Molecular cloning of Bombyx cerebral opsin (Boceropsin) and cellular localization of its expression in the silkworm brain

We have cloned a cDNA for a novel opsin from the larval brain of the silkworm Bombyx mori in which the photoperiodic photoreceptor had been supposed to reside in the cephalic central nervous system (CNS). Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins and showed the greatest degree of homology (84%) with the green visual photoreceptor in the sphingid moth. We have designated this Bombyx cerebral opsin as Boceropsin. Southern blotting experiments indicated that the Boceropsin gene is present in a single copy, and RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain but not in the subesophageal ganglion (Sg) or thoracic ganglion (Tg). Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of Bombyx larvae. This is the first report of expression of an opsin-based protein in CNS of an insect. The possibility that the Boceropsin functions as the photoperiodic receptive pigment in the silkworm is also discussed.     

A new sugar chain of the proteinase inhibitor from latex of Carica papaya

The structure of a sugar chain of the proteinase inhibitor from the latex of Carica papaya was studied. Sugar chains liberated on hydrazinolysis were N-acetylated, and their reducing-end residues were tagged with 2-aminopyridine. One major sugar chain was detected on size-fractionation and reversed-phase HPLC analyses. The structure of the PA-sugar chain was determined by two-dimensional sugar mapping combined with sequential exoglycosidase digestion and partial acid hydrolysis, and by 750 MHz 1H-NMR spectroscopy. The structure found was Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc. This sugar chain represents a new plant-type sugar chain with five mannose residues.     

Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana

A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+ -ATPases and V-type H+ -PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.     

A biochemically defined system for mammalian nonhomologous DNA end joining

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.     

Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.     

Migration from plant to plant: an important factor controlling densities of the epiphytic cladoceran <Emphasis Type="Italic">Alona</Emphasis> (Chydoridae,Anomopoda) on lake vegetation

We investigated seasonal changes in the density of epiphytic cladocerans Alona spp. (Chydoridae, Anomopoda) in two habitats, emergent and submerged aquatic plants, in Lake Suwa, Japan, from April to August 1998 and from April to November 2000. Alona had a density peak in early June on reeds (emergent) and in late June on Potamogeton malaianus (submerged). In summer, Alona density remained low in both habitats. Although density was positively correlated with the abundance of epiphytic algae, the birth rate was constant and no correlation between algal abundance and clutch size was detected. In a field experiment using ropes as an artificial substrate covered with high and low densities of epiphytic algae as food, more Alona attached to the ropes with the high density of algae. These results suggest that Alona may select food-rich habitats and migrate seasonally, and that migration is an important factor in the population dynamics of epiphytic chydorid cladocerans such as Alona. In Lake Suwa, Alona may migrate from the reed zone to the submerged macrophyte zone in June.     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.