Proper Induction Research Articles

A novel, putative MEK kinase controls developmental timing and spatial patterning in Dictyostelium and is regulated by ubiquitin-mediated protein degradation.

We have identified a developmentally regulated, putative MEK kinase (MEKKalpha) that contains an F-box and WD40 repeats and plays a complex role in regulating cell-type differentiation and spatial patterning. Cells deficient in MEKKalpha develop precociously and exhibit abnormal cell-type patterning with an increase in one of the prestalk compartments (pstO), a concomitant reduction in the prespore domain, and a loss of the sharp compartment boundaries, resulting in overlapping prestalk and prespore domains. Overexpression of MEKKalpha or MEKKalpha lacking the WD40 repeats results in very delayed development and a severe loss of compartment boundaries. Prespore and prestalk cells are interspersed throughout the slug. Analysis of chimeric organisms suggests that MEKKalpha function is required for the proper induction and maintenance of prespore cell differentiation. We show that the WD40 repeats target MEKKalpha to the cortical region of the cell, whereas the F-box/WD40 repeats direct ubiquitin-mediated MEKKalpha degradation. We identify a UBC and a UBP (ubiquitin hydrolase) that interact with the F-box/WD40 repeats. Our findings indicate that cells lacking the ubiquitin hydrolase have phenotypes similar to those of MEKKalpha null (mekkalpha-) cells, further supporting a direct genetic and biochemical interaction between MEKKalpha, the UBC, and the UBP. We demonstrate that UBC and UBP differentially control MEKKalpha ubiquitination/deubiquitination and degradation through the F-box/WD40 repeats in a cell-type-specific and temporally regulated manner. Our results represent a novel mechanism that includes targeted protein degradation by which MAP kinase cascade components can be controlled. More importantly, our findings suggest a new paradigm of spatial and temporal control of the kinase activity controlling spatial patterning during multicellular development, which parallels the temporally regulated degradation of proteins required for cell-cycle progression.

TRACHEAL INTUBATION WITHOUT NEUROMUSCULAR BLOCK IN PATIENTS WITH MYASTHENIA GRAVIS

S44 A wide range of sensitivities has been reported for patients with Myasthenia Gravis (MG) to neuromuscular blocking agents. It is difficult to estimate its proper induction dose and use antagonist. It has been reported that propofol may provide adequate conditions for intubation without the need for neuromuscular relaxants. This study was designed to assess intubating conditions with propofol and fentanyl without neuromuscular relaxant in patients with MG. PATIENTS AND METHODS: Hospital ethics committee approval was obtained to study 6 patients with MG aged 24-68 yr, scheduled for extended thymectomy. All patients were premedicated with midazolam 1.5-2.5 mg [center dot] kg-1 i.m. and atropine 0.5 mg [center dot] kg-1 i.m. 30min before anesthesia. Three minutes after administration of fentanyl 2 [micro sign]g [center dot] kg-1, a standard induction dose of propofol 2-3 mg [center dot] kg-1 was given over 30sec and Train-of-four-ratio (TOFR) of the adductor pollicis muscle by ulner nerve stimulation was measured (Table 2). Tracheal intubation was attempted 60sec after administration of propofol and three aspects of intubating conditions were assessed on a four-point scale (Table 1): ease of intubation, vocal cord position and degree of coughing on insertion of the tracheal tube.Table 2Table 1: Intubating condition scoreRESULTS: (Table 2) We found satisfactory intubating conditions in all patients. CONCLUSIONS: It was concluded that anesthesia induction with propofol 2-3 mg [center dot] kg-1 and fentanyl 2 [micro sign]g [center dot] kg-1 provided a satisfactory condition for tracheal intubation without neuromuscular relaxant in patients with MG.

The Function ofsilberblickin the Positioning of the Eye Anlage in the Zebrafish Embryo

In zebrafish, as in other vertebrates, an initially singular eye field within the neural plate has to split during morphogenesis to allow the development of two separated eyes. It has been suggested that anterior progression of midline tissue within the neural plate is involved in the bilateralization of the eye field. Mutations in the recently identifiedsilberblick(slb) gene cause an incomplete separation of the eyes. During gastrulation and early somitogenesis, the ventral midline of the central nervous system (CNS) together with the underlying axial mesendoderm is shortened and broadened inslbembryos. While in wild-type embryos the ventral CNS midline extends to the anterior limit of the neural plate at the end of gastrulation, there is a gap between the anterior tip of the ventral CNS midline and the anterior edge of the neural plate inslb.To investigate the cause for the shortening of the ventral CNS midline inslbwe determined the fate of labeled ventral CNS midline cells in wild-type andslbembryos at different stages of development. Inslb,anterior migration of ventral CNS midline cells is impaired, which indicates that migration of these cells is needed for elongation of the ventral CNS midline. The anterior shortening of the ventral CNS midline inslbleads to medial instead of bilateral induction of optic stalks followed by a partial fusion of the eyes at later developmental stages. The analysis of theslbphenotype indicates that anterior migration of midline cells within the neural plate is required for proper induction and subsequent bilateralization of an initially singular eye field. These findings may therefore provide a starting point in elucidating the role of neural plate morphogenesis in positioning of the eyes.

Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

Molecular analysis of a steroid-induced regulatory hierarchy: the Drosophila E74A protein directly regulates L71-6 transcription.

Steroid hormones orchestrate the growth and development of higher organisms by directing spatially and temporally coordinated programs of gene expression. These changes in gene activity can be visualized in Drosophila by virtue of its giant salivary gland polytene chromosomes. A small set of early puffs are induced directly by the steroid hormone ecdysone. The proteins encoded by these puffs appear to induce many late secondary-response puffs as the animal begins to undergo metamorphosis. Here we report that the ETS domain DNA-binding protein encoded by the E74A early gene directly induces L71-6 late gene transcription. We identify four strong E74A binding sites within the 5' region of L71-6 demonstrate that these sites are essential for proper L71-6 induction at puparium formation. These studies provide a direct link between a steroid-induced transcription factor and the activation of a secondary-response promoter, indicating that steroid signals in higher organisms can be transduced and amplified through regulatory hierarchies.

Guanosine 3',5'-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi.

Cells of Escherichia coli which enter a phase of starvation for Pi induce the synthesis of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp). This induction is relA independent but depends on the spoT gene product. A mutant unable to produce ppGpp is impaired in the expression of two genes which belong to the pho regulon, a defect which is dependent on the product of spoT. We suggest that ppGpp is essential for the proper induction of the genes which belong to the pho regulon.

Mesenchymal and Neuronal Cell Expression of the p75 Neurotrophin Receptor Gene Occur by Different Mechanisms

The p75 neurotrophin receptor serves as a receptor for all known neurotrophins, including NGF, BDNF, NT-3, and NT-4/5. The expression pattern of p75 is known to be widespread and extends outside the nervous system, suggesting that neurotrophins may have functions beyond neuronal cell survival and differentiation. To investigate the expression of p75, a human minigene was constructed that contained the full-length receptor cDNA directed by its own promoter and 4 kb of 5′ upstream sequence. This minigene was used to generate transgenic mice and was found to be expressed in selective neuronal populations such as the cerebellar external granular layer. Expression of the transgene was not observed in sensory or sympathetic ganglia or in the basal forebrain. Transection of the sciatic nerve revealed that the human transgene was appropriately upregulated after nerve injury, indicating that sequences responsible for proper induction during nerve regeneration reside in the minigene construct. In contrast, the p75 transgene was found to be extensively expressed in mesenchymal cells during development, closely mimicking the in vivo pattern of expression. The detection of p75 receptors in the mesenchyme implies that neurotrophins are likely to exert effects during morphogenesis of mesodermal tissues and that separate signals are likely to direct neuronal versus nonneuronal expression of the p75 gene.

Polyamine translocation following photoperiodic flowering induction in soybean

Radioactivity translocation after [14C]‐spermidine application over the third trifoliate leaf of soybean plants (Glycine max. [L.] Merr, cv. Williams) was checked during the first 72 h of short day (SD) treatment to study the involvement of polyamines (PAs) in photoperiodic flowering induction. PAs and/or their metabolites were translocated from the supplied leaf to all parts of the plant. Radioactivity reached its highest concentration in the upper portion of the stem, i.e. the apical bud and the youngest leaf. After the beginning of the first inductive night, the detected radioactivity showed two peaks of maximal concentration. The first arose after the first inductive night, coinciding with the proper flowering induction process; the second one arose after the third inductive night, coinciding with the first morphological symptoms of the transition of vegetative meristems to the reproductive condition. Soluble free PAs showed a different balance in the apical bud of SD‐induced plants compared with LD‐non induced control plants. Soluble conjugated PAs were detected as traces. It is suggested that under flowering inductive conditions, PAs play a different role according to the stage of the flowering process. Thus, their translocation from the leaves to the axillary and apical buds might be related, in a first step, to the fact that they were part of the complex mechanism of the flowering signal, and in a second step, to the flower transition of vegetative buds.

The preregistration alternative

I was told that I was only “a clerking machine.” The comment was meant to reassure me that as a new junior house physician responsibility for patient care did not rest on my shoulders alone. Far from finding the comment reassuring I found it devaluing. It was an underestimation of the reality and highly ironical in the face of current General Medical Council recommendations on the educational content of the preregistration year. After the euphoria of qualifying as a doctor, my first day as a house officer had brought me crashing down from cloud nine. I was the unfortunate house physician on take holding the crash bleep; but I was worried about where to run to if it went off. The introductory talk for new house officers came on the second day - a whole day too late. Some of the anxiety I had experienced would have dissipated if there had been a proper induction, including a tour of the hospital, before the job started. Our formal medical education took the form of a series of weekly one hour seminars, which were of a good quality and relevant to managing several medical conditions seen daily on the wards. But effective education calls for more than quality tuition; it requires receptive and undistracted pupillage. This was impossible as we were continually being bleeped away …

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnosperm Picea glauca (white spruce). Promoter expression was assayed in three different tissues capable of in vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducible Arabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter-beta-glucuronidase-nopaline synthase 3' fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the beta-glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.

Ti plasmid containing Rhizobium meliloti are non-tumorigenic on plants, despite proper virulence gene induction and T-strand formation

We examined the expression of the vir genes of the Agrobacterium tumefaciens Ti plasmid in Rhizobium meliloti, which remains non-tumorigenic on plants after introduction of a Ti- or Ri-plasmid. Both the levels of virulence (vir) gene expression, induced by the plant phenolic compound acetosyringone, and of subsequent T-strand formation were comparable to what is observed in Agrobacterium. In contrast to the situation in Agrobacterium, though, vir induction in R. meliloti did not require a low pH (5.3) of the induction medium and the optimum temperature for induction in R. meliloti was significantly lower than in Agrobacterium. At 37°C no induction of the vir genes was found both in Agrobacterium and R. meliloti. We postulate that the lack of tumorigenicity of Ti carrying R. meliloti strains is due either to a lack of proper attachment of the bacteria to plant cells, or to an improper assembly of a virB-determined essential structure in the cell wall of R. meliloti.

The induction of ALU-sequence transcripts by glucocorticoid in rat liver cells

Two cDNA clones isolated from a library prepared from dexamethasone-treated rat hepatoma cells have permitted us to detect the presence and the induction of heterogeneous, mainly short, RNA molecules in hepatoma cells and in rat liver, but not in several other rat tissues. The induction by dexamethasone is inhibited by 100 × progesterone. Pulse label experiments suggest that it occurs in part at least, at the level of transcription and may be mediated by RNA polymerase III. The induction of the RNAs is stimulated by cycloheximide, even in the absence of hormone, but not significantly by other stressful conditions. One line of hepatoma cells spontaneously lost its ability to induce these RNAs and synthesized them constitutively. These altered cells showed proper induction of another dexamethasone-mediated response, indicating that the glucocorticoid receptor was functionally normal in these cells. The two clones contain a type 2 Alu-like sequence. The short RNAs can be distinguished from 7SL RNA, which also contains Alu-sequences. We hypothesize that the synthesis of these RNAs may be regulated by an inhibitor of transcription which is inactivated by dexamethasone. Accordingly, cycloheximide relieves the inhibition by preventing synthesis of the inhibitor and the altered cell line has spontaneously lost the function of the inhibitor. The function of these RNAs for the cell is not known. We believe this to be the first report of hormone-regulated tissue specific synthesis of repeat-sequence transcripts.

Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.

Political socialization in the schools: Latin America

In most of the world where much of education is formal and institutionalized, the role of the school in political socialization is considered a major dimension of its task. A concern for the proper induction of youth into the political values and practices of society is pervasive and capable of penetrating every aspect of the curriculum. In modernizing nations education is viewed as a major mechanism for progress of all kinds, and its potential for leading the march from inadequate to modern political institutions is clearly recognized. Here a snag arises in the political fabric of the world, however, for accompanying a necessary and earnest quest for political structures commensurate with the needs of the contemporary world is an equally intense and jealously guarded nationalistic fervor. It is a paradox of our times that the demands of modernity which have provoked recognition of the need for regional and international cooperation have simultaneously created the political fragmentations which abound. The world changes, political institutions evolve, alliances rise and fall, power coalesces and disintegrates, and on a national and increasingly global level a case for political connection must be made not once and for all but continuously for each new generation and each new period.1 Attempts to incorporate a world view and cross-national sense of civic responsibility into programs of political socialization in the schools are few and feeble but increasingly