Showing papers by "Torrey Pines Institute for Molecular Studies published in 1993"

Transgenic systems for in vivo mutation analysis.

Abstract: Transgenic mice carrying shuttle vectors containing the lacI gene as the target permit the in vivo measurement of mutations in multiple tissues and have been used to test the mutagenic effects of several compounds. Tissue-specific and time-dependent responses have been observed, and the spectrum of mutations determined by sequencing allows analysis of the role of expression time in mutagenesis. The results obtained from sequencing analysis have demonstrated spectra paralleling those observed in alternative in vivo assays. In addition to color screening, modifications to this system have permitted direct selection for mutations in the lacI target by a variety of methods. Transgenic rats containing the same lambda/lacI shuttle vector have been developed for inter-species comparison of mutagenesis testing results, which may offer a better understanding of the specific mechanisms involved in mutagenesis at the molecular level in vivo.

Identification of substrate-analog trypsin inhibitors through the screening of synthetic peptide combinatorial libraries.

Abstract: Synthetic peptide combinatorial libraries (SPCLs), which are made up in total of tens to hundreds of millions of peptides, enable the systematic screening for biologically active peptides in virtually all in vitro and even in vivo assay systems. In the current study, the applicability of this method to the identification of peptide enzyme inhibitors was investigated using trypsin as the model enzyme. A specifically designed library of hexapeptide mixtures was synthesized on cotton carriers and screened. The synthetic approach, using cotton as a solid support, was modified so that the deprotected peptides remained attached to the cotton carrier until they were released into solution directly prior to being assayed. Following an iterative process of synthesis and screening, in which all of the positions of the sequence were successively defined, a number of individual hexapeptides with trypsin inhibitory activity were identified. The most active, defined individual peptide sequence was then reincorporated into a new library, now made up of dodecapeptide mixtures. The iterative screening and synthesis of this library led to a dodecapeptide with improved inhibitory activity when compared to the hexapeptide from which it was derived.

High output genetic mapping of polyploids using PCR-generated markers.

Abstract: The polymerase chain reaction (PCR) with arbitrarily selected primers has been established as an efficient method to generate fingerprints that are useful in genetic mapping and genomic fingerprinting. To further increase the productivity of mapping and fingerprinting efforts, we have altered existing protocols to include the use of the Stoffel fragment, which is derived from genetically engineered Taq polymerase. We also optimized the thermal profile of the reaction to increase the number of useful primers. In mapping of the genome of Saccharum spontaneum 'SES 208', a polyploid wild relative of sugarcane, these modifications allowed for an increase of 30% in the number of loci screened per primer, and an 80% increase in the number of polymorphisms per primer. Furthermore, the enzyme cost per reaction was decreased approximately 1.6-fold. Finally, there was an increase from about 70% to about 97% in the number of primers that were useful (i.e., gave a reproducible fingerprint) using our protocol. We have placed some of these markers into linkage groups.

Acetalins: opioid receptor antagonists determined through the use of synthetic peptide combinatorial libraries

Abstract: A synthetic peptide combinatorial library made up of 52,128,400 hexapeptides, each having an acetyl group at the N terminus and an amide group on the C terminus, was screened to find compounds able to displace tritiated [D-Ala2,MePhe4,Gly-ol5]enkephalin from mu opioid receptor binding sites in crude rat brain homogenates. Individual peptides with mu receptor affinity were found using an iterative process for successively determining the most active peptide mixtures. Upon completion of this iterative process, the three peptides with the highest affinity were Ac-RFMWMT-NH2, Ac-RFMWMR-NH2, and Ac-RFMWMK-NH2. These peptides showed high affinity for mu and kappa 3 opioid receptors, somewhat lower affinity for delta receptors, weak affinity for kappa 1 receptors, and no affinity for kappa 2 receptors. They were found to be potent mu receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These peptides represent a class of opioid receptor ligands that we have termed acetalins (acetyl plus enkephalin).

Retrospective: 12 years of antigenic determinant predictions, and more

Abstract: The Hopp and Woods hydrophilicity method for locating antigenic determinants was published in 1981. In the years since then, the method has been used widely and has played a vital role in many antigenic structure studies. The method has been criticized occasionally and replacements for it have been proposed. However, at this time, the Hopp and Woods method remains a method of choice for identifying antigenic sites and other protein interaction sites, because it has a higher success rate than other similar methods. Key to the success of this method is its cautious approach to charge-charge interactions, giving equal weight to positively and negatively charged residues, whereas other methods tend to favor one or the other. It has become clear that sites chosen by our method tend to be highly exposed, charged regions of the protein's surface which project into the environment and therefore have ample opportunity to contact other proteins. We have been exploring new uses for the method, and have found some applications in locating sites for other types of interactions, including those with other macromolecules such as DNA and RNA. It seems likely that this simple and reliable procedure will continue to find use in predicting the locations of major antigenic epitopes, and may also find use as a general prediction method to identify interaction sites on proteins that make charge-dependent contacts with a variety of other biological macromolecules.

Cytotoxic T lymphocyte (CTL) low-responsiveness to the Plasmodium falciparum circumsporozoite protein in naturally-exposed endemic populations: analysis of human CTL response to most known variants.

Abstract: Cytotoxic T lymphocytes (CTL) specific for epitope(s) within the circumsporozoite (CS) protein of malaria sporozoites have been shown to play an important role in protective immunity against malaria, at least in murine models. Their role in sporozoite immunity in the human host has, however, not yet been elucidated. Immunological non-responsiveness and antigenic diversity within T cell epitopes of the CS protein have been identified as potential problems in producing a sporozoite vaccine. These factors may contribute to the widespread lack of sporozoite immunity in endemic populations. In this study, 137 individuals with a history of natural endemic exposure to falciparum sporozoites (119 resident in north west Thailand and 18 resident in coastal Papua New Guinea) were tested for a CTL response to the Plasmodium falciparum CS protein. Fifty-four overlapping peptides, spanning the entire sequence of the CS protein of P. falciparum including most known variants, were studied. While most individuals had antibodies to the immunodominant B cell repeat, (NANP)n, and while CTL specific for an influenza virus matrix synthetic peptide could be generated from five of 23 Karen Thai individuals tested, no CS protein-specific CTL could be detected in these populations. Our data have important implications for vaccine programs.

Mutations in the p53 suppressor gene do not correlate with C-k-ras oncogene mutations in colorectal-cancer.

Abstract: Mutations in the p53 tumor suppressor gene have been analyzed in 196 colorectal tumors previously analyzed for mutations at codons 12 and 13 of the c-K-ras and N-ras oncogenes by a combination of Single Strand Conformation Polymorphism (SSCP) and Cycle Sequencing (CS) using total cellular RNA. Mutations were detected in 3 of 21 adenomas, 84 of 149 primary carcinomas, and 11 of 18 hepatic metastases. Over half of the tumors were homozygous for the mutant p53 allele at the mRNA level. Although deletions were detected in 5 tumors, missense mutations were the most frequent. The spectrum of 63 point mutations was heterogeneous, with all possible nucleotide substitutions ocurring at least once. No correlation was found between the spectrum of p53 gene mutations and the age, sex, race of the cancer patients or the anatomical localization of the tumors, although mutations were significantly more frequent in tumors of the distal colon. Mutations in the p53 gene did not correlate with mutations in the c-K-ras gene, indicating that colorectal cancer can develop through pathways independent not only of the presence of mutations in any of these genes but also of their cooperation.

Fucosidosis: Four New Mutations and a New Polymorphism

Abstract: Fucosidosis is a rare lysosomal storage disease due to a nearly complete deficiency of alpha-L-fucosidase (EC 3.2.1.51). In this study, all 8 exons of the alpha-L-fucosidase structural gene (FUCA-1) were amplified by PCR methods, and the amplified products were subcloned and sequenced. Five patient groups with fucosidosis were selected according to their ethnic backgrounds and haplotypes for RFLPs in FUCA-1. Four presumptive disease causing mutations were detected: 1) A major deletion of DNA containing the last two exons of FUCA-1 in two Algerian siblings. 2) A G to T mutation in exon 6 resulting in an in-frame termination codon (E375X) in eight Hispanic patients from Colorado and New Mexico. 3) A G to A mutation (G60D) in exon 1 in four Italian patients and in three related French-American (Cajun) patients. This G60D mutation creates a unique site for AflIII. 4) A frameshift mutation resulted from a two-base deletion in exon 2 (K151fs) in an Italian patient. This deletion obliterates a unique BstXI site and creates a new BpmI site, and was found in only this patient and in only one allele. The rationale for proposing these defects as disease causing mutations includes pedigree analysis and the predicted consequences of each defect upon the activity and the concentration of the enzyme. An A to G transition (Q281R) in exon 5 was found to be present in homozygous form in affected patients and also in normal subjects; it appears to be a newly identified polymorphism. It causes a charge change and may be responsible for the electrophoretic variant phenotype of fucosidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Probability plots for assessing multivariate normality

Abstract: A method of assessing multivariate normality by decomposition of measures of multivariate skewness and kurtosis into orthogonal components is described. The components can readily be inspected analytically, as well as graphically with probability plots. Examination of the individual components can often reveal departures from multivariate normality that may be masked in their underlying measures of skewness and kurtosis. There exist many techniques for assessing multivariate normality; see, for example, extensive reviews by Gnanadesikan (1977), Cox and Small (1978), Mardia (1980) and Koziol (1986). One graphical method devolves from the 'radius and angles' approach, which is based on the well-known transformation of Euclidean to spherical coordinates. Gnanadesikan (1977) in particular describes various informal tests for multivariate normal- ity with graphical procedures for examining the radii and angles following spherical coordinate transformation; Small (1978), Koziol (1982, 1983), Royston (1983), Szkutnik (1987) and Quiroz and Dudley (1991) among others have considered more formal procedures related to this approach. A different paradigm for assessing multivariate normality is based on the attributes of multivariate moments. In a series of seminal papers (Mardia, 1970, 1974, 1975; Mardia & Foster, 1983; Mardia & Kanazawa, 1983), Mardia introduced affine invariant measures of multivariate skewness and kurtosis, and comprehensively examined their properties under various circumstances. From a different perspective, Koziol (1986, 1987) considered measures of multivariate skewness and kurtosis suggested from the notion of Neyman's smooth tests; upon deriving smooth tests for assessing multivariate normality, he further found that the smooth test for multivariate skewness coincides with Mardia's measure of multivariate skewness, but the smooth test for multivariate kurtosis differed somewhat from Mardia's measure of multivariate kurtosis. Koziol noted that the smooth tests may be decomposed into individual components, which are distributed as independent %2 random variables, each with one degree of freedom. See also Mardia (1987) and Mardia and Kent (1991) for a unifying treatment devolving from Rao's score tests. The purpose of this note is to describe graphical techniques for depicting the components, and to demonstrate that examination of the individual components can be a valuable tool for assessing multivariate normality. For completeness, the smooth tests and their components are briefly reviewed in Section 2; examples of assessing multivariate normality with the components are found in

Dimeric oligopeptide mixture sets

Abstract: Dimeric oligopeptide mixture sets, their synthesis and use in determining the sequence of an oligopeptide dimer ligand that optimally binds to a receptor are disclosed. A dimeric oligopeptide mixture set has two oligopeptide portions bonded together by a disulfide bond. Each oligopeptide of a first oligopeptide portion has the same number of 3 to about 10 residues including an oxidized mercaptan-containing residue that forms part of the disulfide bond and an amino acid residue sequence that includes at least one of at least six residues in addition to the oxidized mercaptan-containing residue at the same one or more predetermined positions of the oligopeptide chain. The second portion has an oligopeptide chain having a length of 4 to about 10 residues, including an oxidized mercaptan-containing residue that forms part of the disulfide bond. The second portion is a mixture whose chains have equimolar amounts of those at least six amino acid residues at the same one or more other positions of the oligopeptide chain.

Peptides of the formula (KFmoc) ZZZ and their uses

Abstract: Peptides having Anti-microbial activity and having the formula (KFmoc)ZZZ-NH 2 , wherein Z is an amino acid are disclosed. Also disclosed are peptides having anti-trypsin activity and having the formula Ac-rypwz-NH 2 , wherein z is a D-amino acid. Also disclosed are compositions containing these peptides and methods of using them.

The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp super-repressors in vivo

Abstract: The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challenge-phage assay, an assay based on a positive genetic selection for DNA binding. These proteins, which differ by single amino acid changes from the wild type (Glu13-->Lys, Glu18-->Lys, Glu49-->Lys and Ala77-->Val), require less L-tryptophan than wild-type repressor for activation in vivo, and are super-aporepressors. However, none of the four mutant repressors binds DNA in a corepressor-independent manner. Three of the four mutant repressors (with Glu-->Lys changes) are more active when complexed with tryptophan, and are superholorepressors. Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding studies in vitro. Challenge-phage assays with limiting tryptophan reveal additional phenotypic differences among the mutant proteins. These results show that the challenge-phage assay is a robust assay for measuring the relative affinities of specific protein-DNA interactions in vivo.

A european interlaboratory evaluation study of an in vitro ocular irritation model (skin2TM model ZK1100) using 15 chemicals and 3 shampoos

Abstract: The aim of this interlaboratory study was to ascertain the feasibility of the use of the in vitro Skin2™ Model ZK1100 kit as a tissue substrate for cytotoxicity studies. Eighteen compounds — 15 raw chemicals and 3 finished products (commercial shampoos) — were tested in four different laboratories on two separate occasions, and the data obtained were compared with the data available in the literature on eye irritancy potential. The kit was easy to manage, and no technical difficulties were encountered in the estimation of MTT reduction, LDH and PGE2 release in the medium. Similar MTT50 values were recorded in two independent experiments in each laboratory, and the rankings of irritation potency within the same chemical class were comparable. Both LDH and PGE2 were important parameters to be taken into account along with MTT50 values in establishing the irritation potential of some categories of compounds.