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.     

Biosynthesis of glyantrypine by Aspergillus clavatus

The biosynthesis of glyantrypine from radiolabelled amino acid precursors has been shown experimentally to involve anthranilic acid, tryptophan and glycine. Low values for percentage incorporation of radiolabel into glyantrypine were partly influenced by a complex array of other novel alkaloids shown by the radiolabelling experiments to be related to glyantrypine. Interpretation of radiolabel incorporation from [14C-carboxyl]-anthranilic acid into microbial metabolites seen to contain an anthranilyl moiety in various biosynthetic arrangements is discussed. The possibility of diversion of anthranilic acid from the kynurenine pathway to glyantrypine biosynthesis is recognised.     

Specific and nonspecific T-cell recruitment in viral meningitis: Possible implications for autoimmunity

Specific and nonspecific T-cell invasion into cerebrospinal fluid has been investigated in the nonfatal viral meningoencephalitis induced by intracerebral inoculation of mice with vaccinia virus. At the peak of the inflammatory process on Day 7 approximately 5 to 10% of the Lyt 2⁺ T cells present are apparently specific for vaccinia virus. Concurrently, in mice primed previously with influenza virus, 0.5 to 1.0% of the appropriate T-cell set located in cerebrospinal fluid is reactive to influenza-infected target cells. This vaccinia virus-induced inflammatory exudate may thus contain as many as 500 influenza-immune memory T cells. These findings are discussed from the aspect that such nonspecific T-cell invasion into the central nervous system during aseptic viral meningitis could result in exposure of potentially brain-reactive T cells to central nervous system components.     

The enzymic estimation of glutamate and glutamine

A method of estimating glutamic acid is described, based on its dehydrogenation by glutamate dehydrogenase coupled, by means of N-methylphenazine methosulphate, to the reduction of tetrazolium salts. The method is suitable for the estimation of 0-0.3mumole of glutamic acid. The response is linear, but not stoicheiometric: possible reasons for this are discussed. If suitable precautions are taken, the use of a partially purified preparation of glutaminase makes it possible to estimate glutamine also.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Subject: Navigation and orientation

Research Notes on Avian Biology 1994: Selected Contributions from the 21st International Ornithological CongressMorphology and Physiology: Orientation

Subject: Navigation and orientation