Yuuki Kawamura

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early‐onset familial Alzheimers disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β‐protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro‐blastoma (Neuro 2a) cell lines expressing the human wild‐type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N‐terminally truncated variant(s) Aβx‐42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx‐42, whereas extracellular levels of Aβ1–42 and Aβx‐42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1‐induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx‐42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx‐42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post‐Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42‐specific γ‐secretase cleavage that occurs in the normal β‐amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β‐secretase cleavage or (b) in the distinct sites where Aβx‐42 and Aβ1–42 are generated.

Connectin, giant elastic protein, in giant sarcomeres of crayfish claw muscle

SummaryIn the giant sarcomeres (sarcomere length, 10 μm at rest) of crayfish claw muscle, 3000 kDa connectin-like protein but not projectin (mini-titin) appears to be responsible for passive tension generation. Proteolysis of crayfish connectin in skinned fibres was parallel with disappearance of resting tension. Immunofluorescence observations using the antiserum to crayfish connectin showed that crayfish connectin linked the A band to the Z line ina giant sarcomere. It appears that crayfish connectin exerts a centering force on the A band in a sarcomere. Very thin filaments in the I band were visualized after the actin filaments had been removed by the treatment with plasma gelsolin. Crayfish connectin was partially purified and its rotary shadowed image was a very long filament. Projectin was localized on the A band of crayfish giant sarcomeres and remained unmoved during stretch or contraction. However, on dissolution of myosin filaments, projectin moved to the Z line together with crayfish connectin. It seems that projectin binds to connectin on the myosin filament. In regular size of sarcomeres (sarcomere lengths, 3–4 μm at rest) of crayfish stretcher muscle, projectin linked the A band to the Z line, as in insect flight muscle.

Characterization of connectin-like proteins of obliquely striated muscle of a polychaete (Annelida)

SummaryIn obliquely striated muscle of polychaete, Neanthes sp., three kinds of connectin (titin)-like high molecular weight proteins, ∼4000 kDa, ∼1200 kDa and ∼700 kDa, were detected by SDS gel electrophoresis and immunoblots using antibodies to vertebrate skeletal muscle connectin and antiserum to the protein in question. The 700 kDa protein was isolated and characterized as a β sheet-rich filament 170 nm long and 4 nm wide. Using polyclonal antibodies to the 700 kDa protein, the binding of the immunogold to the thick filament was only demonstrated in high ionic strength relaxing solution which solubilized some myosin. This observation suggested that the 700 kDa protein was localized below the layers of myosin in the thick filament and this localization is different from that of twitchin of C. elegans bodywall muscle that is on the surface of thick filament. The 4000 kDa protein was identified as a very thin filament linking the thick filament to the dense body. The very thin filaments were visualized in gelsolin-treated actin filament-free fibres. The 1200 kDa protein was located in the periphery of the dense body. A model of the elastic filament in polychaete bodywall muscle is presented.

Biodiversity of the localization of the epitopes to connectin antibodies in the sarcomeres of lamprey, electric ray, and horse mackerel skeletal muscles

SDS gel electrophoresis showed the presence of connectin, approximately 3000 kDa, in skeletal muscles of fishes, lamprey, electric ray and horse mackerel. The antibodies to avian or mammalian skeletal muscle connectin, 3B9 and Pc1200, reacted with all the fish connectins. However, a monoclonal antibody SM1 recognized electric ray connectin but did not react with lamprey and horse mackerel connectins. Immunoelectron microscopy revealed that the epitope to Pc1200 was localized at the periphery of the Z line of all the fish muscle sarcomeres and that to SM1 was located at the N2 line in the I band of ray muscle. These two localizations are the same as those in rabbit, chicken and frog skeletal muscles. On the other hand, the positions of epitopes to 3B9 were variable in the three classes of fishes, although all of them were localized in the A band. Some of the epitope positions were common to those in chicken skeletal muscle. Thus the present work demonstrates biodiversity of connectin in fish skeletal muscles, distinct from avian and mammalian skeletal muscles.

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of α-actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with α-actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.

Chicken leg muscle α-connectin as studied by a monoclonal antibody to the1200 kDa fragment

Abstract Chicken leg gracills muscle contained only α-connectin ( ca 3000 kDa) without β-connectin. When myofibrils were kept standing for 20 hr at 4°C, α-connectin was degraded to β-connectin ( ca 2000 kDa) and 1200 kDa peptide. The latter was prepared from myofibrils and purified by gel filtration in the presence of SDS. A monoclonal antibody, α7, to this 1200 kDa fragment was prepared. The antibody reacted with the 1200 kDa fragment and its mother molecule α-connectin, but not with β-connectin. Immunoelectron microscopy using α7, as well as other antibodies to chicken breast muscle β-connectin, revealed that the 1200 kDa peptide covered the portion of α-connectin from the Z line to the N2 line region in the I band of chicken leg gracilis muscle sarcomeres. The results were in good agreement with those observed in rabbit skeletal muscle.

216 Production and characterization of mice devoid of the glutamic acid decarboxylase (67 kDa isoform-GAD67)

Hideo Asada’, Yuuki Kawamura’ , Kei Maruyama I, Hideaki Kumel , Nobuko Kanbara’ , Hiroko Kuzumei , Takeshi Yagi2, Kunihiko Obata’ In additionto its role as an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is presumed to be involved in development and plasticity of the nernous system. Glutamic acid decarboxylase (GAD) catalyzes the formation of GABA from glutamic acid. There are two isoforms, GAD65 and GAD67, which are encoded by two different genes. Recently we produced GAD65 null mice (-/-) and demonstrated that lack of GAD65 does not change brain GAD activity or GABA contents. Here we report the production of GAD67 null mice (-/-). GAD activities and GABA contents in the brain of the GAD67 -/mice were greatly reduced. Furthermore GAD67 -/mice died of severe cleft palate within 24 hrs. after birth, even though they were born at the expected Mendelian frequency. This indicate a role of GAD67-derived GABA in the development of non-neuronal tissue.