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 (