Showing papers by "Torrey Pines Institute for Molecular Studies published in 1992"
Journal Article•
TL;DR: A conceptually unique set of individual synthetic peptide combinatorial libraries (SPCLs) that can be used for the rapid identification of peptide sequences that bind with high affinity to antibodies, receptors or other acceptor molecules are described.
Abstract: We describe here a conceptually unique set of individual synthetic peptide combinatorial libraries (SPCLs), termed a positional scanning SPCL (PS-SPCL), that can be used for the rapid (i.e., a single day) identification of peptide sequences that bind with high affinity to antibodies, receptors or other acceptor molecules. The PS-SPCL described here is made up of six individual positional peptide libraries, each one consisting of hexamers with a single position defined and five positions as mixtures. As an example of the utility of such PS-SPCLs, the antigenic determinants recognized by two different monoclonal antibodies were correctly identified upon a single screening.
353 citations
TL;DR: Using reversed-phase high-performance liquid chromatography as a means to study the secondary structure of peptides at aqueous/lipid interfaces, a sequence was found to readily adopt an amphipathic alpha-helical conformation upon interacting with the lipid groups of the stationary phase during RP-HPLC.
Abstract: Induced amphipathic alpha-helical conformations play an important role in the biological activity of peptides. By using reversed-phase high-performance liquid chromatography (RP-HPLC) as a means to study the secondary structure of peptides at aqueous/lipid interfaces, a sequence (Ac-LKLLKKLLKKLKKLLKKL-NH2) was found to readily adopt an amphipathic alpha-helical conformation upon interacting with the lipid groups of the stationary phase during RP-HPLC. This peptide exhibited potent antimicrobial activities against both Gram-positive and Gram-negative bacteria. We have prepared a complete set of omission, as well as of leucine and lysine substitution, analogs of this sequence. These analogs were used to investigate the effects of such alterations on the parent sequence's antimicrobial and hemolytic activities relative to each analog's behavior during RP-HPLC. The potential for the formation of ion channels through cell membranes by this amphipathic model peptide was also evaluated through preparation of analogs which varied in length from 8 to 22 residues, while maintaining their amphipathicity.
352 citations
Journal Article•
TL;DR: Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay.
Abstract: The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity peptide ligands using an opiate radio-receptor binding assay.
335 citations
TL;DR: Data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein.
Abstract: Eukaryotic expression vectors designed to produce E. coli Lac repressor protein targeted to the nucleus of mammalian cells were constructed. These constructions carry the lac repressor gene (lacI) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a. When the NLS's were fused to the lacI gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified LacI protein. Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity. When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the LacI protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected. The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacI behind the nine base pair linker, AGC AGC CTG (ser-ser-leu). This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction. The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-terminus of the protein. These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein. This modified lacI gene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells.
119 citations
TL;DR: Overall, these studies provide strong support for a model in which the first three zinc fingers of TFIIIA bind with high affinity between nucleotides +80 and +92 of the internal control region of the 5 S RNA gene.
95 citations
TL;DR: The procedure used to identify, sequence and derive primers for PCR detection of tRNA‐ILPs in Staphylococcus should be applicable to many other genera of eubacteria and could be used on uncultured material such as clinical samples.
Abstract: Summary
The intergenic spacers between some adjacent tRNA genes were shown to be polymorphic in length when closely related Staphylococcus species were compared. A simple procedure was developed to detect and sequence these tRNA intergenic length polymorphisms (tRNA-ILPs). A comparison of homologous tRNA gene sequences flanking these ILPs in three Staphylococcus species was used to derive primers for high-stringency amplification of the ILPs by the polymerase chain reaction (PCR). The detection of tRNA-ILPs by PCR allowed the classification of virtuatly all strains from the five species of Staphylococcus that were examined. The procedure used to identify, sequence and derive primers for PCR detection of tRNA-ILPs in Staphylococcus should be applicable to many other genera of eubacteria. These primers could be used on uncultured material such as clinical samples.
74 citations
TL;DR: In vitro T cell responses of a group of malaria exposed and non‐exposed adult Caucasian donors to recombinant circumsporozoite proteins, to blood‐stage parasites, and to synthetic peptides copying the CS protein and defined blood-stage proteins are examined.
Abstract: A major goal of current candidate malaria vaccines is to stimulate the expansion of clones of malaria-specific lymphocytes. We have examined the in vitro T cell responses of a group of malaria exposed and non-exposed adult Caucasian donors to recombinant circumsporozoite (CS) proteins, one of which is undergoing clinical trials, to blood-stage parasites, and to synthetic peptides copying the CS protein and defined blood-stage proteins. In nearly all individuals tested, CD4 T cell proliferation or lymphokine production occurred in response to whole parasite or CS protein stimulation, and T cells from many individuals responded to synthetic peptides. T cell responses were major histocompatibility complex-restricted, and stimulation of T cells with malaria parasites or CS protein did not appear to expand a population of T cell receptor gamma/delta cells. Malaria-specific responses were independent of prior malaria exposure, and in some cases exceeded the magnitude of response to tetanus toxoid. Specific T cells are present in high frequency in the peripheral blood of many donors who have never been exposed to malaria. Although malaria-specific CD4 T cells play an important role in immunity, these data question whether vaccines need to stimulate such cells, and focus attention on other aspects of malaria immunity which may be more critical to a successful vaccine.
56 citations
TL;DR: Junctional diversity in immunoglobulin (Ig) from an adult mouse contributes significantly to the size of the final Ig repertoire and may play a role in the overexpression of certain Vh genes early in ontogeny.
Abstract: Junctional diversity in immunoglobulin (Ig) from an adult mouse contributes significantly to the size of the final Ig repertoire. In adult pre-B cells, N region addition and deletion of nucleotides form coding regions produces very heterogenous CDR3 sequences. In contrast, Ig from fetal and newborn mice show very restricted junctional diversity. The reasons for this are: (a) the lack of N regions; and (b) the predominance of certain junctional sequences. These common junctional sequences all appear to occur by targeted rearrangement to short stretches of sequence homology near the ends of the segments to be joined. Targeted rearrangement may play a role in the over-expression of certain Vh genes early in ontogeny. These non-random junctional sequences in the neonate will reproducibly create certain Ig, for example, the dominant T15 anti-PC antibodies. Thus the immune system first creates a small repertoire of predictable Ig sequences. To the extent that these Ig are expressed in long-lived B cells, these ...
47 citations
TL;DR: It is concluded that bystander B cells may generate tumors in CLL-reconstitutedSCID mice that emulate the EBV-associated lymphoproliferations noted in SCID mice reconstituted with normal human PBL.
41 citations
TL;DR: It is concluded that recombinant pseudorabies virus (PRV) worked successfully as a live vaccine vector to induce both antibodies and CTL, albeit non-protective in vivo, and the herpesviruses should be considered as subunit vaccines where T- and B-cell immunity is required.
39 citations
TL;DR: The antimicrobial activities of Ac‐LLKLLKKLLKK LLKKLKK‐NH2 and its 18 proline substitution analogues were found to be inversely correlated with their RP HPLC retention times.
Abstract: Reversed-phase high performance liquid chromatography (RP HPLC) has been found to be a convenient and powerful tool for the study of the secondary structure of peptides. Here, the ability of proline to perturb the secondary structures of peptides induced at aqueous-lipid interfaces and the induced conformation of polyproline peptides were investigated by means of RP HPLC. For these studies, four different complete sets of substitution analogues of model peptides expected to have specific induced conformations were used. In the first two studies, a single lysine was "walked" through two 18-residue polyproline sequences (one N-acetylated, the other not). In the remaining two studies, a proline was "walked" through two different sequences that had been found earlier to be induced into an alpha-helical conformation during RP HPLC (an 18-residue polyalanine sequence and the amphipathic 14-residue sequence Ac-LLKLLKKLLKKLKK-NH2). Sixty-eight individual analogues were synthesized for this study and the effect of the respective substitutions on retention times was determined. The results are consistent with the concept that, upon interaction with the C-18 of the stationary phase during RP HPLC, polyproline is induced into a type II helical conformation, polyalanine into an alpha-helical conformation, and Ac-LLKLLKKLLKKLKK-NH2 into an amphipathic alpha-helical array. In an extension of this study, the antimicrobial activities of Ac-LLKLLKKLLKKLKK-NH2 and its 18 proline substitution analogues were found to be inversely correlated with their RP HPLC retention times.
TL;DR: Strong peptide-peptide interactions, occurring through interchain hydrophobic forces, resulted in a presenting face to the C18 group, consisting primarily of lysine residues and, in turn, in early retention times.
TL;DR: Upon the completion of an iterative screening and selection process utilized in the SPCL approach, a number of peptide sequences that were related to the known antigenic determinant region were found.
Abstract: Synthetic peptide combinatorial libraries (SPCLs) are composed of tens of millions of peptide sequences from which specific sequences can be systematically determined. The broad versatility of SPCLs residues in the fact that nonsupport-bound peptides are generated in quantities sufficient for use in the majority of existing bioassays. In the present study, an SPCL composed of more than 52 million peptides was screened with an antipeptide monoclonal antibody for the identification of peptide sequences that inhibit the interaction of the monoclonal antibody with its known antigenic peptide. Upon the completion of an iterative screening and selection process utilized in the SPCL approach, a number of peptide sequences that were related to the known antigenic determinant region were found.
TL;DR: H-2O is a recently described non-polymorphic mouse MHC class II molecule and the discovery of the H-2Ob and H- 2Oa loci and the pattern of expression is described, and the possible function of this unusual molecule is speculated.
TL;DR: It appears that high levels of antigen result in selective stimulation of Th1 cells which produce IFN-gamma, and diminished activation of IL-4-producing Th2 cells, which help explain observations regarding the influence of antigen dose on the generation of experimental and clinical IgE antibody responses in vivo.
TL;DR: The development of new antimicrobial peptides through the isolation and characterization of active substances in insect, mammalian, human tissues, or through the design of well defined structures remains difficult and limited by the need to screen and synthesize large numbers of peptides.
Abstract: Publisher Summary This chapter discusses the antimicrobial peptides derived from natural sources and summarizes the recent approach consisting of the systematic screening of peptide libraries that represents a new source of antimicrobial peptides Cecropins represent a class of antimicrobial peptides originally found in the humoral immune response of the North American silk moth Hyalophora cecropia In a search to improve the biological activity of the natural cecropins, hybrids of cecropin and melittin have been synthesized The mechanism of action of cecropins has been studied through the use of L- or D-amino acid-containing model peptides Magainins consist of a family of basic antimicrobial peptides produced in the granular glands present in amphibian skin They exhibit a broad spectrum of antimicrobial activity including gram-positive and gram-negative bacteria, fungi, and protozoa As determined by Raman spectroscopy and NMR, the secondary structure of magainin 2 changes from unfolded in aqueous solution to helical when bound to lipid vesicles An endopeptidase that cleaves the magainins into half-peptides in vivo has been isolated and characterized Defensins, a family of cationic peptides, were first identified in mammalian phagocytic cells To understand the mechanism of defense in action, the crystal structure of HNP-3 has been determined Gramicidins are hydrophobic peptides produced by Bacillus brevis, which exhibits antimicrobial activity primarily against gram-positive bacteria but are known to be toxic to humans The development of new antimicrobial peptides through the isolation and characterization of active substances in insect, mammalian, human tissues, or through the design of well defined structures remains difficult and limited by the need to screen and synthesize large numbers of peptides Natural or synthetic model peptides represent a fertile source of novel chemotherapeutic agents
TL;DR: In this paper, the title compound was prepared in excellent yield in four steps starting from inosine(1) starting from the root root of the compound, and was then processed in four stages.
TL;DR: The linearity was found to be dependent on the number of cycles used in the PCR amplification, and the best linearity, at a target level of a few copies, was achieved using a low number ofcycles.
TL;DR: It is demonstrated that activation of IgE-receptor mediated exocytosis of RBL cells does not require AA metabolites, and strongly suggest that PLA2 activation and release of AA and lysophospholipid may be involved in triggering this response.
TL;DR: All of the peptides which eluted anomalously late during RP-HPLC included the regions of these proteins known from earlier studies to be antigenic T cell sites, including the areas of hen egg lysozyme and circumsporozoite protein.
TL;DR: Advances in pulsed-field gel electrophoresis are presented for the isolation of high molecular weight DNA from embedded soybean protoplasts and the conditions for separating large DNA fragments using PFE.
Abstract: Summary
The technique of pulsed-field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DMA from embedded soybean protoplasts and the conditions for separating large DMA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.
TL;DR: Cross‐reactivity of cryptic self Ig with non‐self epitopes could be a mechanism to increase the T cell repertoire for foreign antigens.
Abstract: The adult T cell repertoire is the result of positive and negative intrathymic selective processes. Since non-self antigens are not present in the thymus, self peptides are believed to play a role in the thymic T cell selection. T cell lines raised against a peptide from the second complementarity-determining region (CDR2) of the S107 germ-line immunoglobulin (Ig) do not respond to the intact Ig, indicating that in adult animals this CDR2 epitope is cryptic in the context of intact S107 Ig. To show that the response to self Ig peptide is the result of positive selection, the fetal expression of the S107 germ-line Ig was suppressed by maternal anti-idiotype (Id) injection. Id S107-suppressed mice responded poorly to the CDR2 peptide, indicating that S107 Ig selects positively T cells responding to a polymorphic self epitope. Cross-reactivity of cryptic self Ig with non-self epitopes could be a mechanism to increase the T cell repertoire for foreign antigens.
TL;DR: Examining the sensitivities of several mouse strains to interleukin (IL)-4 and interferon-gamma shows that BALB/c mice have a non-H-2-linked gene which decreases B cell sensitivity to IFN-Gamma, but the gene effect is not associated with the expression of IFN -gamma R mRNA on B cells.
TL;DR: The effect of in vivo administration of the I7 and I9 antibodies to mice is examined, with implications for understanding the nature of network regulation of the immune system and for those attempting idiotypic vaccination.
TL;DR: In this paper, the title compound was prepared in excellent yield in four steps starting from inosine(1) starting from the root root of the compound, and was then processed in four stages.
Abstract: The title compound is prepared in excellent yield in four steps starting from inosine(1).