Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (␣) and 40-kDa () AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.Human acid ceramidase ((AC) 1 N-acylsphingosine amidohydrolase, EC 3.5.1.23) catalyzes the hydrolysis of ceramide to free fatty acid and sphingosine (1). An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease (FD), also called Farber lipogranulomatosis (2). Patients with FD accumulate ceramide in most tissues, leading to painful swelling of the joints and tendons, pulmonary insufficiency, and a shortened life-span. The clinical diagnosis of FD is usually confirmed by biochemical methods, including the determination of lysosomal ceramide accumulation and/or the deficiency of AC activity. To date, seven FD subtypes have been described with varying degrees of clinical involvement; notably, a direct correlation between the amount of ceramide accumulation and the clinical severity of FD patients has recently been demonstrated (3).In addition to its central role in disease pathogenesis, sphingolipid biosynthesis, and membrane formation, ceramide is an important cell-signaling molecule involved in a variety of diverse processes such as neurite growth, monocyte differentiation, and Fas (Apo1/CD95)-induced apoptosis (for reviews, see Refs. 4 and 5). Moreover, sphingosine (the catabolic product of ceramide degradation) has been shown to inhibit protein kinase C activity and can exert a variety of effects on cell growth and differentiation (6 -8). Since ceramide degradation is the only catabolic source of intracellular sphingosine (9, 10), AC activity may be the rate-limiting step in determining the intracellular levels of this compound.About 2 years ago, AC was purified...
Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.
Influenza A virus (IAV) infection poses a serious health threat and novel antiviral strategies are needed. Defective interfering particles (DIPs) can be generated in IAV infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. The antiviral activity of DIPs is exerted by a DI genomic RNA segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. DI-244 is a naturally occurring prototypic segment 1-derived DI RNA in which most of the PB2 open reading frame has been deleted and which is currently developed for antiviral therapy. At present, coinfection with wt virus is required for production of DI-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. Here, we show that cocultures of 293T and MDCK cell lines stably expressing codon optimized PB2 allow production of DI-244 particles solely from plasmids and in the absence of helper virus. Moreover, we demonstrate that infectivity of these particles can be quantified using MDCK-PB2 cells. Finally, we report that the DI-244 particles produced in this novel system exert potent antiviral activity against H1N1 and H3N2 IAV but not against the unrelated vesicular stomatitis virus. This is the first report of DIP production in the absence of infectious IAV and may spur efforts to develop DIPs for antiviral therapy.
The human cytomegalovirus (HCMV) basic phosphoprotein pp 150, encoded by the UL32 gene, together with the two other major phosphoproteins, pp65 (ppUL83) and pp71 (ppUL82) and several minor structural proteins, form the tegument around the viral nucleocapsid. Experiments were undertaken to locate the area of assembly of tegument proteins ppl50 and pp65 and nucleocapsids in fibroblasts, in order to assess the functional role of these two structural proteins in HCMV morphogenesis. Whereas ppl50 expression starts during the cytoplasmic maturation of HCMV, pp65 is expressed in the early and late phases of HCMV gene transcription. Western blot analysis of isolated cell fractions showed that ppl50 is initially (48 h postinfection) localized in the nucleus, associated either with the nuclear membrane or with viral assembly regions, and later (72 h post-infection) in the cytoplasm. By indirect immunofluorescence, ppl50 and pp65 could be detected in nuclear subcompartments and were strongly associated with the nuclear membrane. Using immunogold analysis by electron microscopy, pp65 was exclusively detected within the matrix of cytoplasmic and extracellular dense bodies and of dense body-like structures in the nucleoplasm. These were localized in close contact with hypertrophic nucleoli, in the proximity of developing nucleocapsids and in special patches at the inner nuclear membrane L Positive immunostaining of ppl50 was observed at the surface of developing nucleocapsids concentrated within viral assembly regions in the nucleoplasm. Additionally, the tegument of cytoplasmic and extracellular virions was stained, whereas dense bodies or nuclear dense body-like structures did not react. Thus, the acquisition of the tegument protein pp 150 seems to start in special nuclear subcompartments of the HCMV-infected fibroblasts.
Epidemiological studies exploring the connection between hypertension and cancer demonstrate a higher cancer incidence, especially of kidney cancer, and a higher cancer mortality in hypertensive patients. Hormones elevated in hypertension, i.e., aldosterone and angiotensin II, which exert genotoxic effects in vitro, could contribute to carcinogenesis in hypertension. The present study was conducted to investigate the possible DNA-damaging effect of aldosterone receptor activation in vivo. Crl:CD (Sprague-Dawley) rats were treated for 6 wk with desoxycorticosterone acetate (DOCA) and salt to induce a mineralocorticoid-dependent hypertension. DOCA-salt treatment caused increased blood pressure (؉26 mmHg) compared to untreated rats, elevated markers of kidney failure (up to 62-fold for Kim-1), and the induction of several proinflammatory genes and proteins (up to 2.6-fold for tissue MCP-1). The mineralocorticoid receptor (MR) antagonist spironolactone (MR IC 50 24 nM) and the novel nonsteroidal antagonist BR-4628 (MR IC 50 28 nM) decreased these damage markers. DOCA-salt treatment also caused 8.8-fold increased structural DNA damage, determined with the comet assay, double-strand breaks (3.5-fold), detected immunohistochemically, and oxidative stress. Furthermore, the oxidatively modified mutagenic DNA base 7,8-dihydro-8-oxoguanine (8-oxodG), quantified by LC-MS/MS, was almost 2-fold higher in DOCA-salt-treated kidneys. Our results suggest a mutagenic potential of high mineralocorticoid levels, frequent in hypertensive individuals.-Schupp, N., Kolkhof, P., Queisser, N., Gärtner, S., Schmid, U., Kretschmer, A., Hartmann, E., Oli, R G., Schäfer, S., Stopper, H. Mineralocorticoid receptor-mediated DNA damage in kidneys of DOCA-salt hypertensive rats. FASEB J. 25, 968 -978 (2011). www.fasebj.org Key Words: 8-oxodG ⅐ double-strand breaks ⅐ hypertension ⅐ oxidative DNA damage ⅐ oxidative stressAldosterone is produced in the adrenal zona glomerulosa on stimulation by angiotensin II, potassium, or the adrenocorticotropic hormone. Classically, it regulates sodium excretion and thereby blood pressure homeostasis through mineralocorticoid receptor (MR) dependent, genomic effects in the distal nephron of the kidney. Together with angiotensin II, it is the major effector of the renin-angiotensin-aldosterone system. Recent studies suggest that 10 -15% of hypertensive individuals have an elevated aldosterone to renin ratio, a biochemical measure for hyperaldosteronism (1). The plasma concentration of aldosterone in these patients ranges from 0.5 to 6.3 nM, with patients suffering from congestive heart failure reaching 8 nM (2, 3). Studies demonstrating the effectiveness of MR antagonism in patients with resistant hypertension, also support the high prevalence (20%) of hyperaldosteronism in this population (1, 4). Aldosterone plays an important role in the pathophysiology of heart failure. Clinical trials examining the effect of combined angiotensin converting enzyme (ACE) inhibition and MR antagonism showed a reduction of mortalit...
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