Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.     

Immunohistochemical characterization of cell types expressing the cellular prion protein in the small intestine of cattle and mice

The gastrointestinal tract is thought to be the main site of entry for the pathological isoform of the prion protein (PrP^Sc). Prion diseases are believed to result from a conformational change of the cellular prion protein (PrP^c) to PrP^Sc. Therefore, PrP^c expression is a prerequisite for the infection and spread of the disease to the central nervous system. However, the distribution of PrP^c in the gut is still a matter of controversy. We therefore investigated the localization of PrP^c in the bovine and murine small intestine. In cattle, most PrP^c positive epithelial cells were detected in the duodenum, while a few positive cells were found in the jejunum. PrP^c was expressed in serotonin producing cells. In bovine Peyer’s patches, PrP^c was distributed in extrafollicular areas, but not in the germinal centre of the jejunum and ileum. PrP^c was expressed in myeloid lineage cells such as myeloid dendritic cells and macrophages. In mice, PrP^c was expressed in some epithelial cells throughout the small intestine as well as in cells such as follicular dendritic cell in the germinal centre of Peyer’s patches. In this study, we demonstrate that there are a number of differences in the localization of PrP^c between the murine and bovine small intestines.     

Expression of Myostatin in Neural Cells of the Olfactory System

Recent studies show that myostatin mRNA expression is found in some regions of the brain. However, the functional significance of this is currently unknown. We therefore investigated myostatin expression and function in the brain. In this study, we used immunohistochemistry, in situ hybridization, and RT-PCR analysis to reveal that myostatin is expressed in the mitral cells in the olfactory bulb (OB) and in neurons in the olfactory cortex (OC). Using 3D reconstruction, mitral cells positive for myostatin were positioned in the lateral and ventral regions of the OB. In contrast, myostatin-positive mitral cells were detected in mice at 2 weeks of age, but not on days 0 and 7 after birth. Activin receptor IIB, a myostatin receptor, was expressed in the OB, OC, hippocampus, and paraventricular thalamic nucleus. Moreover, c-Fos immunostaining in granule cells in the OB was augmented after intracerebroventricular injection of myostatin. These findings suggest that myostatin is localized in specific cells associated with the olfactory system of the brain and may act as a key inhibitor in cell and/or signal development of the olfactory system.     

Laser confocal scanning microscopy of the surface membrane/T-tubular system and the sarcoplasmic reticulum in insect striated muscle stained with DilC18(3)

The structure of the surface membrane/transverse tubular (T-tubular) system and of the sarcoplasmic reticulum (SR) of the labial adductor muscle of the honey bee (Apis mellifera) was examined by laser confocal scanning microscopy, after staining with the fluorescent membrane probe DiIC18(3). The following components of the surface membrane/T-tubular system were visualized: transverse tubular networks that are located in the A-band close to the A-I junction and form dyads with the SR, longitudinal tubules that link the T-tubular networks within the between sarcomeres, and surface invaginations of larger diameter that contain tracheoles. The well developed SR forms a dense network of branching and anastomosing tubules in the A-band. A few tubular elements in the interfibrillar space in the I-band link the SR of adjacent sarcomeres. This study demonstrates the advantages of the laser confocal microscope and lipophilic fluorescent dyes for studying the 3-D structure of cellular membrane systems.     

Discovery of two rare species of stalked crinoids from Okinawa Trough, southwestern Japan, and their systematic and biogeographic implications

A recent submersible dive on the southern slope of Aguni Knoll, central Okinawa Trough has led to a discovery of two rare species of stalked crinoids, Proisocrinus ruberrimus and Naumachocrinus hawaiiensis, both of uncertain taxonomic affinity. The specimens of Proisocrinus ruberrimus, here termed "Moulin rouge" as a pseudonym due to its conspicuous red color, were collected from depths of approximately 1,800 m and are characterized by a long, slender stalk. The proximal columnals are pentalobate in cross section, with isocrinid-like articular facets; several bear rudimentary cirri. The following columnals lose the cirri and rapidly become almost circular; their facets bear radiating crenulations similar to those of Millericrinida. The characters observed in the proximal stalk indicate that the 'Moulin Rouge' (P. ruberrimus) belongs to the order Isocrinida rather than to the order Millericrinida. Naumachocrinus hawaiiensis was collected at a depth of 1,440 m. The long, cylindrical crown and number of arms indicate that Naumachocrinus should be classified in the Bathycrinidae.     

Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adheres to human colonic mucin

Aims:  To characterize the adhesion molecule of Lactobacillus plantarum LA 318 that shows high adhesion to human colonic mucin (HCM).
Methods and Results:  The adhesion test used the BIACORE assay where PBS-washed bacterial cells showed a significant decrease in adherence to HCM than distilled water-washed cells. A component in the PBS wash fraction adhered to the HCM and a main protein was detected as a c . 40-kDa band using SDS-PAGE. Using homology comparisons of the N-terminal amino acid sequences compared with sequence databases, this protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The DNA sequence of LA 318 GAPDH was 100% identical to the GAPDH ( gapB ) of L. plantarum WCFS1. The purified GAPDH adhered to HCM.
Conclusions:  We found the adhesin of L. plantarum LA 318 to HCM in its culture PBS wash fraction. The molecule was identified as GAPDH. Because LA 318 possesses the same adhesin as many pathogens, the lactobacilli GAPDH may compete with pathogens infecting the intestine.
Significance and Impact of the Study:  This is the first report showing GAPDH expressed on the cell surface of lactobacilli adheres to mucin suggesting L. plantarum LA 318 adheres to HCM using GAPDH binding activity to colonize the human intestinal mucosa.     

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM). and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.     

The role of xanthine oxidase in paraquat intoxication.

The role of xanthine oxidase in the mechanism of paraquat toxicity was assessed by in vitro and in vivo experiments. Paraquat stimulated the reduction of cytochrome c by xanthine-xanthine oxidase system in vitro. Paraquat, when added in vitro, stimulated hypoxanthine-dependent superoxide production in the cytosol of rat lung. Tungsten-feeding inhibits xanthine oxidase activity in a variety of tissues in experimental animals. Its therapeutic effect on paraquat intoxication was studied in this paper. In rats fed a tungsten-enriched diet for 5 weeks prior to intraperitoneal injection of 50 mg/kg paraquat dichloride, the mortality decreased significantly compared with rats fed a standard diet. Pretreatment with oxypurinol (1000 mg/kg, s.c.) also ameliorated the paraquat toxicity in rats. We conclude that xanthine oxidase plays an important role in paraquat toxicity and that xanthine oxidase inhibitors may become antidotes for paraquat intoxication.     

Hormonal control of the orange coloration of diapause pupae in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

Diapause pupae of Papilio xuthus show color polymorphism, represented by diapause-green, orange, and brownish-orange types that are each associated with specific pupation sites. We investigated the role of the site of pupation on the induction of the development of orange types (or brownish-orange types), and the endocrine mechanism underlying the control of color polymorphism in short-day pupae. All short-day larvae of the wandering stage developed into orange or brownish-orange type pupae when they were placed in rough-surfaced containers after gut-purge. Utilizing a pharate pupal ligation between the thorax and abdomen, the endocrine mechanism underlying the control of color polymorphism was shown to involve a head-thorax factor (Orange-Pupa-Inducing Factor: OPIF) that induced orange types in short-day pupae. OPIF was bioassayed using the ligated abdomens of short-day pharate pupae. OPIF was extractable with 2% NaCl solution from 5th-instar larval ganglia complexes following the mesothoracic complex (TG(2,3)-AG(1-7)), but it could not be extracted with either acetone or 80% ethanol solution. OPIF may not exist in the brains of day-0 pupae or in brain-subesophageal ganglion and prothoracic ganglion complexes of 5th-instar larvae. The short-day pharate pupae responded to OPIF in a dose-dependent manner.