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1.
Neurology ; 59(2): 210-4, 2002 Jul 23.
Article in English | MEDLINE | ID: mdl-12136059

ABSTRACT

OBJECTIVE: To assess alterations in brain metabolites in patients with late-onset ornithine transcarbamylase deficiency (OTCD). METHODS: Six unrelated, asymptomatic Japanese late-onset OTCD patients were analyzed by proton MRS ((1)HMRS) using a point-resolved spectroscopy technique (repetition and echo times, 5000 and 30 ms). Localized spectra for the centrum semiovale were acquired and absolute metabolite concentrations were calculated using an LCModel. RESULTS: Compared with age-matched controls, N-acetylaspartate and creatine concentrations were normal in all patients. The glutamine (Gln) plus glutamate concentration was increased in four patients, which progressed in proportion to the clinical stage. myo-inositol (mI) could not be detected in five symptomatic patients. A decreased choline (Cho) concentration was detected in two clinically severe patients. (1)HMRS after liver transplantation in one patient revealed the normalization of all metabolites. CONCLUSION: These findings suggest progression of neurochemical events in OTCD, i.e., mI depletion and Gln accumulation followed by Cho depletion, which is reverse of that in hepatic encephalopathy, i.e., Cho depletion followed by mI depletion and Gln accumulation.


Subject(s)
Brain/metabolism , Liver Transplantation , Ornithine Carbamoyltransferase Deficiency Disease/metabolism , Adolescent , Adult , Age of Onset , Brain/enzymology , Case-Control Studies , Child , Child, Preschool , Choline/metabolism , Female , Glutamine/metabolism , Humans , Inositol/metabolism , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy/methods , Male , Ornithine Carbamoyltransferase Deficiency Disease/enzymology , Ornithine Carbamoyltransferase Deficiency Disease/surgery , Protons
2.
Neurology ; 58(2): 237-41, 2002 Jan 22.
Article in English | MEDLINE | ID: mdl-11805250

ABSTRACT

OBJECTIVE: To assess alterations in brain metabolites of patients with Pelizaeus-Merzbacher disease (PMD) with the proteolipid protein gene 1 (PLP1) duplications using quantitative proton MRS. METHODS: Five unrelated male Japanese patients with PMD with PLP1 duplications were analyzed using automated proton brain examination with the point resolved spectroscopy technique (repetition and echo time of 5,000 and 30 msec). Localized spectra in the posterior portion of the centrum semiovale were acquired, and absolute metabolite concentrations were calculated using the LCModel. RESULTS: Absolute concentrations of N-acetylaspartate (NAA), creatine (Cr), and myoinositol (MI) were increased by 16% (p < 0.01), 43% (p < 0.001), and 31% (p < 0.01) in patients with PMD as compared with age-matched controls. There was no statistical difference in choline concentration. CONCLUSION: The increased concentration of NAA, which could not be detected by previous relative quantitation methods, suggests two possibilities: axonal involvement secondary to dysmyelination, or increased cell population of oligodendrocyte progenitors. Elevated Cr and MI concentrations may reflect the reactive astrocytic gliosis. Our study thus emphasizes the importance of absolute quantitation of metabolites to investigate the disease mechanism of the dysmyelinating disorders of the CNS.


Subject(s)
Aspartic Acid/analogs & derivatives , Aspartic Acid/metabolism , Brain/metabolism , Myelin Proteolipid Protein/genetics , Pelizaeus-Merzbacher Disease/genetics , Pelizaeus-Merzbacher Disease/metabolism , Brain/pathology , Humans , Japan , Magnetic Resonance Spectroscopy/methods , Male
3.
J Cereb Blood Flow Metab ; 21(11): 1295-302, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11702044

ABSTRACT

SUMMARY: Slowly progressive degeneration of the hippocampal CA1 neurons was induced by 3-minute transient global ischemia in gerbils. Sustained degeneration of hippocampal CA1 neurons was evident 1 month after ischemia. To investigate the effects of an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain (18MP) on this sustained neuronal degeneration, an intracerebroventricular 18MP infusion was initiated 3 days after ischemia. Histopathologic and behavior evaluations were conducted 1 week and 1 month after induction of ischemia. When compared with the vehicle infusion, 18MP treatment significantly increased the response latency time in a passive avoidance task. Increased neuronal density was also evident, as was the number of intact synapses in the hippocampal CA1 region at 1 week and 1 month after ischemia. 18MP treatment also significantly decreased the number of TUNEL-positive CA1 neurons 1 week after ischemia. Subsequent in vitro experiments using cultured neurons demonstrated that the 18MP at optimal extracellular concentrations of 1 to 100 fg/mL prevented nitric oxide-induced neuronal damage as expected and significantly up-regulated the expressions of bcl-x(L) mRNA and its translated protein. These results suggest that the gerbil model of 3-minute ischemia is useful in studying the pathogenesis of slowly progressive neuronal degeneration after stroke and in evaluating effects of novel therapeutic agents. It is likely that the 18MP at low extracellular concentrations prevents neuronal apoptosis possibly through up-regulation of the mitochondrial antiapoptotic factor Bcl-x(L).


Subject(s)
Glycoproteins/pharmacology , Ischemic Attack, Transient/drug therapy , Nerve Degeneration/drug therapy , Peptide Fragments/pharmacology , Amino Acid Sequence , Animals , Cell Count , Cells, Cultured , Cerebral Cortex/cytology , Gene Expression/drug effects , Gerbillinae , Hippocampus/pathology , In Situ Nick-End Labeling , Male , Molecular Sequence Data , Neurons/cytology , Neuroprotective Agents/pharmacology , Nitric Oxide Donors/pharmacology , Nitroprusside/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/analysis , Saposins , Synapses/physiology , bcl-X Protein
4.
Radiology ; 216(2): 576-81, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10924588

ABSTRACT

PURPOSE: To determine the prevalence of abnormalities of the acetabular labrum in asymptomatic hips by means of magnetic resonance (MR) imaging and to correlate such abnormalities with age and the portion of the labrum. MATERIALS AND METHODS: MR imaging was performed in 71 asymptomatic hips that were radially sectioned perpendicular to the acetabular labrum at 30 degrees intervals. RESULTS: The shape of the labrum was triangular in 80% (304 of 382) of the labral segments, round in 13% (49 of 382), irregular in 7% (27 of 382), and not identified in 1% (two of 382). A homogeneous low signal intensity was observed in 56% (212 of 382). The frequencies of labral irregularity or its absence and of high signal intensity increased both with subject age and with a more anterior anatomic labral location. CONCLUSION: In asymptomatic hips, abnormal findings regarding the shape and signal intensity of the acetabular labrum can be detected by means of MR imaging. The fact that the findings vary according to age and labral portion should be considered in interpreting MR images in patients suspected of having a labral lesion.


Subject(s)
Acetabulum/pathology , Hip Joint/pathology , Magnetic Resonance Imaging , Adolescent , Adult , Age Factors , Aged , Bone Diseases/diagnosis , Cartilage, Articular/pathology , Chi-Square Distribution , Female , Humans , Image Processing, Computer-Assisted/methods , Magnetic Resonance Imaging/methods , Male , Middle Aged , Sex Factors , Single-Blind Method
5.
Hum Immunol ; 60(6): 539-49, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10408804

ABSTRACT

We have established a precise, rapid, simple and economical subtyping method for alleles encoding the HLA-A2 and -B40 antigens using microtiter plate-reverse hybridization assay (MRHA), which is based on the general principle of HLA oligotyping by reverse dot blot hybridization. Amino-modified sequence-specific oligonucleotide (SSO) probes were immobilized covalently onto a carboxylate-modified microtiter plate. In order to perform high-resolution subtyping of the HLA-A2 and -B40 antigen groups, the alpha1 and alpha2 domain regions were amplified using a pair of group-specific primers composed of an unlabeled sense primer and a biotinylated antisense primer. PCR-amplified products were hybridized with SSO probes in hybridization buffer containing formamide for 1 hour at 37 degrees C. After washing with 2 X SSC at room temperature, the bound PCR products were detected by alkaline phosphatase-conjugated streptavidine followed by color development. All of 8 HLA-B40 suballeles, all of 2 HLA-B47 suballeles (B40 group-specific primers used in this study allowed also B47 amplification) and 17 out of 21 HLA-A2 suballeles were discriminated. The remaining four HLA-A2 suballeles were determined by analysis after exon 4 amplification. HLA-DNA typing by this method was easily and exactly performed regardless of sample number. The greatest advantages of this technique are strong positive signals obtained, reproducibility and the ease of thermoregulation for hybridization and washing as compared to previously reported microtiter plate hybridization methods.


Subject(s)
Genes, MHC Class I , HLA-A2 Antigen/classification , HLA-B Antigens/classification , Nucleic Acid Hybridization/methods , DNA Probes , HLA-A2 Antigen/genetics , HLA-B Antigens/genetics , HLA-B40 Antigen , Humans , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Time Factors
6.
J Biochem ; 125(2): 334-42, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9990131

ABSTRACT

Smooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogeneously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.


Subject(s)
Muscle, Smooth, Vascular/metabolism , Myosin Light Chains/metabolism , Protein Isoforms/metabolism , Animals , Antibody Specificity , Aorta/metabolism , Cells, Cultured , Fluorescent Antibody Technique , In Vitro Techniques , Molecular Weight , Muscle, Smooth, Vascular/cytology , Rabbits , Rats , Subcellular Fractions/metabolism , Tissue Distribution
7.
J Biochem ; 124(3): 609-14, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9722673

ABSTRACT

To detect structural changes around the reactive Cys707 (SH1) in the myosin heavy chain during the ATPase reaction, the reactivity of SH1 in rabbit skeletal myosin subfragment-1 (S-1) was measured using a fluorescent reagent, 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, in the presence of various ATP analogs: adenosine 5'-(3-thiotriphosphate) (ATPgammaS), ADP-vanadate (ADP-Vi), ADP-BeFx, and ADP-AlF4. The SH1 reactivities in the S-1 complexes with ATPgammaS and ADP-BeFx, analogs of the E-ATP state, were very high, as well as that in the E-ADP state. In contrast, the SH1 reactivities in the S-1 complexes with ADP-Vi and ADP-AlF4, analogs of the E-ADP-P state, were extremely low. The structural changes around SH1 can be correlated to changes in the structure of the gamma-phosphate of ATP during the ATPase reaction or to the structure of the corresponding part of ATP analogs at the active site of ATPase. This is consistent with the crystal structure of S-1 in which the heavy chain structure around SH1 of S-1-ADP-BeFx is significantly different from those of S-1-ADP-Vi and S-1-ADP-AlF4 [Fisher et al. (1995) Biochemistry 34, 8960-8972; Smith and Rayment (1996) Biochemistry 35, 5404-5417].


Subject(s)
Cysteine/metabolism , Myosins/metabolism , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Fluorescent Dyes , Myosins/chemistry , Naphthalenesulfonates , Protein Conformation , Rabbits
8.
Neurosci Res ; 30(4): 361-4, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9678641

ABSTRACT

An attempt was made to apply functional magnetic resonance imaging (fMRI) to reveal cortical areas activated upon presentation of two groups of Chinese characters in six normal right-handed, male, Japanese subjects. Presentation of the characters representing 'abstract concepts' activated the bilateral occipital region without a significant difference between the bilateral occipital and temporal regions. Presentation of the characters representing 'concrete objects' resulted in significantly stronger activation in the left occipital and temporal regions. These results suggest that recognition of concrete characters involves a stronger initial process in the left occipital temporal cortices than recognition of abstract characters.


Subject(s)
Cognition/physiology , Evoked Potentials, Visual/physiology , Magnetic Resonance Imaging , Pattern Recognition, Visual/physiology , Reading , Adult , Functional Laterality/physiology , Humans , Male , Visual Perception/physiology
9.
J Biochem ; 124(1): 163-70, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9644259

ABSTRACT

Single-headed myosin was prepared by digestion of porcine aorta smooth muscle myosin with Staphylococcus aureus V8 protease in the presence of actin. The single-headed myosin preparation contained intact light chains, a rod fragment of a heavy chain, and a heavy chain of which only a minor fraction contained a nick in the head segment. Below 0.2 M NaCl, the single-headed myosin showed a decrease in Ca2+-ATPase activity and an increase in the elution time on gel filtration HPLC in a phosphorylation-dependent manner, indicating a phosphorylation-dependent conformational transition between the extended and folded forms. These conformations were confirmed by electron microscopic observation of rotary-shadowed samples of single-headed myosin. However, the conformational transition of single-headed myosin occurred in a narrower range with lower salt concentrations than that of double-headed myosin. The filament assembly of single-headed myosin was thus facilitated and phosphorylation-independent. The single-headed myosin also showed high actin-activated ATPase activity independent of phosphorylation. These results indicate that the two-headed structure of smooth muscle myosin is not essential for the conformational transition, but is required for the phosphorylation-dependent regulation of enzymatic activity and filament assembly.


Subject(s)
Muscle, Smooth, Vascular/chemistry , Myosins/chemistry , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Microscopy, Electron , Molecular Sequence Data , Myosins/metabolism , Phosphorylation , Protein Conformation , Swine
10.
J Physiol ; 508 ( Pt 3): 871-81, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9518739

ABSTRACT

1. CPI-17 has recently been identified as a novel protein in vascular smooth muscle. In vitro , its phosphorylation and thiophosphorylation by protein kinase C (PKC) specifically inhibits the type 1 class of protein phosphatases, including myosin light chain (MLC) phosphatase. 2. Both of the phosphorylated CPI-17 states dose-dependently potentiated submaximal contractions at constant [Ca2+] in beta-escin-permeabilized and Triton X-100-demembranated arterial smooth muscle, but produced no effect in intact and less intensely permeabilized (alpha-toxin) tissue. Thiophosphorylated CPI-17 (tp-CPI) induced large contractions even under Ca2+-free conditions and decreased Ca2+ EC50 by more than an order of magnitude. Unphosphorylated CPI-17 produced minimal but significant effects. 3. tp-CPI substantially increased the steady-state MLC phosphorylation to Ca2+ ratios in beta-escin preparations. 4. tp-CPI affected the kinetics of contraction and relaxation and of MLC phosphorylation and dephosphorylation in such a manner that indicates its major physiological effect is to inhibit MLC phosphatase. 5. Results from use of specific inhibitors in concurrence with tp-CPI repudiate the involvement of general G proteins, rho A or PKC itself in the Ca2+ sensitization by tp-CPI. 6. Our results indicate that phosphorylation of CPI-17 by PKC stimulates binding of CPI-17 to and subsequent inhibition of MLC phosphatase. This implies that CPI-17 accounts largely for the heretofore unknown signalling pathway between PKC and inhibited MLC phosphatase.


Subject(s)
Enzyme Inhibitors/metabolism , Muscle, Smooth, Vascular/enzymology , Phosphoprotein Phosphatases/metabolism , Protein Kinase C/metabolism , Proteins/metabolism , Signal Transduction/physiology , Animals , Aorta/cytology , Calcium/pharmacology , Dose-Response Relationship, Drug , Femoral Artery/cytology , Guanosine Diphosphate/analogs & derivatives , Guanosine Diphosphate/pharmacology , Male , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Smooth, Vascular/chemistry , Myosin-Light-Chain Phosphatase , Peptide Fragments/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/pharmacology , Protein Phosphatase 2 , Rabbits , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Sulfur Compounds/metabolism , Swine , Thionucleotides/pharmacology , Vasoconstriction/drug effects , Vasoconstriction/physiology
11.
Neurosci Res ; 30(1): 83-6, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9572582

ABSTRACT

An attempt was made to apply functional magnetic resonance imaging (fMRI) to reveal cortical areas activated upon presentation of two groups of Chinese characters in six normal right-handed, male, Japanese subjects. Presentation of the characters representing 'abstract concepts' activated the bilateral occipital region without a significant difference between the bilateral occipital and temporal regions. Presentation of the characters representing 'concrete objects' resulted in significantly stronger activation in the left occipital and temporal regions. These results suggest that recognition of concrete characters involves a stronger initial process in the left occipital temporal cortices than recognition of abstract characters.


Subject(s)
Brain Mapping , Cerebral Cortex/physiology , Language , Pattern Recognition, Visual , Writing , Adult , China , Functional Laterality , Humans , Japan , Magnetic Resonance Imaging/methods , Male
12.
FEBS Lett ; 410(2-3): 356-60, 1997 Jun 30.
Article in English | MEDLINE | ID: mdl-9237662

ABSTRACT

The cDNA encoding a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147-residue polypeptide, which we termed CPI17, a 17-kDa PKC-potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC50 = 0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non-muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.


Subject(s)
Enzyme Inhibitors/analysis , Muscle Proteins/analysis , Muscle, Smooth, Vascular/chemistry , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoproteins , Amino Acid Sequence , Animals , Aorta/chemistry , Base Sequence , Cloning, Molecular , DNA, Complementary , Molecular Sequence Data , Muscle Proteins/genetics , Phosphorylation , Protein Phosphatase 1 , RNA, Messenger , Sequence Homology, Amino Acid , Swine
13.
J Biochem ; 121(2): 225-30, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9089394

ABSTRACT

Telokin, which is a candidate for one of the factors stabilizing dephosphorylated myosin filaments in smooth muscle cells, was subjected to a chemical crosslinking study to determine its binding location on the myosin molecule. Telokin labeled with 5-iodoacetamidofluorescein (Fl-telokin) retained the nature of unlabeled telokin: it bound to dephosphorylated gizzard myosin with a stoichiometry of 1 mol/mol myosin and induced filament assembly. The carboxyl groups of Fl-telokin were activated with 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide and N-hydroxysuccinimide and then crosslinked to myosin. The production of three fluorescent peptides was observed on an SDS-gel, accompanying a decrease in the amount of regulatory light chain (LC20). The molecular weights of these products estimated to be > 200, 62, and 41 kDa. When unlabeled telokin was crosslinked to myosin of which LC20 was exchanged with 5-[[2-[(iodoacetyl)amino]ethyl]amino]-naphthalene-1-sulfonic acid-labeled LC20, the 62- and 41-kDa bands were also fluorescent. These results suggest that the > 200, 62, and 41-kDa species are telokin crosslinked with a heavy chain, with 2 LC20, and with 1 LC20, respectively. Myosin crosslinked with unlabeled telokin showed an extra structure with a small projection at the head-rod junction on electron microscopy and this structure was proved to be telokin by decorating it with anti-telokin antibodies. In addition, dephosphorylated myosin crosslinked with Fl-telokin was incapable of folding into the 10S conformation at 0.2 NaCl in the presence of MgATP, and assembled into filaments at 0.15 M NaCl in the presence of MgATP. Thus, telokin may bind to LC20 and a heavy chain region at the head-rod junction and suppress folding into the 10S conformation, leading to the assembly of myosin into filaments.


Subject(s)
Muscle Proteins/metabolism , Muscle, Smooth/metabolism , Myosin-Light-Chain Kinase/metabolism , Myosins/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Carbodiimides/metabolism , Chickens , Chromatography, High Pressure Liquid , Cross-Linking Reagents/metabolism , Fluoresceins , Fluorescent Dyes , Gizzard, Avian/metabolism , Microscopy, Electron , Muscle Proteins/ultrastructure , Peptide Fragments , Peptides , Protein Conformation , Protein Folding , Succinimides/metabolism
14.
J Biochem ; 121(1): 56-62, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9058192

ABSTRACT

Porcine aorta smooth muscle myosin contains two essential light chain (LC17) isoforms and the light chain was replaced with one of the LC17 isoforms, rabbit skeletal muscle myosin alkali light chain 2 (A2), or scallop striated muscle myosin essential light chain (SHLC). The myosin containing either an LC17 isoform or A2 showed phosphorylation-dependent properties in the monomer conformation, filament formation, ATPase activities, and superprecipitation, behaving in essentially the same way as native myosin. On the other hand, the replacement of LC17 with SHLC destabilized the 10S conformation and the myosin was predominantly filamentous under physiological conditions, irrespective of the phosphorylation state. This myosin containing dephosphorylated regulatory light chain showed higher actin-activated ATPase activity than native dephosphorylated myosin and was further activated by Ca2+, resulting in a decrease of phosphorylation-dependent regulation. Superprecipitation for the myosin was observed only when the regulatory light chain was phosphorylated. Superprecipitation for myosin containing SHLC was significantly slow in the absence of Ca2+ in comparison with that for myosin containing LC17, and was further activated by the presence of Ca2+. On the basis of the differences in amino acid sequences of these essential light chains, it appears that the N-terminal domain of LC17 may be implicated in these phosphorylation-dependent properties of smooth muscle myosin.


Subject(s)
Muscle, Smooth, Vascular/chemistry , Myosins/chemistry , Myosins/metabolism , Actin Cytoskeleton/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chemical Precipitation , Isoenzymes , Molecular Sequence Data , Muscle, Skeletal/chemistry , Myosin Light Chains/chemistry , Myosin Light Chains/metabolism , Phosphorylation , Protein Conformation , Protein Folding , Rabbits , Sequence Homology, Amino Acid , Swine
16.
J Biochem ; 120(3): 580-6, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8902624

ABSTRACT

A 2-kDa peptide (2K peptide) which was derived from the neck region of porcine aorta smooth muscle myosin heavy chain binds to actin competitively with skeletal myosin subfragment 1 (S1) in the absence of ATP and inhibits acto-S1 ATPase activity [Katoh, T. and Morita, F. (1993) J. Biol. Chem. 268, 2380-2388]. Using this and other peptides, myosin-binding sites on actin were mapped and their functions were studied. The 2K peptide inhibited the acto-S1 ATPase activity without inhibiting the binding of S1 to actin in the presence of ATP. On the other hand, the dansylated 2K peptide (DNS-2K peptide) inhibited not only the acto-S1 ATPase activity but also the binding of S1 to actin in the presence of ATP. Then, DNS-2K peptide was crosslinked to actin with 1-ethyl-3[3-(dimethylamino)propyl] carbodiimide. Amino acid composition and sequencing analyses of the fluorescent lysylendopeptidase-peptides of the crosslinked product indicated that DNS-2K peptide was crosslinked to acidic residues within residues 1-18 (Asp1, Glu2, Asp3, Glu4, and/or Asp11), 19-50 (Asp25), and 85-113 (Glu99 or Glu100) of actin. A competition experiment for the crosslinking with unlabeled 2K peptide showed that the crosslinking to residues 85-113 of actin was specific for DNS-2K peptide. In addition, isolated actin peptide 85-113 was found to show the competitive inhibition of actin-activated ATPase activity of S1 with respect to actin. These results suggest that the site within residues 1-28 of actin participates in the actin-activation of myosin ATPase activity, and the site within residues 85-113 of actin participates in the weak binding of myosin to actin in the presence of ATP.


Subject(s)
Actins/chemistry , Actins/metabolism , Actomyosin/chemistry , Actomyosin/metabolism , Myosins/antagonists & inhibitors , Myosins/metabolism , Actomyosin/antagonists & inhibitors , Amino Acid Sequence , Animals , Cross-Linking Reagents , Dansyl Compounds , Kinetics , Molecular Sequence Data , Muscle, Skeletal/metabolism , Myosins/chemistry , Peptide Fragments/chemistry , Rabbits
17.
No Shinkei Geka ; 24(8): 765-9, 1996 Aug.
Article in Japanese | MEDLINE | ID: mdl-8741414

ABSTRACT

This is the first report of rhinorrhea detected by special modulation of the magnetization (SPAMM)-tagging method. A 54-year-old male was admitted to our hospital for treatment of right rhinorrhea which had continued for 2 months and a half. The CSF leakage was induced by specific head position and the volume was more than 50 ml a day. Metrizamide CT cisternography revealed the contrast medium in the lateral extension of the right sphenoid sinus. MRI demonstrated liquorrhea with abnormal intensity of the sinus. 3D-CT revealed bony defects at the temporal base. Operation revealed herniated brain through the same bony defect of the temporal base into the extended sphenoid sinus. Post-operative diagnosis was Morley's rhinorrhea. The leakage point was closed and patched with the femoral fascia. Preoperative SPAMM-tagging image with cine MRI was useful to identify the responsible leakage point as a disorder of lattice tags. This method is a kind of flowmetry with MRI and is very effective because it can detect non-invasively slight CSF motion into the sinuses. It can also detect the direction and rough flow volume of rhinorrhea, so may also predict the risk of meningitis. The disadvantages were also discussed.


Subject(s)
Cerebrospinal Fluid Rhinorrhea/diagnosis , Magnetic Resonance Imaging/methods , Cerebrospinal Fluid Rhinorrhea/cerebrospinal fluid , Cerebrospinal Fluid Rhinorrhea/surgery , Humans , Male , Middle Aged , Tomography, X-Ray Computed
18.
J Biochem ; 120(1): 189-92, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8864863

ABSTRACT

The dissociation constant for the binding of myosin subfragment 1 (S1) and chromatographed actin in the presence and absence of nucleotide was measured at various ionic strengths and various temperatures. The dissociation constant was of nM order in the absence of nucleotide and increased by approximately 100- and approximately 100,000-fold in the presence of ADP and ATP, respectively. The dissociation constant also increased with increasing ionic strength, irrespective of the presence of nucleotide, and its dependence on the ionic strength was increased by the presence of ATP but decreased by the presence of ADP. The standard enthalpy change and entropy change for the binding of S1 to actin were both positive, irrespective of the presence of nucleotide, indicating that the binding was entropy-driven. The standard entropy change was essentially unaffected by the presence of ADP but was greatly decreased by ATP, suggesting that the large increase in the dissociation constant in the presence of ATP was due to the decrease of hydrophobic interactions. On the other hand, the increase in the dissociation constant for acto-S1 in the presence of ADP might be induced by the decrease of electrostatic interactions.


Subject(s)
Actins/chemistry , Myosin Subfragments/chemistry , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Kinetics , Muscle, Skeletal , Osmolar Concentration , Protein Binding , Rabbits , Temperature , Thermodynamics
19.
J Biol Chem ; 271(17): 9992-6, 1996 Apr 26.
Article in English | MEDLINE | ID: mdl-8626639

ABSTRACT

The 20-kDa regulatory (LC20) and 17-kDa essential (LC17) light chain subunits could be removed from porcine aorta smooth muscle myosin by the use of trifluoperazine and ammonium chloride. The isolated heavy chain rebound both light chains, resulting in the restoration of native properties. Experiments on reconstitution of the isolated heavy chain with LC17 and/or LC20 showed that both light chains were required for folding into the 10 S conformation and thus for the phosphorylation-dependent filament formation of smooth muscle myosin. However, LC17 was not essential for the phosphorylation-dependent regulation of actin-activated ATPase activity and superprecipitation but was required for full regulation. LC17 and phosphorylated LC20 were found to act as activators, and dephosphorylated LC20 was found to act as a repressor of the motor activities of smooth muscle myosin.


Subject(s)
Muscle, Smooth, Vascular/enzymology , Myosin Light Chains/chemistry , Myosins/metabolism , Actins/metabolism , Animals , Aorta/enzymology , Chemical Precipitation , Chromatography, High Pressure Liquid , Enzyme Activation , Microscopy, Electron , Myosins/chemistry , Protein Conformation , Structure-Activity Relationship , Swine
20.
J Biochem ; 119(4): 783-90, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8743582

ABSTRACT

In a porcine aorta extract, we observed two protein kinase activities which specifically phosphorylate the 204-kDa heavy chain isoform of aorta myosin in the absence of conventional kinase activators. We referred to these two protein kinases, eluted at 0.15 and 0.2 M KCl from a DEAE-column, as myosin kinases I (MKI) and II (MKII), respectively. The phosphorylation site for MKI was determined using a purified phosphopeptide derived from porcine aorta myosin phosphorylated with MKI. By comparison with the deduced amino acid sequence for smooth muscle myosins, the site corresponded to a Ser located at 3 amino acids upstream from a Pro, the putative end of the alpha-helical segment of the 204-kDa heavy chain tail. A homologous Ser is only present in smooth muscle myosins, i.e. not in nonmuscle myosins. MKI was purified 130-fold, but not separated from a kinase activity phosphorylating Ser1 or Ser2 in the 20-kDa regulatory light chain of aorta myosin. In contrast, MKII was purified to near homogeneity. MKII phosphorylated the porcine aorta myosin heavy chain at a Ser 19 amino acids downstream from the MKI site. The amino acid sequence around the Ser shared a consensus sequence of the phosphorylation site. The amino acid sequence around the Ser shared a consensus sequence of the phosphorylation site for casein kinase II and was homologous to that reported for bovine aorta myosin [Kelley, C.A. and Adelstein, R.S. (1990) J Biol. Chem. 265, 17876-17882]. MKII was identified as a multifunctional protein kinase, casein kinase II.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Muscle, Smooth, Vascular/metabolism , Myosin Heavy Chains/metabolism , Protein Serine-Threonine Kinases/metabolism , Acid Phosphatase , Amino Acid Sequence , Animals , Aorta , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/isolation & purification , Casein Kinase II , Cattle , Molecular Sequence Data , Molecular Weight , Myosin Heavy Chains/chemistry , Myosin Light Chains/metabolism , Peptide Mapping , Phosphopeptides/analysis , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/isolation & purification , Protozoan Proteins , Sequence Analysis , Sequence Homology, Amino Acid , Serine/metabolism , Substrate Specificity , Swine
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