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.     

Thioredoxin 1 as a serum marker for breast cancer and its use in combination with CEA or CA15-3 for improving the sensitivity of breast cancer diagnoses

Background

Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection.

Findings

All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure.

Conclusions

When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3′ splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation.     

Functional reconstitution and regulation of IL-18 activity by the IL-18R beta chain

IL-18 and IL-12 are major IFN-gamma-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18R alpha, and IL-18R beta are expressed constitutively but IL-18 did not induce IFN-gamma unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1 beta or TNF-alpha, do not respond to IL-18, despite abundant expression of the IL-18R alpha chain. COS-1 cells lack expression of the IL-18R beta chain. The IL-18R beta cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18R beta chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18R alpha chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18R beta chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18R alpha and IL-18R beta; the production of IFN-gamma corresponded to IL-12-induced IL-18R alpha and IL-18R beta chains. We conclude that functional reconstitution of the IL-18R beta chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-gamma production is, in part, due to IL-12 up-regulation of both IL-18R alpha and IL-18R beta chains, although postreceptor events likely contribute to IFN-gamma production.     

Multiple actions of dimethylsphingosine in 1321N1 astrocytes

N,N-dimethyl-D-erythro-sphingosine (DMS) is an N-methyl derivative of sphingosine and an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In the present study, we examined the effects of DMS on intracellular Ca2+ concentration, pH, and glutamate uptake in human 1321N1 astrocytes. DMS increased intracellular Ca2+ concentration and cytosolic pH in a concentration-dependent manner. Pretreatment of the cells with the Gi/o protein inhibitor PTX and the PLC inhibitor U73122 had no obvious effect. However, removal of extracellular Ca2+ with the Ca2+ chelator EGTA or depletion of intracellular Ca2+ stores with thapsigargin impeded the DMS-induced increase of intracellular Ca2+ concentration. Pretreatment of cells with NH4Cl or monensin reduced the DMS-induced Ca2+ increase. However, inhibition of the DMS-induced Ca2+ increase with BAPTA did not influence the DMS-induced pH increase. DMS also inhibited glutamate uptake by the 1321N1 astrocytes in a concentration-dependent manner. It also increased intracellular Ca2+ and pH in PC12 neuronal cells. Our observations on the effects of DMS on 1321N1 astrocytes and PC12 neuronal cells point to a physiological role of DMS in the brain.     

Structure-activity relationship of leucyladenylate sulfamate analogues as leucyl-tRNA synthetase (LRS)-targeting inhibitors of Mammalian target of rapamycin complex 1 (mTORC1)

Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1 signaling, which is known to be associated with cellular metabolism and proliferation. Therefore, LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate sulfate analogues by extensively modifying three different pharmacophoric regions comprising adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1 activation assays and further tested for anticancer activity. The selected compounds mostly exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic approaches.     

Draft Genome Sequence of Escherichia coli W26, an Enteric Strain Isolated from Cow Feces

An enteric bacterium, Escherichia coli W26 (KACC 16630), was isolated from feces from a healthy cow in South Korea. Here, we report the draft genome sequence of the isolate, which is closely affiliated with commensal strains belonging to E. coli phylogroup B1.     

Altered regulation of renal nitric oxide and atrial natriuretic peptide systems in angiotensin II-induced hypertension

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO) and atrial natriuretic peptide (ANP) systems in the kidney in association with the angiotensin (Ang) II-induced hypertension. Male Sprague-Dawley rats were used. Ang II (100 ng·min?1·kg?1) was infused through entire time course. Thirteenth day after beginning the regimen, kidneys were taken. The protein expression of NO synthase (NOS) and nitrotyrosine was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed hypertension and decreased creatinine clearance in the experimental group. The protein expression of eNOS, nNOS and nitrotyrosine was increased in the cortex, while that of iNOS remained unaltered. The urinary excretion of NO increased in Ang II-induced hypertensive rats. The catalytic activity of soluble guanylyl cyclase was blunted in the glomerulus in Ang II-induced hypertensive rats. The mRNA expression of ANP was increased in Ang II-induced hypertensive rats. Neither the expression of NPR-A nor that of NPR-C was changed. The protein expression of neutral endopeptidase was decreased and the activity of particulate guanylyl cyclase was blunted in the glomerulus and papilla in Ang II-induced hypertensive rats. In conclusion, the synthesis of NO and ANP was increased in the kidney of Ang II-induced hypertension, while stimulated cGMP response was blunted. These results suggest desensitization of guanylyl cyclase in the kidney of Ang II-induced hypertensive rats, which may contribute to the associated renal vasoconstriction and hypertension.