Microtubule destruction induces tau liberation and its subsequent phosphorylation

Neurofibrillary tangle-bearing neurons, a pathological hallmark of Alzheimer’s disease, are mostly devoid of normal microtubule (MT) structure and instead have paired helical filaments that are composed of abnormal hyperphosphorylated tau. However, a causal relationship between tau phosphorylation and MT disruption has not been clarified. To examine whether MT disruption induces tau phosphorylation, stathmin, an MT-disrupting protein, was co-expressed with tau in COS-7 cells. Stathmin expression induced apparent MT catastrophe and tau hyperphosphorylation at Thr-181, Ser-202, Thr-205, and Thr-231 sites. In contrast, c-Jun N-terminal kinase activation, or phosphatase inhibition, led to significant tau phosphorylation without affecting MT structure. These findings suggest that MT disruption induces subsequent tau phosphorylation.     

Mitinokuidrilus excavatus n. g., n. sp., a Marine Tubificid (Oligochaeta) with a Unique Mode of Reproduction

Mitinokuidrilus excavatus n. g., n. sp. is described from shallow subtidal coarse sands in northern Japan (NW Pacific). The species is characterized by a unique mode of sexual reproduction. Each of the two types of mature worms represents the opposite sex: "male" worms have testes, seminal vesicles, male ducts and small spermathecae; "female" individuals possess clitellum, ovaries, ovisac, female ducts and fully developed spermathecae. No mature worms with intermediate sexual condition were collected. The taxonomic position and the possibility of consecutive hermaphroditism of the present species are discussed.     

Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

The crystal structure of the nitrile hydratase (NHase) from Bacillus smithii SC-J05-1 was determined. Our analysis of the structure shows that some residues that seem to be responsible for substrate recognition are different from those of other NHases. In particular, the Phe52 in the beta subunit of NHase from B. smithii covers the metal center partially like a small lid and narrows the active site cleft. It is well known that the NHase from B. smithii especially prefers aliphatic nitriles for its substrate rather than aromatic ones, and we can now infer that the Phe52 residue may play a key role in the substrate specificity for this enzyme. This finding leads us to suggest that substitution of these residues may alter the substrate specificity of the enzyme.     

Relationship between dopamine content and its secretion in PC12 cells as a function of cell growth

The PC12 cell line derived from a rat adrenal medullary tumor is known to synthesize dopamine and to release it in response to cholinergic agonists or depolarizing agents. In this report, we have studied the relationship between dopamine biosynthesis and its stimulus-induced secretion in PC12 cells as a function of cell growth. The endogenous dopamine content was found to depend on cell growth, and reached a maximum in the stationary phase. This increase was associated both with an increase in the specific activity of tyrosine 3-monooxygenase, and with an increase of DOPA-decarboxylase in the cells. On the other hand, the maximal release of dopamine occurred in the late exponential phase before the endogenous dopamine was maximally synthesized in the cells. Moreover, the uptake of 45Ca2+ stimulated with either carbamylcholine or high K+ was also regulated by cell division: the maximal uptake took place in the same period of culture in which the maximal release of dopamine was observed. Thus, this report offers new evidence that the biosynthesis and secretion of dopamine are separately regulated in PC12 cells.     

Neuroepithelial cells supply an initial transient wave of MSC differentiation

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1+ cells give rise to oligodendrocytes by expression of PDGFRbeta and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins.     

Studies on the physical states of human platelet myosin in crude extracts

The physical properties of human platelet myosin in crude extracts were studied by means of Sepharose 4B gel filtration and sucrose density gradient centrifugation in the presence or absence of Mg-ATP. Platelet myosin extracted with a buffer containing 0-0.15 M KCl gave a Stokes radius of about 12.0-12.5 nm irrespective of the presence or absence of Mg-ATP. The sedimentation coefficients obtained in the presence of Mg-ATP were about 10-11 and 8.5S at 0.05-0.10 and 0.15 M KCl, respectively, whereas the values obtained in the absence of Mg-ATP were about 16, 9-12, and 8.5S at 0.05, 0.10, and 0.15 M KCl, respectively. The apparent molecular weight in the presence of Mg-ATP, therefore, was about 500,000 and 420,000 at 0.05-0.10 and 0.15 M KCl, respectively, while the molecular weight in the absence of Mg-ATP was about 790,000, 460,000-620,000, and 440,000 at 0.05, 0.10, and 0.15 M KCl, respectively. The purified monomeric platelet myosin that had been solubilized with Mg-ATP at 0.10 M KCl had a Stokes radius of about 12.5 nm, a sedimentation coefficient of about 9S, and an apparent molecular weight of 460,000. On the other hand, while crude platelet myosin extracted at 0.6 M KCl with Mg-ATP gave a Stokes radius of about 20 nm, a sedimentation coefficient of about of 6S, and an apparent molecular weight of about 490,000, each of these physical parameters obtained in the absence of Mg-ATP was much larger than that obtained in the presence of Mg-ATP because the myosin was associated with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.     

Periaxin mutations cause recessive Dejerine-Sottas neuropathy

The periaxin gene (PRX) encodes two PDZ-domain proteins, L- and S-periaxin, that are required for maintenance of peripheral nerve myelin. Prx(-/-) mice develop a severe demyelinating peripheral neuropathy, despite apparently normal initial formation of myelin sheaths. We hypothesized that mutations in PRX could cause human peripheral myelinopathies. In accordance with this, we identified three unrelated Dejerine-Sottas neuropathy patients with recessive PRX mutations-two with compound heterozygous nonsense and frameshift mutations, and one with a homozygous frameshift mutation. We mapped PRX to 19q13.13-13.2, a region recently associated with a severe autosomal recessive demyelinating neuropathy in a Lebanese family (Delague et al. 2000) and syntenic to the location of Prx on murine chromosome 7 (Gillespie et al. 1997).     

Single-molecule PCR using water-in-oil emulsion

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.