Aspergillus fumigatus RasA regulates asexual development and cell wall integrity

The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The DeltarasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The DeltarasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The DeltarasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.     

Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus

Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37 degrees C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a DeltacgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the DeltacgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although DeltacgrA conidia germinated normally at 22 degrees C, they swelled excessively when incubated at 37 degrees C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.     

Developmental expression patterns of cuticular protein genes with the R&R Consensus from Anopheles gambiae

CPR proteins are the largest cuticular protein family in arthropods. The whole genome sequence of Anopheles gambiae revealed 156 genes that code for proteins with the R&R Consensus and named CPRs. This protein family can be divided into RR-1 and RR-2 subgroups, postulated to contribute to different regions of the cuticle. We determined the temporal expression patterns of these genes throughout post-embryonic development by means of real-time qRT-PCR. Based on expression profiles, these genes were grouped into 21 clusters. Most of the genes were expressed with sharp peaks at single or multiple periods associated with molting. Genes coding for RR-1 and RR-2 proteins were found together in several co-expression clusters. Twenty-five genes were expressed exclusively at one metamorphic stage. Five out of six X-linked genes showed equal expression in males and females, supporting the presence of a gene dosage compensation system in A. gambiae. Many RR-2 genes are organized into sequence clusters whose members are extremely similar to each other and generally closely associated on a chromosome. Most genes in each sequence cluster are expressed with the same temporal expression pattern and at the same level, suggesting a shared mechanism to regulate their expression.     

Rhodobacter sphaeroides: complexity in chemotactic signalling

Most bacteria have much more complex chemosensory systems than those of the extensively studied Escherichia coli. Rhodobacter sphaeroides, for example, has multiple homologues of the E. coli chemosensory proteins. The roles of these homologues have been extensively investigated using a combination of deletion, subcellular localization and phosphorylation assays. These studies have shown that the homologues have specific roles in the sensory pathway, and they differ in their cellular localization and interactions with other components of the pathway. The presence of multiple chemosensory pathways might enable bacteria to tune their tactic responses to different environmental conditions.     

Measurement of mRNA of 11 biomarkers by RT-PCR to detect lymph node involvement in cervical cancer

Lymph node metastases are a major prognostic factor in cervical carcinomas. The aim of this study was to characterize the expression of 11 markers in cervical tumors and negative lymph nodes and to determine which ones could be helpful for improving the specificity of molecular diagnosis of nodal involvement. Using TaqMan RT-PCR, we studied the expression of CK19, MUC1, HER1-HER4, VEGF, VEGF-C, uPA, MMP9, and PRAD1 in uterine cervical tumors and in histologically nonmetastatic lymph nodes of 8 patients diagnosed with locally advanced cervical cancer. We observed that CK19, MUC1, HER1-HER3, uPA, and VEGF had a significantly higher expression in cervical tumors than in the negative nodes, whereas VEGF-C expression level was higher in the negative nodes than in the tumors. PRAD1 harbored similar expression levels in the tumors and in the negative nodes. Interestingly, 1 of the 4 patients who presented a clinical recurrence, showed elevated HER1, HER2, uPA, and VEGF in the histologically negative nodes. Our results suggest that CK19, MUC1, HER1-3, uPA, and VEGF are biomarkers that have a higher expression in tumoral cervical tissues compared with the negative lymph nodes and could be useful to diagnose nodal involvement in uterine cervical carcinoma. Our results should encourage us in continue to investigate a greater number of patients, including patients with histologically involved nodes.     

Genome integrity is regulated by the Caenorhabditis elegans Rad51D homolog rfs-1

Multiple mechanisms ensure genome maintenance through DNA damage repair, suppression of transposition, and telomere length regulation. The mortal germline (Mrt) mutants in Caenorhabditis elegans are defective in maintaining genome integrity, resulting in a progressive loss of fertility over many generations. Here I show that the high incidence of males (him)-15 locus, defined by the deficiency eDf25, is allelic to rfs-1, the sole rad-51 paralog group member in C. elegans. The rfs-1/eDf25 mutant displays a Mrt phenotype and mutant animals manifest features of chromosome fusions prior to the onset of sterility. Unlike other Mrt genes, rfs-1 manifests fluctuations in telomere lengths and functions independently of telomerase. These data suggest that rfs-1 is a novel regulator of genome stability.     

Trichinella spiralis induces de novo expression of group IVC phospholipase A2 in the intestinal epithelium

Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.     

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.     

Oxidative pentose phosphate pathway-dependent sugar sensing as a mechanism for regulation of root ion transporters by photosynthesis

Root ion transport systems are regulated by light and/or sugars, but the signaling mechanisms are unknown. We showed previously that induction of the NRT2.1 NO(3)(-) transporter gene by sugars was dependent on carbon metabolism downstream hexokinase (HXK) in glycolysis. To gain further insights on this signaling pathway and to explore more systematically the mechanisms coordinating root nutrient uptake with photosynthesis, we studied the regulation of 19 light-/sugar-induced ion transporter genes. A combination of sugar, sugar analogs, light, and CO(2) treatments provided evidence that these genes are not regulated by a common mechanism and unraveled at least four different signaling pathways involved: regulation by light per se, by HXK-dependent sugar sensing, and by sugar sensing upstream or downstream HXK, respectively. More specific investigation of sugar-sensing downstream HXK, using NRT2.1 and NRT1.1 NO(3)(-) transporter genes as models, highlighted a correlation between expression of these genes and the concentration of glucose-6-P in the roots. Furthermore, the phosphogluconate dehydrogenase inhibitor 6-aminonicotinamide almost completely prevented induction of NRT2.1 and NRT1.1 by sucrose, indicating that glucose-6-P metabolization within the oxidative pentose phosphate pathway is required for generating the sugar signal. Out of the 19 genes investigated, most of those belonging to the NO(3)(-), NH(4)(+), and SO(4)(2-) transporter families were regulated like NRT2.1 and NRT1.1. These data suggest that a yet-unidentified oxidative pentose phosphate pathway-dependent sugar-sensing pathway governs the regulation of root nitrogen and sulfur acquisition by the carbon status of the plant to coordinate the availability of these three elements for amino acid synthesis.