Inhibition of ras-induced oocyte maturation by peptides from ras-p21 and GTPase activating protein (GAP) identified as being effector domains from molecular dynamics calculations.

In the accompanying article, using molecular dynamics calculations, we found that the 66-77 and 122-138 domains in ras-p21 and the 821-827, 832-845, 917-924, 943-953, and 1003-1020 domains in GAP have different conformations in complexes of GAP with wild-type and oncogenic ras-p21. We have now synthesized peptides corresponding to each of these domains and coinjected them into oocytes with oncogenic p21, which induces oocyte maturation, or injected them into oocytes incubated with insulin that induces maturation by activating wild-type cellular ras-p21. We find that all of these peptides inhibit both agents but do not inhibit progesterone-induced maturation that occurs by a ras-independent pathway.

Differences in patterns of activation of MAP kinases induced by oncogenic ras-p21 and insulin in oocytes.

Oncogenic ras (Val 12-containing)-p21 protein induces oocyte maturation by a pathway that is blocked by peptides from effector domains of ras-p21, i.e., residues 35-47 (that block Val 12-p21-activated raf) and 96-110 and 115-126, which do not affect the ability of insulin-activated cellular p21 to induce maturation.

Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation.

Purpose: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21.

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

Purpose: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway.

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain.

We have previously found that a peptide corresponding to residues 35-47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras-p21 peptide corresponding to residues 96-110 that blocks ras-jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation.

Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the guanine-nucleotide exchange protein, SOS, identified from molecular dynamics calculations. Selective inhibition of oncogenic ras-p21.

In the preceding paper we performed molecular dynamics calculations of the average structures of the SOS protein bound to wild-type and oncogenic ras-p21. Based on these calculations, we have identified four major domains of the SOS protein, consisting of residues 631-641, 676-691, 718-729, and 994-1004, which differ in structure between the two complexes. We have now microinjected synthetic peptides corresponding to each of these domains into Xenopus laevis oocytes either together with oncogenic (Val 12)-p21 or into oocytes subsequently incubated with insulin.

Action of chlorogenic acid in vegetables and fruits as an inhibitor of 8-hydroxydeoxyguanosine formation in vitro and in a rat carcinogenesis model.

Various plant extracts, such as carrot, burdock (gobou), apricot and prune, showed inhibitory effects in an in vitro assay of lipid peroxide-induced 8-hydroxydeoxyguanosine (8-OH-dG) formation. The major inhibitor purified from various plants extracts was identified as chlorogenic acid (CA), on the basis of UV- and mass-spectra and comparison with a standard sample. To examine whether CA also inhibits 8-OH-dG formation in animal organs, an oxygen radical-forming carcinogen, 4-nitroquinoline-1-oxide, was administered to rats, with or without CA.

Modified nucleosides in the first positions of the anticodons of tRNA(Leu)4 and tRNA(Leu)5 from Escherichia coli.

Minor leucine tRNA species, tRNA(Leu)4 and tRNA(Leu)5, from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M.

Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the ras-binding domain of the raf-p74 protein, identified from molecular dynamics calculations.

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein-protein interaction interface, e.g.

Selective inhibition of oncogenic ras-p21 in vivo by agents that block its interaction with jun-N-kinase (JNK) and jun proteins. Implications for the design of selective chemotherapeutic agents.

We have obtained evidence that oncogenic and activated normal ras-p21 proteins utilize overlapping but distinct signal transduction pathways. Recently, we found that ras-p21 binds to both jun and its kinase, jun kinase (JNK). We now present evidence that suggests that oncogenic but not normal activated p21 depends strongly on early activation of JNK/jun.

Identification of new mutagenic heterocyclic amines and quantification of known heterocyclic amines.

2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine (4'-OH-PhIP) was mutagenic, inducing 180 revertants of Salmonella typhimurium TA98 per 100 micrograms with S9 mix and was formed by heating a mixture of creatine, tyrosine and glucose. It was detected in broiled beef at a level of 21.0 ng per g of broiled beef, which is comparable to the level of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

Structural determination of a new mutagenic heterocyclic amine, 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), present in beef extract.

We previously found two new mutagens, compounds I and II, in bacteriological-grade beef extract by monitoring the mutagenicity to a new Salmonella strain, YG1024; compound I was identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx). In the present study, we isolated compound II from the beef extract, which accounted for 2% of the total mutagenicity of materials adsorbed on blue cotton. Further, we found that a large quantity of compound II was produced by heating a mixture of creatine, threonine and glucose (1:1:0.

Recognition of UUN codons by two leucine tRNA species from Escherichia coli.

Codon recognition by Escherichia coli tRNA(Leu)4 and tRNA(Leu)5 was investigated by analysis of the competition between two aminoacyl-tRNA species in an in vitro protein synthesis. Both tRNA species strictly obey the wobble rule when they are in competition with other tRNA species. This is probably due to the post-transcriptional modifications at the first position of the anticodon of these tRNA(Leu) species, supporting the proposal that the conformational rigidity of post-transcriptionally modified pyrimidine nucleotides guarantees the correct codon recognition.

The hydration of Ras p21 in solution during GTP hydrolysis based on solution X-ray scattering profile.

The small-angle X-ray scattering technique was used to characterize the structure in solution of wild type ras p21 as well as the oncogenic proteins mutated at residue 12, 59, or 61. In the presence of GDP, the radius of gyration, Rg, determined for wild type ras p21 was 16.89 +/- 0.

Isolation and identification of a new mutagen, 2-amino-4-hydroxy-methyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx), from beef extract.

By monitoring the mutagenicity to a new Salmonella tester strain, YG1024, which has a much higher level of O-acetyltransferase activity than S.typhimurium TA98, we found two new mutagenic compounds in bacteriological-grade beef extract. One of them (compound I), which had a similar UV spectrum to that of 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), was isolated and shown to account for approximately 2% of the total mutagenicity of the materials adsorbed to blue cotton, and its concentration was estimated to be 6.

A modified uridine in the first position of the anticodon of a minor species of arginine tRNA, the argU gene product, from Escherichia coli.

The argU (dnaY) gene product, a minor tRNA(Arg), from Escherichia coli has the anticodon N*CU with an unidentified modified nucleoside N* in position 34 [Kiesewetter, S., Fisher, W. & Sprinzl, M.

A nickel-binding serpin, pNiXa, induces maturation of Xenopus oocytes and shows synergism with oncogenic ras-p21 protein.

A nickel-binding serine proteinase inhibitor, pNiXa (43 kDa), was isolated from Xenopus ovary and assayed for effects on oocyte maturation. Microinjection of pNiXa (0.12 pmol/50 nl) induced maturation in 60% of Xenopus oocytes, beginning at 4 hours and reaching completion by 9 hours.

Pathways for activation of the ras-oncogene-encoded p21 protein.

The ras-oncogene-encoded p21 protein is known to cause a large number of human tumors. This protein differs from its normal counterpart protein, which is present in all eukaryotic cells, in that it contains a single amino acid substitution at critical positions in the polypeptide chain, such as at Gly 12, Gly 13, Ala 59, and Gln 61. Using computer-based molecular modeling, it has been found that one region of this protein that is a candidate for interacting with other intracellular proteins is the region from residues 35 to 47.

X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis.

RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes.

Evidence that the ras oncogene-encoded p21 protein induces oocyte maturation via activation of protein kinase C.

The ras oncogene-encoded p21 protein is known to induce cell maturation of Xenopus laevis oocytes and malignant transformation of NIH 3T3 mouse fibroblasts. The pathways involved in oocytes and NIH 3T3 cells appear to be similar to one another. For example, in both cases, the ras p21-induced cellular events involve increased intracellular levels of the second messengers diacylglycerol and inositol phosphates, the former of which activates protein kinase C (PKC).

Photosensitized formation of 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in DNA by riboflavin.

Potosensitized formation of 8-hydroxyguanine in DNA by riboflavin was observed. A reaction mechanism involving guanine radical cation and hydration reaction was proposed. This hypothesis was confirmed by the incorporation of [18O]-atom within guanine moiety in isotopic experiments using [18O]-H2O.

A peptide from the GAP-binding domain of the ras-p21 protein as well as azatyrosine block ras-induced maturation of Xenopus oocytes.

The ras-oncogene-encoded p21 protein is known to produce malignant transformation of NIH 3T3 cells as well as maturation of Xenopus oocytes when microinjected into these cells. p21 protein is known to bind a GTPase activating protein (GAP) intracellularly; residues 32-45 have been implicated in interacting with GAP. We demonstrate here that a peptide corresponding to residues 35-47 of p21 as well as the antibiotic azatyrosine inhibit the ras-induced maturation of Xenopus oocytes in a dose-related manner upon microinjection.

A peptide from the GAP-binding domain of the ras-p21 protein and azatyrosine block ras-induced maturation of Xenopus oocytes.

The ras-oncogene-encoded p21 protein causes malignant transformation of NIH 3T3 cells and maturation of Xenopus oocytes when microinjected into these cells. P21 is known to interact with GTPase activating protein (GAP) intracellularly. Residues 32-45 of p21 have been implicated in interacting with GAP.

Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain.

Normal RAS proteins play a key role of molecular switch in the transduction of the growth signal from extracellular to intracellular space. The state of the switch is "on" when GTP is bound and "off" when GDP is bound to the protein. The crystal structure of a complex between a nonhydrolyzable GTP analog and the catalytic domain of a RAS protein has been determined by a rotation-translation search method.

5-Formyldeoxyuridine: a new type of DNA damage induced by ionizing radiation and its mutagenicity to salmonella strain TA102.

An aqueous solution of calf thymus DNA was irradiated by 60Co gamma-rays and modified nucleosides produced in DNA were analyzed by high-pressure liquid chromatography coupled with a photodiode array UV detector. A new product with UV absorption maxima at 230 nm and 280 nm was observed. The structure of this compound was proposed to be 5-formyldeoxyuridine (f5dU) based on the mass spectrum of its trimethylsilyl derivative (M+, m/z472) and the structure was confirmed by chemical synthesis.