fraction is incubated with 1 mM Ca2+. The lysosomes can then be separated from the swollen mitochondria by Percoll density gradient centrifugation. The lysosomal fraction obtained by our method was enriched more than 120-fold in terms of the marker enzymes with a yield of 25%. The electron microscopic examination and the measurement of the activities of marker enzymes for various subcellular organelles indicated that our lysosomal preparation was essentially free from contamination by other organelles.     

The effect of chemicals on the activity of deglycosylated Aureobasidiumβ-fructofuranosidases

The effects of chemicals such as metal ions and typical organic enzyme inhibitors on the activity of deglycosylated β-fructofuranosidases ( P -1 and P -2) from Aureobasidium were observed and compared with those of native enzymes. P -1 and P -2 enzymes became sensitive to metal ions, such as Fe²⁺, Co²⁺, Ni²⁺ and Al³⁺, after deglycosylation. The enzymes were also inhibited by a sulphydryl reagent (monoiodoacetic acid), a peptide-hydrolysing reagent (hydroxylamine) and chelating reagents (sodium citrate, EDTA and sodium azide) after deglycosylation. The importance of deglycosylation for the determination of the true characteristics of the enzymes against chemicals is discussed.     

Calcium- and Calmodulin-Dependent Phosphorylation of Diphosphoinositide in Acetylcholine Receptor-Rich Membranes from Electroplax of Narke japonica

The phosphorylation of phosphoinositides in the acetylcholine receptor (AChR)-rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR-rich membranes were incubated with [gamma-32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri- and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR-rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P-incorporation into these lipids was realized by the membrane-bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane-bound DPI kinase was activated by Ca2+ (greater than 10(-6) M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+-dependent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the phosphorylation of DPI in the AChR-rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+- and calmodulin-dependent DPI kinase responding to the change in the intracellular Ca2+ level.     

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(−)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 – 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(−)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2–6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(−)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5–6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.     

Substrate recognition mechanism of the peptidase domain of the quorum-sensing-signal-producing ABC transporter ComA from Streptococcus

ComA of Streptococcus is a member of the bacteriocin-associated ABC transporters, which is responsible for both the processing of the propeptide ComC and secretion of the mature quorum-sensing signal. The quorum-sensing system is a bacterial intercellular communication system implicated in various functions including biofilm formation. In this study, the peptidase domains (PEPs) of the ComAs from six species of Streptococcus and ComCs from four species were expressed, purified, and characterized to address the mechanism of the substrate recognition of PEP. PEPs specifically cleaved ComCs after the Gly-Gly site in all the PEP-ComC combinations examined. The N-terminal leader region of ComC was found to form an amphiphilic alpha-helix structure upon binding to the PEP. Furthermore, mutagenesis studies revealed that four conserved hydrophobic residues in this leader region of ComC extending from -15 to -4 positions are critical in the interaction with PEP. Together with the double glycine motif, these structural features of ComC would explain the strict substrate specificity of the PEP.     

Amylase in human lungs and the female genital tract. Histochemical and immunohistochemical localization

We investigated the localization of amylase in normal human lungs and the female genital tract using immunohistochemical and histochemical methods. Immunohistochemical procedures were applied to formaldehyde-fixed, paraffin-embedded specimens as well as to cryostat sections of periodate/lysine/paraformaldehyde (PLP)-fixed tissues. The starch-substrate-film method was used for the histochemical investigation of unfixed frozen sections. Amylase immunoreactivity was observed in ciliated epithelial cells of the bronchus and in serous cells of the bronchial glands but not in the alveolar epithelium. Immunoreactive amylase was also found in the cytoplasm of the ciliated epithelium of the fallopian tubes, especially in the apical part of the cytoplasm and in ciliary vesicles. Immunoreactive amylase was also found to be present in the surface epithelial cells and glands of the uterine cervix, as well as in the superficial part of the endometrial glands. The distribution of amylase activity revealed using histochemistry was similar to that observed in cryostat sections of PLP-fixed tissues after immunohistochemical staining. Amylase antigenicity was better preserved in cryostat sections of PLP-fixed materials than in formaldehyde-fixed, paraffin-embedded specimens. The results are discussed in relation to pulmonary and female-genital-tract diseases.