Rod Portion Research Articles

New insights into the molecular basis of desmoplakinand desmin-related cardiomyopathies

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

Uniquely Stable 40 kDa Subfragment-2 in Carp Myosin

Digestion of carp myofibrils at 30 degrees C in 0.5 M KCl medium with calcium ion generated unique 135 kDa heavy meromyosin (HMM). The HMM was not produced when digested at 10 degrees C. A further digestion of the 135 kD HMM isolated in the absence of calcium ion generated uniquely short subfragment-2 (S-2) with a size of 40 kDa (40 kDa S-2) together with subfragment-1 (S-1). The 40 kDa S-2 was identified by N- and C-end sequencing, and demonstrated to locate the amino end of the rod portion. The unfolding temperature for the 40 kDa S-2 was around 52 degrees C as studied by circular dichroism measurement. The same unfolding peak was also detected with the intact rod together with a large unfolding peak at around 36 degrees C coming from the rest of the rod portion, light meromyosin. The unfolding peak for the 40 kDa S-2 in myosin was a little lower (48 degrees C) than that in free form, suggesting the involvement of the head portion in the stability of the 40 kDa S-2 in the structure.

The mammalian cardiac muscle thick filament: Backbone contributions to meridional reflections

Information about the structure of the vertebrate striated muscle thick filament backbone is important for understanding the arrangement of both the rod portion of the myosin molecule and the accessory proteins associated with the backbone region of the filament. Although models of the backbone have been proposed, direct data on the structure of the backbone is limited. In this study, we provide evidence that electron micrographs of isolated negatively stained cardiac thick filaments contain significant information about the filament backbone. Computed Fourier transforms from isolated cardiac thick filaments show meridional (or near meridional) reflections on the 10th and 11th layer lines that are particularly strong. Comparison of Fourier filtrations of the filaments that exclude, or include, these reflections, provide evidence that these reflections originate at least in part from a series of striations on the backbone at a ∼4 nm spacing. The striations are likely to result either from the packing of the myosin rods, or from proteins such as titin associated with the filament backbone.

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Repeated irradiation with suberythemal ultraviolet B reduces the number of epidermal Langerhans cells.

The influence of repeated low-dose ultraviolet B (UVB) radiation, to which we are exposed in daily life, has not been fully clarified, although the damage caused by exposure to high-dose UVB radiation has been well-studied in recent years. To investigate skin damage caused by repeated low-dose exposure, we evaluated the extent of injury to the Langerhans cells which are known to be involved in the cutaneous immune system. The backs of hairless mice were exposed to the following doses of UVB radiation: 100 mJ/cm(2) once, 50 mJ/cm(2) twice, 25 mJ/cm(2) four times or 10 mJ/cm(2) ten times. Skin specimens were taken for histochemical and electron microscopic examination 24 h after the final irradiation. Epidermis exposed to UVB radiation demonstrated a decrease in the number of Langerhans cells which showed less dendricity. The population of these cells in specimens exposed to repeated suberythemal doses was reduced to 40%, whereas exposure to a single high dose of UVB with the same energy resulted in a reduction of only 33%. These results indicate that repeated suberythemal doses injure Langerhans cells more than a single high-dose exposure. Furthermore, Birbeck granules in Langerhans cells of UVB-irradiated epidermis were reduced and tended to show shortening of their rod portion. The present study suggested that repeated challenge with suberythemal UVB radiation, to which we are all exposed in daily life, can cause substantial damage to Langerhans cells.

The Tip of the Coiled-coil Rod Determines the Filament Formation of Smooth Muscle and Nonmuscle Myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.

Early structural changes in myosin rod upon heating of carp myofibrils.

Upon heating carp myofibrils at 40 degrees C, the amount of myosin that is soluble and monomeric dropped very quickly, roughly 5 times faster than the ATPase inactivation. This rapid decrease of solubility was well explained by a rapid denaturation of the rod portion as measured by chymotryptic digestibility. Chymotryptic digestion of heated myofibrils in a low-salt medium with EDTA generated a reduced amount of rod and subfragment-1 (S-1). The decrease of S-1 produced from the heated myofibrils was consistent with the ATPase inactivation. The decrease of rod produced from the heated myofibrils was explained by the increased susceptibility of the heavy meromyosin (HMM)/light meromyosin (LMM) junction to chymotryptic. It was, therefore, concluded that the fastest event occurring in the myosin molecule upon heating of myofibrils is the irreversible exposure of the HMM/LMM junction.

Experimental autoimmune myocarditis and its pathomechanism.

The pathomechanisms of autoimmune myocarditis are quite different from viral infection. In this type of myocarditis, cardiac myosin fragments, proper dendritic cells and autoreactive T cells are the 3 major elements in initiating and promoting the inflammation. The causative epitope is locating on the S2 rod portion of the myosin heavy chain (MHC). Through our recombinant study, the peptide was found to be located on the latter half of MHC residues 1070 to 1165. Activity of antigenicity was not different between alpha and beta chain. The cardiac dendritic cell presents a unique structure with large mononuclear and interdigitating processes. This antigen presenter is quickly activated and suppressed by the antigen. The autoreactive T cell is closely linked with cytokine production. In the initial stage of myocarditis, IL-2 and IL-12 are increased. According to the progression of inflammatory changes, a great amount of IL-1b, INF-gamma and TNF-alpha is released around the diseased tissue. At the same time, NO is massively produced from infiltrating macrophages. Cytokines secreted from inflammatory cells accelerate T cell induction from Th0 to Th1. In the convalescent stage, production of TGF-beta 1 and IL-10 become dominant. They contribute to cell induction from Th0 to Th2.

FTIR-Spectroscopy of multistranded coiled coil proteins.

Coiled coils of different order were investigated using infrared (IR) spectroscopy. Recently, we demonstrated that dimeric coiled coils display unique vibrational spectra with at least three separable bands instead of only one band of a classical alpha-helix in the amide I region. This was attributed to a distortion of the helical structure by the supercoil bending, giving rise to bands that are not observed in the undistorted helix. Here, we investigated coiled coils forming trimers, tetramers, and pentamers. These higher order coiled coils, in general, possess larger superhelical pitches, resulting in a smaller helical distortion. We found that all coiled coils studied, including the native dimeric GCN4 leucine zipper and its variants leading to parallel trimers and tetramers as well as the rod portions of fibritin (parallel trimer), alpha-actinin (antiparallel spectrin type trimer), and COMP (parallel pentamer), displayed the typical three band pattern of the coiled coil amide I spectra. However, the separation of these three bands and their positional deviation from the classical alpha-helical band position was correlated to the extent of the helical distortion as reflected by the pitch values of the supercoils. The most pronounced spectral anomaly was found for the tropomyosin dimer with a reported helical pitch of 137 A, whereas the smallest spectral distortion was found for the pentameric COMP complex and the tetrameric leucine zipper mutant, both with a pitch of about 205 A.

Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments

The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded α-helical coiled coil formation of their ∼350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct ∼56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a ∼72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a ∼310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, ∼24.5 nm intervals, thereby giving them a “beaded” appearance with an axial periodicity of about 24.5 nm, which is ∼3 nm longer than the corresponding ∼21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.

Peptide-specific antibodies localize the major lipid binding sites of talin dimers to oppositely arranged N-terminal 47 kDa subdomains

Using ultrastructural analysis and labeling with polyclonal antibodies that recognize peptide sequences specific for phospholipid binding, we mapped the functional domain structure of intact platelet talin and its proteolytic fragments. The talin dimer, which is crucial for actin and lipid binding, is built of a backbone containing the 200 kDa rod portions, at both ends of which a 47 kDa globular domain is attached. Peptide-specific polyclonal antibodies were raised against three potential lipid binding sequences residing within the N-terminal 47 kDa domain (i.e. S19, amino acids 21–39; H18, amino acids 287–304; and H17, amino acids 385–406). Antibodies H17 and H18 localize these lipid binding sequences within the N-terminal 47 kDa globular talin subdomains opposed at the outer 200 kDa rod domains within talin dimers. Hence, we conclude that in its dimeric form, which is used in actin and lipid binding, talin is a dumbbell-shaped molecule built of two antiparallel subunits.

Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice.

Immunization with cardiac myosin induces T cell-mediated myocarditis in genetically predisposed mice and serves as a model for autoimmune heart disease. This study was undertaken to identify pathogenic epitopes on the myosin molecule. Our approach was based on the comparison of the pathogenicity between cardiac (alpha-)myosin and soleus muscle (beta-)myosin. We show that alpha-myosin is the immunodominant isoform and induces myocarditis at high severity and prevalence whereas beta-myosin induces little disease. Therefore the immunodominant epitopes of alpha-myosin must reside in regions of different amino acid sequence between alpha- and beta-myosin isoforms. Cardiac myosin peptides corresponding to these regions of difference were synthesized and tested for their ability to induce inflammatory heart disease. Three pathogenic peptides were identified. One peptide that is located in the head portion of the molecule induced severe myocarditis, whereas two others that reside in the rod portion possessed only minor pathogenicity. The identification of pathogenic epitopes on the cardiac myosin molecule will allow detailed studies on the recognition of this antigen by the immune system and might be used to downmodulate ongoing heart disease.

Self-Assembly of [n]Rotaxanes Bearing Dendritic Stoppers

We have developed a synthetic approach to the self-assembly of [2]-, [3]-, and [4]rotaxanes, incorporating bis-p-phenylene-34-crown-10 as the ring component(s) surrounding bipyridinium-based dumbbell-shaped components bearing dendritic stoppers at both ends. As a result of the hydrophobic dendritic framework, these [n]rotaxanes are soluble in a wide range of organic solvents, despite the polycationic natures of their bipyridinium-based backbones. In all cases, they could be purified by means of column chromatography employing relatively low polar eluants. The molecular shuttling action of the [2]rotaxane containing two bipyridinium units on the rod portion of the dumbbell-shaped component has been investigated by variable-temperature 1H-NMR spectroscopy in a range of solvents [CDCl3, CD2Cl2, THF-d8, and (CD3)2CO] for the first time. This investigation reveals a marked dependence of the rate of the shuttling process upon the polarity of the media. On going from CDCl3 to (CD3)2CO, the rate constant increases from ca. 200 to 33000 times per second. Molecular dynamics simulations, performed in CHCl3 and Me2CO on the [2]rotaxane, suggest that significant conformational changes occur upon changing the polarity of the medium resulting in both steric and electronic hindrance of the shuttling process in CHCl3. Three-dimensional representations, as well as the approximate sizesi.e. overall lengths and molecular volumes which range from 3 to 6 nm and from 4 to 6 nm3, respectivelyof these molecular compounds, were obtained by means of molecular modeling studies. Thus, these nanometer-scale dendritic rotaxanes resemble naturally-occurring chemical systems incorporating an active component, in so far as the rotaxane-like core with its distinctive recognition features is surrounded by a molecular shell in the form of the dendritic framework.

A new method for quantification of the dynamics of dark adaptation.

A new method of quantifying the dynamics of the human dark adaptation function is presented. This method is based on nonlinear regression analysis and allows the derivation of a number of clinically useful indices of dark adaptation ability. The analysis method is applied to literature data describing the effect of ageing on dark adaptation and to clinical data from a retinitis pigmentosa patient. Ageing was seen to decrease the rate of adaptation in the cone portion of the dark adaptation function, and to have little effect on the rate of adaptation in the rod portion of the dark adaptation function. One case of retinitis pigmentosa was associated with increased rod and cone thresholds but little change in the rate of adaptation of the cone portion of the dark adaptation function. It is suggested that this analysis method may offer advantages in quantifying changes in dark adaptation dynamics.

The Drosophila homeotic target gene centrosomin (cnn) encodes a novel centrosomal protein with leucine zippers and maps to a genomic region required for midgut morphogenesis

The products of the homeotic genes in Drosophila are transcription factors that are necessary to impose regional identity along the anterior-posterior axis of the developing embryo. However, the target genes under homeotic regulation that control this developmental process are largely unknown. We have utilized an immunopurification method to clone target genes of the Antennapedia protein (ANTP). We present here the characterization of centrosomin (cnn), one of the target genes isolated using this approach. The spatial and temporal expression of the cnn gene in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS) of wild-type and homeotic mutant embryos is consistent with the idea that cnn is a homeotic target. In the VM, Antp and abdominal-A (abd-A) negatively regulate cnn, while Ultrabithorax (Ubx) shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Characterization of a cDNA encoding CNN predicts a novel structural protein with three leucine zipper motifs and several coiled-coil domains exhibiting limited homology to the rod portion of myosin. Immunocytochemical results demonstrate that the cnn encoded protein is localized to the centrosome and the accumulation pattern is coupled to the nuclear and centrosome duplication cycles of cleavage. In addition, evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut construction. The centrosomal localization of CNN and the involvement of microtubules in midgut morphogenesis suggest that this protein may participate in mitotic spindle assembly and the mechanics of morphogenesis through an interaction with microtubules, either directly or indirectly.

Cast titanium as implant material

The tissue response in rats to implants made of machined and cast titanium was evaluated after 1 and 12 weeks. The implants consisted of a circular plate portion, located in the abdominal wall, and a cylindrical rod portion protruding into the peritoneal cavity. The chemical and topographical surface properties of the two types of implants differed considerably. The implants with surrounding tissue were processed en bloc for light and electron microscopy. The bulk metal was removed by an electrochemical procedure which permitted the sectioning and evaluation of the intact implant-tissue interface. The general distribution of macrophages and fibroblasts was the same around the plate portion of both types of implants. Macrophages constituted the predominating cell type with the highest concentration in the innermost cell zone closest to the implant. The number of macrophages per section area was significantly higher around machined implants. Multinuclear giant cells, always located at the implant surface, were more frequent around cast implants. The majority of the intraperitoneal rod portions were partially (1 week) or completely (12 weeks) covered by tissue; partial or complete overgrowth of tissue was rare for machined rod portions. Imaging electron energy loss spectroscopy demonstrated the presence of titanium in macrophages in the peripheral part of the tissue capsule around cast, but not machined implants. We conclude that the tissue responses to the two types of titanium implants differed considerably in the two biological environments (soft tissue in abdominal wall; peritoneal cavity) examined and that the response in one environment does not predict the response in the other. We also believe that improvements have to be made in the casting procedure in order to reduce surface roughness and contamination before cast implants can be used in clinical applications.

Trans-splicing of a Meloidogyne incognita mRNA encoding a putative esophageal gland protein

A monoclonal antibody, 7A 9, specific for antigens in the subventral esophageal glands of adult female Meloidogyne incognita and for antigens in the longitudinal muscles of second-stage juveniles, was used to isolate a clone from a M. incognita cDNA expression library. The corresponding genomic DNA was isolated by hybridization and the gene designated sec-1. DNA sequence analysis of sec-1 revealed the presence of 9 introns having structural similarities to introns from the free-living nematode Caenorhabditis elegans. The sec-1 message was also trans-spliced and the leader sequence differed in one position from the sequence of the C. elegans trans-spliced leader SL1. Sequences analogous to sec-1 were specific to the genus Meloidogyne, and regulation of sec-1 expression did not involve differences in overall rates of transcript accumulation. The deduced amino acid sequence of the protein encoded by sec-1 had some similarities to the rod portions of several myosin heavy chains.

Ultrastructure of Human Skin by a Rapid Freezing Technique: Structural Preservation and Antigenicity

To investigate the natural fine structure of skin, human skin biopsies were studied by electron microscopy and immunoelectron microscopy using rapid freezing and freeze-substitution techniques. Well-preserved structures were observed within a depth of 20 microns from the slammed surface of the samples. Epidermal cells were always closely located with very narrow intercellular spaces. Keratin filaments were distributed densely and homogeneously without forming bundles in the cytoplasm of epidermal keratinocytes. Langerhans cell granules were composed of a flat vesicular portion and an oval rod portion. The most striking finding was in the dermal-epidermal junction, where the lamina densa was present but no lamina lucida was observed. Further, by immunoelectron microscopy with anti-keratin monoclonal antibody, the 20-microns-deep part of the samples revealed the most significant staining. These findings indicate that the ultrastructure of routinely processed skin samples may be modified during the procedure for conventional chemical fixation and dehydration and that the ultrastructurally most preserved specimens, reflecting the conditions close to natural in vivo structures of the skin, can be obtained by the techniques described in this paper.

Analysis of Repeated Motifs in the Talin Rod

The amino acid sequence of the rod portion of talin contains strong periodic patterns with a long period of 32 to 34 residues superimposed on short periods of 7 and 7/2 residues. The rod includes 50 to 60 copies of an irregular repeated motif approximately 34 residues long. The motif itself consists of three sections: a short, "leader" segment of about six residues, which has a high proportion of the prolines and acidic residues; a relatively well-conserved hydrophobic "core" pattern of approximately 21 residues; and a highly variable "linker" region of seven residues which joins onto the next leader. The core section sequence has many of the characteristics of an amphipathic helix. The extensive hydrophobic side of this postulated helix has a characteristic surface pattern of large and small hydrophobic residues (mainly Leu and Ala), with a strong periodicity of seven residues. It also has a narrow hydrophilic edge with a highly variable sequence. The core sequence is unlike either a normal helical coiled coil or a leucine zipper, because it contains several helical ridges and grooves. The helical cores probably form a tightly packed hydrophobic central strand for the fibrous tail. The leader and linker sections are highly variable in length, so that, the spacing between the starting points of adjacent cores varies between 20 and 40 residues. The most common spacing is 34, and many spacings are close to this length.

Human smooth muscle myosin heavy chain gene mapped to chromosomal region 16q12.

The partial nucleotide sequence encoding the rod portion of the entire amino acid sequence of human smooth muscle myosin heavy chain (MHC) which corresponds to MYH11, according to Human Gene Mapping nomenclature, has been determined by cloning a complementary DNA (cDNA) and sequencing the cDNA (UMYHSM). Northern blot analysis with the UMYHSM fragment (4.3 Kb) showed that the smooth muscle MHC of the human umbilical artery is expressed in the human umbilical artery, bladder, esophagus and trachea. Southern blot analysis of human genomic DNA from human-mouse or human-Chinese hamster somatic cell hybrids demonstrated that the human smooth muscle MHC was mapped to human chromosome 16. Regional mapping of UMYHSM was performed using human cell lines with partial deletion and trisomy of chromosome 16. As a result, the human smooth muscle MHC gene segregated with 16p11-q12. In situ hybridization of biotin-labeled human smooth muscle MHC probe (UMYHSM fragment) to normal human metaphase chromosome independently showed that the human smooth muscle MHC gene (MYH11) is assigned to chromosome region 16q12. Analysis of early metaphase chromosomes showed that hybridization signals were in 16q12.1. In the human, although skeletal, cardiac, smooth muscle, and nonmuscle MHC genes are mapped to chromosomes 17, 14, 16, and 22, respectively, structural similarities of these MHC genes strongly suggest the common origin of these genes.