A novel device to prevent osteoporosis by promoting bone metabolism using a newly developed double-loading stimulation with vibration and shaking.

In Japan, 13 million people have osteoporosis, including approximately 9 hundred thousand people who are bedridden owing to bone fractures from falls. Preventing osteoporosis is considered to be an important and effective way of preventing fall-related fractures. Thus, we developed a novel method of locomotor stimulation and analyzed its effectiveness in mice.

Effect of shaking and vibration stimulation on lumbar vertebrae in ovariectomized mice.

Objectives: Bone fractures affect the activities of daily living and lower quality of life, so investigating preventative measures is important. We developed novel stimulation equipment that combined a vibration stimulus with a shaking stimulus for preventing osteoporosis (one of the causes of bone fractures). We aimed to investigate the effect of this equipment on ovariectomized mice.

Effectiveness of exercise-induced cytokines in alleviating arthritis symptoms in arthritis model mice.

Recently, health awareness in Japan has been increasing and active exercise is now recommended to prevent lifestyle-related diseases. Cytokine activities have many positive effects in maintaining the health of a number of organs in the body. Myokines are cytokines secreted by skeletal muscles in response to exercise stimulation, and have recently generated much attention.

Groped for a novel stimulation method for the prevention of lumbar vertebral compression pressure bone fracture and verification using a bone density drop model mouse.

Osteoporosis is leaving bones more fragile and susceptible to fracture. It has a massive impact, both physically and mentally, markedly diminishing quality of life. A new form of therapeutic exercise or physical therapy that mitigates the abrupt decrease in bone density in postmenopausal women must quickly be developed to avoid those problems.

Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.

Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn(2+)-Phos-tag SDS-PAGE).

The neuronal connexin36 interacts with and is phosphorylated by CaMKII in a way similar to CaMKII interaction with glutamate receptors.

Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of (35)S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase.

Crystal structure of human mono-phosphorylated ERK1 at Tyr204.

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204.

Biochemical characterization of rab proteins from Bombyx mori.

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified.

Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE.

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. The phosphate-affinity site is a polyacrylamide-bound Phos-tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c-Abl, MET, and Fyn.

Neural complex-specific expression of xylosyl N-glycan in Ciona intestinalis.

We herein report N-glycosylation profiles of the individual tissues derived from the ascidian Ciona intestinalis. Multidimensional HPLC mapping revealed that the C. intestinalis expresses high-mannose-type oligosaccharides as major N-glycans, along with paucimannose-type and complex-type oligosaccharides, in a tissue-specific manner.

Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori.

The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro.

Structure of human Fyn kinase domain complexed with staurosporine.

The tyrosine kinase Fyn is a member of the Src kinase family. Besides the role of Fyn in T cell signal transduction in concert with Lck, its excess activity in the brain is involved with conditions such as Alzheimer's and Parkinson's diseases. Therefore, inhibition of Fyn kinase may help counteract these nervous system disorders.

The interaction between PSD-95 and Ca2+/calmodulin is enhanced by PDZ-binding proteins.

In this study, we evaluate the interaction between the postsynaptic scaffolding protein, PSD-95, and calmodulin. Surface plasmon resonance spectroscopy was used to characterize the binding of PSD-95 to calmodulin that had been immobilized on a sensor chip. Additionally, soluble calmodulin was found to inhibit the binding of PSD-95 to immobilized calmodulin.

Myristoyl moiety of HIV Nef is involved in regulation of the interaction with calmodulin in vivo.

Human immunodeficiency virus Nef is a myristoylated protein expressed early in infection by HIV. In addition to the well known down-regulation of the cell surface receptors CD4 and MHCI, Nef is able to alter T-cell signaling pathways. The ability to alter the cellular signaling pathways suggests that Nef can associate with signaling proteins.

Demonstration of a role for alpha-synuclein as a functional microtubule-associated protein.

Alpha-synuclein is a major constituent of pathological intracellular inclusion bodies, a common feature of several neurodegenerative diseases. Two missense mutations in the alpha-synuclein gene have been identified in confirmed autosomal-dominant familial Parkinson's disease, which segregate with the illness. However, the physiological function of alpha-synuclein remains unknown.

Crystal structure of a myristoylated CAP-23/NAP-22 N-terminal domain complexed with Ca2+/calmodulin.

A variety of viral and signal transduction proteins are known to be myristoylated. Although the role of myristoylation in protein-lipid interaction is well established, the involvement of myristoylation in protein-protein interactions is less well understood. CAP-23/NAP-22 is a brain-specific protein kinase C substrate protein that is involved in axon regeneration.

Direct involvement of protein myristoylation in myristoylated alanine-rich C kinase substrate (MARCKS)-calmodulin interaction.

MARCKS, a major in vivo substrate of protein kinase C, interacts with plasma membranes in a phosphorylation-, myristoylation-, and calmodulin-dependent manner. Although we have previously observed that myristoylated and non-myristoylated MARCKS proteins behave differently during calmodulin-agarose chromatography, the role of protein myristoylation in the MARCKS-calmodulin interaction remained to be elucidated. Here we demonstrate that the myristoyl moiety together with the N-terminal protein domain is directly involved in the MARCKS-calmodulin interaction.

Regulation of endothelial nitric oxide synthase by protein kinase C.

Endothelial nitric oxide synthase (eNOS) is a key enzyme in nitric oxide-mediated signal transduction in mammalian cells. Its catalytic activity is regulated both by regulatory proteins, such as calmodulin and caveolin, and by a variety of post-translational modifications including phosphorylation and acylation. We have previously shown that the calmodulin-binding domain peptide is a good substrate for protein kinase C [Matsubara, M.

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin.

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction.

Nef of HIV-1 interacts directly with calcium-bound calmodulin.

It was recently found that the myristoyl group of CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is essential for the interaction between the protein and Ca(2+)-bound calmodulin (Ca(2+)/CaM). Based on the N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein-calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca(2+)/CaM in the presence of a myristoylated peptide corresponding to the N-terminal region of Nef.