miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

The canine baculum: the structure and mechanical properties of an unusual bone.

The baculum is an extraskeletal bone located in the penis of a few species in several orders of mammals such as carnivores, insectivores, rodents, bats and primates. This study aims to describe the structure, architecture and mechanical properties of the canine baculum. To this end canine bacula from castrated and uncastrated dogs were collected and examined by light microscopy, micro-computed tomography (microCT) scanning, histological staining, and mechanical testing. Their mineral density and mechanical properties were compared with those of a typical skeletal bone (the radius) in the same dog. Furthermore, a numerical model of a representative baculum was created and its mechanical performance analyzed using the finite element method, in order to try to elucidate its function. Examination of light microscopy images of transverse sections shows that the baculum consists of a typical sandwich structure, with two cortical plates separated, and joined, by loose cancellous bone. MicroCT scans reveal that the mineral density is lower in the baculum than in the radius, both in castrated as well as in uncastrated dogs, resulting in much lower stiffness. Castration was found to decrease the mineral density in both the baculum and the radius. The most likely function of the baculum of the dog is to stiffen the penis to assist intromission, and its much lower mineral density compared to that of the radius may be a mechanism designed to decrease the stiffness somewhat, and thus reduce the risk of fracture during copulation.

Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene.

Transplantation of muscle precursor cells (MPCs) is a promising approach for the treatment of muscular dystrophies. However, preclinical and clinical results have shown that the technology is not yet efficient enough for most therapeutic applications. Among the problems that remain unsolved are low cellular survival, poor proliferation and lack of migration of the transplanted cells. One major technical hurdle for the optimization of transplantation protocols is how to follow precisely the fate of the cells after transplantation. In this study, we examined the use of a secreted form of the mouse alkaline phosphatase (mSeAP) enzyme as the reporter system transduced into MPCs using a retroviral vector. We show that circulating mSeAP could be detected in the serum of the transplanted mice at different time points after MPC transplantation. We also found that the level of circulating mSeAP is highly correlated with the number of transplanted cells and that mSeAP is an excellent histological marker. Further, studying the levels of circulating mSeAP compared with the number of muscle fibers positive to mSeAP and to dystrophin, enabled detailed analyses of bottleneck steps for successful transplantation. Taken together, our results show that mSeAP is an excellent quantitative 'real-time' reporter gene for cell therapy preclinical studies.

Myogenic program induction in mature fat tissue (with MyoD expression).

MyoD exerts a master transcriptional control over the myogenic differentiation cascade. Here, we study different approaches to induce myogenic transdifferentiation in mature adipocytes utilizing MyoD gene transfer. Organotypic cultures of fat tissue and a long-term culture of in vitro differentiated adipocytes deduced that MyoD provoked morphological changes in mature adipocytes that can be summarized as loss of fat content, acquisition of a fusiform shape and eventual fusion with committed neighbor cells. In vivo, MyoD gene transfer into rat interscapular and inguinal fat pads demonstrated that while structural proteins of muscle lineage were expressed, they co-existed with specific adipocyte proteins. Expression of these proteins diminished over time likewise the fat content. The transdifferentiation process initiated by MyoD did not require cell cycle progression and was well tolerated by the fully differentiated and mature adipocytes.

[Retinal injury induced by laser pointers].

Laser pointers originally designed for use during presentations are ubiquitous and are even sold as toys (such as pens or on key chains) in drug stores. Though reported as safe, the laser pointers still carry the risk of potential damage to the eye. We report a 16-year-old boy with bilateral retinal injury caused by 20-30 seconds of exposure to a laser pointing-device. Immediately thereafter, vision was blurred bilaterally and he noted a central red scotoma in each eye. Symptoms resolved spontaneously within 2 days but the retinal scars remained all during the 10 months of follow-up. It is clear from our report and 3 other publications that retinal damage can develop from misusing laser pointers. Laser hazards and safety should be stressed for the general public. We recommend that laser-pointers should not be available as toys to children and teenagers.

Association of the C677T polymorphism in the MTHFR gene with breast and/or ovarian cancer risk in Jewish women.

The C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with reduced enzyme activity, hyperhomocysteinaemia and increased risk for atherosclerosis in homozygotes. We examined the frequency of this mutation and its association with disease pattern in 491 Jewish women with either sporadic (n = 355; 72%) or hereditary (n = 136; 28%) breast and/or ovarian cancer and in 69 asymptomatic BRCA1/2 mutation carriers, genotyped for the three predominant Jewish founder BRCA1/2 mutations (185delAG, 5382insC and 6174delT). 677T homozygotes were equally distributed among women with sporadic breast and/or ovarian cancer (71/355; 20.0%) and among BRCA1/2 mutation carriers (43/205; 21.0%) (P=non-significant). 677T homozygotes were equally distributed among women diagnosed with breast cancer prior to (22/122; 18.0%) and after 42 years of age (42/243; 17.3%). Among BRCA1/2 carriers, the rate of 677T homozygotes in manifesting cancer (32/136; 23.5%) and asymptomatic individuals (11/69; 15.9%) was not significantly different. The rate of 677T homozygotes (24/72; 33.3%) was higher (P=0.0026) among women with bilateral breast cancer and those with both breast and ovarian carcinoma than among those with unilateral breast cancer (64/365; 17.5%). Differences in morbidity (one versus multiple breast/ovarian tumours) are mainly attributed to 677T homozygosity and partly to BRCA1/2 mutations. Confirmation of these data, namely, that the 677T allele is significantly more common in cases of bilateral breast cancer or combined breast and ovarian cancer would have important clinical implications.

Behavioral alterations associated with apoptosis and down-regulation of presenilin 1 in the brains of p53-deficient mice.

Presenilin 1 (PS1) expression is repressed by the p53 tumor suppressor. As shown herein, wild-type PS1 is an effective antiapoptotic molecule capable of significantly inhibiting p53-dependent and p53-independent cell death. We analyzed, at the functional and molecular levels, the brains of p53 knockout mice. Surprisingly, we found that lack of p53 expression induces apoptotic brain lesions, accompanied by learning deficiency and behavioral alterations. p53-deficient mice show an unexpected overexpression of p21(waf1) with subsequent down-regulation of PS1 in their brains. This process is progressive and age-dependent. These data indicate that the p53 pathway, besides affecting tumor suppression, may play a major role in regulating neurobehavioral function and cell survival in the brain.

SIAH-1 promotes apoptosis and tumor suppression through a network involving the regulation of protein folding, unfolding, and trafficking: identification of common effectors with p53 and p21(Waf1).

We have previously described biological model systems for studying tumor suppression in which, by using H-1 parvovirus as a selective agent, cells with a strongly suppressed malignant phenotype (KS or US) were derived from malignant cell lines (K562 or U937). By using cDNA display on the K562/KS cells, 15 cDNAs were now isolated, corresponding to genes differentially regulated in tumor suppression. Of these, TSAP9 corresponds to a TCP-1 chaperonin, TSAP13 to a regulatory proteasome subunit, and TSAP21 to syntaxin 11, a vesicular trafficking molecule. The 15 cDNAs were used as a molecular fingerprint in different tumor-suppression models. We found that a similar pattern of differential regulation is shared by activation of p53, p21(Waf1), and the human homologue of Drosophila seven in absentia, SIAH-1. Because SIAH-1 is differentially expressed in the various models, we characterized it at the protein and functional levels. The 32-kDa, mainly nuclear protein encoded by SIAH-1, can induce apoptosis and promote tumor suppression. These results suggest the existence of a common mechanism of tumor suppression and apoptosis shared by p53, p21(Waf1), and SIAH-1 and involving regulation of the cellular machinery responsible for protein folding, unfolding, and trafficking.

Activation of p53 and its target genes p21(WAF1/Cip1) and PAG608/Wig-1 in ischemic preconditioning.

A brief, 3 min period of global forebrain ischemia in the rat, induced by bilateral common carotid occlusion combined with hypotension, confers resistance to hippocampal pyramidal neurons against a subsequent 10 min ischemia, which is normally lethal to these cells. The molecular mechanisms underlying this ischemic preconditioning, or tolerance, are poorly understood. The tumor suppressor p53 is a transcription factor implicated in neuronal death following various insults, including cerebral ischemia. p53 is activated in response to cellular stress, e.g. hypoxia and DNA damage. Using in situ hybridization, we investigated the hippocampal mRNA expression of p53, and two of its target genes, p21(WAF1/Cip1) and the recently cloned PAG608/Wig-1, in a two-vessel occlusion model of ischemic preconditioning. We also evaluated changes in the protein levels of p53 and PAG608/Wig-1 using immunohistochemistry. The mRNA levels of all three genes increased in the ischemia sensitive CA1 region both following 3 min (non-lethal) preconditioning and 10 min of (lethal) nonconditioned ischemia. In contrast, after 10 min of ischemia preconditioned by a 3 min ischemic insult 48 h earlier, no upregulation of these genes was detected in the CA1. Following 10 min of nonconditioned ischemia, increased neuronal immunostaining of p53 and PAG608/Wig-1 was observed in the hippocampus, which was less pronounced following 3 min of preconditioning ischemia and 10 min of preconditioned ischemia. Our results demonstrate that activation of p53 and its response genes p21(WAF1/Cip1) and PAG608/Wig-1 occurs in the brain following lethal as well as non-lethal ischemic insults, and that ischemic preconditioning markedly diminishes this activation.

The c-fos proto-oncogene is a target for transactivation by the p53 tumor suppressor.

The p53 tumor suppressor gene is mutated in over 50% of human cancers, resulting in inactivation of the wild-type (wt) p53 protein. The most notable biochemical feature of p53 is its ability to act as a sequence-specific transcriptional activator. Through use of the suppression subtractive hybridization differential screening technique, we identified c-fos as a target for transcriptional stimulation by p53 in cells undergoing p53-mediated apoptosis. Overexpression of wt p53 induces c-fos mRNA and protein. Moreover, in vivo induction of c-fos in the thymus following whole-body exposure to ionizing radiation is p53 dependent. p53 responsiveness does not reside in the basal c-fos promoter. Rather, a distinct region within the c-fos gene first intron binds specifically to p53 and confers upon the c-fos promoter the ability to become transcriptionally activated by wt p53. Identification of c-fos as a specific target for transcriptional activation by p53 establishes a direct link between these two pivotal regulatory proteins and raises the possibility that c-fos contributes to some of the biological effects of p53.

Computerised dystrophic muscle simulator: prospecting potential therapeutic strategies for muscle dystrophies using a virtual experimental model.

Inherited muscle diseases are often characterised by widespread muscle damage in the body, limiting the clinical relevance of cell or gene therapy based upon direct injections into muscles. Recent studies have shown, however, that cells originating from the bone marrow are able to target necrosis-regeneration sites as they occur and, in addition, may also participate in the muscle regeneration after undergoing myogenic differentiation. Here, we present a computerised dystrophic muscle simulator that allows the prospecting of different scenarios of both disease evolution and appropriate employment of blood-borne cells as therapeutic shuttles. It provides the option of examining their use either to transfer a healthy gene into the tissue or to impart substances designed to boost its regeneration. One of the major advantages of this tool is that it offers the opportunity of visualising and composing therapeutic strategies in virtual paradigms in which severe clinical situations, not necessarily available in animal models, can be created. The dystrophic muscle simulator is freely accessible via the Genethon web site (www.genethon.fr), and in the online version via http:@www.wiley.co.uk/genmed.

p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs.

Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wild-type (wt), p53 mutant (mt), and p53 deficient (p53(-/-)) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability of p53(-/-) Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene. wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.

Paranasal implants for correction of midface concavity.

Central midface concavity was corrected with the placement of porous polyethylene implants in the paranasal area. This simulated the effect of skeletal osteotomies and advancement without altering dental occlusion. Implants were placed to correct congenital, posttraumatic, and cleft-related skeletal midface retrusion in nine patients. In seven of the patients, paranasal augmentation was performed in conjunction with rhinoplasty. There have been no implant-related complications during a mean 33-month follow-up (range 5 to 83 months). Screw fixation of these implants ensures stable positioning and allows precise final contouring during surgery.

Inhibition of presenilin 1 expression is promoted by p53 and p21WAF-1 and results in apoptosis and tumor suppression.

Previously, we cloned a cDNA fragment, TSIP 2 (tumor suppressor inhibited pathway clone 2), that detects by northern blot analysis of M1-LTR6 cells a 3-kb mRNA downregulated during p53-induced apoptosis. Cloning the full-length TSIP 2 cDNA showed that it corresponds to the presenilin 1 (PS1) gene, in which mutations have been reported in early-onset familial Alzheimer's disease. Here we demonstrate that PS1 is downregulated in a series of model systems for p53-dependent and p53-independent apoptosis and tumor suppression. To investigate the biological relevance of this downregulation, we stably transfected U937 cells with antisense PS1 cDNA. The downregulation of PS1 in these U937 transfectants results in reduced growth with an increased fraction of the cells in apoptosis. When injected into mice homozygous for severe combined immunodeficiency disease (scid/scid mice), these cells show a suppression of their malignant phenotype. Our results indicate that PS1, initially identified in a neurodegenerative disease, may also be involved in the regulation of cancer-related pathways.

Use of bilateral folded radial forearm free flaps for reconstruction of a midface gunshot wound.

Optimal treatment of midfacial gunshot wounds includes early definitive reconstruction of the bony scaffold to prevent soft tissue contraction. When this is not possible, secondary reconstruction is more difficult. The authors present a case of delayed reconstruction of a midface gunshot wound. Two months following a self-inflicted, submental gunshot wound and eventual rigid fixation of the remaining midfacial bony anatomy, two simultaneous radial forearm free flaps were utilized in the reconstruction. The first flap was folded onto itself to recreate the hard palate in conjunction with a split rib graft; the second flap filled the remaining soft-tissue defect and simultaneously provided lining for an eventual staged nasal reconstruction. The second stage of the nasal reconstruction was completed 5 weeks later with a calvarial bone graft and forehead flap. This dual microsurgical approach allowed for one-step reconstruction of both surfaces of the hard palate, resulting in separate oral and nasal cavities, and optimizing the patient's ability to speak and eat. Extensive soft-tissue contraction encountered in late reconstructions underscores the importance of an early, definitive, surgical approach in these difficult wounds.

A novel p53-inducible gene, PAG608, encodes a nuclear zinc finger protein whose overexpression promotes apoptosis.

The biological effects of the p53 tumor suppressor protein are elicited, at least in part, through sequence-specific transactivation of a battery of target genes. The differential display method was employed towards identifying additional p53 target genes, with emphasis on genes whose induction may contribute to p53-mediated apoptosis. We report here the cloning of a novel p53-inducible gene, designated PAG608. PAG608 transcripts are induced by DNA damage in a p53-dependent manner. PAG608 encodes a nuclear zinc finger protein, which appears to localize preferentially to nucleoli when expressed at moderate levels in transfected cells. Transient overexpression of PAG608 in human tumor-derived cells leads to distinctive changes in nuclear morphology, and can promote apoptosis. Together with additional p53 target genes, PAG608 may therefore play a role in mediating the biological activities of p53.

Partial sequencing of the rat steroidogenic acute regulatory protein message from immortalized granulosa cells: regulation by gonadotropins and isoproterenol.

Steroidogenic acute regulatory protein (StAR), a 30-kDa protein involved in the transport of cholesterol to inner mitochondrial membrane during stimulation of steroid hormone biosynthesis, has recently been cloned from human adrenals and MA-10 mouse Leydig tumor cells. We examined the regulation of StAR mRNA accumulation upon induction of steroidogenesis in immortalized rat granulosa cells. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene and LH/CG receptor (GLHR15) or with FSH receptor (GFSHR17) or with the beta 2-adrenergic receptor (G beta 2AR13) expression plasmids. Cells were cultured to confluency and then stimulated for 24 h with oFSH (4 nM), hCG (2.4 nM), isoproterenol (10 microM) or forskolin (50 microM). By quantitative RT-PCR, StAR mRNA was undetectable in non-steroidogenic cells (transfected with SV40 DNA alone, POGS5) either in the presence or in the absence of forskolin. In contrast, variable amount of the message was detected in all steroidogenic cell lines cotransfected with SV40 DNA and Ha-ras. Moreover, an increase in the StAR mRNA expression was evident in all steroidogenic cells upon stimulation with their respective agonists, concomitantly with enhanced progesterone production. The RT-PCR product was sequenced and the 379 base pairs of rat StAR were found to be 93% and 86% identical to mouse and human cDNA, respectively. The deduced 126 amino acid sequence was 95%, 88% and 88% identical to the mouse, human and bovine deduced protein sequences. We conclude that StAR message is expressed only in the steroidogenic rat granulosa cells and can be upregulated by FSH, hCG, isoproterenol and forskolin in the appropriate cell lines. In addition, we find that the rat StAR cDNA exhibit a high degree of homology with the mouse and human sequences.

Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression.

Developmentally regulated genes in Drosophila, which are conserved through evolution, are potential candidates for key functions in biological processes such as cell cycle, programmed cell death, and cancer. We report cloning and characterization of the human homologue of the Drosophila seven in absentia gene (HUMSIAH), which codes for a 282 amino acids putative zinc finger protein. HUMSIAH is localized on human chromosome 16q12-q13. This gene is activated during the physiological program of cell death in the intestinal epithelium. Moreover, human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA. A similar pattern of expression is also displayed by the p21waf1. These results suggest that mammalian seven in absentia gene, which is a target for activation by p53, may play a role in apoptosis and tumor suppression.