Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems

Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1–7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting.     

Induction of aneuploidy by oncodazole (nocodazole), an anti-tubulin agent, and acetone

Oncodazole (nocodazole) is a compound which interacts with yeast and bovine tubulin. We have shown that it induces aneuploidy in the yeast Saccharomyces cerevisiae at very low concentrations. In the course of a search for an appropriate solvent for oncodazole we observed that acetone also induces mitotic aneuploidy in yeast. This effect of acetone was greatly enhanced when the treatment of growing cells at 28 degrees C was interrupted by a period of holding at ice-bath temperature.     

A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots

Calcium (Ca²⁺), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca²⁺] elevations are early events in the interaction between the plant growth‐promoting fungus Piriformospora indica and Arabidopsis thaliana. A cell wall extract (CWE) from the fungus promotes the growth of wild‐type seedlings but not of seedlings from P. indica‐insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca²⁺ signalling‐related functions. The CWE induces a transient cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum) plants, as well as in BY‐2 suspension cultures expressing the Ca²⁺ bioluminescent indicator aequorin. Nuclear Ca²⁺ transients were also observed in tobacco BY‐2 cells. The Ca²⁺ response was more pronounced in roots than in shoots and involved Ca²⁺ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca²⁺ response by staurosporine and the refractory nature of the Ca²⁺ elevation suggest that a receptor may be involved. The CWE does not stimulate H₂O₂ production and the activation of defence gene expression, although it led to phosphorylation of mitogen‐activated protein kinases (MAPKs) in a Ca²⁺‐dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica. Thus, Ca²⁺ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.     

Borderline typical symptoms in adult patients with attention deficit/hyperactivity disorder

Adult attention deficit/hyperactivity disorder (ADHD) and borderline personality disorder (BPD) share several clinical features, e.g. emotional lability and impulsivity. This study aimed to delineate differences and similarities between ADHD and BPD with respect to borderline typical symptomatology and gender specifics. Borderline symptomatology was assessed in 60 adult patients with ADHD with the borderline symptom list (BSL) and compared to both 60 gender- and age-matched BPD patients and control subjects. The BSL is a standardized instrument including 95 items on 7 subscales (self-perception, affect regulation, self-destruction, dysphoria, loneliness, intrusions and hostility). Adult ADHD patients showed significantly higher BSL total scores and all of the seven subscales compared to healthy controls (p < 0.001) but lower scores than BPD patients (p < 0.001). With respect to the seven subscales, the largest differences between ADHD and BPD patients were found with respect to self-destruction (d = 1.12) and affect dysregulation (d = 0.90), whereas the smallest difference was found with respect to loneliness (d = 0.36). In females, the BSL subscales "loneliness" and "hostility" did not differentiate between BPD and ADHD. Borderline typical symptoms are common in adult patients with ADHD but seem to be less pronounced than in patients with BPD. Females with ADHD and BPD share more clinical features than males. However, symptoms of self-destruction and affect dysregulation appear to be more severe in BPD patients.     

The hen's egg test for micronucleus induction (HET-MN): novel analyses with a series of well-characterized substances support the further evaluation of the test system

The hen's egg test for micronucleus induction (HET-MN) combines the use of the commonly accepted genetic endpoint "formation of micronuclei" with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics -- including those that are mutagens or promutagens. This assay procedure is in line with demands for animal protection. In three previous publications we presented the scientific rationale and methodological aspects for this assay as well as results for some well-characterized mutagens and promutagens. Here we present the results of new experiments involving further genotoxic and non-genotoxic model substances. Making a comparison with published data we have to date not found any false negatives or false positives in the experiments presented here and in trials published before, thus demonstrating a promising predictivity of genotoxic effects with this assay. We could confirm relevant genotoxicity for the following substances in the HET-MN: acetylamino-fluorene (2-AAF), acrylamide (ACM), cytarabine (AraC), methotrexate (MTX), cadmium chloride (CD), dipotassium monochromate (DPC), and epirubicine (EPI). Negative results were obtained for azorubin (E122), orange G (OG) and starch (STRC). The micronucleus frequencies (MNE II) of the concurrent negative controls were in agreement with the values of the historical negative control (0.87 per thousand+/-0.87; average+/-s.d.). This value is based upon the scoring of 556,500 erythrocytes from 445 eggs. In historical positive controls the administration of 0.05mg cyclophosphamide/egg at d8 resulted in an MNE II-frequency of 12.4 per thousand+/-6.8 (average+/-s.d.) at d 10.5. This value is based upon the scoring of 249,250 erythrocytes from 223 eggs.     

Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase

As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.