Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies

Atypical forms of bovine spongiform encephalopathy (BSE) may be caused by different prions from classical BSE (C-BSE). In this study, we examined the susceptibility of mice overexpressing mouse and hamster chimeric prion protein (PrP) to L-type atypical BSE (L-BSE). None of the transgenic mice showed susceptibility to L-BSE, except mice overexpressing hamster PrP. We also examined the transmission properties of L-BSE in hamsters. The incubation period of hamsters intracerebrally inoculated with L-BSE was 576.8 days, and that of the subsequent passage was decreased to 208 days. Although the lesion and glycoform profiles and relative proteinase K resistant core fragment of the abnormal isoform of PrP (PrPcore) of L-BSE were similar to that of C-BSE, the deposition of the abnormal isoform of PrP (PrP^Sc) and the molecular weight of PrPcore of L-BSE was different from than that of C-BSE. In hamster models, some prion strain characteristics of L-BSE were indistinguishable from those of C-BSE.Key words: prion, atypical, L-BSE, PrPcore, hamster, transmission     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts, spheroplast membrane vesicles and chromatophores.

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K+ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures ("chromatophores") were obtained by treating spheroplast membrane vesicles by French press or sonication. The membrane structures with either sidedness showed the same light-induced change of the "red shift" type. However, the absorbance change by K+ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, "blue shift" in the former and "red shift" in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-gamma-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects of MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N5-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N5-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

Synthesis and antinematodal activity of 3-n-alkylphenols

Several 3-alkylphenols including 3-undecylphenol, which was isolated from a Sumatran rainforest plant, were synthesized to investigate their antinematodal activity against the phytopathogenic nematodes, Bursapherencus xylophilus. A three-step synthesis involving the treatment of 2-cyclohexen-1-one with the Grignard reagent, oxidation of the resulting 1-alkyl-2-cyclohexen-1-ol and subsequent aromatization of 3-alkyl-2-cyclohexen-1-one successfully afforded such phenols. Among the 3-alkylphenols, 3-nonylphenol showed the highest activity, while 3-decylphenol and 3-undecylphenol also showed high activity.     

Low-intensity pulsed ultrasound (LIPUS) induces RANKL, MCP-1, and MIP-1beta expression in osteoblasts through the angiotensin II type 1 receptor

Constant mechanical stress is essential for the maintenance of bone mass and strength, which is achieved through the cooperative functions of osteoblasts and osteoclasts. However, it has not been fully elucidated how these cell types mediate mechanical signals. Low-intensity pulsed ultrasound (LIPUS) therapy is a recently developed method for application of mechanical stress, and is used clinically to promote bone fracture healing. In the present study, we applied LIPUS to osteoblasts at different stages of maturation and analyzed their chemokine and cytokine expression. In comparison with their immature counterparts, mature osteoblasts expressed significantly higher levels of mRNAs for the receptor activator of nuclear factor kappa B ligand (RANKL), monocyte chemoattractant protein (MCP)-1, and macrophage-inflammatory protein (MIP)-1beta after a few hours of LIPUS treatment. Intriguingly, protein and mRNA expression of angiotensin II type 1 receptor (AT1), a known mechanoreceptor in cardiomyocytes, was detected in osteoblasts, and the level of expression increased significantly during cell maturation. Furthermore, LIPUS-induced extracellular signal-regulated kinase (ERK) phosphorylation and RANKL/chemokine expression was abrogated by a specific AT1 inhibitor. Thus, AT1 may play one of the essential roles in bone metabolism as a mechanoreceptor of osteoblasts.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

Augmentation of antitumor resistance by a strain of unicellular green algae,Chlorella vulgaris

Summary Growth of Meth-A tumor in CDF1 mice was inhibited significantly by injection of a hot water extract of a strain of Chlorella vulgaris (CE) into the tumor or into the subcutaneous tissue near the tumor. The augmentation of resistance by CE may require the participation of T cells and macrophages, since it was abolished or reduced in athymic nude mice or mice treated with carrageenan, a macrophage blocker. Mice treated with CE exhibited antigen-specific augmented resistance against rechallenge with tumor. Moreover, the antitumor effect of CE was comparable with that of Corynebacterium parvum, but its mechanism of effect might be different.     

Underwater observations of the rare deep-sea fish <Emphasis Type="Italic">Triodon macropterus</Emphasis> (Actinopterygii,Tetraodontiformes, Triodontidae), with comments on the fine structure of the scales

Underwater observations of two living individuals and an examination of a freshly dead individual of the rare deep-sea fish Triodon macropterus revealed that they usually have the large ventral flap completely uplifted and seamlessly retracted into the abdominal region of the trunk: one individual was collected at a depth of 280 m by hook and line east of Tonaki-jima Island, Ryukyu Islands, and kept in a tank at the Okinawa Churaumi Aquarium for six years; the second living individual was observed by the ROV Hakuyo 2000 at a depth of 275 m east of Ishigaki-jima Island, Ryukyu Islands; and another individual collected at depths of 250?350 m by hook and line off Funchu Point, Yoron-jima Island (27°00.15’N, 128°26.06’E), Ryukyu Islands, provided an opportunity to demonstrate that the ventral flap could be manually uplifted by rotating the pelvis. When the ventral flap was uplifted in all of these specimens, whether naturally or manually, there were no scaleless linear bands or streaks and gaps evident in the skin between the retracted ventral flap and the abdominal region. The fine structure of the scales of the body and the ventral flap was observed by light microscopy, scanning electron microscopy, and X-ray CT scanning. The scales of the ventral flap are arranged in rows radiating from the antero-dorsal part of the ventral flap where the anterior part of the pelvis articulates with and rotates around the pectoral girdles. The orientation of the rows on the ventral flap changes from a posterior direction dorsally to a posteroventral direction more ventrally. A ridge with many sharp serrations is raised and bent from the edge of each scale on the body and ventral flap. The raised ridges on the lower scales on the ventral flap become packed together with the upper scales to form a seamless surface when the ventral flap is uplifted into the abdominal region.     

Cystatin 10, a novel chondrocyte-specific protein,may promote the last steps of the chondrocyte differentiation pathway

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.