Masticatory function and properties of masseter muscle fibers in microphthalmic (mi/mi) mice during postnatal development.

It is well known that teeth do not erupt in microphthalmic (mi/mi) mice, a type of osteopetrotic mice, due to bone resorption failure. Therefore, we surmise that the masticatory function of these mice is different from that of normal mice. In this study, the differences to the properties of masseter muscle fibers were clarified morphologically and immunohistochemically in the mi/mi and normal mice. Morphological observations revealed that the muscle fibers in the mi/mi mice were smaller than those in normal mice at 9 weeks of age. However, no marked differences between mi/mi and normal mice at 2 and 4 weeks of age. Immunohistochemical observations showed myosin heavy chain (MHC) slow type fibers, which were usually seen at only early stages of development, in 4-week old mi/mi mice. There were also differences in isoform compositions between the mi/mi and normal mice. These results suggest that differences in masticatory function affect the properties of its muscle fibers.     

Identification of the cell type origin of odontoma-like cell masses in microphthalmic (mi/mi) mice by in situ hybridization

Tooth abnormalities occur in microphthalmic ( mi/mi ) mice. The elongated odontogenlc epithelium is interrupted by un-resorbed bone at the basal end of the mi/mi Incisor, with the epithelium gathered into cell clusters. These clusters develop to odontoma-like masses. To identify the origin of the cell types of these odontoma-like masses, the localization of osteonectin (Osn), osteocalcin (Osc), osteopontin (Osp), matrix Gla protein (MGP) and amelogenin (Am) mRNA in the process of tooth development in mi/mi and +/+ mice was investigated by means of in situ hybridization. Decalcified mandibles of neonatal, 5-, 10-, 14-day-old mice were examined. Osn and Osc mRNA, which localized in osteoblasts and odontoblasts, were also detected in the cells of odontoma-like masses in mi/mi mice. The cells expressing these mRNA were short, columnar and odontoblast-like. Am mRNA was detected In ameloblasts. In mi/mi mice, Am mRNA was also detected in ameloblastic cell clusters, which were formed by the tall columnar cells in the odontoma-like masses. No apparent Osp mRNA expression was detected in the masses. These results indicated that even In odontogenlc abnormal cells resulting from physical obstruction in mi/mi mice, the genes that are involved in normal tooth development were still expressed.     

Differential modification of myosin heavy chain expression by tenotomy in regenerating fast and slow muscles of the rat

We have examined the effect of tenotomy on the expression of myosin heavy chains (MyHC) in regenerating fast and slow skeletal muscles. Degeneration/regeneration of the left soleus and plantaris of Wistar male rats was induced by an injection into the muscle belly of a myotoxin (snake venom: Notechis scutatus scutatus). MyHC isoform content of regenerating plantaris and soleus muscles were studied 21 days after muscle injury using an electrophoretic technique. Tenotomy of the regenerating plantaris (mechanical underload) did not alter its MyHC expression (P > 0.05). In contrast, tenotomy of the regenerating soleus increased its relative levels of MyHC-2b (P < 0.05) and MyHC-2x/d (P < 0.01), and decreased its relative level of MyHC-1 (P < 0.01). Tenotomy of the synergistic gastrocnemius (overload) tended to decrease the relative level of MyHC-2b in regenerating plantaris (P < 0.07). The effect of tenotomy of the synergistic gastronecmius on the regenerating soleus was different: a decrease in the relative levels of MyHC-1 (P < 0.05) and an increase in the relative level of MyHC-neonatal (P < 0.01). In conclusion, and in contrast to a regenerating slow muscle, a change of mechanical loading by tenotomy did not seem to markedly alter the expression of mature MyHC phenotype in a fast regenerating muscle.     

Myosin heavy chain composition of the human sternocleidomastoid muscle

The sternocleidomastoid (SCM) muscle is one of the neck muscles responsible for head posture and control of head movement. It functions in rotation, inclination, protraction, extension and flexion of the head, whilst chewing and in exerting increased respiratory efforts. This study is the first one describing the myosin heavy chain (MyHC) isoform composition of the SCM muscle of presumably healthy young males for the purpose of better understanding the contractile properties of the muscle as well as to help in evaluation of pathologically altered structure of the muscle. Autopsy samples were processed immunohistochemically to reveal the MyHC isoform composition. The muscle fibres expressed MyHC-1 (31.5%), -2a (29.7%) and -2x (4.3%) or co-expressed MyHC-2a with MyHC-2x (26.8%), MyHC-1 with MyHC-2a (4.1%) and/or MyHC-1, -2a with -2x (1.1%). In addition to the MyHC isoforms, characteristic of adult limb muscles, a very low percentage of muscle fibres (0.2-2.7%) expressed MyHC-neo, which is normally not found in adult limb muscles. Only two samples exhibited MyHC-neo at a rather higher percentage (6.3% and 7.5%) of muscle fibres. The high share of hybrid fibres and the presence of MyHC-neo in the SCM muscle differ from that of adult limb muscles where hybrid fibres are rare and the expression of immature MyHC isoforms occurs only in pathological or experimental conditions. Since the SCM muscle shares the same embryogenic potential as limb muscles, its distinct MyHC expression appears to be associated with twin innervation and with the intrinsic specialisation to perform multiple functions.     

Deficient Differentiation of Mast Cells in the Skin of mi/mi Mice: Usefulness of in Situ Hybridization for Evaluation of Mast Cell Phenotype

The staining property of skin mast cells changed from Alcian blue⁺/berberine sulfate^- to Alcian blue ⁺/berberine sulfate⁺ in the skin of normal (+/+) and W^v/W^v mice. In contrast, this change did not occur in the skin of mi/mi mice. Heparin content and histamine content per a mi/mi skin mast cell were estimated to be 34% and 18% those of a +/+ skin mast cell, respectively. The low heparin content of mi/mi skin mast cells seemed to be consistent with the Alcian blue⁺/berberine sulfate^- staining property. Expression of genes encoding mast cell-specific proteolytic enzymes was examined by Northern blotting and in situ hybridization. Messenger RNA of mast cell carboxypeptidase A was expressed most of all by +/+, WV W^v/W⁺ and mi/mi skin mast cells, but mRNA of mouse mast cell protease (MMCP)-6 was expressed by approximately a half of +/+ and W^v/W^v skin mast cells and by only 3% of mi/mi skin mast cells. A significant amount of MMCP-2 mRNA was not expressed in the skin of all +/+, W^v/W^v and mi/mi mice. This shows the presence of at least three phenotypes in skin mast cells of mice: berberine sulfate⁺/MMCP-6⁺, berberine sulfate⁺/MMCP-6^-, and berberine sulfate^-/ MMCP-6^-. The in situ hybridization of mRNA of mast cell-specific proteolytic enzymes seemed to be useful to describe abnormalities of mast cell differentiation in the skin of mi/mi mice.     

Delayed tooth eruption in membrane type-1 matrix metalloproteinase deficient mice

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed highly in mineralizing tissues including bones and teeth. Mice deficient in MT1-MMP (-/-) display severe defects in skeletal development including dwarfism, osteopenia, and craniofacial abnormalities. Death occurs in these mice by about 3 weeks of age. Since MT1-MMP is expressed by the ameloblasts of the enamel organ and by the odontoblasts of the dental papilla, we asked if the developing teeth were adversely affected in the knockout animals. Molars from MT1-MMP -/- mice and controls were examined by histological, X-ray, and SEM analysis at 4, 18-20, and 25 days of postnatal development. At 4 days of development the molars from the -/- mice appeared histologically normal. At 18-20 days of development, the first molars of the -/- mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. In contrast to the -/- mice, the first molars of the 18-20-day control animals had erupted. SEM analysis of a -/- first molar and incisor revealed a normal enamel prism pattern. However, X-ray analysis demonstrated that tooth eruption was delayed by approximately 5 days and that the tooth roots were abnormally short in the knockout animals. Since MT1-MMP-deficient mice have been demonstrated to display a generalized increase in bone resorption, these data suggest that inefficient growth of bone surrounding the tooth root complex causes a delay in tooth eruption.     

A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice.     

Effect of parathyroid hormone and heparin on dental development in mice homozygous for the microphthalmia mutation

Cutting of the teeth is disturbed in mice homozygous for the microphthalmia gene (mi/mi) and osteopetrosis develops. The effect of heparin and of parathyroid hormone on cutting of the teeth was studied in mi/mi mice. Administration of heparin from the 2nd to the 20th day in a dose of 5 units/g body weight was shown to stimulate cutting of the teeth in mi/mi mice. Combined administration of heparin, in the same doses, and parathyroid hormone led to eruption of practically the same number of teeth as heparin alone. This may be because the heparin level is deficient in mi/mi mice but secretion of parathyroid hormone is undisturbed.     

Mechanical and kinetic properties of β-cardiac/slow skeletal muscle myosin

We aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the ??-cardiac/slow skeletal muscle myosin heavy chain (??-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the ??-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (??-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the ??-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the ??-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.     

Delayed Tooth Eruption in Membrane Type-1 Matrix Metalloproteinase Deficient Mice

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed highly in mineralizing tissues including bones and teeth. Mice deficient in MT1-MMP ( &#109 / &#109 ) display severe defects in skeletal development including dwarfism, osteopenia, and craniofacial abnormalities. Death occurs in these mice by about 3 weeks of age. Since MT1-MMP is expressed by the ameloblasts of the enamel organ and by the odontoblasts of the dental papilla, we asked if the developing teeth were adversely affected in the knockout animals. Molars from MT1-MMP &#109 / &#109 mice and controls were examined by histological, X-ray, and SEM analysis at 4, 18-20, and 25 days of postnatal development. At 4 days of development the molars from the &#109 / &#109 mice appeared histologically normal. At 18-20 days of development, the first molars of the &#109 / &#109 mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. In contrast to the &#109 / &#109 mice, the first molars of the 18-20-day control animals had erupted. SEM analysis of a &#109 / &#109 first molar and incisor revealed a normal enamel prism pattern. However, X-ray analysis demonstrated that tooth eruption was delayed by approximately 5 days and that the tooth roots were abnormally short in the knockout animals. Since MT1-MMP-deficient mice have been demonstrated to display a generalized increase in bone resorption, these data suggest that inefficient growth of bone surrounding the tooth root complex causes a delay in tooth eruption.     

Osteoclasts in the dental microenvironment: a delicate balance controls dental histogenesis

The impact of osteoclast activity on dental development has been previously analyzed but in the context of severe osteopetrosis. The present study sought to investigate the effects of osteoclast hypofunction,present in Msx2 gene knockin mutant mice (Msx2-/-), and hyperfunction, in transgenic mice driving RANK over-expression in osteoclast precursors (RANK(Tg)), on tooth development. In Msx2-/- mice, moderate osteopetrosis was observed, occurring exclusively in the periodontal region. Microradiographical and histological analyses revealed an abnormal dental epithelium histogenesis that gave rise to odontogenic tumor-like structures. This led to impaired tooth eruption, especially of the third mandibular molars. In RANK(Tg) mice, root histogenesis showed site-specific upregulation of dental cell proliferation and differentiation rates. This culminated in roots with a reduced diameter and pulp size albeit of normal length. These two reverse experimental systems will enable the investigation of distinctive dental cell and osteoclast communication in normal growth and tumorigenesis.     

miR-206及Bcl-2蛋白在非小细胞肺癌中的表达及意义

目的探讨mi R-206及Bcl-2蛋白在非小细胞肺癌(NSCLC)的表达,以及抑制或过表达mi R-206对Bcl-2蛋白表达的影响。方法实时定量PCR方法检测NSCLC组织mi R-206的表达水平,Western blot方法检测NSCLC组织Bcl-2蛋白表达;将mi R-206 inhibitors及mi R-206 mimics转染至A549细胞,通过CCK-8法测定A549细胞增殖能力的变化,Western blot方法检测转染后Bcl-2蛋白表达变化。结果 mi R-206在非小细胞肺癌组织中的表达量较癌旁组织明显降低(0.01),Bcl-2蛋白在癌组织中较癌旁组织表达明显增高(0.05);转染mi R-206 inhibitor的细胞增殖能力与对照组相比显著升高,细胞内Bcl-2蛋白水平亦明显升高(0.05),转染mi R-206 mimics的细胞增殖能力与对照组相比显著降低,细胞内Bcl-2蛋白水平亦明显降低(0.05)。结论mi R-206在NSCLC组织中低表达,mi R-206抑制能够促进A549细胞增殖和Bcl-2蛋白表达。     

The effects of protein malnutrition on the pathogenesis of murine cytomegalovirus disease.

BALB/c and CBA mice maintained on low (4%) or normal (18%) protein diets for 2 weeks after weaning were infected with a sublethal dose of murine cytomegalovirus, adjusted in proportion to body weight. Viral replication, histopathological changes and humoral responses to the virus were compared between the dietary groups 2-42 days post infection (p.i.). Higher numbers of viral antigen-positive cells and/or more prominent tissue necrosis were noted in the livers, spleens, hearts, adrenal glands, kidneys and bone marrows of infected protein-deficient mice. These mice also showed a delayed onset of leucocytic exudation in their livers and salivary glands, relative to infected mice on the normal diet. Second peaks of viral replication were detected by plaque assays in livers and spleens from protein-deficient mice and in livers from normal mice 12-18 days p.i., but few antigen-positive cells and no tissue necrosis were observed. Virus also persisted at higher titres in the salivary glands from protein-deficient mice. Although cellular immunity may be defective in these mice, humoral IgG and IgM responses to the virus were not inhibited. The influence of genetic factors on the pathogenesis of murine cytomegalovirus disease in protein-deficient mice is discussed.     

Distribution of BMP6 in the alveolar bone during mouse mandibular molar eruption

Eruption requires synchrony of the tooth with the surrounding tissues, particularly the bone. One important step during eruption is remodelling of the alveolar bone at the base of the tooth and along the roots. Expression of BMP6 was reported to be increased in the basal half of the dental follicle prior to eruption and inhibition of BMP6 affected bone formation at the base of the alveolar crypt. The aim of this study was to further investigate BMP6 protein in relation to tooth eruption and the corresponding bone remodelling using temporospatial correlations of BMP6 localization with morphogenetic events (proliferation, differentiation, apoptosis and bone apposition/resorption), other BMPs (BMP2 and BMP7) and three-dimensional images of tooth–bone development. BMP6 expression pattern was mapped in the mandibular molar teeth and related structures around eruption. Localization of BMP6 dominated in osteoblasts, in regions of bone formation within the alveolar crypt. These findings positively correlated with proliferation at the tooth base region, osteocalcin expression in the osteoblasts/osteocytes and BMP2 and BMP7 presence in the alveolar bone surrounding the tooth. Osteoclast activity and apoptotic elimination in the root region gradually decreased before eruption and totally ceased at eruption stages. Generally, BMP6 positively correlated with BMP2, BMP7 and osteocalcin-positive osteoblasts, and areas of bone remodelling. Moreover, BMP6 was found in the periodontium and cementoblasts. BMP6 expression in the alveolar bone accompanied tooth eruption. Notably, the expression pattern of BMP6 in the bone did not differ around individual molar teeth at the same stage of development. The expression of BMP6 in periodontal ligaments may contribute to interaction between the tooth and bone during the eruption and anchoring process.     

A study of osteoclasts on calvaria of normal and osteopetrotic (mi/mi) mice by vital staining with acridine orange.

A novel staining procedure for enumerating osteoclasts on neonatal mouse calvaria with the vital fluorescent dye acridine orange is described. It has the advantage over Barnicot''s neutral-red method in that the nuclei and cytoplasm of the osteoclast are stained differentially. The osteopetrotic calvarium (mi/mi) has fewer multinucleate osteoclasts than its normal counterpart (mi/+) and they are differently distributed. The osteopetrotic calvarium has more mononucleate cells which stain like osteoclasts with acridine orange than the normal calvarium and these cells also are differently distributed. These mononuclear cells may be mononuclear osteoclasts or their precursors. These observations suggest that the defect resulting in this osteopetrosis lies with osteoclast differentiation.     

The blood-vessel thrust theory of tooth eruption and migration

The Blood-Vessel Thrust Theory is a new hypothesis regarding the forces which produce the normal eruption of teeth, and the movement of 'nonerupted' teeth through bone away from their normal position in the jaws. It points out that the flow of blood through the vessels of the dental pulp, and of the tissues surrounding the tooth, must produce hydrodynamic and hydrostatic forces within the blood vessels, and that these forces have a resultant towards the tooth crown, thus causing the tooth to move, crown first, through the bone during normal eruption or abnormal tooth migration.