Identification of the C-terminal portion of a protein by comparative peptide mapping

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.     

Confirmation of yessotoxin and 45,46,47-trinoryessotoxin production by Protoceratium reticulatum collected in Japan

Two different strains of the dinoflagellate Protoceratium reticulatum collected at Harima Nada and Yamada Bay in Japan were cultured and analyzed by fluorometric HPLC for yessotoxin production. Only the Yamada Bay strain produced yessotoxin. The toxin together with its analog, 45,46,47-trinoryessotoxin, were isolated from larger scale culture and unambiguously confirmed by (1)H NMR and MS measurements. This is the first confirmation of the biogenetic origin of yessotoxin in Japan, where the toxin was first reported. The results also indicate that the production of yessotoxins by P. reticulatum differs from strain to strain, in a similar way to that observed in many other toxigenic dinoflagellates such as Dinophysis spp. and Alexandrium spp.     

Preparation of optically active allothreonine by separating from a diastereoisomeric mixture with threonine

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH(3)) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH(3) by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.     

Lung surfactant secretion by interalveolar Ca2+ signaling

Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.     

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.     

Temporal change in nutrient concentrations and phytoplankton biomass in short time scale local upwelling around the Izu Peninsula, Japan*

Temporal changes in nitrate plus nitrite and chlorophyll concentrationswere observed by continuous shipboard measurements in upwelledand coastal water masses covering an area 20 x 30 km² alongthe Izu Peninsula between May 22 and 26, 1982. Two localizedupwelling events were observed, one which had occurred beforeMay 22 and the other on May 26. Nutrient input into the surfacefrom upwelling and subsequent phytoplankton growth were clearlydocumented. Phytoplankton growth, estimated from the disappearanceof nutrients, agreed well with modelling estimates based uponsimulated culture experiments done previously in the same area.Phytoplankton growth terminated when nutrients were depleted.Similar stimulation of phytoplankton growth supported by localizedupwelling can be expected in other coastal regions where similarconditions exist.     

Three-dimensional architecture of the tubular endocytic apparatus and paramembranous networks of the endoplasmic reticulum in the rat visceral yolk-sac endoderm

The three-dimensional architecture of the tubular endocytic apparatus and the endoplasmic reticulum in the rat yolk-sac endoderm was investigated after loading with horseradish peroxidase-conjugated concanavalin A by intrauterine administration. After 30 min, small vesicles (50–150 nm in diameter), small tubules (80–100 nm in diameter) and large vacuoles (0.2–1.0 m in diameter) in the apical cytoplasm were labeled with the tracer, but lysosomes (1.0–3.5 m in diameter) in the supranuclear cytoplasm were not labeled until 60 min after loading. Stereo-viewing of the labeled small tubules in thick sections revealed that they were not isolated structures but formed three-dimensional anastomosing networks, which were also confirmed by scanning electron microscopy after maceration with diluted osmium tetroxide. Their earlier labeling with the endocytic tracer, localization in the apical cytoplasm and three-dimensional network formation indicated that the labeled small tubules represented tubular endosomes (tubular endocytic apparatus). These well-developed membranous networks provided by the tubular endosomes are suggested to facilitate the receptor-mediated endocytosis and transcytosis of the maternal immunoglobulin in the rat yolk-sac endoderm. Scanning electron microscopy further revealed lace-like networks of the smooth endoplasmic reticulum near the lateral plasma membrane. Their possible involvement in transport of small molecules or electrolytes is discussed.