GALl, GAL4 and GALIO transcription [5,6]. The sequence 5'-SYGGRG-Y been proposed as a consensus for CreA-binding [7]. Unlike MIG1, however, CreA contains an additional domain downstream of the zinc-finger, which has been reported to bear high similarity to S. cerevisiae RGR1 [8,9], and whose function is unknown. Since its cloning and sequencing, molecular evidence has been presented for an involvement of CreA in the catabolite repression of transcription of genes involved in proline utilization [7], ethanol metabolism [10,11] and polysaccharide hydrolysis [12] in A. nidulans.Nothing is known as yet on the mechanism of carbon catabolite repression in other fungi. The filamentous fungus Trichoderma reesei is an industrially important producer of several extracellular enzymes, including a highly active cellulase [13] and hemicellulase enzyme system [14]. The formation of some of these enzymes (e.g. cellobiohydrolase I; endo-fl-l,4-xylanase I) is repressed by glucose [15,16]. It has been reported that the 5'-upstream nt-sequence of the T. reesei gene encoding cellobiohydrolase I (cbhl) shows consensus sequences for binding of a potential CreA-homologue [17]. Deletion of these sequences resulted in glucose derepressed transcription of cbhl [17]. It is therefore possible that carbon catabolite repression in T. reesei occurs by a mechanism similar to that existant in Aspergillus. However, the presence of a DNA-binding protein in T. reesei similar to CreA has not yet been published. As a first step towards understanding the mechanisms and cloning of the genes involved in carbon catabolite repression in T. reesei, we demonstrate here the presence of a creA homologue in T. reesei Crel --and provide evidence that the native gene product is a DNA-binding protein, thereby showing that the mechanisms of carbon catabolite repression have been basically conserved in the ascomycetous classes of Pyrenomycetes and Plectomycetes.Carbon catabolite repression in microorganisms is a means I~) control the synthesis of a range of enzymes required for the t~tilization of less favoured carbon sources when more readily t~tilized carbon sources are present in the medium. Several genes participating in this process have been identified in Sactzaromyces cerevisiae [1,2]. In the multicellular fungi, the creA gene cloned from Aspergillus nidulans [3] and A. niger [4] is the ~nly hitherto regulatory gene known to mediate carbon cat~l bolite repression. It encodes a DNA-binding protein containi Mlg a two-zinc-finger domain of the C2H2 class, which mediates ,~4% similarity to MIG1 from S. cerevisiae, which is also *Corresponding author. Fax: (43)(1) 581-6266. l-mail: jos@eichow.tuwien.ac.at Experimental Strain, cloning vector and plasmidTrichoderma reesei strain QM 9414 (ATCC 26921 ) was used throughout this study and maintained on malt agar. Bluescript II/SK+ (Stratagene, La Jolla, CA) and E. coli LC 137 (Pharmacia-LKB, Uppsala, Sweden) were used as cloning and plasmid vectors, respectively. Cloning of the T. reesei crel geneFungal genomic DNA...

BackgroundH5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals.Methodology/Principal FindingsWe demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH≤5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice.Conclusion/SignificanceOur finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

BACKGROUND. The nonstructural protein NS1 of influenza virus counteracts the interferon-mediated immune response of the host. By deleting the open reading frame of NS1, we have generated a novel type of influenza vaccine. We studied the safety and immunogenicity of an influenza strain lacking the NS1 gene (DeltaNS1-H1N1) in healthy volunteers. METHODS. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the DeltaNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log(10) median tissue culture infective dose) (n = 36 recipients) or placebo (n = 12 recipients). RESULTS. Intranasal vaccination with the replication-deficient DeltaNS1-H1N1 vaccine was well tolerated. Rhinitis-like symptoms and headache were the most common adverse events identified during the 28-day observation period. Adverse events were similarly distributed between the treatment and placebo groups. Vaccine-specific local and serum antibodies were induced in a dose-dependent manner. In the highest dose group, vaccine-specific antibodies were detected in 10 of 12 volunteers. Importantly, the vaccine also induced neutralizing antibodies against heterologous drift variants. CONCLUSIONS. We show that vaccination with an influenza virus strain lacking the viral interferon antagonist NS1 induces statistically significant levels of strain-specific and cross-neutralizing antibodies despite the highly attenuated replication-deficient phenotype. Further studies are warranted to determine whether these results translate into protection from influenza virus infection. TRIAL REGISTRATION. ClinicalTrials.gov identifier: NCT00724997 .

It is well established that high expression of the antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL can significantly contribute to chemoresistance in a number of human malignancies. Much less is known about the role the more recently described Bcl-2 family member Mcl-1 might play in tumor biology and resistance to chemotherapy. Using an antisense strategy, we here address this issue in melanoma, a paradigm of a treatment-resistant malignancy. After in vitro proof of principle supporting an antisense mechanism of action with specific reduction of Mcl-1 protein as a consequence of nuclear uptake of the Mcl-1 antisense oligonucleotides employed, antisense and universal control oligonucleotides were administered systemically in combination with dacarbazine in a human melanoma SCID mouse xenotransplantation model. Dacarbazine, available now for more than three decades, still remains the most active single agent for treatment of advanced melanoma. Mcl-1 antisense oligonucleotides specifically reduced target protein expression as well as the apoptotic threshold of melanoma xenotransplants. Combined Mcl-1 antisense oligonucleotide plus dacarbazine treatment resulted in enhanced tumor cell apoptosis and led to a significantly reduced mean tumor weight (mean 0.16 g, 95% confidence interval 0.08-0.26) compared to the tumor weight in universal control oligonucleotide plus dacarbazine treated animals (mean 0.35 g, 95% confidence interval 0.2-0.44) or saline plus dacarbazine treated animals (mean 0.39 g, 95% confidence interval 0.25-0.53). We thus show that Mcl-1 is an important factor contributing to the chemoresistance of human melanoma in vivo. Antisense therapy against the Mcl-1 gene product, possibly in combination with antisense strategies targeting other antiapoptotic Bcl-2 family members, appears to be a rational and promising approach to help overcome treatment resistance of malignant melanoma.

Systemic tumor-targeted gene delivery is attracting increasing attention as a promising alternative to conventional therapeutical strategies. To be considered as a viable option, however, the respective transgene has to be administered with high tumor specificity. Here, we describe novel polyethylenimine (

Summary In Aspergillus nidulans, the genes coding for nitrate reductase (niaD) and nitrite reductase (niiA), are transcribed divergently from a common promoter region of 1200 basepairs. We have previously characterized the relevant cis‐acting elements for the two synergistically acting transcriptional activators NirA and AreA. We have further shown that AreA is constitutively bound to a central cluster of four GATA sites, and is involved in opening the chromatin structure over the promoter region thus making additional cis‐acting binding sites accessible. Here we show that the asymmetric mode of NirA–DNA interaction determined in vitro is also found in vivo. Binding of the NirA transactivator is not constitutive as in other binuclear C6‐Zn2+‐cluster proteins but depends on nitrate induction and, additionally, on the presence of a wild‐type areA allele. Dissecting the role of AreA further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly exert its effect on NirA via inducer exclusion. We have tested this possibility in a strain accumulating nitrate in the absence of areA. We found that in such a strain the intracellular presence of inducer is not sufficient to promote either chromatin rearrangement or NirA binding, implying that both processes are directly dependent on AreA.

Hepatocytes adopt an invasive and metastatic phenotype caused by the cooperation of transforming growth factor (TGF)-b and oncogenic Ha-Ras. In the initial phase of this process, c-Fos is rapidly induced by TGF-b, but then decreases to undetectable levels. Here, we investigated the functional implications of c-Fos activation and its contribution to hepatocellular tumorigenesis. By employing conditional c-Fos expression, we observed that continuous activation of c-Fos and consequently AP-1 activity leads to depolarization of differentiated murine epithelial hepatocytes. Most remarkably, this change in morphology was associated with inhibition of proliferation and induction of cell death. Coexpression of antiapoptotic Bcl-X L or scavenging of reactive oxygen species was sufficient to prevent the c-Fos-mediated phenotype. In contrast, the cooperation of c-Fos with oncogenic Ha-Ras or a Ras mutant selectively activating the MAPK pathway even enhanced c-Fos-induced effects. Showing the negative role in hepatocellular tumorigenesis, c-Fos repressed oncogenic Ras-driven anchorage-independent growth in vitro and strongly suppressed tumour formation in vivo. Taken together, we demonstrate that c-Fos modulates plasticity of epithelial hepatocytes and acts tumour suppressive in neoplastic hepatocytes by stimulating cell cycle inhibition and cell death.

BackgroundWe developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses.Methodology/Principal FindingsWe applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5∶3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log10 TCID50/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains.Conclusion/SignificanceOur findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.