Neumonía grave causada por Haemophilus influenzae no tipificable en un lactante: Reporte de un caso / Non-typable Haemophilus influenzae severe pneumonia in an infant: Case report

Previo a la introducción de la vacuna conjugada, Haemophilus influenzae b (Hib) representó una de las causas importantes de neumonía en niños. Recientemente cepas no b y no tipificable (nt) han emergido como agentes importantes de enfermedad. Reportamos el caso de una lactante afectada por una neumonía consolidante extensa con empiema y bacteriémica, con el antecedente de haber recibido antes vacuna contra Hib. Los cultivos de sangre y líquido pleural revelaron la presencia de H. influenzae nt. La evolución clínica fue favorable, retirándose el drenaje pleural al segundo día. Fue tratada con cefotaxima endovenosa durante cinco días y luego 9 días de amoxicilina en forma ambulatoria. Se revisa la literatura acerca de esta inusual forma de presentación en nuestro medio y se advierte la emergencia de cepas no tipificables de H. influenzae como responsables de algunas formas graves de neumonía.

Genetic dissection of the Drosophila Cubitus interruptus signaling complex.

Much of our understanding of the Hedgehog (Hh) signaling pathway comes from Drosophila, where a gradient of Hh signaling regulates the function of the transcription factor Cubitus interruptus (Ci) at three levels: protein stabilization, nuclear import, and activation. Regulation of Ci occurs in a cytoplasmic complex containing Ci, the kinesin-like protein Costal-2 (Cos2), the serine-threonine kinase Fused (Fu), and the Suppressor of Fused [Su(fu)] protein. The mechanisms by which this complex responds to different levels of Hh signaling and establishes distinct domains of gene expression are not fully understood. By sequentially mutating components from the Ci signaling complex, their roles in each aspect of Ci regulation can be analyzed. The Cos2-Ci core complex is able to mediate Hh-regulated activation of Ci but is insufficient to regulate nuclear import and cleavage. Addition of Su(fu) to the core complex blocks nuclear import while the addition of Fu restores Hh regulation of Ci nuclear import and proteolytic cleavage. Fu participates in two partially redundant pathways to regulate Ci nuclear import; the kinase function plays a positive role by inhibiting Su(fu), and the regulatory domain plays a negative role in conjunction with Cos2.

Nuclear import of cubitus interruptus is regulated by hedgehog via a mechanism distinct from Ci stabilization and Ci activation.

The Hedgehog (Hh) signal is transduced via Cubitus interruptus (Ci) to specify cell fates in the Drosophila wing. In the absence of Hh, the 155 kDa full-length form of Ci is cleaved into a 75 kDa repressor. Hh inhibits the proteolysis of full-length Ci and facilitates its conversion into an activator. Recently, it has been suggested that Hh promotes Ci nuclear import in tissue culture cells. We have studied the mechanism of Ci nuclear import in vivo and the relationship between nuclear import, stabilization and activation. We found that Ci rapidly translocates to the nucleus in cells close to the anteroposterior (AP) boundary and this rapid nuclear import requires Hh signaling. The nuclear import of Ci is regulated by Hh even under conditions in which Ci is fully stabilized. Furthermore, cells that exhibit Ci stabilization and rapid nuclear import do not necessarily exhibit maximal Ci activity. It has been previously shown that stabilization does not suffice for activation. Consistent with this finding, our results suggest that the mechanisms regulating nuclear import, stabilization and activation are distinct from each other. Finally, we show that cos2 and pka, two molecules that have been characterized primarily as negative regulators of Ci activity, also have positive roles in the activation of Ci in response to Hh.

The subcellular localization and activity of Drosophila cubitus interruptus are regulated at multiple levels.

Cubitus interruptus (Ci), a Drosophila transcription factor, mediates Hedgehog (Hh) signaling during the patterning of embryonic epidermis and larval imaginal discs. In the absence of Hh signal, Ci is cleaved to generate a truncated nuclear form capable of transcriptional repression. Hh signaling stabilizes and activates the full-length Ci protein leading to strong activation of downstream target genes including patched and decapentaplegic. A number of molecules have been implicated in the regulation of Ci. Mutations in these molecules lead to changes in Ci protein level, the extent of Ci proteolysis and the expression of Ci target genes. This paper examines the regulation of Ci subcellular localization and activity. We first characterize a bipartite nuclear localization signal (NLS) within Ci. We propose that the subcellular distribution of Ci is affected by two opposing forces, the action of the NLS and that of at least two regions targeting Ci to the cytoplasm. Further our data show that loss of PKA or Costal-2 activity does not fully mimic Hh signaling, demonstrating that Ci proteolysis and Ci activation are two distinct events which are regulated through different paths. Finally, we propose that there are three levels of apparent Ci activity, corresponding to three zones along the AP axis with different sets of gene expression and different levels of Hh signaling.

Cubitus interruptus is necessary but not sufficient for direct activation of a wing-specific decapentaplegic enhancer.

In Drosophila, the imaginal discs are the primordia for adult appendages. Their proper formation is dependent upon the activation of the decapentaplegic (dpp) gene in a stripe of cells just anterior to the compartment boundary. In imaginal discs, the dpp gene has been shown to be activated by Hedgehog signal transduction. However, an initial analysis of its enhancer region suggests that its regulation is complex and depends upon additional factors. In order to understand how multiple factors regulate dpp expression, we chose to focus on a single dpp enhancer element, the dpp heldout enhancer, from the 3' cis regulatory disc region of the dpp locus. In this report, we present a molecular analysis of this 358 bp wing- and haltere-specific dpp enhancer, which demonstrates a direct transcriptional requirement for the Cubitus interruptus (Ci) protein. The results suggest that, in addition to regulation by Ci, expression of the dpp heldout enhancer is spatially determined by Drosophila TCF (dTCF) and the Vestigial/Scalloped selector system and that temporal control is provided by dpp autoregulation. Consistent with the unexpectedly complex regulation of the dpp heldout enhancer, analysis of a Ci consensus site reporter construct suggests that Ci, a mediator of Hedgehog transcriptional activation, can only transactivate in concert with other factors.

Drosophila cubitus interruptus forms a negative feedback loop with patched and regulates expression of Hedgehog target genes.

The Drosophila segment polarity gene cubitus interruptus (ci) encodes a zinc finger protein that is required for the proper patterning of segments and imaginal discs. Epistasis analysis indicates that ci functions in the Hedgehog (Hh) signal transduction pathway and is required to maintain wingless expression in the embryo. In this paper, the role of the Ci protein in the Hh signaling pathway is examined in more detail. Our results show that ectopic expression of ci in imaginal discs and the embryo activates the expression of Hh target genes. One of these target genes, patched, forms a negative feedback loop with ci that is regulated by Hh signal transduction. Activation is also achieved using the Ci zinc finger domain fused to a heterologous transactivation domain. Conversely, repression of Hh target genes occurs in animals expressing the Ci zinc finger domain fused to a repression domain. To examine Ci function in more detail, regions of the Ci protein that are responsible for its ability to transactivate and its subcellular distribution have been identified.

Age-dependent differences in the rat's conditioned defensive burying behavior: effect of 5-HT1A compounds.

The ontogeny of conditioned defensive-burying behavior was studied in rats from 2 to 21 weeks of age. At early ages this parameter shows low values that gradually increase until the 11th week, decreasing steadily thereafter. Reactivity, measured by the burying-behavior latency, appeared increased in 2-week-old rats. The effect of the serotonergic1A compounds: 8-OH-DPAT (0.25 and 0.5 mg/kg), ipsapirone, buspirone, and indorenate (at 2.5 and 5.0 mg/kg) was studied at 3, 7, 11, and 21 weeks of age. All compounds produced a dose-dependent decrease in burying behavior in rats of 7 and 11 weeks, while at the Week 21, only 8-OH-DPAT and indorenate reduced it. At 3 weeks of age, burying-behavior latency was increased by all compounds, but burying behavior was not altered. Motor coordination was affected by buspirone at all ages and by 8-OH-DPAT at the Week 21. Data are discussed on the bases of the development of defensive behaviors.

Pharmacological analysis of acute morphine dependence in infant rats: close molecular relationship of head-shaking precipitated by opiate antagonists and cholinergic neurotransmission.

The influence of drugs affecting different neurotransmitter systems on an acute abstinence head-shaking (AHS) model induced by nalorphine or naloxone was studied in 9-day-old rat pups pretreated (3 h before) with morphine (10 mg/kg, i.p.). One hour after the injection of nalorphine (10 mg/ kg, i.p.) AHS was stopped by a second dose of morphine (10 mg/kg, i.p.) and reinitiated 1 h later by a higher dose of nalorphine (20 mg/kg, i.p.). In other groups AHS was blocked by spiroperidol (0.1 mg/kg, i.p.), clonidine (0.01 mg/kg, i.p.) or scopolamine (50 mg/kg, i.p.). In these groups a second injection of nalorphine did not reinitiate AHS. In dose-effect curve experiments the AHS induced by naloxone or nalorphine was significantly reduced by previous injections of scopolamine, spiroperidol, metergoline or phentolamine in the corresponding groups. Scopolamine was the only antagonist which displaced the AHS dose-effect curves to the right without affecting the maximal response. Since no common receptors exist for a direct competitive interaction between opiate antagonists and scopolamine, these experiments suggest that a direct molecular relationship exists between the tissue concentration of nalorphine (or naloxone) and the endogenous ACh release during abstinence. Thus, the AHS model in 9-day-old rats clearly differentiates specific from non-specific blockade of the abstinence syndrome, and confirms a distinct or primary role of cholinergic neurotransmission in morphine abstinence.

Specification of neuroblast identity in the Drosophila embryonic central nervous system by gooseberry-distal.

The Drosophila central nervous system develops from a segmentally reiterated array of 30 unique neural precursors, called neuroblasts. Each neuroblast goes through a stereotyped cell lineage to produce an invariant clone of neural progeny. It is critical to identify the genes that specify neuroblast identity as these genes control the time of formation, gene expression profile, and cell lineage characteristics of each neuroblast. Here we show that the Pax-type gooseberry-distal gene specifies row 5 neuroblast identity. Initially, four rows of neuroblasts form per segment (1, 3, 5, 7) and gooseberry-distal is expressed in row 5 neuroblasts. By using 10 molecular markers, and by following the number and orientation of neuroblast divisions, we show that lack of gooseberry-distal transforms row 5 neuroblasts into row 3 neuroblasts, whereas ubiquitous gooseberry-distal generates the reciprocal transformation. Thus, gooseberry-distal is necessary and sufficient to specify row 5 neuroblast identity autonomously. The 10 genes coordinately regulated by gooseberry-distal are prime candidates for controlling specific aspects of neuroblast identity.

Expression of the Drosophila gooseberry locus defines a subset of neuroblast lineages in the central nervous system.

The development of the central nervous system is known to require lineage-specific factors that are expressed in neuroblasts and their descendants, as well as molecules involved in cell-cell signaling mechanisms. The transcription factors encoded by the gooseberry locus appear to be required for the proper specification of neuroblasts and their lineages. To examine whether gooseberry expression is lineage-specific, we have utilized the FLP recombinase of yeast to positively mark cell lineages throughout Drosophila development. In this system, the actin5C promoter and the lacZ gene are separated by a polyadenylation signal flanked by two direct repeat FRTs. A heat shock is used to induce a pulse of FLP recombinase which catalyzes a site-specific recombination event between the FRT sites. The resulting excision of the polyadenylation site allows expression of lacZ from the actin5C promoter. The descendants of a cell which has undergone a recombination event are now positively marked, enabling us to compare cell lineages with the pattern of gooseberry gene expression. We find that the expression of the gooseberry locus is lineage-specific, suggesting that gooseberry may function as a selector gene in the patterning of the Drosophila central nervous system.

The Drosophila cubitus interruptus protein and its role in the wingless and hedgehog signal transduction pathways.

The segment polarity gene cubitus interruptus (ci) is required to maintain expression of the wingless gene and to specify naked cuticle within each epidermal segment. Antibodies were generated against the ci protein and used to analyze its pattern of expression. By stage 11, post-transcriptional regulation of ci is observed. ci transcript levels are uniform across the anterior compartment, but protein levels are higher next to the compartment boundaries. The distribution of the ci protein is altered in fused, hedgehog and wingless mutants suggesting cell-cell signaling may regulate ci protein levels. The role of ci in cell-cell signaling and pattern formation was examined in double mutants of ci with patched and zeste-white3/shaggy.

Mutations that alter the timing and pattern of cubitus interruptus gene expression in Drosophila melanogaster.

The cubitus interruptus (ci) gene is a member of the Drosophila segment polarity gene family and encodes a protein with a zinc finger domain homologous to the vertebrate Gli genes and the nematode tra-1 gene. Three classes of existing mutations in the ci locus alter the regulation of ci expression and can be used to examine ci function during development. The first class of ci mutations causes interruptions in wing veins four and five due to inappropriate expression of the ci product in the posterior compartment of imaginal discs. The second class of mutations eliminates ci protein early in embryogenesis and causes the deletion of structures that are derived from the region including and adjacent to the engrailed expressing cells. The third class of mutations eliminates ci protein later in embryogenesis and blocks the formation of the ventral naked cuticle. The loss of ci expression at these two different stages in embryonic development correlates with the subsequent elimination of wingless expression. Adults heterozygous for the unique ciCe mutation have deletions between wing veins three and four. A similar wing defect is present in animals mutant for the segment polarity gene fused that encodes a putative serine/threonine kinase. In ciCe/+ and fused mutants, the deletions between wing veins three and four correlate with increased ci protein levels in the anterior compartment. Thus, proper regulation of both the ci mRNA and protein appears to be critical for normal Drosophila development.

Ectopic expression of either the Drosophila gooseberry-distal or proximal gene causes alterations of cell fate in the epidermis and central nervous system.

Previous studies have shown that the segment polarity locus gooseberry, which contains two closely related transcripts gooseberry-proximal and gooseberry-distal, is required for proper development in both the epidermis and the central nervous system of Drosophila. In this study, the roles of the gooseberry proteins in the process of cell fate specification have been examined by generating two fly lines in which either gooseberry-distal or gooseberry-proximal expression is under the control of an hsp70 promoter. We have found that ectopic expression of either gooseberry protein causes cell fate transformations that are reciprocal to those of a gooseberry deletion mutant. Our results suggest that the gooseberry-distal protein is required for the specification of naked cuticle in the epidermis and specific neuroblasts in the central nervous system. These roles may reflect independent functions in neuroblasts and epidermal cells or a single function in the common ectodermal precursor cells. The gooseberry-proximal protein is also found in the same neuroblasts as gooseberry-distal and in the descendants of these cells.

Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development.

Three mouse genes, Gli, Gli-2, and Gli-3, which share a similar zinc finger domain with the products of the Drosophila segment polarity gene cubitus interruptus and the Caenorhabditis elegans sex-determining gene tra-1 were cloned and characterized. The expression patterns during postimplantation development of the three genes were analyzed by Northern blot, whole-mount, and section in situ hybridizations. Expression was first detected during gastrulation in both the ectoderm and mesoderm. Later in development, their expression became more restricted in various ectoderm- and mesoderm-derived tissues and was not detectable after completion of organogenesis. Interestingly, in the developing neural tube, Gli showed a narrow ventral domain of expression, whereas Gli-2 and Gli-3 showed a broad and dorsally restricted domain. Expression of these three Gli genes in various ectoderm- and mesoderm-derived tissues suggests that they play multiple roles during postimplantation development. Consistent with this hypothesis, a naturally occurring Gli-3 mutation, the mouse extra-toes mutant; shows defects in both mesoderm- and ectoderm-derived tissues.

Invariant tryptophan at a shielded site promotes folding of the conformational unit of spectrin.

The tryptophan that is highly conserved among repeating structural units of spectrin is reported to promote the conformational stability of one such unit of chicken brain alpha-spectrin. Four constructs were inserted into pET vectors for overexpression in Escherichia coli of the following spectrin peptides: (i) two adjacent but separately expressed "conformationally phased" repeating units, R16 and R17, one of which (R17) contains a single tryptophan; (ii) a mutant, M17, of the single tryptophan-containing unit with alanine substituted for the tryptophan; and (iii) a conformationally unphased unit, 1617, composed of half of each of the phased units. Both the mutant unit and the unphased unit were much more readily digested by chymotrypsin and by elastase than the phased units and exhibited only 38% and 54% as much alpha-helical structure, respectively, as the phased units by their far UV CD spectra; 90 degrees light scattering measurements revealed the folded peptides to be predominantly monomeric in solution, whereas the unfolded, protease-sensitive peptides consisted of dimers and/or trimers. This trend was corroborated by their dynamic light scattering. Both the blue-shifted wavelength of maximal emission and the relative inaccessibility to acrylamide of the single tryptophan in the folded unit indicate that the invariant tryptophan occupies a site that is shielded from the aqueous phase.

A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster.

The GATA transcription factors are a family of C4 zinc finger-motif DNA-binding proteins that play defined roles in hematopoiesis as well as presumptive roles in other tissues where they are expressed (e.g., testis, neuronal and placental trophoblast cells) during vertebrate development. To investigate the possibility that GATA proteins may also be involved in Drosophila development, we have isolated and characterized a gene (dGATAa) encoding a factor that is quite similar to mammalian GATA factors. The dGATAa protein sequence contains the two zinc finger DNA-binding domain of the GATA class but bears no additional sequence similarity to any of the vertebrate GATA factors. Analysis of dGATAa gene transcription during Drosophila development revealed that its mRNA is expressed at high levels during early embryogenesis, with transcripts first appearing in the dorsal portion of the embryo just after cellularization. As development progresses, dGATAa mRNA is present at high levels in the dorsal epidermis, suggesting that dGATAa may be involved in determining dorsal cell fate. The pattern of expression in a variety of dorsoventral polarity mutants indicates that dGATAa lies downstream of the zygotic patterning genes decapentaplegic and zerknüllt.

Two inbred rat sublines that differ in spontaneous yawning behavior also differ in their responses to cholinergic and dopaminergic drugs.

This work compares the sensitivities of high-yawning (HY) and low-yawning (LY) sublines of Sprague-Dawley rats to dopaminergic and cholinergic yawning-inducing drugs. HY animals are significantly more sensitive to apomorphine and (-)3PPP than LY animals. Physostigmine is a less effective yawning-inducer in HY than in LY rats. With pilocarpine no differences were detected between both sublines in regard to its yawning-inducing activity. Since yawning behavior is subject to dopaminergic (inhibitory) and cholinergic (excitatory) influences, it is suggested that the genetic differences between these sublines affect the dopaminergic pathways that normally regulate yawning frequency.

gli, a zinc finger transcription factor and oncogene, is expressed during normal mouse development.

The oncogene GLI is amplified and expressed in some cases of human malignant glioma and undifferentiated childhood sarcoma and is the prototype for a gene family characterized by a highly conserved set of five tandem zinc fingers and a consensus cysteine-histidine link. This zinc finger motif has been shown to bind DNA with sequence specificity and may mediate transcriptional regulation. Since GLI is expressed in embryonal carcinoma cell lines but not in most normal adult tissues and shows significant sequence similarity within its zinc finger domain to cubitus interruptus dominant (ciD), a Drosophila segmentation gene known to be important in the morphogenesis of the posterior portion of each larval segment, we established the temporal and tissue expression patterns of the mouse homologue of human GLI in day 10 through 18 mouse embryos with Northern blotting, reverse transcriptase coupled PCR, and in situ hybridization. gli transcripts were demonstrated on days 10 through 18 of mouse embryonic development as well as in normal adult uterus, brain, testis, and limb. Tissue expression of gli during gestation was demonstrated in Meckel's precartilage mesenchyme, the basis occipitus, rib mesenchymal condensations, primordial vertebral bodies, digital mesenchymal condensations in forefoot and hindfoot plates, the ependymal layer of the spinal cord, and the mesoderm of the gastrointestinal tract. Expression persisted throughout gestation in developing bone and cartilage of the extremities, the ribs, and the vertebral bodies, as well as the gastrointestinal tract mesoderm. These findings support a role for gli family genes in normal craniofacial and digital development in mammals first suggested by the demonstration of translocation breakpoints within the GLI3 gene in families with the Greig cephalopolysyndactylyl syndrome and subsequently by reduced gli3 expression in the mouse mutant extra toes. It is surprising that a single gene would be expressed in such a wide range of mesenchymal structures.

The taiep rat: a myelin mutant with an associated oligodendrocyte microtubular defect.

This report describes a new inherited disorder of myelination in the rat, named taiep, in which failure of normal myelination of the CNS and subsequent demyelination result in a progressive neurological disturbance. At two months of age, myelin is present throughout the spinal cord, but is immature in the fasciculus gracilis and corticospinal tracts despite the presence of abundant oligodendrocytes. By 12 months, myelin has largely been lost in these spinal cord tracts and also in more rostral parts of the CNS, such as the cerebellum and optic nerves. Other funiculi of the spinal cord show a more diffuse lack of myelin. Oligodendrocytes develop a unique cellular abnormality, most obviously in older rats, which is characterized by the accumulation of microtubules throughout their cytoplasm. As the mutant rats age, there is a continued protracted breakdown of myelin throughout the CNS, with evidence suggesting either persistent hypomyelination or attempts at remyelination of affected axons. It is proposed that the microtubular defect in oligodendrocytes results in a disruption of the normal myelination process in certain areas of the CNS of this mutant, and eventually leads to failure of maintenance of the myelin sheath.

Age-dependent changes in serotonergic modulation of yawning in the rat.

Serotonin (5-HT) effects on physostigmine (PHY)-induced yawning were studied in LY Sprague-Dawley rats by injecting Lu 10 171 (citalopram), a specific 5-HT uptake blocker, and two antagonists--methiothepine and ritanserin--which differ slightly in the selectivity of their actions on different 5-HT receptor subtypes. Infant and young rats show significant increases in PHY-induced yawning when preinjected with citalopram (5-10 mg/kg). Two-month-old animals show this effect only with 10 mg/kg. With adult animals (3-5 months old), the effect is the opposite: Yawning decreases. The facilitory effect in infant and young rats was counteracted by methiothepine but not by ritanserin, suggesting that it is mediated through 5-HT1A or 5-HT1B receptor subtypes. The inhibitory effect of citalopram in adult rats was unmodified by the two antagonists used, leaving open the possibility that it is mediated by 5-HT3 receptors.