Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

The metabolism of steviol to 13-hydroxylated ent-Gibberellanes and ent-kauranes

Steviol(ent-13-hydroxykaur-16-en-19-oic acid) is rapidly metabolised by the mutant B1-41a of Gibberellafujikuroi. The initial product is the ent- 7-α-hydroxy derivative which is then further metabolised to gibberellins A₁, A₁₈, A₁₉, A₂₀, 13-hydroxy GA₁₂, the ent-6α, 7α, 13- and ent-6β, 7α, 13 (19,6-lactone)-trihydroxykaurenoic acids, and a seco-ring B diacid. This apparently low substrate specificity of the enzymes operative beyond the block in the mutant B1-41a provides a useful model for the biosynthetic pathways to 13-hydroxylated gibberellins of higher plants and a preparative route to these plant gibberellins.     

Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria

Delayed infectivity panning (DIP) is a novel approach for the in vivo isolation of interacting protein pairs. DIP combines phage display and cell surface display of polypeptides as follows: an antigen is displayed in many copies on the surface of F(+) Escherichia coli cells by fusing it to a Lpp-OmpA' hybrid. To prevent premature, non-specific infection by phage, the cells are rendered functionally F(-) by growth at 16 degrees C. The antigen-displaying cells are used to capture antibody-displaying phage by virtue of the antibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37 degrees C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scFv antibodies and the human ErbB2 proto-oncogene and IL2-Ralpha chain as model antibody-antigen pairs, we demonstrate enrichment of those phage that display a specific antibody over phage that display an irrelevant antibody of over 1,000,000 in a single DIP cycle. We further show the successful isolation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthetic human library. The effectiveness of DIP makes it suitable for the isolation of rare clones present in large libraries.Since DIP can be applied for most of the phage libraries already existing, it could be a powerful tool for the rapid isolation and characterization of binders in numerous protein-protein interactions.     

EGFR-Targeted Granzyme B Expressed in NK Cells Enhances Natural Cytotoxicity and Mediates Specific Killing of Tumor Cells

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells'' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.     

Liposome-based Systems for Anti-tumor Vaccination: Influence of Lipopeptide Adjuvants

We have developed liposome-based synthetic constructs incorporating peptide epitope(s) (ErbB2 p63-67 CTL which is overexpressed in many tumors and/or HA 307-319 T-helper) and lipopeptide adjuvants (Pam3CysSerSer, Pam3CysAlaGly) in order to elicit an anti-tumor immune response. The epitopes, derivatized with a linker containing a cysteine residue, were conjugated on preformed vesicles (dia. ～ 100 nm) containing lipopeptides functionalized with thiol reactive groups (maleimide or bromoacetyl). The therapeutic efficacy of these constructs was evaluated on a Balb/c mice tumor model inoculated with syngenic murine renal carcinoma (Renca) cells expressing human ErbB2 (Her2/neu) receptor. A successful therapeutic vaccination was obtained which was antigen specific. Furthermore, it appeared that the nature of the polar head group of the lipopeptide adjuvant and also its type of functionalization influence the efficacy of the construct. In our study, the best results were obtained with formulations containing a Pam₃CSS anchor in association with the CTL and Th epitopes. Considering these promising results studies are in progress with a new generation of liposomes that incorporate a neutral lipid – lacking adjuvant properties – that serves as anchor of the peptide epitopes and new adjuvants synthesized in our laboratory, which are screened for their antitumour activity in a therapeutic setting.     

Transformation and mineralization of benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene by microbial cultures enriched on mixtures of three- and four-ring polycyclic aromatic hydrocarbons

Microorganisms originating from a soil contaminated by low levels of polycyclic aromatic hydrocarbons (PAHs) were enriched with three- and four-ring PAHs as primary substrates in the presence of benzo[a]pyrene (BaP). Most enrichment cultures, isolated in the presence or absence of a sorptive matrix, significantly transformed BaP. Evidence of BaP mineralization was obtained with cultures enriched on phenanthrene and anthracene. Our findings supplement literature data suggesting the wide occurrence of microbial activity against BaP. Journal of Industrial Microbiology & Biotechnology (2002) 28, 70–73 DOI: 10.1038/sj/jim/7000211 Received 11 December 2000/ Accepted in revised form 04 September 2001     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.