ABSTRACT The dual roles of baculovirus for the control of natural insect populations as an insecticide, and for foreign gene expression and delivery, have called for a comprehensive understanding of the molecular mechanisms governing viral infection. Here, we demonstrate that the Bombyx mori Niemann-Pick C1 (BmNPC1) is essential for baculovirus infection in insect cells. Both pretreatment of Bombyx mori embryonic cells (BmE) with NPC1 antagonists (imipramine or U18666A) and down-regulation of NPC1 expression resulted in a significant reduction in baculovirus BmNPV ( Bombyx mori nuclear polyhedrosis virus) infectivity. Furthermore, we show that the major glycoprotein gp64 of BmNPV, responsible for both receptor binding and fusion, is able to interact predominantly with the BmNPC1 C domain, with an enhanced binding capacity at low pH conditions, indicating that NPC1 most likely plays a role during viral fusion in endosomal compartments. Our results, combined with previous studies identifying an essential role of hNPC1 in filovirus infection, suggest that the glycoprotein of several enveloped viruses possess a shared strategy of exploiting host NPC1 proteins during virus intracellular entry events. IMPORTANCE BmNPV is one of the most important members of the Baculoviridae ; many viruses in this family have been frequently employed as viral vectors for foreign gene delivery or expression and as biopesticides, but their host receptors still remain unclear. Here, we describe that the intracellular cholesterol transporter BmNPC1 is indispensable for BmNPV infection in insect cells, and it interacts with the major viral glycoprotein gp64. Our study on the role of BmNPC1 in baculovirus infection has further expanded the list of the enveloped viruses that require host NPC1 proteins for entry, and will ultimately help us to uncover the molecular mechanism of the involvement of NPC1 proteins in the entry process of many enveloped viruses.
The ubiquitin-proteasome system (UPS) is the major protein turnover mechanism that plays an important role in regulating various cellular functions. F-box proteins are the key proteins of the UPS, responsible for the specific recognition and ubiquitination of downstream targets. Our previous studies showed that the F-box protein Fbp1 plays an essential role in the virulence of C. neoformans. However, the molecular mechanism of Fbp1 regulating the virulence of C. neoformans is still unclear. In this study, we analyzed the potential Fbp1 substrates using an iTRAQ-based proteomic approach and identified the zinc-binding protein Zbp1 as a substrate of Fbp1. Protein interaction and stability assays showed that Zbp1 interacts with Fbp1 and is a downstream target of Fbp1. Ubiquitination analysis in vivo showed that the ubiquitination of Zbp1 is dependent on Fbp1 in C. neoformans. Subcellular localization analysis revealed that the Zbp1 protein was localized in the nucleus of C. neoformans cells. In addition, both deletion and overexpression of the ZBP1 gene led to the reduced capsule size, while overexpression has a more significant impact on capsule size reduction. Fungal virulence assays showed that although the zbp1Δ mutants are virulent, virulence was significantly attenuated in the ZBP1 overexpression strains. Fungal load assay showed that the fungal burdens recovered from the mouse lungs decreased gradually after infection, while no yeast cells were recovered from the brains and spleens of the mice infected by ZBP1 overexpression strains. Thus, our results revealed a new determinant of fungal virulence involving the post-translational regulation of a zinc-binding protein.
The ubiquitin-proteasome system is one of the major protein turnover mechanisms that plays important roles in the regulation of a variety of cellular functions. It is composed of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 ubiquitin ligases that transfer ubiquitin to the substrates that are subjected to degradation in the 26S proteasome. The Skp1, Cullin, F-box protein (SCF) E3 ligases are the largest E3 gene family, in which the F-box protein is the key component to determine substrate specificity. Although the SCF E3 ligase and its F-box proteins have been extensively studied in the model yeast Saccharomyces cerevisiae, only limited studies have been reported on the role of F-box proteins in other fungi. Recently, a number of studies revealed that F-box proteins are required for fungal pathogenicity. In this communication, we review the current understanding of F-box proteins in pathogenic fungi.
Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence.
Autophagy (macroautophagy) is an essential and highly conserved protein degradation mechanism in eukaryotes. In this process, cytoplasmic organelles, old proteins and other macromolecules are sequestered into a double membrane vesicle called autophagosome and delivered to a degradative organelle for degradation and recycling. Extensive studies have revealed that autophagy plays an essential role in the cellular processes such as cell differentiation, nutrient homeostasis and pathogenicity in pathogenic fungi. In this review, we introduce the process of autophagy and describe the role of autophagy in regulation of the fungal sexual reproduction, using the human fungal pathogen Cryptococcus neoformans as an example. Furthermore, we summarize the autophagy related genes studied so far and the deduced physiological functions of autophagy for proper asexual and/or sexual reproduction in model pathogenic fungi. We also discuss perspectives on autophagy function in fungal reproduction.
ABSTRACT Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1 Δ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1 Δ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1 Δ mutant is due to its inability to leave the lung. The fbp1 Δ mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus -macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus -macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.
Objective
To investigate the efficacy and safety of radiofrequency catheter ablation(RFCA) treatment for children with tachyarrhythmia of various types.
Methods
Two hundred and sixty-one cases with tachyarrhythmia who received RFCA at Shandong Provincial Hospital Affiliated to Shandong University from Aug. 2000 to Dec. 2012 were selected. All electrocardiogram(ECG) and echocardiography data were obtained. All of the 261 patients underwent electrophysiological study and RFCA. The clinical data of the pediatric patients with tachyarrhythmia after RFCA in Shandong Provincial Hospital Affiliated to Shandong University were retrospectively analyzed and the curative effect and the complication rate of RFCA treatment for children with tachyarrhythmia of various types were investigated.
Results
(1)Among the 261 cases, 4 cases had tachyarrhythmia associated with tachycardia induced cardiomyopathy, and 1 case had tachycardia associated with heart failure. (2)One hundred and forty-six cases had atrioventricular reentrant tachycardia(AVRT); 74 cases had atrioventricular nodal reentrant tachycardia(AVNRT); 32 cases had idiopathic ventricular tachycardia(IVT); 6 cases had atrial tachycardia(AT); and 3 cases had atrial flutter(AF). Ten children with tachyarrhythmia associated with organic heart disease received RFCA successfully. (3) The average operation time was(101.23±51.37) minutes and the average X-ray exposure time was(21.85±17.10) minutes. (4)The total successful rate of RFCA was 98.08%(256/261 cases), 1 case(0.38%) suffered from pneumothorax after operation, and recovered after treatment. There was no serious complications nor deaths of all the patients. (5) Twenty-two cases recurred, and the total recurrence rate was 8.43%(22/261 cases), time to relapse was 3 days to 5 years, and the average time was 7 months. There were 9 cases in IVT(9/32 cases, 28.13%), 7 cases in AVRT(7/146 cases, 4.79%), 4 cases in AVNRT(4/74 cases, 5.41%), 2 cases in AT(2/6 cases, 33.33%). Eighteen cases received successful RFCA for second time, and 4 cases had good effect after drug control.
Conclusions
(1)RFCA in pediatric patients of tachyarrhythmia is relatively convenient, and this therapy can be performed safely and effectively that can cure certain tachyarrhythmia. (2) AVRT is the common type of tachyarrhythmia in children, followed by AVNRT, IVT, AT and AF. (3) The total recurrence rate of RFCA in children is low, but is relatively high in IVT and AT. (4) The success rate of RFCA is the same in children combined organic heart disease.
Key words:
Radiofrequency catheter ablation; Tachyarrhythmia; Child
To identify single nucleotide polymorphisms (SNP) of the angiotensin II type 2 receptor (AGTR2) gene, and to determine whether the AGTR2 polymorphisms are associated with essential hypertension in a male Chinese population.Direct DNA sequencing was performed in 20 subjects. 96 male hypertensive patients and 107 normal controls were included to assess the contribution of the SNP of AGTR2 gene to hypertension.Seven SNP of the AGTR2 gene were identified, of which 4 were reported for the first time. A case-control study including two polymorphisms (A1675G and T1334C) showed a significant increase in the A1675 allele frequency among male hypertensive subjects as compared with normotensive subjects (49.0% vs 34.6%, P < 0.05).The AGTR2 A1675G polymorphism might be involved in the development of essential hypertension in male Chinese.
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