Human c-Myc isoforms differentially regulate cell growth and apoptosis in Drosophila melanogaster.

The human c-myc proto-oncogene, implicated in the control of many cellular processes including cell growth and apoptosis, encodes three isoforms which differ in their N-terminal region. The functions of these isoforms have never been addressed in vivo. Here, we used Drosophila melanogaster to examine their functions in a fully integrated system. First, we established that the human c-Myc protein can rescue lethal mutations of the Drosophila myc ortholog, dmyc, demonstrating the biological relevance of this model. Then, we characterized a new lethal dmyc insertion allele, which permits expression of human c-Myc in place of dMyc and used it to compare physiological activities of these isoforms in whole-organism rescue, transcription, cell growth, and apoptosis. These isoforms differ both quantitatively and qualitatively. Most remarkably, while the small c-MycS form truncated for much of its N-terminal trans-activation domain efficiently rescued viability and cell growth, it did not induce detectable programmed cell death. Our data indicate that the main functional difference between c-Myc isoforms resides in their apoptotic properties and that the N-terminal region, containing the conserved MbI motif, is decisive in governing the choice between growth and death.

Neuronal promoter of human aromatic L-amino acid decarboxylase gene directs transgene expression to the adult floor plate and aminergic nuclei induced by the isthmus.

In order to analyze the regulatory sequences involved in the neuronal expression of aromatic L-amino acid decarboxylase (AADC), we have generated transgenic mice carrying the LacZ gene under the control of a 3.6-kb human aadc genomic fragment flanking the neuronal alternative first exon. A series of double labeling experiments were performed to compare the pattern of transgene expression to that of specific markers for catecholaminergic and serotonergic neurons. In the adult brain parenchyma, transgene expression was observed in the substantia nigra (SN), the ventral tegmental area (VTA) and the dorsal, medial and pontine raphe nuclei. A large degree of co-expression was observed with tyrosine-hydroxylase (TH) in the SN and VTA, and with serotonin (5-HT) in the dorsal raphe nucleus. Moreover, expression was observed in cells that were both TH- and 5-HT-negative, in particular in the ventral tegmental decussation and the dorsal tip of the VTA. Transgene expression was also observed in the walls of central cavities. Cells positive for both beta-gal and PSA-NCAM were localized in the ventral ependyma of the third and fourth ventricle, and of the central canal of the spinal cord, in what appears to be the adult floor plate. Transgene expressing, PSA-NCAM negative, cells located along the ventral midline of the spinal cord seemed to have migrated out of the ependyma. Our data thus reveal the complexity of aadc gene regulation. The present transgene provides a unique marker for monoaminergic nuclei induced by the isthmus and for the adult floor plate.

Regulated control by granulocyte-macrophage colony-stimulating factor AU-rich element during mouse embryogenesis.

In vitro studies have indicated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression is regulated at the posttranscriptional level by the AU-rich element (ARE) sequence present in its 3' untranslated region (UTR). This study investigated the importance of the ARE in the control of GM-CSF gene expression in vivo. For this purpose, transgenic mice bearing GM-CSF gene constructs containing or lacking the ARE (GM-CSF AU(+) or GM-CSF AU(-), respectively) were generated. Both transgenes were under the transcriptional control of the immediate early promoter of the cytomegalovirus (CMV) to ensure their early, widespread, and constitutive expression. The regulation imposed by the ARE was revealed by comparing transgene expression at day 14 of embryonic development (E14); only the ARE-deleted but not the ARE-containing construct was expressed. Although GM-CSF AU(+) embryos were phenotypically normal, overexpression of GM-CSF in E14 GM-CSF AU(-) embryos led to severe hematopoietic alterations such as abnormal proliferation of granulocytes and macrophages accompanied by an increased number of peroxidase-expressing cells, their putative progenitor cells. These abnormalities compromise development because no viable GM-CSF AU(-) transgenic pups could be obtained. Surprisingly, by E18, significant accumulation of transgene messenger RNA was also observed in GM-CSF AU(+) embryos leading to similar phenotypic abnormalities. Altogether, these observations reveal that GM-CSF ARE is a developmentally controlled regulatory element and highlight the consequences of GM-CSF overexpression on myeloid cell proliferation and differentiation.

Healthy mice with an altered c-myc gene: role of the 3' untranslated region revisited.

c-Myc is a protooncogene involved in the control of cellular proliferation, differentiation and apoptosis. Like many other early response genes, regulation of c-myc expression is mainly controlled at the level of mRNA stability. Multiple cis-acting destabilizing elements have been described that are located both in the protein-coding region and in the 3' untranslated region (3' UTR). However, it is not known when they function during development and whether they act as partly redundant or independent elements to regulate c-myc mRNA level of expression. To begin to address these questions, we created a series of c-myc alleles modified in the 3' UTR, using homologous recombination and the Cre/loxP system, and analysed the consequences of these modifications in ES cells and transgenic animals. We found that deletion of the complete 3' UTR, including runs of Us and AU-rich elements proposed, on the basis of cell-culture assays, to be involved in the control of c-myc mRNA stability, did not alter the steady-state level of c-myc mRNA in any of the various situations analysed in vivo. Moreover, mice homozygous for the 3' UTR-deleted gene were perfectly healthy and fertile. Our results therefore strongly suggest that the 3' UTR of c-myc mRNA does not play a major role in the developmental control of c-myc expression.

c-myc Internal ribosome entry site activity is developmentally controlled and subjected to a strong translational repression in adult transgenic mice.

The expression of c-myc proto-oncogene, a key regulator of cell proliferation and apoptosis, is controlled at different transcriptional and posttranscriptional levels. In particular, the c-myc mRNA contains an internal ribosome entry site (IRES) able to promote translation initiation independently from the classical cap-dependent mechanism. We analyzed the variations of c-myc IRES activity ex vivo in different proliferating cell types, and in vivo in transgenic mice expressing a bicistronic dual luciferase construct. c-myc IRES efficiency was compared to that of encephalomyocarditis virus (EMCV) IRES under the same conditions. The c-myc IRES was active but with variable efficiency in all transiently transfected cell types; it was also active in the 11-day- old (E11) embryo and in some tissues of the E16 embryo. Strikingly, its activity was undetected or very low in all adult organs tested. In contrast, EMCV IRES was very active in most cell types ex vivo, as well as in embryonic and adult tissues. These data suggest a crucial role of IRES in the control of c-myc gene expression throughout development, either during embryogenesis where its activity might participate in cell proliferation or later on, where its silencing could contribute to the downregulation of c-myc expression, whose deregulation leads to tumor formation.

Fixation in endoscopic forehead plasty.

Bioabsorbable screws are a simple and cost-effective method of obtaining stable and durable fixation of brow position in endoscopic forehead plasty without permanent hardware or the need for screw removal. The author describes a technique for using this method of fixation.

Synchronous and regulated expression of two AU-binding proteins, AUF1 and HuR, throughout murine development.

The AUF1 (hnRNPD) and HuR (ELAV-like) proteins, potential trans-acting factors for regulated mRNA decay, bind in vitro to A+U-rich elements (AREs) found in the 3' untranslated region (3' UTR) of many labile transcripts. In an effort to determine whether these trans-acting factors are likely to play a role in embryogenesis, we have analysed their expression during mouse development both at the mRNA and protein levels. We show that AUF1 and HuR are expressed at all the developmental stages analysed from day 8.5 of embryonic development to adulthood. Expression levels are dynamic, varying between tissues and developmental stages. However, a strong positive correlation between AUF1 and HuR protein levels was observed in all examined tissues. Finally, we compared AUF1 and HuR expression with accumulation of one common target mRNA, c-myc. The similar spatio-temporal distribution of these proteins and of c-myc mRNA is in agreement with a potential concerted role in ARE-mediated control of mRNA stability.

Fibroblast growth factor 2 internal ribosome entry site (IRES) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation.

Fibroblast growth factor 2 (FGF-2) is a powerful mitogen involved in proliferation, differentiation, and survival of various cells including neurons. FGF-2 expression is translationally regulated; in particular, the FGF-2 mRNA contains an internal ribosome entry site (IRES) allowing cap-independent translation. Here, we have analyzed FGF-2 IRES tissue specificity ex vivo and in vivo by using a dual luciferase bicistronic vector. This IRES was active in most transiently transfected human and nonhuman cell types, with a higher activity in p53 -/- osteosarcoma and neuroblastoma cell lines. Transgenic mice were generated using bicistronic transgenes with FGF-2 IRES or encephalomyocarditis virus (EMCV) IRES. Measurements of luciferase activity revealed high FGF-2 IRES activity in 11-d-old embryos (E11) but not in the placenta; activity was high in the heart and brain of E16. FGF-2 IRES activity was low in most organs of the adult, but exceptionally high in the brain. Such spatiotemporal variations were not observed with the EMCV IRES. These data, demonstrating the strong tissue specificity of a mammalian IRES in vivo, suggest a pivotal role of translational IRES- dependent activation of FGF-2 expression during embryogenesis and in adult brain. FGF-2 IRES could constitute, thus, a powerful tool for gene transfer in the central nervous system.

lacZ sequences prevent regulated expression of housekeeping genes.

In order to dissect the MHC class I H-2K gene regulatory sequences, we p reviously generated transgenic mice containing various H-2K/lacZ fusion genes. However contrary to transgenes where H-2K sequences were fused to other coding sequences, none of the lacZ fusion transgenes was widely ex pressed like H-2K gene. We now show that this silencing also occurs when lacZ is inserted into a larger H-2K genomic construct including promoter and other regulatory elements. Because the 5'H-2K region contains a CpG island, we suspected that the presence of lacZ coding sequences was inte rfering with the mechanism by which the H-2K promoter region is normally unmethylated and transcriptionally active. Indeed, we show that in high ( >10) copy number transgenic mice, insertion of lacZ sequences in the v icinity of the H-2K promoter results in partial or complete methylation of the H-2K CpG island. However, in low (1-3) copy number transgenic mic e no methylation was observed but the transgene was still silent, sugges ting that the silencing effect of lacZ does not only rely on abnormal CpG methylation. Intriguingly, when the H -2/lacZ construct was introduced via embryonic stem (ES) cells, regulate d transgene expression was observed in several chimaeric embryos derived from independent ES clones, but never in adult chimeras. Combined with t he fact that, despite much effort, it has been very difficult to generat e 'blue' mice, our results highlight the transcription-silencing effect of lacZ sequences when they are associated with regulatory sequences of ubiquitously expressed genes.

Complex treatment of dens invaginatus type III in maxillary lateral incisor.

The complex anatomy and diagnosis of dens invaginatus make endodontic treatment of such teeth difficult. This case describes combined nonsurgical and surgical treatment of a maxillary lateral incisor with a normally shaped canal and a dens invaginatus type III with a lateroradicular lesion. The root canal was treated conventionally with gutta-percha and a zinc oxide-eugenol sealer. The root was surgically exposed and the canal of the dens invaginatus was cleaned, instrumented and obturated with gutta-percha and a zinc oxide-eugenol sealer. At follow-up 3 years 6 months later, the tooth was asymptomatic and radiographically showed repair of the lesion in the region of the dens invaginatus.

Developmental expression of AUF1 and HuR, two c-myc mRNA binding proteins.

Several proteins that may regulate c-myc mRNA post-transcriptionally were previously isolated and characterized. Two of them, HuR and AUF1, bind specifically to the 3' untranslated region (UTR) of c-myc mRNA. Because c-myc is regulated post-transcriptionally in various mouse tissues, including quiescent tissues, fetal liver and regenerating liver, we investigated whether HuR and AUF1 expression was also regulated in these tissues. Concerning AUF1, we analysed the expression of various mRNA and protein isoforms. We discovered a new AUF1 mRNA variant with a long AU-rich 3' UTR. We show that AUF1 expression, regardless of the RNA isoform considered, and HuR mRNA expression parallel c-myc expression in quiescent tissues and during liver development; their expression is high in lymphoid tissues and fetal liver and low in adult liver. However, no upregulation of HuR or AUF1 accompanies the upregulation of c-myc mRNA following partial hepatectomy. We discuss our results in relation to the current hypothesis that HuR and AUF1 act as mRNA destabilizing factors.

An introduction to ambulatory surgery facility accreditation.

"Managing Your OR" focuses on various aspects of aesthetic surgery in the ambulatory surgical setting.

Patient safety in accredited office surgical facilities.

The medical profession is besieged by concerns about cost containment. This in turn has focused attention on the use of ambulatory surgical facilities. However, the costs of hospital outpatient surgery programs usually prevent them from being competitive when compared with the costs of using office surgical facilities. To address the question of patient safety in office surgical facilities, the American Association for Accreditation of Ambulatory Surgery Facilities (AAAASF) sent a questionnaire to its accredited facilities. Two-hundred and forty-one (57.7 percent) of the 418 accredited facilities returned the anonymous questionnaires, a very high response rate. Or interest are the following findings: 400,675 operative procedures were reported during a 5-year period. Significant complications (hematoma, hypertensive episode, wound infection, sepsis, hypotension) were infrequent, occurring in 1 in every 213 cases. Return to the operating room within 24 hours and preventive hospitalization were less frequent. A death occurred in 1 in 57,000 cases (0.0017 percent). The overall risk is comparable in an accredited office (plastic surgical facility) and in a free-standing or hospital ambulatory surgical facility. This study documents an excellent safety record for plastic surgery done in accredited office surgical facilities by board-certified plastic surgeons.

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation.

Genomic structure and precise mapping of a thymic regulatory region on mouse chromosome 17 revealed by a c-myc transgene insertion.

In one transgenic strain harboring a human c-myc proto-oncogene construct, the transgene was actively and exclusively expressed in the thymus, where it contributed to the development of lymphoma that corresponded to CD4(+)CD8(+) cells. Here, we have pursued the analysis of transgene expression in healthy transgenic mice and show that transgene activation occurs in the thymus 3 days before birth, at a time when CD4(+)CD8(+) lymphocytes emerge. In the adult, its expression is restricted to the CD4(+)CD8(+) cells. The region flanking the transgene insertion site was isolated and made it possible to map the preintegration locus, hereafter called Tsil (for thymus-specific integration locus) on chromosome 17 between D17Rp11e and Ras12-3. A YAC that contains both Tsil and the Pim2 locus, previously shown to be involved in progression of T-cell lymphoma, was isolated. Analysis of Tsil offers a unique opportunity to identify a regulatory region or a gene that might play an important role in T-cell maturation.

Liver regeneration 7. Prometheus' myth revisited: transgenic mice as a powerful tool to study liver regeneration.

In this review, we present examples of the contribution of transgenic mice to our knowledge concerning the type of cells that are able to repopulate a damaged liver and information on the factors and mechanisms involved in postnatal liver growth and regeneration. The transgenic technology offers the opportunity to evaluate the physiological consequences of perturbating expression of a given gene in vivo. It has provided insights into the concerted action of extracellular (HGF/SF, TGF-alpha, EGF, TGF-beta) and intracellular factors (c-myc, c-fos, c-jun, p53, c-met, and others) in liver regeneration. Transgenic mice can also contribute to the dissection of the molecular mechanisms responsible for the regulated expression of these factors, both at the transcriptional and the posttranscriptional level. An illustration of such a strategy is given by the study of the sequences involved in the posttranscriptional regulation of the c-myc proto-oncogene. The recent improvement of gene targeting, in which endogenous genes are inactivated by homologous recombination, represents a further step toward the study of the function of a particular gene. Inactivation of most of the factors described in this review has been undertaken. However, further studies of their role in liver growth control are impeded by the fact that the corresponding knockout mice die prematurely. This problem could be overcome by the advent of new techniques, which will be briefly presented, aimed at turning genes on and off at will and in a tissue-specific manner.

Developmental expression of H-2K major histocompatibility complex class I transgenes requires the presence of proximal introns.

The pattern of expression of the H-2K major histocompatibility complex (MHC) class I gene is complex. During embryonic development H-2K mRNA, detectable from midgestation, is poorly expressed. In the adult, H-2K expression is nearly ubiquitous but transcriptional regulation occurs leading to different mRNA levels in the different organs of the mouse. In vitro studies have shown that most of the regulatory elements controlling H-2K gene transcription are located in the 5' region of the gene. However, using fusion transgenes in which reporter genes were under the control of 2 kb of H-2K 5' regulatory region, we have previously shown that this region was not sufficient to ensure correct developmental transgene expression. By contrast, a native 9.25 kb H-2K transgene was expressed appropriately both in the adult and in the embryo. In order to localise more precisely the cis-acting regulatory sequences involved in H-2K developmental expression, we have now constructed new transgenic lines containing H-2Kb transgenes that were deleted from specific parts of the H-2Kb gene. We show that deletion of either the H-2K 3' flanking region or the 5 (out of 7) distal introns results in an expression of the transgenes which is similar to that of the endogenous H-2K gene, both in the adult and during embryonic development. By contrast, deletion of all the introns or of the two proximal ones abrogates H-2K transgene expression. Our data reveal the complexity of H-2K regulation and highlight the crucial role of proximal introns in H-2K expression in the living organism.

Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition.

In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2Kb class I regulatory sequences, we have shown that the 5' and the 3' noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3' untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation.

Characterization of the 5' region of the CD10/neutral endopeptidase 24.11 gene.

We have characterized the 5' region of the CALLA/CD10 gene which has been shown to be identical to the membrane-associated enzyme neutral endopeptidase 24.11 (NEP). There is no CAAT or TATA box in the 5' flanking region, upstream of exon 1, but a GC rich region with several Sp1 binding sites. We have detected several putative initiation transcription sites by primer extension and by nuclease S1 analysis. Moreover by reverse transcriptase-polymerase chain reaction, we demonstrated the existence of a new exon: exon 1bis. This exon can be alternatively spliced as has already been described for exon 1 and exon 2.