Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2+, although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 microM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.

CgDN24: A gene involved in hyphal development in the fungal phytopathogen Colletotrichum gloeosporioides

A cDNA corresponding to a transcript induced in culture by N starvation, was identified in Colletotrichum gloeosporioides by a differential hybridisation strategy. The cDNA comprised 905 bp and predicted a 215 aa protein; the gene encoding the cDNA was termed CgDN24. No function for CgDN24 could be predicted by database homology searches using the cDNA sequence and no homologues were found in the sequenced fungal genomes. Transcripts of CgDN24 were detected in infected leaves of Stylosanthes guianensis at stages of infection that corresponded with symptom development. The CgDN24 gene was disrupted by homologous recombination and this led to reduced radial growth rates and the production of hyphae with a hyperbranching phenotype. Normal sporutation was observed, and following conidia inoculation of S. guianensis, normal disease development was obtained. These results demonstrate that CgDN24 is necessary for normal hyphal development in axenic culture but dispensable for phytopathogenicity. (c) 2005 Elsevier GmbH. Alt rights reserved.

THE STELLAR CONTENT OF OBSCURED GALACTIC GIANT H II REGIONS. VII. W3

Spectrophotometric distances in the K band have been reported by different authors for a number of obscured Galactic H II regions. Almost 50% of them show large discrepancies compared to the classical method using radial velocities measured in the radio spectral region. In order to provide a crucial test of both methods, we selected a target that does not present particular difficulty for any method and which has been measured by as many techniques as possible. The W3 star-forming complex, located in the Perseus arm, offers a splendid opportunity for such a task. We used the Near-Infrared Integral Field Spectrograph on the Frederick C. Gillett Gemini North telescope to classify candidate ""naked photosphere"" OB stars based on Two Micron All Sky Survey photometry. Two of the targets are revealed to be mid-O-type main-sequence stars leading to a distance of d = 2.20 kpc. This is in excellent agreement with the spectrophotometric distance derived in the optical band (d = 2.18 pc) and with a measurement of the W3 trigonometric parallax (d = 1.95 kpc). Such results confirm that the spectrophotometric distances in the K band are reliable. The radio-derived kinematic distance, on the contrary, gives a distance twice as large (d = 4.2 kpc). This indicates that this region of the Perseus arm does not follow the Galactic rotation curve, and this may also be the case for other H II regions for which discrepancies have been found.

Book review of Mark McGillivray and Gary Smith's (eds.) "Australia and Asia", Melbourne: Oxford University Press, 1997. vii + 248 pp

The political capital invested in Australia's engagement with Asia over the past decade has sparked a lively discussion in the Australian academic community. The back cover of the book under review suggests that there are 'few bigger contemporary issues facing Australia than its relationship with Asia'. If the volume of scholarly material being produced on this issue is any indication, they are right. Like a number of similar works covering the shift in Australian foreign, defence, and trade policies towards Asia over the last decade, this book acknowledges a particular debt of gratitude to the Keating government for establishing regional engagement at the forefront of our national consciousness. Unlike some others however, this book seeks to place Australia's more recent 'discovery' of Asia into a broader historical framework.

Annexin 1 mimetic peptide protects against renal ischemia/reperfusion injury in rats

Inflammation is currently recognized as a key mechanism in the pathogenesis of renal ischemia-reperfusion (I/R) injury. The importance of infiltrating neutrophil, lymphocytes, and macrophage in this kind of injury has been assessed with conflicting results. Annexin 1 is a protein with potent neutrophil anti-migratory activity. In order to evaluate the effects of annexin A1 on renal I/R injury, uninephrectomized rats received annexin A1 mimetic peptide Ac2-26 (100 mu g) or vehicle before 30 min of renal artery clamping and were compared to sham surgery animals. Annexin A1 mimetic peptide granted a remarkable protection against I/R injury, preventing glomerular filtration rate and urinary osmolality decreases and acute tubular necrosis development. Annexin A1 infusion aborted neutrophil extravasation and attenuated macrophage infiltration but did not prevent tissue lymphocyte traffic. I/R increased annexin A1 expression (assessed by transmission electron microscopy) in renal epithelial cells, which was attenuated by exogenous annexin A1 infusion. Additionally, annexin A1 reduced I/R injury in isolated proximal tubules suspension. Annexin A1 protein afforded striking functional and structural protection against renal I/R. These results point to an important role of annexin A1 in the epithelial cells defense against I/R injury and indicate that neutrophils are key mediators for the development of tissue injury after renal I/R. If these results were confirmed in clinical studies, annexin A1 might emerge as an important tool to protect against I/R injury in renal transplantation and in vascular surgery.

Annexin A1 subcellular expression in laryngeal squamous cell carcinoma

Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.

Expression of genes that encode the annexin-1 and galectin-1 proteins in nasal polyposis and their modulation by glucocorticoid

Rhinosinusal polyps physiopathology is not fully understand, despite numerous hypotheses regarding its inflammatory process. Aims: a prospective study regarding the gene expression of proteins: anexin-1 and galectin-1, which has an anti-inflammatory action and is modulated by steroids. Materials and Methods: eleven patients with rhinosinusal polyps suffered a biopsy of their polyps at two moments: in the absence of systemic steroids and during its use. In the two samples we assessed the expression of these genes and compared it to the normal nasal mucosa in the middle meatus. Results: We noticed that the mean expression of the genes which code anexin-1 and galectin-1 was predominantly increased, regardless of the use of steroids in relation to the control nasal mucosa. Notwithstanding, in polyps without the use of steroids, the mean gene expression of anexin-1 was significantly higher than in the polyps which were under the use of steroids. Regarding galectin-1, there was no significant difference between the expression mean values before and after the use of systemic steroids. Conclusion: The genes present an expression increase in the polyp mucosa, regardless of the use of steroids; nonetheless, the relationship of these two genes of anti-inflammatory proteins with steroids did not happen the same way.

Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca2+-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (∼1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability. © 2002 Elsevier Science B.V. All rights reserved.

Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes-but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells.

Aspectos ecológicos da tripanossomíase americana: VII. Permanência e mobilidade do Triatoma sordida em relação aos ecótopos artificiais

Nas colônias desenvolvidas espontaneamente em ecótopos artificiais, representados por galinheiros experimentais, procurou-se observar a permanência e a mobilidade de Triatoma sordida. Para tanto, procedeu-se à marcação individual de adultos e ninfas de 4.° e 5.° estádios, bem como à instalação de abrigos ao redor daqueles biótopos. Os dados obtidos permitiram observar a curta permanência dos primeiros na colônia, não ultrapassando o período de dois meses para a maioria dessas formas. A mobilidade evidenciou-se pela presença de ovos e ninfas de 1.° estádio, nos esconderijos instalados, e as evidências sugeriram ser devidas à passagem dos adultos por esses ecótopos. Concluiu-se que esta espécie é dotada de elevada capacidade de disseminação, em concordância com sua apreciável valência ecológica.

Estudo das condições de saúde das crianças do Município de São Paulo (Brasil), 1984/1985: VII - Parasitoses intestinais

Como parte de estudo populacional sobre condições de saúde na infância, uma amostra representativa das crianças menores de cinco anos residentes no Município de São Paulo, SP (Brasil) (n=695) foi submetida a exames parasitológicos de fezes. Os exames foram realizados através da técnica de sedimentação e, quando as fezes tinham consistência amolecida ou liquefeita, também pela técnica do exame direto. A prevalência de enteroparasitoses em geral foi de 30,9%, sendo de 16,4%, 14,5% e 12,5% as prevalências específicas da ascaridíase, giardíase e tricuríase. Prevalências inferiores foram assinaladas para os enteroparasitas E. histolytica, H. nana e S. stercoralis, respectivamente 2,0%, 0,9% e 0,3%. Em apenas uma criança foram encontrados ovos de ancilostomídeos e em nenhuma delas ovos de Schistosoma mansoni. Das crianças examinadas, 13,1% apresentaram duas ou mais espécies de enteroparasitas e 4,8% três ou mais. As prevalências atuais, comparadas às prevalências encontradas em 1973/74 por outro inquérito populacional realizado no município, indicam queda expressiva da ascaridíase e tricuríase, mas não da giardíase. A estratificação das prevalências segundo faixa etária revelou aumento significativo com a idade da criança, chamando atenção o aumento que ocorre do primeiro para o segundo ano de vida. As enteroparasitoses aumentam também significativamente sua freqüência à medida que piora o nível socioeconômico, chegando a ser de nove vezes a diferença de prevalência existente entre os estratos socioeconômicos extremos da população. No caso específico da giardíase o gradiente socioeconômico foi consideravelmente menor do que o encontrado para as demais enteroparasitoses, o que confirma a maior complexidade epidemiológica do problema.

Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the b-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.

Analysis of a 17.9 kb region from Saccharomyces cerevisiae chromosome VII reveals the presence of eight open reading frames, including BRF1 (TFIIIB70) and GCN5 genes

We report the nucleotide sequence of a 17,893 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5' portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat-shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins. YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases.