Gene dosage studies with pleiotropic mutants of Serratia marcescens superactive in the synthesis of marcescin A and certain other exocellular proteins

Summary Pleiotropic mutants of Serratia marcescens have been isolated. They synthesize greater quantities of the bacteriocin marcescin A and exocellular lipase and exhibit higher rates of spontaneous induction of prophage than does the wild-type strain. These mutants were found to contain more marcescin A plasmid DNA than the parent strain and, furthermore, this increase in plasmid DNA was observed to be proportional to the increase in synthesis of marcescin A. From these results it is proposed that the mutation functions via a gene-dosage effect (at least in the case of bacteriocin synthesis) and causes an elevated synthesis of bacteriocin plasmid DNA.A preliminary report of this work was presented to the 1972 Summer Meeting of the Gesellschaft für Physiologische Chemie held in Bochum, Germany (Timmis and Winkler, 1972).     

An efficient chemical approach to bispecific antibodies and antibodies of high valency

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (d-Lys⁶)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (d-Lys⁶)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (d-Lys⁶)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.     

Mechanisms of neisserial serum resistance

Pathogenic Neisseria use a variety of mechanisms to survive the bactericidal action of the complement system. Serum resistance is a crucial virulence factor for the development of severe meningococcal disease, meningococcal meningitis and disseminated gonococcal infection. Furthermore, local inflammation at the site of gonococcal infection exposes the bacteria to moderate concentrations of complement factors. We review current concepts of neisserial serum resistance with emphasis on porins and polysaccharides exposed on the neisserial surface and their interaction with components of normal human serum.     

Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1:2 complex, namely Cu₂(indomethacin)₄ · L₂, in the unit cell. Suprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu₂(acetate)₄ was seen.Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. The superoxide dismutating activity was successfully demonstrated in Me₂SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 · 10⁹ M^?1 · s^?1 in strictly aqueous systems dropped only slightly to 1.1 · 10⁹ M^?1 · s^?1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO₂-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 · 10^?10 mol · ml^?1 of Cu₂(indomethacin)₄. The inhibitory action dropped to 25% when Cu₂Zn₂superoxide dismutase was employed. The formation of copper · indomethacin in rat serum after administration of indomethacin was shown in vitro and in vivo.     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.