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Biomedical subjects

T Mikawa

Publications and source records attributed to T Mikawa.

At least 37 records · Page 2Linked to original sources
PubMed1999

Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage.

The cardiac pacemaking and conduction system sets and maintains the rhythmic pumping action of the heart. Previously, we have shown that peripheral cells of the conduction network in chick (periarterial Purkinje fibers) are selected within a cardiomyogenic lineage and that this recruitment occurs as a result of paracrine cues from coronary arteries. At present, the cellular derivation of other elements of this specialized system (e.g. the nodes and bundles of the central conduction system) are controversial, with some proposing that the evidence supports a neurogenic and others a myogenic origin for these tissues. While such ontological questions remain, it is unlikely that progress can be made on the molecular mechanisms governing patterning and induction of the central conduction system. Here, we have undertaken lineage-tracing strategies based on the distinct properties of replication-incompetent adenoviral and retroviral lacZ-expressing constructs. Using these complementary approaches, it is shown that cells constituting both peripheral and central conduction tissues originate from cardiomyogenic progenitors present in the looped, tubular heart with no detectable contribution by migratory neuroectoderm-derived populations. Moreover, clonal analyses of retrovirally infected cells incorporated within any part of the conduction system suggest that such cells share closer lineage relationships with nearby contractive myocytes than with other, more distal elements of the conduction system. Differentiation birthdating by label dilution using [(3)H]thymidine also demonstrates the occurrence of ongoing myocyte conscription to conductive specialization and provides a time course for this active and localized selection process in different parts of the system. Together, these data suggest that the cardiac conduction system does not develop by outgrowth from a prespecified pool of 'primary' myogenic progenitors. Rather, its assembly and elaboration occur via processes that include progressive and localized recruitment of multipotent cardiomyogenic cells to the developing network of specialized cardiac tissues.

Adenoviridae↗
PubMed1998

Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament.

Self-assembly of RecA protein in solution and on single-stranded DNA exerts a significant effect on the catalytic activities of this protein. To manipulate the self-association reaction, we examined the effects of various salts on the self-association of RecA from Thermus thermophilus (ttRecA) by circular dichroism spectroscopy and gel-filtration analysis. We showed that the self-association of ttRecA strongly depends on the kind and concentration of the salt, as well as on the protein concentration. Chaotropic ions were especially useful for obtaining RecA in its hexameric and monomeric states. On the basis of these observations, we were able to regulate the oligomeric states of ttRecA and we then examined the activity of RecA in various oligomeric states. Monomeric ttRecA bound to ssDNA and formed a nucleoprotein filament, which showed ssDNA-dependent ATPase activity. These results suggest that the monomeric form of RecA is an intermediate in filament formation on ssDNA.

Chromatography, Gel↗
PubMed1998

Endothelin-induced conversion of embryonic heart muscle cells into impulse-conducting Purkinje fibers.

A regular heart beat is dependent on a specialized network of pacemaking and conductive cells. There has been a longstanding controversy regarding the developmental origin of these cardiac tissues which also manifest neural-like properties. Recently, we have shown conclusively that during chicken embryogenesis, impulse-conducting Purkinje cells are recruited from myocytes in spatial association with developing coronary arteries. Here, we report that cultured embryonic myocytes convert to a Purkinje cell phenotype after exposure to the vascular cytokine, endothelin. This inductive response declined gradually during development. These results yield further evidence for a role of arteriogenesis in the induction of impulse-conducting Purkinje cells within the heart muscle lineage and also may provide a basis for tissue engineering of cardiac pacemaking and conductive cells.

Animals↗
PubMed1998

Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa. Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased. The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T. thermophilus MutM protein was active in E.coli. The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity. Size-exclusion chromatography indicated that T. thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution. Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30%. Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C. Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.

Amino Acid Sequence↗
PubMed1998

Trabecular myocytes of the embryonic heart require N-cadherin for migratory unit identity.

The myocardial wall of the vertebrate heart changes from a simple epithelium to a trabeculated structure during embryogenesis. This process occurs when epithelioid cardiomyocytes migrate toward the endocardium, which we show is coincident with up-regulation of the cell adhesion molecule, N-cadherin. To study the role of N-cadherin expressed at the trabeculation stage, a replication-defective retrovirus expressing a dominant negative mutant of N-cadherin (delta N-cadherin) was engineered. Control viruses were designed to express beta-galactosidase or a full-length N-cadherin. Viruses were introduced into epithelioid presumptive myocytes at the time they initiate the epithelial-mesenchymal transformation. Individual cells infected with control viruses generated daughter myocytes which migrated toward endocardium as a tight cluster, thereby generating a clone that forms a single or at most two trabeculae. In contrast, myocytes expressing delta N-cadherin were sparsely distributed within the myocardium and failed to form the ridge-shaped clone. Thus, in addition to its known roles in myocyte epithelialization and intercalated disc formation, N-cadherin appears to play a role in homotypic interactions between nonepithelial migratory myocytes during trabecular formation of the embryonic heart.

Animals↗
PubMed1998

Neural crest cells in outflow tract septation of the embryonic chicken heart: differentiation and apoptosis.

The heart consists of cells deriving from the cardiogenic plate and also from extracardiac sources. One of the major extracardiac contributions is given by the neural crest. The differentiation pathway and fate of the neural crest cells in the outflow tract have been followed over a prolonged period during outflow tract septation. We studied the role of the neural crest in remodeling the outflow tract by long-term cell tracing, differentiation markers and apoptosis. The pattern of neural crest cells migrating to the heart was investigated by heterospecific chicken quail chimeras and by retroviral infection of the reporter gene LacZ to the stem cells. The tagged neural crest cells move to areas that are morphogenetically active, such as the outflow tract, the semilunar valves, the wall of the arteries and the cardiac ganglia. Two differentiated subpopulations are discerned on the basis of immunohistochemical characterization with antibodies against smooth muscle cells in the arterial vessel wall and against ganglionic cells that were scattered around the vessels of the arterial pole and the heart. A third subpopulation did not stain with these antibodies, but presented locally with the phenomenon of apoptosis as shown with the TUNEL approach. In a developmental series of chicken embryos the populations were followed until stage 40. It was evident that the outflow tract septum in the early phase of development consisted mainly of mesenchymal neural crest cells. In a later phase neural crest cells were still detected at semilunar valve level, but nearly absent in the outflow tract septum below valve level. The septum at that time had become myocardialized. It is evident that neural crest cells are actually removed from this part of the heart by apoptosis. We are pursuing the hypothesis that an important function of apoptotic cells in heart development might be to activate the cardiomyocytes to muscularize the outflow tract septum through mobilizing or delivering growth factors at the time and place that septum formation is initiated.

Animals↗
PubMed1998

RecA protein has extremely high cooperativity for substrate in its ATPase activity.

The single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein, especially its cooperativity for ATP, was investigated. To measure the ATPase activity in detail, the methods and reaction conditions for the ATPase assay were reexamined. Under conditions where RecA protein always showed a maximal rate of ATP hydrolysis, its poly(dT)-dependent ATPase activity was measured. At 25 degrees C, increasing the concentration of RecA protein from 0.3 to 1.0 microM increased the turnover number (kcat) from 0.16 to 0.19 s-1 and the Hill coefficient (nH) for ATP from 9.3 to 11.6. At 0.5 microM RecA protein, increasing the temperature from 25 to 37 degrees C increased kcat from 0.18 to 0.35 s-1 but decreased nH from 9.8 to 6.6. Interestingly, the ATPase activity of RecA protein measured in this study showed much higher cooperativity for ATP than those reported to date. Furthermore, the nH value of 11.6 for ATP obtained here was the highest of any ATPase reported so far. These results suggest that the binding of an ATP molecule to a RecA molecule within a nucleoprotein helical filament causes structural change of many other neighboring RecA molecules. This implies that ATP binding induces structural change of the whole nucleoprotein helical filament. Finally, we demonstrated that analysis of cooperativity is useful for revealing how a protein composed of many subunits functions as a whole.

Adenosine Triphosphatases↗
PubMed1998

FGF1 patterns the optic vesicle by directing the placement of the neural retina domain.

Patterning of the bipotential retinal primordia (the optic vesicles) into neural retina and retinal pigmented epithelium depends on its interaction with overlaying surface ectoderm. The surface ectoderm expresses FGFs and the optic vesicles express FGF receptors. Previous FGF-expression data and in vitro analyses support the hypothesis that FGF signaling plays a significant role in patterning the optic vesicle. To test this hypothesis in vivo we removed surface ectoderm, a rich source of FGFs. This ablation generated retinas in which neural and pigmented cell phenotypes were co-mingled. Two in vivo protocols were used to replace FGF secretion by surface ectoderm: (1) implantation of FGF-secreting fibroblasts, and (2) injection of replication-incompetent FGF retroviral expression vectors. The retinas in such embryos exhibited segregated neural and pigmented epithelial domains. The neural retina domains were always close to a source of FGF secretion. These results indicate that, in the absense of surface ectoderm, cells of the optic vesicles display both neural and pigmented retinal phenotypes, and that positional cues provided by FGF organize the bipotential optic vesicle into specific neural retina and pigmented epithelium domains. We conclude that FGF can mimic one of the earliest functions of surface ectoderm during eye development, namely the demarcation of neural retina from pigmented epithelium.

Animals↗
PubMed1997

Local folding of the N-terminal domain of Escherichia coli RecA controls protein-protein interaction.

To obtain structural information about the self-association of the protein RecA, we studied urea denaturation of RecA by circular dichroism spectroscopy and gel filtration. Gel filtration analysis showed that urea at low concentrations, 1.0-1.2 M, dissociated the RecA oligomer to almost a monomeric state prior to the unfolding of each molecule. Upon treatment with 1.0 M urea, the circular dichroism spectrum showed a decrease in the alpha-helical content of RecA. A similar decrease was observed in the absence of urea for RecA at an extremely low protein concentration; the RecA oligomer dissociated to an almost completely monomeric state. The properties of RecA at low urea concentrations were similar to those of a truncated RecA lacking the first 33 N-terminal residues (Delta33RecA). Addition of a synthetic peptide corresponding to the 33 N-terminal residues to Delta33RecA increased the alpha-helical content. These results suggest that local folding of the N-terminal domain is coupled to protein-protein interactions of monomeric RecA, which are involved in the regulation of filament formation. The dissociation constant for interaction between RecA monomers was determined from the ellipticity data to be 0.1 microM.

Adenosine Triphosphate↗
PubMed1997

Isoform-specific interaction of the myosin-binding proteins (MyBPs) with skeletal and cardiac myosin is a property of the C-terminal immunoglobulin domain.

Full-length cDNAs encoding chicken and human skeletal MyBP-H and MyBP-C have been isolated and sequenced (1-5). All are members of a protein family with repetitive immunoglobulin C2 and fibronectin type III motifs. The myosin binding domain was mapped to a single immunoglobulin motif in cardiac MyBP-C and skeletal MyBP-H. Limited alpha-chymotryptic digestion of cardiac MyBP-C generated three peptides, similar in relative mobility to those of skeletal MyBP-C: approximately 100, 40, and 15 kDa. Tryptic digestion of MyBP-H yielded two peptides: approximately 50 and 14 kDa. Partial amino acid sequences proved that the 15- and 14-kDa fragments are located at the C termini of cardiac MyBP-C and skeletal MyBP-H, respectively. Only the 14- and 15-kDa peptides bound to myosin. Thus, the myosin binding site in all three proteins resides within an homologous, C-terminal immunoglobulin domain. Binding reactions (2) between the skeletal and cardiac MyBPs and corresponding myosin isoforms demonstrated saturable binding of the MyBP proteins and their C-terminal peptides to myosin, but there are higher limiting stoichiometries with the homologous isoform partners. Evidence is presented indicating that MyBP-H and -C compete for binding to a discrete number of sites in myosin filaments.

Amino Acid Sequence↗
PubMed1997

Skeletal muscle-specific myosin binding protein-H is expressed in Purkinje fibers of the cardiac conduction system.

Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of approximately 86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.

Animals↗
PubMed1997

Retroviral techniques for studying organogenesis with a focus on heart development.

The study of development has been revolutionized by the application of molecular techniques, which make it possible to identify factors involved in the developmental process. However, in order to correctly assess the contribution of these growth factors, transcription factors, receptors or signaling molecules, it is necessary to study them in the animal as a whole; it is not enough to conclude that they must be important based on their expression patterns.

Animals↗
PubMed1996

The fate diversity of mesodermal cells within the heart field during chicken early embryogenesis.

In gastrulation stage embryos of birds and mammals, the heart field is established as mesodermal crescents flanking the area rostrolateral to Hensen's node. Subsequent fusion of the bilateral heart primordia gives rise to a single tubular heart consisting of two epithelial layers: an outer myocardium and an inner endocardium. To date, it is uncertain whether these two distinct cell types of the heart arise from common or separate progenitor populations of mesodermal cells within the heart field. By retroviral single cell marking and tracking, we examined the diversity of cell populations present in the heart field of stage 4 chicken embryos. Here we demonstrate that individual mesodermal cells in the heart field gave rise to a clone consisting only of one cell type, either endocardial or myocardial cells; i.e., 95.1% of the mesoderm-derived clones were localized in the myocardium, while 4.9% of them were found in endocardium. No clones containing both of these two cell types were detected. The results suggest that the heart field mesoderm at stage 4 consists of at least two distinct subpopulations, containing more premyocardial cells than preendocardial cells. If there exists a common precursor of both myocardial and endocardial cells, the lineage diversification must occur at or prior to the arrival of mesodermal cells to the heart field.

Animals↗
PubMed1996

Smooth muscle alpha-actin downregulation in cultured chick aortic smooth muscle and neural crest cells is associated with altered cell shape.

A modified CXL retrovirus was used to clone an antisense smooth muscle alpha-actin ribozyme sequence adjacent to the reporter lacZ sequence. The virus was applied to downregulate alpha-actin expression in cultured smooth muscle cells obtained from chicken aortic arch and cultured neural crest cells. After infection with the ribozyme-containing CXL retrovirus both the smooth muscle and neural crest cells showed beta-galactosidase activity accompanied by a reduction of smooth muscle alpha-actin-positive fibers. Double staining of beta-galactosidase and smooth muscle alpha-actin using immunohistochemistry revealed that single cells infected with the CXL/ribozyme showed little to no smooth muscle alpha-actin protein. The absence of smooth muscle alpha-actin was associated with a distinct change in cellular morphology of the cultured cells, suggesting that expression of smooth muscle alpha-actin in cultured neural crest cells may be associated with cytoskeletal elements rather than vascular smooth muscle phenotype.

Actins↗
PubMed1996

Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ.

The vascular smooth muscle cells of coronary arteries are distinguished from those of the proximal aorta by a number of structural and functional criteria which may include an increased propensity for atherosclerotic transformation. At present, the source of this variation between smooth muscle subpopulations is uncertain. Whilst smooth muscle of the proximal aorta is thought to be derived from neural crest, the origin of coronary vascular smooth muscle remains uncharacterized. We have previously shown that precursors of the coronary vasculature enter the tubular heart on the same day as the epicardial mantle starts to envelop the myocardium and that coronary vessels form by ingrowth of these migratory precursors and not by outgrowth from the aorta (Mikawa and Fischman, 1992). To study the origin of coronary smooth muscle cells, the proepicardial organ, from which epicardial cells arise, was tagged with either a vital dye (DiI) or replication-defective retroviruses encoding beta-galactosidase. Cellular lineage marking was achieved by either direct targeting of putative vasculogenic cells in the proepicardium in ovo or tagging dissected proepicardial cells in vitro followed by transplantation to stage-matched host embryos. Monitoring of tagged cells during coronary vasculogenesis indicate incorporation of proepicardial-derived cells into three vessel-associated populations; coronary smooth muscle, perivascular connective tissue, and endothelial cells. Immunoconfocal microscopy identified both endothelial and smooth muscle cell populations within the proepicardial organ. The results demonstrate that: (1) the proepicardium contains a progenitor population of coronary smooth muscle cells that migrates into the heart along with ingrowth of the epicardium and (2) prior to the migration, the coronary smooth muscle lineage is established.

Animals↗
PubMed1996

Ependymal/subependymal zone cells of postnatal and adult songbird brain generate both neurons and nonneuronal siblings in vitro and in vivo.

The songbird forebrain continues to generate neurons in adulthood, from precursor cells located in the ependymal /subependymal zone (SZ) over the mediocaudal neostriatum. Precursor mitosis is followed by migration of neuronal daughter cells into the underlying forebrain, along radial fibers derived from the SZ. To define the ontogeny of both the new neurons and their radial guide cells, we employed retroviral insertion of the lacZ gene into neostriatal SZ precursor cells derived from postnatal and adult songbirds. We found that single SZ cells generate both neurons and substrate glia in vitro, and in an analogous fashion, both neurons and radial cells in vivo. This suggests that newly generated neurons and radial cells of the adult avian brain derive from a common pluripotential progenitor.

Animals↗