Overexpression and purification of rat peroxisomal membrane protein 22, PMP22, in Pichia pastoris

Peroxisomal membrane protein 22, PMP22, is an integral membrane protein that has four putative transmembrane-spanning regions. First reported as a major component of rat liver peroxisomal membranes and suggested to be involved in the metabolism of reactive oxygen species, its function and structure are still unknown owing to a lack of biochemical and structural experiments. Here we report the overproduction and purification of rat PMP22 (rPMP22) with the use of a methylotrophic yeast, Pichia pastoris, as a host. rPMP22 was localized not to peroxisomal membranes but to membrane compartments, such as the nuclear envelope. Highly pure rPMP22 was obtained in two steps. Several physicochemical assays indicated that the purified preparation should retain its functional structure. Furthermore, fed-batch fermentation yielded 90 mg of rPMP22 protein from 4L of culture. This is the first report to demonstrate the overproduction of a recombinant rPMP22 in the membrane compartments of P. pastoris.     

Vascular permeability-increasing activity possessed byTreponema phagedenis (Reiter strain)

Treponema phagedenis possessed the ability to induce an increase in vascular permeability (blueing) in guinea pig skin, which was exerted not only by cell-free preparations but also directly by intact cells. Cell-free preparations induced maximum blueing 0 h after sample injection, while whole cells did so after 1 h. Log dose-response curves for cell-free preparations were linear within the range of blue spots with diameters of 10–24 mm, with slopes ( ) of 8.5–8.7. The vascular permeability-increasing activity was not ascribable to hyaluronidase and probably to -galactosidase; this organism did not produce any detectable hyaluronidase activity.     

Two new species ofConiochaeta with a cephalothecoid peridium wall

Two new species ofConiochaeta, isolated from Japanese soils, are described and illustrated:C. cephalothecoides, which is characterized by dark brown ascomata clothed with short setae, cylindrical asci and ovoid to almond-shaped or pyriform ascospores with a longitudinal germ slit; andC. dumosa, which is characterized by dark brown ascomata clothed with short setae and dense hyphal hairs, cylindrical asci and ellipsoid-fusoid ascospores with a longitudinal germ slit. These species are distinguished from most species of the genus by the unique cephalothecoid peridium of their ascomata. The associated anamorphs of both species are assignable to the form-genusLecythophora.     

Two new species ofHeleococcum withAcremonium anamorphs

Two new species ofHeleococcum (a cleistothecial nectrioid genus in the Hypocreaceae) are described and illustrated.Heleococcum alatosporum, isolated from Indonesian soil, is recognized by the production of salmon-colored ascomata, cylindrical asci, and hyaline, small, bicellular ascospores with walls that are verruculose and ornamented with longitudinal ridges.Heleococcum inapertum, isolated from Philippine soil, is characterized by yellow ascomata, clavate asci, and pale yellow, middle-sized, bicellular, verruculose to weakly striate ascospores surrounded with a hyaline sheath. Anamorphs of the new species are included inAcremonium. A key to the accepted species of the genus is provided.     

Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.     

Isolation ofRoumegueriella rufula as a new record from East Asia

Roumegueriella rufula (Ascomycota; Hypocreales) isolated from a soil sample collected in Taiwan is described as a new record from East Asia. Some observations are recorded, particularly on cultural characteristics of aGliocladium anamorph of this fungus, which has scarcely been reported since Bainier described both ascosporic and conidial forms as species ofGliocladium.     

Use of a surface plasmon resonance biosensor for the determination of binding constants of transiently expressed recombinant antibody to its antigen

Summary By using a commercially available surface plasmon resonance (SPR) biosensor, the values of the association rate constant (kass), dissociation rate constant (kdiss), and association constant (KA = kass / kdiss) for binding to the antigens were determined. They were almost the same for the recombinant antibody expressed in COS cells, CHO cells, and mouse hybridoma cells. The system of transient expression of the recombinant antibody (Ab) in COS cells and SPR analysis of the supernatant should be useful for rapid expression and evaluation of the binding ability of large numbers of engineered Abs.     

A sensitive method to screen for hydroxyl radical scavenging activity in natural food extracts using competitive inhibition ELISA for 8-hydroxy deoxyguanosine

Summary Representative endogenous antioxidants and natural food extracts were screened for hydroxyl radical scavenging activity by an ELISA. Whereas conventional assays for hydroxyl radical scavenging activity use either spin traps following the induction of Fenton reaction or measure thiobarbituric acid-reactive substances, this assay measures 8-hydroxy deoxyguanosine liberated from the hydroxylation of deoxyguanosine by Cu²⁺/ascorbate system.