Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues: highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T-191. and Y-193 in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T-187 showed reduced enzymatic activity against exogenous substrates but retained autophosphorylationactivity. Phosphorylation was confirmed by the use of a phospho-specific T-187 antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed. (C) 2002 Elsevier Science (USA). All rights reserved.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

Identification and characterization of the hepatic stellate cell transferrin receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [H-3]Thymidine incorporation, and real-time RT-PCR for procollagen 1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, Candidatus Accumulibacter phosphatis was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions. (C) 2003 Wiley Periodicals, Inc.

The use of 16s rDNA restriction fragment length polymorphism analysis for the identification of non-fermenting gram negative bacilli in a cystic fibrosis microbiology referral service

The utility of 16s rDNA restriction fragment length polymorphism (RFLP) analysis for the partial genomovar differentiation of Burkholderia cepacia complex bacterium is well documented. We compared the 16s rDNA RFLP signatures for a number of non-fermenting gram negative bacilli (NF GNB) LMG control strains and clinical isolates pertaining to the genera Burkholderia, Pseudomonas, Achromobacter (Alcaligenes), Ralstonia, Stenotrophomonas and Pandoraea. A collection of 24 control strain (LMG) and 25 clinical isolates were included in the study. Using conventional PCR, a 1.2 kbp 16s rDNA fragment was generated for each organism. Following restriction digestion and electrophoresis, each clinical isolate RFLP signature was compared to those of the control strain panel. Nineteen different RFLP signatures were detected from the 28 control strains included in the study. TwentyoneyTwenty- five of the clinical isolates could be classified by RFLP analysis into a single genus and species when compared to the patterns produced by the control strain panel. Four clinical B. pseudomallei isolates produced RFLP signatures which were indistinguishable from B. cepacia genomovars I, III and VIII. The identity of these four isolates were confirmed using B. pseudomallei specific PCR. 16s rDNA RFLP analysis can be a useful identification strategy when applied to NF GNB, particularly for those which exhibit colistin sulfate resistance. The use of this molecular based methodology has proved very useful in the setting of a CF referral laboratory particularly when utilised in conjunction with B. cepacia complex and genomovar specific PCR techniques. Species specific PCR or sequence analysis should be considered for selected isolates; especially where discrepancies between epidemiology, phenotypic and genotypic characteristics occur.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and

Emergence of joint attention: Relationships between gaze following, social referencing, imitation, and naming in infancy

The authors investigated the extent to which the joint-attention behaviors of gaze following, social referencing, and object-directed imitation were related to each other and to infants vocabulary development in a sample of 60 infants between the ages of 8 and 14 months. Joint-attention skills and vocabulary development were assessed in a laboratory setting. Split-half reliability analyses on the joint-attention measures indicated that the tasks reliably assessed infants' capabilities. In the main analysis, no significant correlations were found among the joint-attention behaviors except for a significant relationship between gaze following and the number of names in infants' productive vocabularies. The overall pattern of results did not replicate results of previous studies (e.g., M. Carpenter, K. Nagell, & M. Tomasello, 1998) that found relationships between various emerging joint-attention behaviors.

Pyramidal cell heterogeneity in the visual cortex of the nocturnal new world owl monkey (aotus trivirgatus)

Recent studies have revealed marked variation in pyramidal cell structure in the visual cortex of macaque and marmoset monkeys. In particular, there is a systematic increase in the size of, and number of spines in, the arbours of pyramidal cells with progression through occipitotemporal (OT) visual areas. In the present study we extend the basis for comparison by investigating pyramidal cell structure in visual areas of the nocturnal owl monkey. As in the diurnal macaque and marmoset monkeys, pyramidal cells became progressively larger and more spinous with anterior progression through OT visual areas. These data suggest that: 1. the trend for more complex pyramidal cells with anterior progression through OT visual areas is a fundamental organizational principle in primate cortex; 2. areal specialization of the pyramidal cell phenotype provides an anatomical substrate for the reconstruction of the visual scene in OT areas; 3. evolutionary specialization of different aspects of visual processing may determine the extent of interareal variation in the pyramidal cell phenotype in different species; and 4. pyramidal cell structure is not necessarily related to brain size. Crown Copyright (C) 2003 Published by Elsevier Science Ltd on behalf of IBRO. All rights reserved.

Dendritic branching of pyramidal cells in the visual cortex of the nocturnal owl monkey: A fractal analysis

The branching structure of neurones is thought to influence patterns of connectivity and how inputs are integrated within the arbor. Recent studies have revealed a remarkable degree of variation in the branching structure of pyramidal cells in the cerebral cortex of diurnal primates, suggesting regional specialization in neuronal function. Such specialization in pyramidal cell structure may be important for various aspects of visual function, such as object recognition and color processing. To better understand the functional role of regional variation in the pyramidal cell phenotype in visual processing, we determined the complexity of the dendritic branching pattern of pyramidal cells in visual cortex of the nocturnal New World owl monkey. We used the fractal dilation method to quantify the branching structure of pyramidal cells in the primary visual area (V1), the second visual area (V2) and the caudal and rostral subdivisions of inferotemporal cortex (ITc and ITr, respectively), which are often associated with color processing. We found that, as in diurnal monkeys, there was a trend for cells of increasing fractal dimension with progression through these cortical areas. The increasing complexity paralleled a trend for increasing symmetry. That we found a similar trend in both diurnal and nocturnal monkeys suggests that it was a feature of a common anthropoid ancestor.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs. (C) 2002 Wiley-Liss, Inc.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

SICV: sistema integrado de comunica????o visual

A necessidade de identificar os blocos e os ambientes de forma que os usu??rios internos e externos n??o tenham dificuldade de encontrar o que precisa. De forma clara e bem especificada mesmo que em alguns casos sejam usadas apenas siglas e n??meros. Assim sendo a comunica????o visual atrav??s de placas ficou din??mica e de f??cil compreens??o

Manual de identidade visual: Reposit??rio Institucional da Escola Nacional de Administra????o P??blica (Enap)

A identidade visual contida neste manual ?? o conjunto de informa????es gr?????cas que formalizam a personalidade visual do Reposit??rio institucional da Escola Nacional de Administra????o P??blica - ENAP. O projeto de identidade visual envolve a cria????o do logotipo e s??mbolo e tamb??m um conjunto de elementos institucionais e regras que regem a aplica????o da identidade em impressos administrativos, letreiros, ve??culos, uniformes, entre outros elementos, de acordo com a necessidade de cada institui????o. Neste manual est??o as informa????es que disciplinam o uso da marca Reposit??rio institucional da ENAP e seus elementos, garantindo a identidade visual junto ao p??blico alvo, institui????es e o mercado.