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664 records · Page 37Linked to original sources
PubMed2003

Flightin is a myosin rod binding protein.

The assembly of striated muscle myosin into thick filaments of precise and regular length requires the assistance of accessory proteins. Drosophila indirect flight muscle (IFM) contain flightin, a 20-kDa protein that has been shown to be essential for flight, for maintenance of sarcomeric integrity in active muscle, and informative in length determination of thick filaments during IFM development. Additionally, a point mutation in the myosin rod (Mhc(13)) negates flightin accumulation in the IFM in vivo. The manner in which flightin interacts with thick filaments is not known. Here, two different solid-state binding assays demonstrate that flightin binds to myosin and to a recombinant fragment of the myosin rod that include the COOH-terminal 600 amino acids (zone 19 to tail piece). The interaction of flightin and myosin is abolished by the single amino acid substitution in Mhc(13) at position 1e of zone 27 of the rod (residue 1554). The molar ratio of flightin to myosin is approx 1 : 1 to 1 : 2. Thus, the instability of thick filaments seen in vivo in the absence of flightin suggests that the flightin myosin interaction is critical for maintaining sarcomere integrity in active muscle.

Animals↗
PubMedDate not supplied

Electrophoretic studies of muscle proteins in Duchenne muscular dystrophy and other neuromuscular disorders--with special reference to the change of dystrophin.

We studied total SDS-solubilized muscle proteins (TMP) of Duchenne muscular dystrophy (DMD) and other neuromuscular disorders, with special attention to the change of dystrophin suspected of being the product of DMD locus. SDS gel electrophoresis of DMD patients showed an absence of band 5 and an extreme faintness of band 2 with a decrease of band 4', 5', and 5''. Immunoblot analysis, using anti-dystrophin antibodies (anti-30 kd and anti-60 kd polyclonals), showed an absence of dystrophin in all 6 DMD cases. In other neuromuscular disorders, there was no change of TMP, and dystrophin was clearly detectable. To elucidate the degenerative mechanism of DMD muscle, further studies, including the problem of clarifying the physiological role of dystrophin, are necessary.

Adult↗
PubMed2002

[Search for interacting proteins of esophageal cancer related gene-1 encoded protein through the yeast two-hybrid system].

OBJECTIVE: To understand the role that esophageal cancer related gene-1 (ECRG-1) plays and to search for ECRG-1-interacting proteins. METHODS: A DNA fragment encoding the carboxy-terminus of ECRG-1 (amino acids 40 - 418) was inserted into pGBKT7-DNA-BD vector and fused in-frame to the DNA-binding domain of GAL4. Then, it was used as a bait to screen the human fetal liver cDNA library by yeast two-hybrid, with the cDNA fragment inserted into pACT2 vector and fused in-frame to the Gal4 activation domain. If ECRG-1 interacted with a protein encoded by a cDNA fragmant in the yeast, the transcription of reporter Gene could be activated. With the false positive clonies eliminated, the inserts in the positive plasmids were sequenced and compared to those in the GenBank. RESULTS: In approximately 3 x 10(6) independent tansformants screened, 23 clonies exhibited the expression of reporter gene. After eliminating the false positive clonies, two cDNA fragments were obtained. DNA sequencing revealed that one encoded Miz-1 (Myc-interacting Zn finger protein-1), and another encoded FLNA (actin-binding protein-280), Miz-1, being a Zn finger protein, could be bound to p15 promotor and activated the transcription. FLNA, being an actin-binding protein took part in the TGF-beta pathway via interaction with Smad. CONCLUSION: ECRG-1 is able to be specifically bound to Miz-1 and FLNA in the yeast. It may play a role in the regulation of cell cycle via interaction with Miz-1 and FLNA.

Contractile Proteins↗
PubMed2004

Evaluation of sarcoglycans, vinculin-talin-integrin system and filamin2 in alpha- and gamma-sarcoglycanopathy: an immunohistochemical study.

The sarcoglycan subcomplex (SGC) is a well-known system of interaction between extracellular matrix and sarcolemma-associated cytoskeleton in skeletal and cardiac muscle. The SGC is included in the DGC made up of sarcoplasmic subcomplex and a dystroglycan subcomplex. Recent developments in molecular genetics have demonstrated that the mutation of each single sarcoglycan gene, causes a series of recessive autosomal muscular dystrophies, dystrophin-positive, called sarcoglycanopathies or limb girdle muscular dystrophies. Our recent studies have demonstrated that costameres are a proteic machinery made up of DGC and vinculin-talin-integrin system, also revealing the colocalization of sarcoglycans and integrins in adult human skeletal muscle. These results may support the hypothesis of the existence of a bidirectional signalling between sarcoglycans and integrins in cultured L6 myocytes. The hypothesis of bidirectional signalling between sarcoglycans and integrins could be supported by the identification of a skeletal and cardiac muscle filamin2 as a gamma-sarcoglycan, delta-sarcoglycan and, hypothetically, beta1 integrin interacting protein. Our results, acquired with an immunofluorescence study on adult human skeletal muscle affected by LGMD type 2D and 2C, showed that in LGMD2D: a) alpha-sarcoglycan staining is severely reduced; b) the beta-gamma-delta-sarcoglycan subunit and all tested integrins staining are clearly detectable; c) filamin2 is normal and shows a costameric distribution. In LGMD2C: a) alpha-sarcoglycan staining is preserved; b) the beta-gamma-delta-sarcoglycan subunit staining is severely reduced; c) the alpha7B-integrin is slightly reduced and beta1D-integrin is severely reduced; d) filamin2 is severely reduced. Other tested proteins of the two systems show a normal staining pattern in both sarcoglycanopathies. Our study seems to confirm, for the first time on adult human skeletal muscle of subjects affected by LGMDs, the hypo-theses of: a) the existence, in mouse myotubes in culture, of two distinct subunits in sarcoglycans subcomplex; b) the presence of a bidirectional signalling between sarcoglycans and integrins, previously demonstrated on rat cultured L6 myocytes; c) the interaction of FLN2 with both sarcoglycans and integrins. These results may stimulate the search of yet unidentified common interactors of both fiber-extracellular matrix interaction systems.

Biopsy↗
PubMedDate not supplied

[Heterotopia as the cause of seizures. A case report of familial periependymal heterotopia].

INTRODUCTION: Cortical development disorders constitute a group of entities resulting from an interruption in the development of the central nervous system. Most of them derive from the stoppage of migration, but proliferation and organisation can also be affected. Heterotopia is the most frequent of all migration disorders. CASE REPORTS: We describe three cases of familial periependymal heterotopia consisting in three sisters who, after having seizures in infancy, were submitted to a magnetic resonance (MR) scan that revealed isointense nodules in the cortical grey matter in all the MR sequences. They were diagnosed as having periependymal heterotopia. The existence of a history of early onset seizures in the family of the mother supported the diagnosis of familial heterotopia. CONCLUSIONS: Heterotopia is the most frequently occurring anomaly affecting cortical development and, of these, the periependymal form is the most common. Periependymal heterotopia may be determined by sex-linked inheritance (X chromosome). It is considered to be one of the most common congenital disorders in familial and early onset epilepsy. MR is the preferred diagnostic technique, since its high resolution allows it to identify and characterise heterotopias.

Agenesis of Corpus Callosum↗
PubMed1995

Role of gelsolin in the formation and organization of triton-soluble F-actin during myeloid differentiation of HL-60 cells.

Structurally and functionally distinct F-actin pools coexist with globular (G)-actin in a variety of eukaryotic cells, including polymorphonuclear leukocytes (PMNs). In PMNs, a Triton-soluble F-actin pool (TSF) exists as short cytoplasmic filaments capped with gelsolin, while Triton-insoluble F-actin (TIF) is a three-dimensional meshwork of F-actin associated with actin-binding protein 280 (ABP-280), alpha-actinin, and tropomyosin. The unique association of gelsolin with the TSF suggests a role for gelsolin in creation or regulation of TSF. To evaluate gelsolin's role in TSF formation, the quantities of actin and gelsolin were determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots in uninduced HL-60 cells (U-HL-60) and in HL-60 cells induced to myeloid differentiation with 1.25% dimethyl sulfoxide for 4 to 5 days (I-HL-60). U-HL-60 cells contain 17.76 +/- 6.01 pmol actin per 10(6) cells (TIF, 5.3 +/- 1.5; TSF, 2.17 +/- 0.37; G, 10.3 +/- 5.7; n = 5) and 0.073 pmol gelsolin per 10(6) cells (TIF, 0; TSF, 0.002 +/- 0.005; G, 0.07 +/- 0.01; n = 3), representing molar actin to gelsolin (A:G) ratios of 1,085:1 for TSF and 147:1 for G. After myeloid differentiation, the actin content increases 1.80-fold (31.94 +/- 6.14 pmol/10(6) cells) equally in each actin pool (TIF, 9.36 +/- 2.35; TSF, 3.29 +/- 0.62; G, 19.29 +/- 4.83). Gelsolin increases 2.4-fold overall (0.178 +/- 0.02 pmol/10(6) cells) but 19-fold in TSF (0.038 +/- 0.009) and only 1.9-fold in G pool (0.139 +/- 0.006), resulting in A:G ratios of 87:1 in TSF and 139:1 in G. The findings of an increase in TSF gelsolin with decreased A:G ratios (1,085:1 v 87:1) with myeloid differentiation suggest shortening of TSF filaments, while the A:G ratios of unbound gelsolin are unchanged (147:1 v 139:1). Measurement of EGTA-resistant gelsolin/actin complexes in HL-60 cells shows that 95% to 100% of complexes exist in the TSF-actin pool only. These findings are consistent with a role for gelsolin in formation and organization of Triton-soluble F-actin. Furthermore, the apparent shortening of TSF-actin filaments with myeloid cellular differentiation and maturation may represent one mechanism of conversion of the nonmotile myeloblast to the motile PMN.

Actins↗
PubMed1996

Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation.

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP-280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb-IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb-IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb-IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.

Actin Cytoskeleton↗