Gnathodiaphyseal dysplasia with a novel R597I mutation of ANO5: Mandibular reconstruction strategies.

Gnathodiaphyseal Dysplasia (GDD) is a rare, often misdiagnosed, autosomal-dominant disorder due to point mutations in the ANO5 gene. GDD combines craniofacial fibro-osseous lesions, dental loss and progressive curvature and cortical thickening of long bones and vertebra, causing pathological fractures. Diagnosis is based on bone pathology and mutation screening. Here we report three GDD cases within a single family with a novel ANO5 mutation: c.1790 G > T (p.Arg597Ile, i.e. R597I) on exon 16. Microsurgical mandibular reconstructions were performed in the three cases. We reviewed the literature on jaw reconstruction in this condition and discussed the challenges of craniofacial reconstruction in GDD due to the diffuse bone anomalies affecting potential flap donor zones and a specific risk for jawbone osteomyelitis.

The kyphoscoliosis (ky) mouse is deficient in hypertrophic responses and is caused by a mutation in a novel muscle-specific protein.

The ky mouse mutant exhibits a primary degenerative myopathy preceding chronic thoraco-lumbar kyphoscoliosis. The histopathology of the ky mutant suggests that Ky protein activity is crucial for normal muscle growth and function as well as the maturation and stabilization of the neuromuscular junction. Muscle hypertrophy in response to increasing demand is deficient in the ky mutant, whereas adaptive fibre type shifts take place. The ky locus has previously been localized to a small region of mouse chromosome 9 and we have now identified the gene and the mutation underlying the kyphoscoliotic mouse. The ky transcript encodes a novel protein that is detected only in skeletal muscle and heart. The identification of the ky gene will allow detailed analysis of the impact of primary myopathy on idiopathic scoliosis in mice and man.

A novel subgenomic murine leukemia virus RNA transcript results from alternative splicing.

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD') within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD' was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD' without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.

Effects of nitric oxide on the contraction of skeletal muscle.

A review of the literature suggests that the effects of nitric oxide (NO) on skeletal muscles fibers can be classified in two groups. In the first, the effects of NO are direct, due to nitrosation or metal nitrosylation of target proteins: depression of isometric force, shortening velocity of loaded or unloaded contractions, glycolysis and mitochondrial respiration. The effect on calcium release channels varies, being inhibitory at low and stimulatory at high NO concentrations. The general consequence of the direct effects of NO is to 'brake' the contraction and its associated metabolism. In the second group, the effects of NO are mediated by cGMP: increase of the shortening velocity of loaded or unloaded contractions, maximal mechanical power, initial rate of force development, frequency of tetanic fusion, glucose uptake, glycolysis and mitochondrial respiration; decreases of half relaxation time of tetanus and twitch, twitch time-to-peak, force maintained during unfused tetanus and of stimulus-associated calcium release. There is negligible effect on maximal force of isometric twitch and tetanus. The general consequence of cGMP-mediated effects of NO is to improve mechanical and metabolic muscle power, similar to a transformation of slow-twitch to fast-twitch muscle, an effect that we may summarize as a 'slow-to-fast' shift.

Effect of nitric oxide on the maximal velocity of shortening of a mouse skeletal muscle.

Maximum velocity of shortening, Vo, was measured by the method of Edman [J Physiol (Lond) 291:143-159, 1979] on extensor digitorum longus muscles of a mouse in vitro at 20 degreesC. Blockers of nitric oxide synthase, 10 mM nitro-l-arginine or 1 mM 7-nitroindazole, reduced Vo by 18% and 22%, respectively. On removal of the inhibitor, Vo returned to the control value. It was found that 10 mM nitro-d-arginine, an enantiomer of nitro-l-arginine inactive against nitric oxide synthase, did not affect Vo. A donor of nitric oxide, 0.1 mM nitroprusside, increased Vo by 15%. It removed the inhibition caused by nitro-l-arginine. Another donor of nitric oxide, 1 microM (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), increased Vo by 8%. An inhibitor of cGMP synthase, 0.01 mM Ly-83583, decreased Vo by 18%. An analogue of cGMP, 0.1 mM 8-bromo-cGMP, increased Vo by 17%. A general inhibitor of phosphodiesterases, 0.02 mM 3-isobutyl-1-methylxanthine (IBMX), increased Vo by 17%. An inhibitor specific of cGMP phosphodiesterase, 0.01 mM dipyridamole, increased Vo by 8%. The maximal isometric force (F0) was not modified by the drugs, except by 7-nitroindazole and Ly-83583, which depressed F0 by 12%. The cGMP level in tetanized muscles decreased by 12-27% in the presence of blockers of nitric oxide synthase. We conclude that the level of intracellular nitric oxide modulates Vo through the cGMP pathway.

Germinal vesicle material is dispensable for oscillations in cdc2 and MAP kinase activities, cyclin B degradation and synthesis during meiosis in Xenopus oocytes.

We have investigated at a molecular level the requirements for germinal vesicle (nuclear) material during the course of meiosis in Xenopus oocytes. We present the localization of some cell cycle proteins in stage VI oocytes; most of those analyzed are cytoplasmic, although some (MAD, 26S proteasome) are distributed between the cytoplasm and the germinal vesicle. By analyzing changes in individual oocytes, we find that the unphosphorylated form of cyclin B2 disappears and the phosphorylated form is then degraded in both nucleated and enucleated oocytes. Enucleated oocytes are also capable of resynthesizing both cyclin B1 and cyclin B2 after the initial degradation and of reactivating cdc2 kinase. Synthesis of mos protein and activation of MAP kinase concomitant with cdc2-cyclin B reactivation are also unaffected by prior removal of the germinal vesicle.

Isoforms of myosin in growing muscles of ky (kyphoscoliotic) mice.

Kyphoscoliotic (ky) mice are spontaneous mutants of the BDL strain whose postural muscles atrophy during post-natal growth, resulting in extensive kyphoscoliosis in adult animals. At 21 days of age, the seven muscles examined were already well differentiated into fast, slow and mixed type on the basis of the proportions of their native myosin isoforms or their subunits. During post-natal growth, from 21 to 120 days of age, the normal pattern of myosin maturation was essentially respected by the ky mutation: fast muscles became faster, slow muscles became slower and mixed muscles specialized in both directions. However, the post-natal increases of myosin heavy chain 2B and fast myosin light chain LC3f were depressed in ky muscles, whilst there was novel expression of slow myosin light chains, LC1s and LC2s in muscles which normally did not express them. Intermediate native myosin IM was absent in adult ky soleus, but it increased in adult ky tibialis anterior. We conclude that the ky mutation depresses the normal post-natal transition towards faster muscles and results in adult muscles whose myosin isoforms are generally shifted in a fast-to-slow direction.

Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene.

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.

Lack of myoblasts migration between transplanted and host muscles of mdx and normal mice.

Extensor digitorum longus muscles of normal mice (C57BL/10ScSn hereafter called C57) were orthotopically transplanted into dystrophin-deficient mice (mdx) and reciprocally, mdx Extensor digitorum longus muscles were transplanted into C57 mice. After an initial phase of degeneration, transplanted muscles regenerate nearly completely, as evaluated from the maximum isometric force of muscles isolated 60 days after the surgery. In other similar experiments, instead of isolating the grafted muscles, we excised the antero-external muscles of the leg, including the grafted muscle. Cryostat cross-sections at three levels along the muscles were immunostained with an anti-dystrophin antibody. No muscle cells of dystrophin-deficient muscles grafted into normal mice took the antibody except a few 'revertant' fibres, while all the muscle cells of the normal host were immunostained. Reciprocally, all the muscles cells of normal grafts were stained, whilst no antibody stained the cells of the surrounding muscles of the dystrophin-deficient host. These experiments show that very few if any of the myoblasts or muscle precursor cells, active during the regeneration of grafted muscle, migrate into the adjacent muscles. These results could be explained by the absence, in our work, of injuries of the grafted and adjacent host muscles epimysium and the absence of extensive inflammatory reactions. This lack of myoblast mobility suggest that when myoblast transfer is applied to muscle therapy, it will be necessary to inject myoblasts within each muscle to obtain an efficient treatment.

[The interaction of actin with myosin in fast and slow muscles of the mouse]. / Izuchenie vzaimodeistviia aktina s miozinom v bystrykh i medlennykh myshtsakh myshi.

Conformational changes of actin, during the transition of glycerinated muscle fibers of fast (EDL) and slow (SOL) mouse muscles from relaxation to rigor, were investigated by the polarized fluorescent technique. Changes in orientation and mobility of the fluorescent probe, i.e. rhodamin-phalloidin complex bound specifically to actin, testified the alteration of actin structure. The results show that during the transition of muscle fibers from relaxation to rigor the flexibility of actin filaments for EDL and SOL changes differently: increases for the former and practically does not change for the latter. The analysis of heavy myosin chains points out that SOL contains 65.43 +/- 7.26% myosin heavy chains 1 (MHC 1) and 34.57 +/- 7.26% myosin heavy chains 2A (MHC 2A). In contrast, EDL has 4.57 +/- 2.56% MHC 2A and 96.43 +/- 2.56% myosin heavy chains 2B (MHC 2B). No MHC 1 were revealed in EDL. A proposal is made that the isoformal composition of myosin heavy chains defines the character of actin-myosin interaction in slow and fast mouse muscles.

[Prevention of ocular infections in the hospital operating room]. / Prévention des infections oculaires en salle d'opération à l'hôpital.

Prevention of ocular nosocomial infections in the operation room requires control of several parameters: contamination of the air, a very important step of cleaning and sterilization and a staff trained to avoid infectious risk. The problem of PRION infection requires a particular attention especially about sterilization.

Mechanical power and myosin composition of soleus and extensor digitorum longus muscles of ky mice.

Muscles of ky/ky homozygote mice exhibit neonatal muscle fiber necrosis and regeneration with subsequent motor nerve sprouting and development of a prominent kyphoscoliosis from approximately 100 days onward. Soleus and extensor digitorum longus (EDL) muscles from ky mice weighted < 50% of control muscles from age-matched NMRI mice. Maximal tetanic force was more reduced in soleus than in EDL. In EDL, the velocity constant of the force-velocity relation, maximal velocity, twitch time-to-peak, and isomyosin content were normal at all ages. The early mechanical changes seen in ky soleus muscles (47 day) were not accompanied by significant alterations in isomyosin or myosin heavy- and light-chain composition, since ky and NMRI expressed slow-twitch native myosin 2 (SM2, type I fibers) and intermediate-twitch native myosin (IM, type IIa fibers). Adult ky soleus (172 day) showed wholesale loss of IM and sole expression of SM2. This is sufficient to account for the markedly slowing of the force-velocity relation and the twitches observed in adult ky soleus. We propose that since shifts in muscle type only occurred in soleus, this reflects the persistent requirement to withstand the force of gravity.

The force-velocity relation of the rabbit inferior oblique muscle; influence of temperature.

The contractile properties of the rabbit inferior oblique muscle (IO) were studied in vitro with direct stimulation at temperatures between 20 and 35 degrees C. Isovelocity releases were used to determine the force/velocity relation. Cooling the muscle from 35 degrees C to 20 degrees C increased contraction and half-relaxation times of single twitches with a temperature coefficient (Q10) of 0.4, but did not affect significantly the twitch tension. The tetanic tension increases with increasing temperature (Q10 = 1.32). Cooling decreased the maximum shortening velocity of the IO with a Q10 of 1.6 and the maximum mechanical power with a Q10 of 2.3. At 35 degrees C, the maximum speed of shortening of the muscle (19 +/- 2 muscle lengths/s, mean +/- SEM) corresponded to a maximum shortening velocity of the sarcomeres of 57 +/- 6 microns/s. This value is similar to data obtained for extraocular muscles (EOM) of smaller rodents (mice and rats). In comparison with mammalian limb muscles the isometric and force-velocity properties of mammalian EOM appear to be virtually independent of the size of the animal. Thus, IO is a fast-twitch muscle endowed with a maximum velocity of shortening higher than that of fast-twitch skeletal muscle, but using a tetanic mechanical power lower than that produced by slow-twitch muscle: the combination of these properties makes it ideally suited to move an ocular globe of low mass at high velocity.

Force-velocity relation and isomyosins in soleus muscles from two strains of mice (C57 and NMRI).

We compared soleus muscles from two strains of mice, NMRI and C57. Soleus muscles from NMRI mice produced slower twitches and lower maximum tetanic force (Fo) but higher maximum tetanic stress (So), (owing to their smaller weight). Their Hill's velocity constant (b) was lower, but their force constant (a/So), their maximum velocity of unloaded shortening (Vu) and their maximal mechanical power (Pmax) were similar. All soleus muscles contained two isomyosins (SM2 and IM) and the two myosin heavy chains (MHC1 and MHC2A) corresponding to type I fibres and type IIA fibres; however, soleus muscles from NMRI strain had higher proportions of isomyosin SM2 and of myosin heavy chain 2A. Regression equations were computed between the mechanical variables and the myosin heavy chain content. Using a simple hypothesis, the results were used to estimate the mechanical properties of type I and type IIA fibres. We conclude that type IIA fibres from soleus muscle are mechanically more similar to slow-twitch type I fibres than to fast-twitch type II fibres. The results also suggest a hypothesis to account for the diversity of isomyosins, by a matching diversity of mechanical properties based on a separate physiological control of the three factors that control Pmax.

Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch.

Absence of dystrophin in mdx muscles may render the muscle more susceptible to damage when submitted to high stress levels. To test this, typically slow (soleus) and fast (EDL) limb muscles of dystrophic (mdx) and normal (C57BL/10) mice were submitted (in vitro) to a series of isometric contractions, followed by a series of contractions with stretches. Muscle injury was assessed by monitoring the force signal. Membrane damage was evaluated by bathing the muscle in Procion Red, a dye that does not penetrate intact fibres, and subsequent analysis by light microscopy. After isometric contractions, only a very small force drop (< 3% of maximal isometric force) was observed which indicated that no injury had occurred in soleus and EDL muscles in either mdx or C57 strains. After contractions with a stretch, a force drop of 10% was observed in soleus muscles from both strains and in EDL muscles from C57 mice. However, in mdx mice EDL muscles displayed an irreversible force drop of 40-60%. Histological analysis of the muscles indicates that force drop is associated with membrane damage. These results show that EDL muscles from mdx mice are more vulnerable than their controls, supporting the structural role hypothesis for dystrophin. Furthermore, they suggest that contractions with stretches may contribute to the muscle damage and degeneration observed in DMD-patients.

Superinduction of c-fos gene expression by estrogen in cultured guinea-pig endometrial cells requires priming by a cycloheximide-dependent mechanism.

The c-fos gene expression is rapidly induced by various mitogenic agents and protein synthesis inhibitors in many cell types. Estradiol-17 beta can induce c-fos gene expression in breast cancer cell lines and in the uterus in vivo, but not in cultured guinea-pig endometrial cells. Using this model, we investigated whether a protein synthesis inhibitor, cycloheximide, could induce the c-fos gene and permit a superinduction by estrogens. In the presence of cycloheximide (10 micrograms/ml), protein synthesis was inhibited at 95% within the first hour. From 190 min after the addition of estradiol-17 beta or diethylstilbestrol (10(-8) M) and cycloheximide (10 micrograms/ml), there was a significant increase (ranging from 3- to 5-fold) of the c-fos messenger RNA level (2.2 kilobase in size), compared with the level in cells treated with cycloheximide alone. Nonestrogenic steroid hormones and estradiol-17 alpha were unable to induce c-fos gene expression in the presence of cycloheximide. The effect of estradiol-17 beta observed in the presence of cycloheximide was completely abolished by 4-hydroxy-tamoxifen or by Ly 156758 or by ICI 164384 (10(-6) M). The c-fos mRNAs were rather stable in cells treated with cycloheximide for 2 h (half-life = 51 +/- 6 min) and there was no further increase in the c-fos messenger RNA stability after the addition of cycloheximide plus estradiol-17 beta (half-life = 40 +/- 3 min). The overall results suggest a response at the transcriptional level. In conclusion, cycloheximide transmits activating signals to the c-fos gene which act as priming elements to allow the estrogen action in cultured guinea-pig endometrial cells.

Regeneration after free muscle grafting in normal and dystrophic (mdx) mice.

Soleus muscles from C57BL/10 and mdx mice were isotransplanted to induce a cycle of degeneration/regeneration. Sixty days post-surgery, transplanted and contralateral soleus muscles were removed for mechanical and biochemical analyses. The regeneration which occurs after transplantation, induces in both mdx and C57BL/10 soleus muscles a decrease in maximal isometric force, together with an increase of the velocity of contraction. This increase in velocity is accompanied by the expression of typically fast-type myosin heavy chains. Thus degeneration/regeneration of both mdx and normal mice are very similar, causing a shift towards physiologically 'faster' muscle. Previous physiological and biochemical studies of mdx muscles have shown that mdx muscle is shifted towards 'slower' muscle compared to normal mice. One explanation of these findings was that the degeneration/regeneration cycles inherent in dystrophin-deficient mdx muscle causes a shift towards 'slow'. Our results argue against this hypothesis: degeneration/regeneration in both normal and mdx mice causes a shift towards 'fast'.

[Influence of arterial hypertension on diabetic retinopathy]. / Influence de l'hypertension artérielle sur la rétinopathie diabétique.

In the industrialized countries, diabetic retinopathy represents the most frequent cause of blindness during the period of active life. It occurs as two distinct clinical entities: non proliferating retinopathy characterized by dilatation of the retinal capillary bed and alterations in their vascular wall responsible for an increase in permeability, and proliferating retinopathy characterized by the appearance of pre-retinal neovessels secondary to the presence of vast zones of retinal ischemia. Numerous risk factors are implicated in the development of diabetic retinopathy: the primordial factor is the optimal equilibration of blood glucose levels. The primum movens of these diabetic lesions could be intoxication of the pericipets and endothelial cells of the retinal capillaries by an accumulation of sorbitol and fructose in this region. Additionally, the hyperglycemia suppresses the functioning of the retinal blood flow feed back system. An increase in systemic blood pressure will therefore be transmitted directly to the damaged capillary bed. In type II diabetes (NID), worsening of the diabetic retinopathy correlates with elevation of the systolic blood pressure. In type I diabetes (ID), worsening of the diabetic retinopathy correlates with an elevated diastolic blood pressure. A diastolic pressure of less than 74 mm Hg is a statistically significant protective factor against the worsening of type I diabetic retinopathy.