Showing papers by "Torrey Pines Institute for Molecular Studies published in 1996"

Crystal structure of the Aequorea victoria green fluorescent protein.

Abstract: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

Abstract: The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 · 10 ‐6 ) < Deep Vent (2.7 · 10 ‐6 ) < Vent (2.8 · 10 ‐6 ) < Taq (8.0 · 10 ‐6 ) << exo ‐ Pfu and UlTma (~5 · 10 ‐5 ). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2‐3 mM MgSO4 and 100‐300 μM each dNTP and at pH 8.5‐9.1. Under these conditions, the error rate of exo ‐ Pfu was ~40-fold higher (5 · 10 ‐5 ) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased ~2-fold, while the error rate of exo ‐ Pfu increased ~9-fold. An increase in error rate with pH has also been noted for the exonucleasedeficient DNA polymerases Taq and exo ‐ Klenow, suggesting that the parameters which influence replication error rates may be similar in pol I- and α-like polymerases. Finally, the fidelity of ‘long PCR’ DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but ~3‐4-fold higher than the error rate of Pfu DNA polymerase.

Interleukin 2 production in vitro by peripheral lymphocytes in response to human papillomavirus-derived peptides: Correlation with cervical pathology

Abstract: Human papillomavirus (HPV) is believed to be the major cause of cervical cancer. To investigate whether a cellular immune response, espe cially a T helper type 1 response, is related to the natural defense against HPV-related cervical lesions, the interleukin 2 response of peripheral blood lymphocytes in vitro to overlapping peptides from HPV-16 E6 and E7 oncoproteins was compared with the degree of cervical cytological abnormality among 140 women in a cross-sectional study. We compared 66 women diagnosed with low-grade squamous intraepithelial lesions (LSIL), 21 with high-grade squamous intraepithelial lesions (HSIL), and 28 with invasive cervical cancer with 25 women who were cytologically normal but previously HPV-l6 DNA positive. The fraction showing strong interleukin 2 production against HPV-16 peptides was greatest among cytologically normal women (35%) and declined with increasing disease seventy ELSILI (20%), HSIL (17%), and cancer patients (7%); @ test P for the trend 0.021, whereas the responses against a recall influenza antigen were not significantly different among groups. Our finding sug gests that a T helper lymphocyte type 1 response to HPV antigens is associated with disease status. This result may reflect a targeted effect of the disease on immune function or a protective effect of the immune response against disease progression.

Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype

Abstract: We have determined the frequency and spectrum of spontaneous mutations at the hprt locus in LoVo, HCT116, LS180 and DLD-1 colon carcinoma cell lines exhibiting microsatellite genetic instability. Each cell line has a different mutator gene. LoVo and HCT116 cells have mutated hMSH2 and hMLH1 genes, respectively, which account for the majority of hereditary non-polyposis colorectal cancer (HNPCC). LS180 cells are wild type for these genes and also for hPMS1 and hPMS2 mismatch repair genes. DLD-1 cells harbor a mutated GTBP mismatch binding factor and a mutated DNA Polymerase delta. The mutation rate at the hprt locus was several hundred fold higher in these cell lines relative to control cell lines without microsatellite instability. The mutations were frameshifts (deletions and insertions of a single nucleotide in short repeats) and single base substitutions (transversions and transitions). Some mutations were shared by these four cell lines. However, every cell line also exhibited a distinctive spectrum of mutations suggesting that each mutator gene induces a particular mutator phenotype. These results also suggest that the frequency and spectrum of somatic mutations in tumor cells of the microsatellite mutator phenotype may have diagnostic applications to discriminate among the diverse underlying mutator genes.

Dimerization of the human cytomegalovirus protease : kinetic and biochemical characterization of the catalytic homodimer

Abstract: The single-chain 28 kDa human cytomegalovirus (HCMV) protease catalytic domain containing the A143Q mutation has been kinetically and conformationally characterized. The specific activity of the HCMV A143Q protease (HCMVp) increases as the protease concentration increases, suggesting that this protease oligomerizes at high protein concentration to form a more active species. Both cross-linking and light-scattering studies of HCMVp show the existence of a homodimer with an apparent molecular mass of 56 kDa under low ionic strength and high protein concentration. The cosolvent and solute effects of glycerol, trisodium citrate, and NaCl as well as the temperature effects on the HCMVp activity and quaternary structure were investigated. The effects induced by cosolvents and temperature can largely be explained by their influences in the dimerization or oligomerization state of HCMVp. The dissociation constant (Kd) for the HCMVp homodimer was determined to be 8 +/- 1 microM with all activity attributed to the dimeric form. Monomeric HCMVp is inactive. This report demonstrates that in vitro, HCMV A143Q protease exists as an obligate catalytic homodimer. This protease dimerization may have regulatory significance during viral replication.

Screening of Differentially Amplified cDNA Products from RNA Arbitrarily Primed PCR Fingerprints Using Single Strand Conformation Polymorphism (SSCP) Gels

Abstract: Arbitrarily primed PCR fingerprinting of RNA and differential display resolved on an acrylamide gel has been extensively used to detect differentially expressed RNAs. However, after a differentially amplified product is detected the next steps are labor-intensive: a small portion of the fingerprinting gel that contains the differentially amplified product is cut out, reamplified and the correct product is determined, typically by cloning and sequencing what is often a mixture of products of similar size. Here we use a native acrylamide gel to separate DNAs in the reamplified mixture based on single-stranded conformation polymorphisms. Reamplifications are performed for the region carrying the differentially amplified product and a corresponding region from an adjacent lane where the product is less prominent or not visible. Denaturation of the reamplified DNA followed by side-by-side comparison on an SSCP gel allows the classification of reamplified material into (i) those that can be directly cloned because the differentially amplified product is relatively pure, (ii) those that need to be reamplified from the SSCP gel before cloning and (iii) those that are too complex for further study. This screen should save considerable effort now wasted on directly cloning unsuitable products from RNA fingerprinting experiments. An example is presented of cloning a gene differentially expressed in Trypanosoma brucei life cycle.

Synthetic combinatorial libraries: novel discovery strategy for identification of antimicrobial agents.

Abstract: Bacterial resistance to existing drugs is a constantly growing process that is reaching alarming levels (for reviews, see references 16 and 35). This, combined with the recent decline in the development of new antibiotics, can be anticipated to lead to the appearance of infectious diseases lacking a ready treatment regimen. For instance, methicillin-resistant Staphylococcus aureus (MRSA) is resistant to all b-lactams, including penicillins, carbapenems, and penems, because of the production of a penicillin-binding protein that has a low affinity for b-lactam antibiotics (31). This resistance positions MRSA as an increasingly dangerous pathogen in the 1990s (10). Vancomycin is currently the agent of choice against MRSA (for a review, see reference 9). However, its widespread use, as expected, is leading to new resistant strains, as has been observed in enterococcal species (43). Classical sources for novel antimicrobial agents include biological sources (e.g., marine waters, insects, mammals, plants, and soil samples [1, 8, 33, 34, 39]), as well as large collections of individual compounds gathered from various laboratories. While these compounds have diverse skeletal and functional arrays, the isolation and characterization of such natural compounds or the synthesis of large collections of individual compounds can be cumbersome and can delay the discovery process. The recent development of synthetic combinatorial libraries (SCLs) represents a major advancement in the discovery of new lead compounds for drug development (for reviews, see references 4, 21, and 37). Although its use for the identification of novel antimicrobial agents has not been fully exploited, the findings reported so far indicate that combinatorial techniques will play an important role in the infectious disease area.

Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping

Abstract: Summary OBJECTIVE Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disorder resulting in target organ resistance to the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In many cases, this disorder has been shown to be due to mutations in the gene encoding vitamin D receptors (VDR). In a patient with characteristic features of this disorder, we investigated the functional defect and sequenced the coding region of the gene for mutations. DESIGN Skin fibroblasts from patient and control were used to measure binding of 1,25(OH)2D3 and functional responses to the hormone. These cells were also used to prepare RNA from which cDNA was prepared and sequenced. Furthermore, genomic DNA was prepared from the fibroblasts and the intronlexon boundarles sequenced. PATIENT A child with classic features of HVDRR with alopecia diagnosed as having rickets due to resistance to 1,25(OH)2D3. MEASUREMENTS Nuclear association of 1,25(OH)2D3 was determined in patient and control cells and the functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamln D-24-hydroxylase(24-hydroxylase) activity. VDR cDNA and genomic DNA prepared from patient and control cells were sequenced. RESULTS Cells from the patient with HVDRR had undetectable amounts of VDR compared to control cells and did not show induction of 24-hydroxylase activity following treatment with 1,25(OH), D3. Sequencing of the VDR coding region after RT-PCR of RNA revealed an absence of exon 4 in patient RNA which was not due to a deletion in genomic DNA but was caused by exon skipping during RNA processing. In addition, the deletion of exon 4 sequences from RNA leads to a frameshift in translation resulting in a premature stop codon. Amplification of genomic DNA around the intron/exon boundary of exon 4 revealed a point mutation in the 5’donor splice site of intron 4. CONCLUSION In this study, we have identified a novel mutation in the gene for vitamin D receptors in a patient with the Characteristic phenotype of hereditary vitamin D resistant rickets. The mutation at the + 5 position in intron 4 is most likely to cause skipping of exon 4 in this patient.

Isoquinoline derivatives and isoquinoline combinatorial libraries

Abstract: The present invention provides the synthesis of heterocyclic compounds based on the isoquinoline ring. More specifically, the invention provides novel isoquinolines as well as novel libraries comprised of many such compounds, and methods of synthesizing the libraries.

Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/lacl transgenic rats and mice following exposure to aflatoxin B1

Abstract: l -treated mice showed a pattern of mutation similar to that of the previously observed spontaneous mouse liver mutational spectrum. In contrast, rats subjected to one-tenth the mouse AFB | dosage responded with an approximate 20fold induction in liver mutant frequency over background. Sequencing of lad mutations also revealed spectral differences between vehicle- and AFBptreated rats. A large increase in G:C—>T:A transversions was observed among lad mutations isolated from the AFBptreated rats. This work is among the first multi-species in vivo mutagenicity studies using transgenic rodents harboring the same shuttle vector. Such multi-species in vivo assays may prove to be valuable in the areas of mechanistic analysis and risk assessment

Rapid identification of compounds with enhanced antimicrobial activity by using conformationally defined combinatorial libraries

Abstract: We have combined the strength of our synthetic combinatorial library approach for the rapid identification of highly active compounds with prior knowledge of the relationship between the antimicrobial activities of individual peptides with specific induced conformations in order to identify new peptides with enhanced activity relative to a starting known antimicrobial sequence In the current study, conformationally defined combinatorial libraries were generated based on an 18-mer antimicrobial peptide known to be induced into an alpha-helical conformation in a lipidic environment Not only were novel sequences readily identified with 10-fold increases in activity, but detailed information about the structure-activity relationships of the peptides studied was also obtained during the deconvolution process By using circular dichroism spectroscopy it was found that the individual 18-mer peptides could be induced into alpha-helical conformations on interaction with the cell lipid layer and/or sialic acids, which could result in bacterial cell lysis due to perturbation of the lipid packing of the cell wall

Generation and use of nonsupport-bound peptide and peptidomimetic combinatorial libraries.

Abstract: Publisher Summary The practical use of nonsupport-bound combinatorial libraries represents an important breakthrough in all the areas of basic research and drug discovery. The use of a wide variety of chemical transformations permits a range of peptidomimetic libraries to be generated that greatly expands the chemical diversity available. The results described in this chapter demonstrate that an existing peptide positional scanning- synthetic combinatorial libraries (PS-SCL) can be chemically transformed to generate a peptidomimetic SCL, from which highly active individual compounds can be identified. The synthesis and deconvolution methods developed for peptide libraries are easily applied to other types of chemical pharmacophores. The soluble nature of the nonsupport-bound combinatorial libraries is a distinct advantage over the other methods in that membrane-bound and whole cell assays can also be used. In addition, the deconvolution methods used allow the chemical structure of peptidic, peptidomimetic, and organic compounds to be determined based solely on the structural similarities of compounds, within each active pool or sublibrary.

Synthesis of quinazolinone libraries and derivatives thereof

Abstract: The present invention provides synthetic combinatorial libraries of organic compounds based on the quinazolinone ring as well as libraries containing styryl derivatives of the same.

Novel alpha-glucosidase inhibitors identified using multiple cyclic peptide combinatorial libraries.

Abstract: Twenty-six cyclic synthetic peptide combinatorial libraries (disulfides and lactams) of varying size and composition, representing 6.8 × 103 to 4.7 × 107 individual peptides, were synthesized along with their respective linear analogs. One of the hexapeptide lactam libraries (cyclo[xXxXxN]) was found to have significant α-glucosidase inhibitory activity. This library was carried through an iterative process of synthesis and screening, during which all of the five mixture positions (x and X) were successively defined. As the result of this process, potent and selective a-glucosidase inhibitors were identified.

Variation and evolution of class I Mhc in sexual and parthenogenetic geckos.

Abstract: We present the first Mhc class I sequences in geckos. We compared Mhc variation in gekkonid species that reproduce sexually ( Hemidactylus frenatus, Lepidodactylus aureolineatus, L . moestus, L . sp. Arno, L . sp. Takapoto) to others reproducing parthenogenetically ( H. garnotii, L. lugubris ). These comparisons include the known maternal ( L. moestus ) and paternal ( L . sp. Arno) ancestors of the asexual L. lugubris . Sequences similar to other vertebrate species were obtained from both nuclear and cDNA templates indicating that these sequences are derived from expressed class I Mhc loci. Southern blot analysis using gecko class I probes, revealed that parthenogenetic clonal lineages of independent evolutionary origin have no within-clone band variation at class I loci and that no detectable recombination between restriction sites had taken place. Variability in the sexual species was similar to mammalian taxa, i. e. class I genes are highly variable in outbreeding sexual populations. Sequence analysis of the alpha-2 domain of class I genes identified point mutations in a clonal lineage of L. lugubris which led to amino acid substitutions. Potential transspecific allelic lineages were also observed. The persistence of asexual lineages with little or no class I diversification over thousands of generations seems to argue against strong selection for Mhc multi-allelism caused by pathogen-Mhc allele specificity. On the other hand, the high level of heterozygosity in the parthenogenetic species (a consequence of their hybrid origin) may provide clonal lineages with adequate antigen presenting diversity to survive and compete with sexual relatives.

Libraries from libraries: generation and comparison of screening profiles.

Abstract: A positional scanning tetrapeptide library was chemically modified through alkylation and/or reduction of the amide bonds, thus generating three new combinatorial libraries with physico-chemical properties very different from the parent peptide library (‘libraries from libraries’). Specific results were obtained with each of these libraries upon screening in κ-opioid receptor binding and microdilution antimicrobial assays, illustrating the potential of the ‘libraries from libraries’ concept for the efficient generation of a variety of chemically diverse combinatorial libraries.

A Cubic-Phase Oral Drug Delivery System for Controlled Release of AG337

Abstract: AG337 is a novel thymidylate synthase inhibitor possessing potent antitumorogenic activity which was designed using protein structure-based techniques. It is currently undergoing clinical trials as both N and oral formulations. Based on AG337's short in vivo half-life, an oral controlled-release formulation is desired. The feasibility of using cubic liquid crystalline phases formed from monoolein for controlled release of AG337 has been investigated in this study. AG337 (m.p. = 298°C) was triturated with glycerol and then dissolved in monoolein using mild heat. The resulting gel was liqu@ed by further heating to 65"C, then cooled to RT to yield a clear viscous solution. Samples of the formulation were exposed to water for up to 48 hr at 25°C. Thermal analysis of this system was undertaken in order to determine the effect of hydration state on the liquid crystalline structure. The differential scanning colorimetry (DSC) pro$le of samples not exposed to water showed no distinct endo- or exothermic transitions. However, samples exposed to water exhibited multiple endothermic transitions from 80" to 120 "C. These data demonstrate a thermal response to time-dependent water uptake in the formulation as might occur in vivo afrer oral dosing, due to changes in physical properties of the system. In vitro release rates of AG337from this fomrcltion were evaluated.