Max H. Schreier

Sdz Rad, A New Rapamycin Derivative: Pharmacological Properties In Vitro and In Vivo

BACKGROUND This report describes the preclinical pharmacological profile of the new rapamycin analog, SDZ RAD, i.e., 40-O-(2-hydroxyethyl)-rapamycin. METHODS The pharmacological effects of SDZ RAD were assessed in a variety of in vitro and in vivo models, which included an autoimmune disease model as well as kidney and heart allotransplantation models using different rat strain combinations. RESULTS SDZ RAD has a mode of action that is different from that of cyclosporine or FK506. In contrast to the latter, SDZ RAD inhibits growth factor-driven cell proliferation in general, as demonstrated for the in vitro cell proliferation of a lymphoid cell line and of vascular smooth muscle cells. SDZ RAD is immunosuppressive in vitro as demonstrated by the inhibition of mouse and human mixed lymphocyte reactions and the inhibition of antigen-driven proliferation of human T-cell clones. The concentrations needed to achieve 50% inhibition in all of these assays fall into the subnanomolar range. SDZ RAD is effective in the in vivo models when given by the oral route in doses ranging between 1 mg/kg/day and 5 mg/kg/day. When compared with rapamycin, the in vitro activity of SDZ RAD is generally about two to three times lower; however, when administered orally, SDZ RAD is at least as active in vivo as rapamycin. CONCLUSIONS In conclusion, SDZ RAD is a new, orally active rapamycin-derivative that is immunosuppressive and that efficiently prevents graft rejection in rat models of allotransplantation. SDZ RAD has therefore been selected for development for use in combination with cyclosporine A to prevent acute and chronic rejection after solid organ allotransplantation.

Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems

A highly efficient mammalian system was developed for the in vitro translation of exogenous rabbit globin messenger RNA. The system consists of purified ribosome subunits from mouse liver, rabbit reticulocytes, or guinea pig brain, partially purified initiation factors from rabbit reticulocytes, and elongation factors, termination factors, aminoacyl-tRNA synthethases and tRNA from rat liver in the form of pH 5-enzymes. (1) Emphasis was put on well-defined, structurally and functionally intact ribosomes, which we found to be the most difficult component of the system in terms of stability of activity. (2) An improved method for extraction of initiation factors from crude reticulocyte ribosomes was developed. Factor preparations of high specific activity were obtained by a simple, partial purification procedure. The crucial point was not to damage the ribosome structure during extraction of the initiation factors and to eliminate inhibitory components during extraction and purification. (3) The efficiency of the system was demonstrated quantitatively by showing that between 34 and one mRNA molecule per ribosome saturates the system and that each ribosome recycles over the mRNA several times. (4) Major uncertainties and ambiguities in the search for and identification of true initiation factors, as opposed to structural ribosome proteins needed for the reconstitution of damaged ribosomes, can be reduced by using this system.

Initiation of mammalian protein synthesis: I. Purification and characterization of seven initiation factors

We have purified seven protein factors from rabbit reticulocytes, all of which are presumed to be involved in the initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIF-4B, eIF-4C and e-IF-5. The purification from the KCl wash of crude ribosomes involves fractionation with ammonium sulphate, ion-exchange chromatography and separation by size. The operational definition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the two elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was also shown to be required for initiation. The initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60% to 70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in molecular weight from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has three subunits of about 35,000, 50,000 and 55,000 molecular weight. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, however, reveals at least nine subunits ranging in molecular weight from about 35,000 to 160,000. Initiation complexes (mRNA · Met-tRNAf · 80 S ribosome), made in the presence of the seven initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the two elongation factors and GTP.

Initiation of mammalian protein synthesis: II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [125I]mRNA, [3H]Met-tRNAf, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNAf to the 40 S ribosomal subunit. The initial Met-tRNAf binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.

Clones of Killer and Helper T Cells: Growth Requirements, Specificity and Retention of Function in Long-Term Culture

Lymphocyte reactions are complex, involving interactions between lymphocyte subsets and other accessory cells. An overall outline of these interactions has emerged from studies of unseparated or partially separated cells from lymphoid organs in culture. However, progression from mapping of events at a population level to an understanding of mechanisms at the level of single cells and of molecules demands the availability of populations which are homogeneous to a degree not achievable by present methods of cell separation. Continuously growing clones of lymphocytes, capable of unlimited expansion in culture while maintaining normal function and responsiveness to regulation, would represent ideal tools for dissection of these mechanisms. Until very recently, such lines were not available. While lymphocytes could be induced to proliferate in culture, their response was limited to a few days, insufficient to provide clonal populations of the required size. The essential breakthrough for T lymphocytes was achieved 3 years ago, when Morgan and coworkers (1976) and Ruscetti et al. (1977) showed that the proliferation of human T cells could be extended for periods of many months if the cells were supplied with medium conditioned by lectinor alloantigen-stimulated peripheral blood cells. The activity of these conditioned media was subsequently attributed to a protein regulator known now as T cell growth factor (TCGF) or Interleukin 2 (IL-2). Growing T cells are now understood to depend on TCGF absolutely for both

Roles of surface-bound immunoglobulin molecules in regulating the replication and maturation to immunoglobulin secretion of b lymphocytes.

Immunoglobulin (Ig) molecules as recognition structures on the surface of lymphocytes have been postulated by the clonal seleetion theory (Jerne 1955, Burnet 1957), and were first experimentally demonstrated by Moller (1961). Clonal selection demands a lymphocyte to synthesize and express on its surface a single antigen-binding specificity, which is getietically determined. This postulate has been supported by the findings that surface-bound Ig molecules show allelic exclusion (Pernis et al. 1965, Sell 1970) and that subsets ofcells among the lymphocytes cover different antigenic specificities. It was expected that binding of antigen at the cell surface would initiate reactions leading to replication and maturation of lymphocytes, resulting in cell-mediated or humoral effector functions. A clone of antigen-specific lymphocytes responding to antigenic stimulation was expected, and later proven, to retain its original specificity and thereby expand its particular recognition capacity within the whole immune system (Dutton & Eady 1964, Makela & Cross 1970). This simple concept of antigen recognition was no longer tenable when it was found that three different types ofcells T-lymphocytes, B-lymphocytes and macrophages had to cooperate in many humoral Immune responses (Claman ct al. 1966, Miller & Mitchell 1967, Mosier 1967). B lymphocytes have the function of producing and secreting the Ig-molecules. But, mitogens were discovered which could activate, by circumventing of the antigen-binding step to surface Ig molecules, a large part of all B cells, irrespective of their antigen-

Cyclosporine: Chemistry, Structure-Activity Relationships and Mode of Action

Cyclosporine is a cyclic peptide with a selective immunosuppressive action. One of its 11 amino acids is a novel amino acid, (4R)-4-((E)-2-butenyl-4,N-dimethyl-L-threonine, the preparation of which allowed the synthesis of cyclosporine and specifically modified analogues. Structure-activity relationships emerging from the study of modified cyclosporins suggest that a large portion of the cyclosporine molecule is involved in interactions with its lymphocyte receptor and that this includes amino acids 1, 2, 3 and 11.

Translation of bacteriophage R17 and Qβ RNA in a mammalian cell-free system☆

Abstract The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E 3 from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.

Purification of seven protein synthesis initiation factors from Krebs II ascites cells

Seven protein synthesis initiation factors were isolated from Krebs II ascites cells using the procedures developed for the purification of the corresponding factors from rabbit reticulocytes. The ascites factors display identical characteristics in ion exchange chromatography and sucrose density gradient sedimentation. Based on their profiles in SDS polyacrylamide gels, the ascites factors have polypeptide profiles and molecular weights identical to those of the reticulocyte factors. Most significantly, each ascites factor is competent in replacing its corresponding reticulocyte factor in a reconstituted in vitro protein synthesizing system which is dependent on all seven factors.

Monoclonal antibodies to cyclosporin are representative of the major antibody populations present in antisera of immunized mice

Two series of mouse antisera raised against cyclosporin (Cs)-carrier conjugates exposing opposite sides of the Cs molecule and more than sixty monoclonal antibodies (mAbs) derived from the same animals were compared in terms of isotype and fine specificity for Cs. The predominant isotypes of the mAbs reflected the in situ distribution of the circulating anti Cs antibodies. The fine specificity of the antibodies was studied by determining their cross-reactivity for a series of Cs-derivatives and Cs-metabolites in competitive ELISA. The antisera raised by different immunizations showed very different cross-reactivity patterns for the Cs-derivatives. However, the in situ anti Cs antibody populations and the majority of mAbs derived from the corresponding animals showed a striking similarity in fine specificity for restricted clusters of residues on the Cs molecule. These results indicate that the mAbs produced against Cs are representative of the major antibody population present in the sera of the mice used for the fusion. By determining the characteristics of antibodies found in the serum of immunized mice it may thus be possible to select animals that are likely to give rise to mAbs of a certain isotype and specificity.