Myogenic bladder defects in mouse models of human oculodentodigital dysplasia.

To date, over 65 mutations in the gene encoding Cx43 (connexin43) have been linked to the autosomal-dominant disease ODDD (oculodentodigital dysplasia). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. BSMCs (bladder smooth muscle cells) from wild-type and two Cx43 mutant lines (Cx43(G60S) and Cx43(I130T)) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs from both mutant mouse strains were defective in their ability to contract, which is indicative of phenotype changes due to harbouring the Cx43 mutants. Upon stretching, Cx43 levels were significantly elevated in controls and mutants containing BSMCs, but the non-muscle myosin heavy chain A levels were only reduced in cells from control mice. Although the Cx43(G60S) mutant mice showed no difference in voided urine volume or frequency, the Cx43(I130T) mice voided less frequently. Thus, similar to the diversity of morbidities seen in ODDD patients, genetically modified mice also display mutation-specific changes in bladder function. Furthermore, although mutant mice have compromised smooth muscle contraction and response to stretch, overriding bladder defects in Cx43(I130T) mice are likely to be complemented by neurogenic changes.

Investigation of ractopamine-imprinted polymer for dispersive solid-phase extraction of trace beta-agonists in pig tissues.

Ractopamine, as an alternative beta-agonist to clenbuterol, is more and more used as leanness-enhancing agent in the swine industry. This work presents a new molecularly imprinted polymer (MIP) using ractopamine as template for dispersive solid-phase extraction of trace ractopamine and the structural related beta-agonists in animal tissues. The binding properties and selectivity of MIP were investigated. High selectivity in polar environment was found, since the extraction capacity of ractopamine with the MIP was 4.5-fold as much as that with the non-imprinted polymer in acetonitrile. Cross-selectivity investigation indicates that the MIP preferentially binds the template and then the structural analogues according to their molecular similarity. Thermodynamic and kinetic investigation was performed to interpret the specific adsorption and molecular recognition of the MIP for ractopamine. Standard free energy, standard enthalpy, and standard entropy were determined. Related information suggested that adsorption of ractopamine onto MIP was an exothermic, spontaneous process. The MIP can be applied as dispersive solid-phase extraction material for enrichment of ractopamine, isoxsuprine, fenoterol and clenbuterol in complex samples before HPLC analysis. The method revealed detection limits of 0.20-0.90 microg/L, recoveries of 83.8-115.2 and 85.2-110.2% for the spiked pig muscle and pig liver, respectively, with the RSD from 2.5 to 8.8%.

A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein.

The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27-kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed.

Assembly of smooth muscle myosin by the 38k protein, a homologue of a subunit of pre-mRNA splicing factor-2.

Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro. However, thick filaments, which are composed of myosin and myosin-binding protein(s), persist in smooth muscle cells, even if myosin is subjected to the phosphorylation- dephosphorylation cycle. The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy. However, smooth muscle cells that are devoid of telokin have been observed. We expected to find another ubiquitous protein with a similar role, and attempted to purify it from chicken gizzard. The 38k protein bound to both phosphorylated and dephosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephosphorylated myosin into filaments, although it had no effect on the phosphorylated myosin. The 38k protein bound to myosin with both COOH-terminal 20 and NH(2)-terminal 28 residues of the 38k protein being essential for myosin binding. The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2. Western blotting showed that the protein was expressed in various smooth muscles. Immunofluorescence microscopy with cultured smooth muscle cells revealed colocalization of the 38k protein with myosin and with other cytoskeletal elements. The absence of nuclear immunostaining was discussed in relation to smooth muscle differentiation.

Development and application of molecularly imprinted polymers as solid-phase sorbents for erythromycin extraction.

Six molecularly imprinted polymers (MIPs) of erythromycin (ERY) were prepared by noncovalent bulk polymerization using methacrylic acid (MAA) as the functional monomer. On the basis of binding analysis, the MIPs with 1:2 optimum ratio of template to MAA were selected for subsequent scanning electron microscopy and Brunauer-Emmett-Teller analyses, which indicated that the MIPs had more convergent porous structures than the nonimprinted polymers. The equilibrium binding experiments showed that the binding sites of MIPs were heterogeneous, with two dissociation constants of 0.005 and 0.63 mg mL(-1), respectively. Furthermore, the performance of the MIPs as solid-phase extraction (SPE) sorbents was evaluated, and the selectivity analysis showed that the MIPs could recognize ERY with moderate cross-reactivity for other macrolides. The overall investigation of molecularly imprinted SPE for cleanup and enrichment of the ERY in pig muscle and tap water confirmed the feasibility of utilizing the MIPs obtained as specific SPE sorbents for ERY extraction in real samples. [figure: see text]

Material-based regulation of the myofibroblast phenotype.

Fibroblast growth factor receptor (FGFR) activation by basic fibroblast growth factor (FGF-2) serves to naturally repress the myofibroblast activation of valvular interstitial cells (VICs). Co-receptors for FGF-2, the heparan sulfate proteoglycans (HSPGs), are key participants in the formation of active FGF-2 signaling complexes. Bioactive environments regulating the myofibroblast phenotype were created by utilizing heparin glycosaminoglycan as a competitive inhibitor of HSPGs. First, soluble heparin was delivered to compete with cell-surface HSPG for the binding of FGF-2. Exogenous soluble heparin prevented serum-dependent activation of the classic mitogen-activated protein kinase (MAPK) and induced myofibroblast alpha smooth muscle actin (alphaSMA) expression and collagen production. Next, heparin-functionalized hydrogel cell substrates were polymerized from vinyl-modified precursors and rendered adhesive through incorporation of RGDS peptide. Culture of VICs on heparin-modified gels induced alphaSMA expression and inhibited MAPK activity compared to control gel substrates lacking heparin. Additionally, heparin-functionalized gels continued to induce alphaSMA expression in serum-free culture conditions, suggesting that bioactivity was independent of exogenous soluble mediators. Biomaterial scaffolds targeting cell surface growth factor receptors are a promising new direction for regulating cell functions in tissue-engineering applications.

[Polymerization of myorod, a surface protein of thick filaments of molluscan smooth muscle].

The study is concerned with the polymerization of myorod, a protein from thick filaments of molluscan smooth muscles, which is an alternative product of the gene of myosin heavy chains. The dependences of the properties and polymer structure of myorod on the conditions of its formation were investigated. It was shown that myorod loses the ability to form viscous polymers after proteolytic removal of the unique sequence. It was supposed that the specificity of polymerization of myorod are determined by its unique N-terminal sequence.

Interaction of smooth muscle calponin with phospholipids.

Analyzing the primary structure we predicted that calponin may interact with phospholipids. In order to check this suggestion we investigated the interaction of calponin with phospholipids by ultracentrifugation, light scattering, vesicles leakage and differential scanning calorimetry. In agreement with our prediction calponin interacts with acidic phospholipids and the phospholipid-binding site was located in the short (13 kDa) N-terminal chymotryptic peptide of calponin. The apparent dissociation constant of calponin-phospholipids complex was less than 0.2 microM and calmodulin competes with phospholipids for calponin binding. Although the interaction of calponin with phospholipids decreases at high ionic strength, calponin binds phospholipids even in the presence of 100-150 mM of the salt. Under certain conditions calponin induced leakage of phospholipid vesicles and affected the cooperativity of lipid phase transition. It is concluded that both electrostatic and hydrophobic interactions provide for calponin-phospholipid complex formation.

Interaction of smooth muscle calponin and desmin.

Interaction of smooth-muscle calponin and desmin was analyzed by means of ultracentrifugation, fluorescent spectroscopy and affinity chromatography. At low and intermediate ionic strength (30-50 mM NaCl) calponin is cosedimented with desmin with an apparent dissociation constant 3-15 microM and stoichiometry of 1 calponin/4-6 desmin. Calmodulin decreases the quantity of calponin bound to desmin. Increase of ionic strength up to 150 mM weakens calponin-desmin interaction, but even at this ionic strength part of calponin remains bound to desmin. Calponin increases the rate and extent of fluorescence quenching induced by polymerization of 5-iodoacetamidofluorescein-labeled desmin. Affinity chromatography data indicate that desmin-binding sites are located in the N-terminal 22 kDa fragment of calponin. Since calponin interacts with desmin with an affinity comparable with that of, e.g., tropomyosin and myosin we suppose that calponin-desmin interaction may be important for cytoskeleton organization.

Engineering of vaginal tissue in vivo.

Congenital vaginal anomalies and cloacal malformations may require extensive surgical reconstruction. Surgical challenges are often encountered because of the limited amounts of native tissue available. We investigated the feasibility of using vaginal epithelial and smooth muscle cells for the engineering of vaginal tissues in vivo. Vaginal epithelial and smooth muscle cells of female rabbits were grown, expanded in culture, and characterized immunocytochemically. Vaginal epithelial and smooth muscle cells were seeded on polyglycolic acid (PGA) scaffolds at 10 x 10(6) and 20 x 10(6) cells/cm(3), respectively. The cell-seeded scaffolds were subcutaneously implanted into nude mice. The animals were killed 1, 4, and 6 weeks after implantation. Immunocytochemical and histochemical analyses were performed with pancytokeratins AE1/AE3 and with smooth muscle-specific alpha-actin antibodies to confirm the reconstituted tissue phenotype. Western blot analyses and electrical field stimulation studies were also performed to further characterize the tissue-engineered constructs. Vaginal epithelial cells were serially identified with anti-pancytokeratins AE1/AE3 at all culture stages. Smooth muscle cells in culture stained positively with alpha-smooth muscle actin antibodies. One week after implantation in vivo, the retrieved polymer scaffolds demonstrated multilayered tissue strips of both cell types, and penetrating native vasculature was also noted. Increased organization of the smooth muscle and epithelial tissue was evident by 4 weeks. There was no evidence of tissue formation in the controls. Immunocytochemical analyses using anti-pancytokeratins confirmed the presence of vaginal epithelial cells in each of the constructs. Anti-alpha-actin smooth muscle antibodies also confirmed the presence of multilayered smooth muscle fibers and tissue at each time point. Western blot analyses of the scaffolds confirmed the expression of cytokeratin and smooth muscle actin proteins when compared with controls. The contractile properties of the tissue-engineered vaginal constructs in response to electrical field stimulation were similar to those of normal vaginal tissue. Vaginal epithelial and smooth muscle cells can be easily cultured and expanded in vitro. Cell-seeded polymer scaffolds are able to form vascularized vaginal tissue in vivo that have phenotypic and functional properties similar to those of normal vaginal tissues. This is the first demonstration in tissue engineering wherein vaginal epithelial and smooth muscle cells are reconstituted in vivo into vaginal tissue. This technology may be pursued further experimentally in order to achieve the engineering of vaginal tissues for clinical applications.

Vomeronasal organ and its associated structures in the opossum Monodelphis domestica.

The opossum Monodelphis domestica possesses a well-developed vomeronasal system. Uptake of chemical stimuli into the vomeronasal organ (VNO) involves a stereotypical "nuzzling" behavior. In the present study, ten animals were examined by light and electron microscopy. The peripheral oro-nasal structures that apparently enhance access of solutes into the VNO include: (1) two lateral grooves of the ventral rhinarium and a network of channels leading into them, (2) dental gap adjacent to the grooves, (3) butterfly-shaped incisive papilla, and (4) unique bristle/cup-shaped filiform papillae on the tongue. The longitudinal axis of the vomeronasal complex is composed of the VNO proper at its rostral end, and an extensive compound serous gland at its caudal end with a distinct transition zone in between. The transition zone is characterized by the following features: merging of the main excretory duct of the large vomeronasal gland with the VNO lumen and drainage of auxiliary glandular clusters into the lumen, irregularities in the sensory epithelium ("rosette" appearance), and the ending of the cartilaginous support surrounding the VNO. Multiple elongated bundles of smooth muscle are positioned between the sensory epithelium and the cartilaginous capsule and more caudally are intertwined with the glandular parenchyma. These bundles become more numerous at the transition zone. Contraction and extension of these muscles may function to enhance the flow of solutes and glandular secretion within the lumen. Two extensive venous sinuses are associated with the opossum VNO complex: the internal vein bordering the sensory epithelium at its rostral end, and the external vein alongside the nonsensory epithelium. This arrangement suggests a unique dual pumping mechanism.

Characterization of several invertebrate muscle cell types: a comparison with vertebrate muscles.

Ultrastructural classification of invertebrate muscles is complex and not always clear. The aim of the present paper was to establish some criteria that might be useful for classification of invertebrate muscles and for a better understanding of the differences between them. The procedures used were: (1) immunochemical evaluation of those proteins that differentiated striated from smooth muscle (troponin, caldesmon, and calponin), and (2) calculations of several myofilament parameters to establish differences among muscles. The muscles studied were: striated muscles from the rat, Drosophila, the crab Callinectes, and the snail Helix (heart); obliquely striated muscles from the earthworm Eisenia foetida and Helix (mouth); and smooth muscles from the rat, and Helix (retractor, body wall, and intestinal wall). Immunochemical studies revealed that troponin was only present in the striated muscles and the obliquely striated muscle from Eisenia, whereas caldesmon and calponin were only present in the smooth muscles and the obliquely striated muscle from Helix. The highest thick filament/thin filament volume ratio was found in the striated muscles, followed by the obliquely striated muscles, and the smooth muscles. This suggests the order in which the contraction strength decreases. The myofilament length is inversely related to the contraction speed, which was higher in the striated muscles than in the obliquely striated muscles. In vertebrates, the smooth muscle seems to be less rapid than the striated muscle because their myofilaments are longer. This assertion cannot be generalized for invertebrate smooth muscle, because myofilament lengths vary widely in both striated and smooth muscles. In smooth muscles, the presence of apparently unordered electron-dense bodies instead of ordered Z lines and the absence of true sarcomeres permit a certain overlapping of thin filaments increasing the range of shortening.

Assessment of the protein quality of the smooth muscle myofibrillar and connective tissue proteins of chicken gizzard.

The objective of this study was to assess the protein quality of the myofibrillar and connective tissue proteins of chicken gizzard. Protein fractions were isolated from White Leghorn chicken gizzards and quantified by detailed amino acid analysis. This quantification involved repeated extractions of ground gizzards first with Triton X-100, then with low ionic strength imidazole-buffered saline (pH 7.1), followed by either 2% SDS or by 5 M guanidine hydrochloride. The total soluble intracellular protein fraction averaged 86.3% of the total protein and the insoluble extracellular connective tissue proteins comprised the remaining 13.7%. These fractions differed significantly in their essential amino acid (EAA) profiles, with the soluble intracellular fraction having the highest percentage EAA9 (48.6 to 49.0%) and the insoluble connective tissue fraction varying from 20.8 to 23%, compared to the FAO/WHO reference pattern value of 33.9% for a 2- to 5-yr-old child. Calculated protein efficiency ratios (PER) for intracellular proteins averaged 3.02 compared with a value of 1.65 for the extracellular matrix proteins. These results provide an accurate assessment of the protein quality of smooth muscle proteins of chicken gizzard and may prove valuable for industrial control of the amount of connective tissue added to formulations of meats and poultry products.

Sialolipoma of the floor of the mouth: a case report.

Intra-oral lipoma is a well-known entity, but lipomatous tumors including salivary gland tissue containing clustered or peripherally located ducts and acinar cells are uncommon. They are a newly recognized entity of salivary gland lipoma, designated sialolipoma. We describe a case of sialolipoma arising in the floor of the mouth presenting with apparently normal salivary gland tissue, as demonstrated by both histologic and immunohistochemical findings, in a 67-year-old female. Complete surgical removal of the tumor with preservation of the sublingual gland was implemented after a careful examination confirming that the lesion did not originate from the sublingual gland.

New biological properties of coffee melanoidins.

Water-soluble melanoidins isolated from roasted coffee induced ex vivo changes in the bioelectric and contractile activity of rat circular gastric smooth muscle tissues. They provoked a depolarization of smooth muscle cellular membranes and an increase in Ca²âº-influx as evidenced by the increase in the frequency and amplitude of Ca²âº-generated spike potentials. In the presence of 1 × 10⁻6 mol L⁻¹ acetylcholine and 1 × 10⁻6 mol L⁻¹ arecoline the melanoidin-evoked contraction was significantly reduced. M-cholinergic receptor blocking agents atropine, ipratropium, pirenzepine, and 4-DAMP also significantly reduced the melanoidin-provoked contraction. Nonspecific N-cholinergic receptor blockers hexamethonium and decamethonium (1 × 10⁻5 mol L⁻¹ each) did not influence the melanoidin-induced mechanical reaction. The melanoidins did not affect the strength of contractions evoked by adrenaline and dopamine (1 × 10⁻6 mol L⁻¹ each). The results obtained support the assumption that melanoidin-evoked contraction is a result of activation of muscarinic-type cholinergic receptors.

Platelet derived growth factor-CC secreted by M2 macrophages induces alpha-smooth muscle actin expression by dermal and gingival fibroblasts.

Dermal wounds can heal detrimentally by formation of excess fibrosis or hypertrophic scarring. These phenomena are normally absent in the oral mucosa. Macrophages play an important role in wound repair, have a marked heterogeneity and are thought to contribute to fibrosis. To investigate to what extend macrophages are involved in the occurrence of fibrosis, the effect of differently activated macrophages on dermal and gingival fibroblasts was studied in vitro. Macrophages were differentiated into a classical (M1) or alternative (M2) phenotype, which was assessed by receptor expression (CD40/mannose receptor) and cytokine secretion (interleukin-4 and -12). Fibroblasts were exposed to these macrophages and/or conditioned medium (cm), and differentiation into α-SMA-expressing myofibroblasts was quantified. M2, but not M1 macrophages induced α-SMA expression in both dermal and gingival fibroblasts. Blocking of transforming growth factor-ß1 did not decrease the α-SMA expression mediated by M2 macrophages. It appeared that this induction was mediated by platelet derived growth factor-CC (PDGF-CC), produced by M2 macrophages. The expression and role of this growth factor was confirmed by ELISA, RT-PCR, and blocking experiments. Our results indicate that M2 macrophages are able to induce myofibroblast differentiation via production of PDGF-CC. Based on our findings we conclude that PDGF-CC may play a hitherto unknown role in the differentiation of myofibroblasts.

Protein solubilization: attend to the choice of lysis buffer.

The efficient extraction of proteins of interest from cells and tissues is not always straightforward. In this process, the use of the optimal lysis buffer for protein solubilization should be considered. Here we demonstrate the use of a urea/thiourea lysis buffer, based on O'Farrell's buffer, and compare its effectiveness for solubilization of proteins from smooth muscle with the often utilized RIPA lysis buffer.

Calcifying epithelial odontogenic tumour of the maxilla: a case report with respect to immunohistochemical findings.

Calcifying epithelial odontogenic tumour (Pindborg tumour) is a rare benign neoplasm with a poorly understood histogenesis. This report describes a single case of a maxillary calcifying epithelial odontogenic tumour including immunohistochemical analysis. The vast majority of tumour cells were positive for CK5/6 and p63. Furthermore, basal cells also displayed moderate reaction for vimentin and strong membranous positivity for podoplanin. Interestingly, the tumour invaded the dental pulp of the partially disintegrated tooth root. While the tumour showed focal connection with the superficial gingival epithelium and revealed intercellular bridges, the findings of this case study seem to support the suggestion of an epithelial origin of a calcifying epithelial odontogenic tumour derived from the dental lamina.

The effects of peptide-based modification of alginate on left ventricular remodeling and function after myocardial infarction.

Adverse cardiac remodeling and dysfunction after myocardial infarction (MI) is associated with (BioLineRx, BL-1040 myocardial implant) excessive damage to the extracellular matrix. Biomaterials, such as the in situ-forming alginate hydrogel, provide temporary support and attenuate these processes. Here, we tested the effects of decorating alginate biomaterial with cell adhesion peptides, containing the sequences RGD and YIGSR, or a non-specific peptide (RGE), in terms of therapeutic outcome soon after MI. The biomaterial (i.e., both unmodified and peptide-modified alginate) solutions retained the ability to flow after cross-linking with calcium ions, and could be injected into 7-day infarcts, where they underwent phase transition into hydrogels. Serial echocardiography studies performed before and 60 days after treatment showed that alginate modification with the peptides reduced the therapeutical effects of the hydrogel, as revealed by the extent of scar thickness, left ventricle dilatation and function. Histology and immunohistochemistry revealed no significant differences in blood vessel density, scar thickness, myofibroblast or macrophage infiltration or cell proliferation between the experimental groups BioLineRx BL-1040 myocardial implant. Our studies thus reveal that the chemical and physical traits of the biomaterial can affect its therapeutical efficacy in attenuating left ventricle remodeling and function, post-MI.

Functional importance of the cytoskeletal protein in acetylcholine-induced contraction of rat bronchial smooth muscle.

The contractile capacity of smooth muscle cells depends on the cytoskeletal framework of the cell. The aim of this study was to determine the functional importance of the microtubule, actin filament and intermediate filament components of the cytoskeleton in acetylcholine (ACh)-induced contractile responses of the rat isolated bronchial smooth muscle. The expressions of alpha-actin, beta-actin, alpha-tubulin, desmin and vimentin were observed by immunoblotting in rat bronchial tissues. alpha-Actin and desmin were immunohistochemicaly observed in smooth muscle layer. Cytochalasin D, latrunculin A (inhibitors of the actin cytoskeleton) and acrylamide (an inhibitor of the intermediate filament) significantly decreased the contractions induced by ACh in concentration-dependent manners. On the other hand, colchicine or nocodazole (inhibitors of the microtubule cytoskeleton) had no effect on the ACh-induced contraction. These findings suggest that the contraction induced by ACh is highly dependent on polymerization of actin and intermediate filament, such as desmin, but not on the polymerization of microtubule in rat bronchial smooth muscle.