Algunos aspectos del manejo del paciente quemado en un servicio de cirugía infantil: A propósito de 47 pacientes pediátricos / Some aspects burned patient management in a department of surgery child: About 47 pediatric patients

Si bien el manejo inicial de los niños quemados es fundamental para el resultado exitoso del tratamiento de esta cohorte de pacientes, el aspecto quirúrgico en el siguiente nivel tiene una importancia similar. En este estudio se presenta un análisis epidemiológico de 47 pacientes, tratados en nuestro Servicio en un período de dos años. Se esboza un análisis de los datos estadísticos obtenidos, se sacan conclusiones y se elaboran normas de profilaxis.

While the initial management of burned children is fundamental to the successful outcome of treatment of this cohort of patients, surgical appearance at the next level has an equal importance. This study provides an epidemiological analysis of 47 patients treated in our department in a period of two years, outlined an analysis of statistical data obtained, conclusions drawn and develop standards for prophylaxis.

Expression of an Nkx3.1-CRE gene using ROSA26 reporter mice.

The genetic locus of Nkx3.1, an early murine marker of sclerotome and prostate development, was disrupted by a knock in of CRE recombinase via homologous recombination in embryonic stem cells. Cell fate mapping revealed previously unidentified cell lineages expanded from Nkx3.1-expressing cell populations and recapitulated reported Nkx3.1 expression patterns. In lineage trace experiments of E18.5 Nkx3.1-CRE; R26R embryos novel staining was observed in areas of the lungs, portions of the duodenum, and vertebral elements of the skeleton. beta-galactosidase activity measured in Nkx3.1-CRE; R26R and Nkx3.2-CRE; R26R embryos was observed in overlapping regions of the sclerotome but no apparent change in Nkx3.1 expression was seen in the Nkx3.2 mutants by in situ hybridization.

Regulation of organ development by the NKX-homeodomain factors: an NKX code.

Mammalian development is a highly coordinated process that involves sequential and time-dependent gene regulation. Deregulation of this process can have functional or morphological consequences, possibly causing lethality or organ dysfunction. Homeotic genes are considered the master regulators of early developmental processes. Of the many homeodomain genes, the NK2 class represents a family of phylogenetically ancient proteins. NK2 homeobox family members are tissue-specific transcription factors distinguished by a common DNA binding structure unique among the homeodomain genes. Increasing evidence indicates that individual Nkx factors are critical regulators of whole organ development. In the sections below, we review the structure, regulation, and expression of the NK2 gene family beginning with their discovery in Drosophila and relating the known features of vertebrate counterparts to the Drosophilaproteins. In particular, we note that each of the vertebrate NK2 proteins are associated with particular genetic anomalies leading to a variety of described disease states. Further, based upon our examination we propose a new paradigm of development based upon the regulatory capacity of the NK2 homeodomain proteins termed the "Nkx Code"

Decreased expression of smooth muscle alpha-actin results in decreased contractile function of the mouse bladder.

PURPOSE: Smooth muscle alpha-actin (SMalphaA) is an important actin isoform for functional contractility in the mouse bladder. Alterations in the expression of SMalphaA have been associated with a variety of bladder pathological conditions. Recently, a SMalphaA-null mouse was generated and differences in vascular tone and contractility were observed between wild-type and SMalphaA-null mice suggesting alterations in function of vascular smooth muscle. We used SMalphaA-null mice to explore the hypothesis that SMalphaA is necessary for normal bladder function. MATERIALS AND METHODS: Reverse transcriptase polymerase chain reaction, Western blotting and immunohistochemical staining were used to confirm the absence of SMalphaA transcript and protein in the bladder of SMalphaA-null mice. In vitro bladder contractility compared between bladder rings harvested from wild-type and SMalphaA-null mice was determined by force measurement following electrical field stimulation (EFS), and exposure to chemical agonists and antagonists including KCl, carbachol, atropine and tetrodotoxin. Resulting force generation profiles for each tissue and agent were analyzed. RESULTS: There was no detectable SMalphaA transcript and protein expression in the bladder of SMalphaA-null mice. Nine wild-type and 9 SMalphaA-null mice were used in the contractility study. Bladders from SMalphaA-null mice generated significantly less force than wild-type mice in response to EFS after KCl. Similarly, bladders from SMalphaA-null mice generated less force than wild-type mice in response to pretreatment EFS, and EFS after carbachol and atropine, although the difference was not significant. Surprisingly, the bladders in SMalphaA-null mice appeared to function normally and showed no gross or histological abnormalities. CONCLUSIONS: SMalphaA appears to be necessary for the bladder to be able to generate normal levels of contractile force. No functional deficits were observed in the bladders of these animals but no stress was placed on these bladders. To our knowledge this study represents the first report to demonstrate the importance of expression of SMalphaA in force generation in the bladder.

Growth and tissue accretion rates of swine expressing an insulin-like growth factor I transgene.

The goal of this research was to determine whether directing expression of an insulin-like growth factor I (IGF-I) transgene specifically to striated muscle would alter the growth characteristics in swine. Transgenic pigs were produced with a fusion gene composed of avian skeletal alpha-actin regulatory sequences and a cDNA encoding human IGF-I. Six founder transgenic pigs were mated to nontransgenic pigs to produce 11 litters of G1 transgenic and sibling control progeny. Birth weight, weaning weight, and proportion of pig survival did not differ between transgenic and control pigs. The ADG of pigs as they grew incrementally from 20 to 60 kg, 60 to 90 kg, and 90 to 120 kg, respectively, did not significantly differ between transgenic and control pigs. Efficiency of feed utilization (gain:feed) was also similar for transgenic and control pigs. Plasma IGF-I and porcine growth hormone (pGH) concentrations were determined at 60, 90, and 120 kg body weight. Plasma IGF-I concentrations were 19% higher in transgenic gilts than control gilts and 11.1% higher in transgenic boars than control boars (P=0.0005). Plasma IGF-I concentrations for boars were also higher than for gilts (P=0.0001). At 60, 90, and 120 kg body weight each pig was scanned by dual energy X-ray absorptiometry (DXA) to derive comparative estimates of carcass fat, lean, bone content of the live animal. Control pigs had more fat and less lean tissue than transgenic pigs at each of the scanning periods and the difference became more pronounced as the pigs grew heavier (P<0.005 at each weight). Transgenic pigs also had a slightly lower percentage of bone than control pigs (P<0.05 at each weight). While daily rates of lean tissue accretion did not differ for transgenic and control pigs, daily rates of fat accretion were lower in transgenic pigs than in control pigs (P<0.05). Based on these results we conclude that expression of IGF-I in the skeletal muscles gradually altered body composition as pigs became older but did not have a major affect on growth performance.

Rho kinases play an obligatory role in vertebrate embryonic organogenesis.

Rho-associated kinases (Rho kinases), which are downstream effectors of RhoA GTPase, regulate diverse cellular functions including actin cytoskeletal organization. We have demonstrated that Rho kinases also direct the early stages of chick and mouse embryonic morphogenesis. We observed that Rho kinase transcripts were enriched in cardiac mesoderm, lateral plate mesoderm and the neural plate. Treatment of neurulating embryos with Y27632, a specific inhibitor of Rho kinases, blocked migration and fusion of the bilateral heart primordia, formation of the brain and neural tube, caudalward movement of Hensen's node, and establishment of normal left-right asymmetry. Moreover, Y27632 induced precocious expression of cardiac alpha-actin, an early marker of cardiomyocyte differentiation, coincident with the upregulated expression of serum response factor and GATA4. In addition, specific antisense oligonucleotides significantly diminished Rho kinase mRNA levels and replicated many of the teratologies induced by Y27632. Thus, our study reveals new biological functions for Rho kinases in regulating major morphogenetic events during early chick and mouse development.

Long-term insulin-like growth factor-I expression in skeletal muscles attenuates the enhanced in vitro proliferation ability of the resident satellite cells in transgenic mice.

Insulin-like growth factor-I (IGF-I) overexpression for 1-month in mouse skeletal muscle increases satellite cell proliferation potential. However, it is unknown whether this beneficial enhancement by IGF-I expression would persist over a longer-term duration in aged mice. This is an important issue to address if a prolonged course of IGF-I is to be used clinically in muscle-wasting conditions where satellite cells may become limiting. Using the IGF-I transgenic (IGF-I Tg) mouse that selectively expresses the IGF-I transgene in striated muscles, we found that 18-months of continuous IGF-I overexpression led to a loss in the enhanced in vitro proliferative capacity of satellite cells from Tg skeletal muscles. Also 18-month-old IGF-I Tg satellite cells lost the enhanced BrdU incorporation, greater pRb and Akt phosphorylations, and decreased p27(Kip1) levels initially observed in cells from 1-month-old IGF-I Tg mice. The levels of those biochemical markers reverted to similar values seen in the 18-months WT littermates. These findings, therefore, suggest that there is no further beneficial effect on enhancing satellite cell proliferation ability with persistent long-term expression of IGF-I in skeletal muscles of these transgenic mice.

beta1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal alpha-actin promoter.

Activation of RhoA GTPase causes actin filament bundling into stress fibers, integrin clustering, and focal adhesion formation through its action on actin cytoskeleton organization. RhoA also regulates transcriptional activity of serum response factor (SRF). Recent studies in NIH 3T3 fibroblasts have shown that SRF activation by RhoA does not require an organized cytoskeleton and may be regulated by G-actin level. In cardiac myocytes, the organization of actin fibers into myofibrils is one of the primary characteristics of cardiac differentiation and hypertrophy. The primary purpose of this study was to examine if RhoA regulates SRF-dependent gene expression in neonatal cardiomyocytes in a manner different from that observed in fibroblasts. Our results show that RhoA-dependent skeletal alpha-actin promoter activation requires beta1 integrin and a functional cytoskeleton in cardiomyocytes but not in NIH 3T3 fibroblasts. Activation of the alpha-actin promoter by RhoA is greatly potentiated (up to 15-fold) by co-expression of the integrin beta1A or beta1D isoform but is significantly reduced by 70% with a co-expressed dominant negative mutant of beta1 integrin. Furthermore, clustering of beta1 integrin with anti-beta1 integrin antibodies potentiates synergistic RhoA and beta1 integrin activation of the alpha-actin promoter. Cytochalasin D and latrunculin B, inhibitors of actin polymerization, significantly reduced RhoA-induced activation of the alpha-actin promoter. Jasplakinolide, an actin polymerizing agent, mimics the synergistic effect of RhoA and beta1 integrin on the actin promoter. These observations support the concept that RhoA regulates SRF-dependent cardiac gene expression through cross-talk with beta1 integrin signal pathway via an organized actin cytoskeleton.

Physical interaction between the MADS box of serum response factor and the TEA/ATTS DNA-binding domain of transcription enhancer factor-1.

Serum response factor is a MADS box transcription factor that binds to consensus sequences CC(A/T)(6)GG found in the promoter region of several serum-inducible and muscle-specific genes. In skeletal myocytes serum response factor (SRF) has been shown to heterodimerize with the myogenic basic helix-loop-helix family of factors, related to MyoD, for control of muscle gene regulation. Here we report that SRF binds to another myogenic factor, TEF-1, that has been implicated in the regulation of a variety of cardiac muscle genes. By using different biochemical assays such as affinity precipitation of protein, GST-pulldown assay, and coimmunoprecipitation of proteins, we show that SRF binds to TEF-1 both in in vitro and in vivo assay conditions. A strong interaction of SRF with TEF-1 was seen even when one protein was denatured and immobilized on nitrocellulose membrane, indicating a direct and stable interaction between SRF and TEF-1, which occurs without a cofactor. This interaction is mediated through the C-terminal subdomain of MADS box of SRF encompassing amino acids 204-244 and the putative 2nd and 3rd alpha-helix/beta-sheet configuration of the TEA/ATTS DNA-binding domain of TEF-1. In the transient transfection assay, a positive cooperative effect of SRF and TEF-1 was observed when DNA-binding sites for both factors, serum response element and M-CAT respectively, were intact; mutation of either site abolished their synergistic effect. Similarly, an SRF mutant, SRFpm-1, defective in DNA binding failed to collaborate with TEF-1 for gene regulation, indicating that the synergistic trans-activation function of SRF and TEF-1 occurs via their binding to cognate DNA-binding sites. Our results demonstrate a novel association between SRF and TEF-1 for cardiac muscle gene regulation and disclose a general mechanism by which these two super families of factors are likely to control diversified biological functions.

GATA-4 and serum response factor regulate transcription of the muscle-specific carnitine palmitoyltransferase I beta in rat heart.

Transcriptional regulation of nuclear encoded mitochondrial proteins is dependent on nuclear transcription factors that act on genes encoding key components of mitochondrial transcription, replication, and heme biosynthetic machinery. Cellular factors that target expression of proteins to the heart have been well characterized with respect to excitation-contraction coupling. No information currently exists that examines whether parallel transcriptional mechanisms regulate nuclear encoded expression of heart-specific mitochondrial isoforms. The muscle CPT-Ibeta isoform in heart is a TATA-less gene that uses Sp-1 proteins to support basal expression. The rat cardiac fatty acid response element (-301/-289), previously characterized in the human gene, is responsive to oleic acid following serum deprivation. Deletion and mutational analysis of the 5'-flanking sequence of the carnitine palmitoyltransferase Ibeta (CPT-Ibeta) gene defines regulatory regions in the -391/+80 promoter luciferase construct. When deleted or mutated constructs were individually transfected into cardiac myocytes, CPT-I/luciferase reporter gene expression was significantly depressed at sites involving a putative MEF2 sequence downstream from the fatty acid response element and a cluster of heart-specific regulatory regions flanked by two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and competition specificity (or supershifts) with oligonucleotides and antibodies. Individual expression vectors for Nkx2.5, serum response factor (SRF), and GATA4 enhanced CPT-I reporter gene expression 4-36-fold in CV-1 cells. Although cotransfection of Nkx and SRF produced additive luciferase expression, the combination of SRF and GATA-4 cotransfection resulted in synergistic activation of CPT-Ibeta. The results demonstrate that SRF and the tissue-restricted isoform, GATA-4, drive robust gene transcription of a mitochondrial protein highly expressed in heart.

Embryonic expression of an Nkx2-5/Cre gene using ROSA26 reporter mice.

Nkx2-5, one of the earliest cardiac-specific markers in vertebrate embryos, was used as a genetic locus to knock in the Cre recombinase gene by homologous recombination. Offspring resulting from heterozygous Nkx2-5/Cre mice mated to ROSA26 (R26R) reporter mice provided a model system for following Nkx2-5 gene activity by beta-galactosidase (beta-gal) activity. beta-gal activity was initially observed in the early cardiac crescent, cardiomyocytes of the looping heart tube, and in the epithelium of the first pharyngeal arch. In later stage embryos (10.5-13.5 days postcoitum, dpc), beta-gal activity was observed in the stomach and spleen, the dorsum of the tongue, and in the condensing primordium of the tooth. The Nkx2-5/Cre mouse model should provide a useful genetic resource to elucidate the role of loxP manipulated genetic targets in cardiogenesis and other developmental processes.

Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2.

Expression patterns of Tbx2, -3, and -5 genes were analyzed during chick embryonic heart development. Transcripts of these three cTbx genes were detected in overlapping patterns in the early cardiac crescent. cTbx2 and cTbx3 expression patterns closely overlapped with that of bmp2. cTbx5 expression diverged from cTbx2 and bmp2 during the elaboration and folding of the heart tube. In comparison, cTbx2 expression overlapped significantly with that of bmp2 and bmp4 during all stages of heart development and during later embryonic stages, suggestive of a specialized role for Tbx2 in septation. Coexpression of cTbx 2 and cTbx3 genes with bmp2 transcripts raised the possibility that these cTbx genes might be downstream bmp2 targets. Application of bmp2 selectively induced cTbx2 and cTbx3 expression in noncardiogenic embryonic tissue, and the bmp antagonist Noggin down-regulated cTbx2 gene activity. Moreover, the appearance of murine mTbx2 was blocked in bmp2 null mouse embryos. cTbx2 and to a lesser extent cTbx3 gene activity appears to be directed by BMPs during early cardiogenesis.

Impaired vascular contractility and blood pressure homeostasis in the smooth muscle alpha-actin null mouse.

The smooth muscle (SM) alpha-actin gene activated during the early stages of embryonic cardiovascular development is switched off in late stage heart tissue and replaced by cardiac and skeletal alpha-actins. SM alpha-actin also appears during vascular development, but becomes the single most abundant protein in adult vascular smooth muscle cells. Tissue-specific expression of SM alpha-actin is thought to be required for the principal force-generating capacity of the vascular smooth muscle cell. We wanted to determine whether SM alpha-actin gene expression actually relates to an actin isoform's function. Analysis of SM alpha-actin null mice indicated that SM alpha-actin is not required for the formation of the cardiovascular system. Also, SM alpha-actin null mice appeared to have no difficulty feeding or reproducing. Survival in the absence of SM alpha-actin may result from other actin isoforms partially substituting for this isoform. In fact, skeletal alpha-actin gene, an actin isoform not usually expressed in vascular smooth muscle, was activated in the aortas of these SM alpha-actin null mice. However, even with a modest increase in skeletal alpha-actin activity, highly compromised vascular contractility, tone, and blood flow were detected in SM alpha-actin-defective mice. This study supports the concept that SM alpha-actin has a central role in regulating vascular contractility and blood pressure homeostasis, but is not required for the formation of the cardiovascular system.

The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor.

An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.

Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators.

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

Determination of airborne isocyanate exposure: considerations in method selection.

To assess worker isocyanate exposures in a variety of processes involving the manufacture and use of surface coatings, polyurethane foams, adhesives, resins, elastomers, binders, and sealants, it is important to be able to measure airborne reactive isocyanate-containing compounds. Choosing the correct methodology can be difficult. Isocyanate species, including monomers, prepolymers, oligomers, and polyisocyanates, are capable of producing irritation to the skin, eyes, mucous membranes, and respiratory tract. The most common adverse health effect is respiratory sensitization, and to a lesser extent dermal sensitization and hypersensitivity pneumonitis. Furthermore, isocyanate species formed during polyurethane production or thermal degradation may also produce adverse health effects. Isocyanate measurement is complicated by the fact that isocyanates may be in the form of vapors or aerosols of various particle size; the species of interest are reactive and therefore unstable; few pure analytical standards exist; and high analytical sensitivity is needed. There are numerous points in the sampling and analytical procedures at which errors can be introduced. The factors to be considered for selecting the most appropriate methodology for a given workplace include collection, derivatization, sample preparation, separation, identification, and quantification. This article discusses these factors in detail and presents a summary of method selection criteria based on the isocyanate species, its physical state, particle size, cure rate, and other factors.

Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway.

Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.