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J P Shi

Publications and source records attributed to J P Shi.

At least 19 recordsLinked to original sources
PubMed1996

Evidence that specificity of microhelix charging by a class I tRNA synthetase occurs in the transition state of catalysis.

Determinants for the identities of tRNAs are located in the acceptor stem and, commonly, in the anticodon as well. Although the anticodon is an important determinant for the identity of methionine tRNA, RNA microhelices whose sequences are based on the acceptor stem alone can be aminoacylated by the class I methionyl-tRNA synthetase. We show here that specific nucleotide substitutions in a microhelix significantly reduced its rate of aminoacylation. In contrast, affinity coelectrophoresis analysis showed that microhelix binding to the enzyme was not significantly affected by the same substitutions. These and additional experiments and considerations imply that specific determinants for microhelix aminoacylation are needed for orientation of the acceptor stem in the transition state of catalysis rather than for enhanced binding interactions. The effect of linking together acceptor stem interactions with those in the anticodon, as occurs in the whole tRNA molecule, was also evaluated. This analysis showed that linkage results in some of the favorable acceptor stem and anticodon interactions being used to offset the free energy cost of straining the structure of the enzyme-tRNA complex.

Anticodon↗
PubMed1996

Identification of a potential physiological substrate for oncogenic Ras-activated protein kinases in activated Xenopus egg extracts: correlation with oncogenic Ras-induced cell cycle arrest.

Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these activated extracts, we previously showed that purified, bacterially expressed oncogenic human RasH protein arrests cell cycle progression. Because oncogenic Ras activates many serine/threonine protein kinases in Xenopus oocytes and egg extracts, it is possible that induction of cell cycle arrest involves the action of oncogenic Ras-activated kinases. Thus, the identification of the physiological substrates for oncogenic Ras-activated kinases is important for elucidating the molecular mechanism underlying oncogenic Ras-induced cell cycle arrest. We used 32P-orthophosphate as a label to identify the potential substrates. Our results demonstrated that the 32P-labeling of both a 32 and a 33 kDa protein were greatly enhanced by oncogenic Ras during the incubation of activated Xenopus egg extracts. The enhanced labeling correlated with the induced cell cycle arrest and was contributed by serine phosphorylation. Moreover, the 33 kDa protein was detected only in the presence of oncogenic Ras and was a serine-hyperphosphorylated form of the 32 kDa protein. Furthermore, new protein synthesis was not required for the enhanced labeling, consistent with the concept that the enhanced serine phosphorylation of the 32 kDa protein is by oncogenic Ras-activated protein kinases. In addition to serine phosphorylation, our results also suggested that an as yet unidentified modification of the 32 kDa protein might also be induced by oncogenic Ras. Our results suggest that the 32 kDa protein is a potential physiological substrate for oncogenic Ras-activated protein kinases.

14-3-3 Proteins↗
PubMed1996

Lung cancer in nonsmoking Chinese women: a case-control study.

The risk factors for lung cancer in lifetime nonsmoking women were investigated in a hospital-based case-control study in the urban area of Shenyang, China, between April 1992 and May 1994. One-hundred thirty-five newly-diagnosed lung cancer cases and an equal number of controls, matched for age and sex, were enrolled and interviewed by trained personnel who administered a standardized questionnaire. The histopathological cell type was predominantly adenocarcinoma (54.5%), followed by small cell carcinoma (20%), squamous cell carcinoma (16.4%), and others (9.1%). The data were analyzed using the Mantel-Haenszel method and by multivariate logistic regression analysis. The odds ratio (OR) and confidence interval (CI) associated with cooking oil vapors and with family history of cancer were 3.79 (95% CI, 2.29-6.27) and 2.29 (95% CI, 1.01-5.17), respectively. No association was found between exposure to environmental tobacco smoke (ETS), presence of previous lung diseases, and "kang" use. Cooking practices, exposure to cooking fumes, and a family history of cancer were found to significantly increase the risk of lung cancer.

Adult↗
PubMed1994

Region of a conserved sequence motif in a class II tRNA synthetase needed for transfer of an activated amino acid to an RNA substrate.

The class II Escherichia coli alanine tRNA synthetase aminoacylates RNA miniduplexes, which reconstruct the acceptor end of alanine tRNA with the critical G3:U70 base pair. A benzophenone photoaffinity label attached adjacent to G3:U70 in a miniduplex substrate was previously cross-linked to a long enzyme peptide that begins at Gly161 between the class-defining motifs 2 and 3 [Musier-Forsyth, K., & Schimmel, P. (1994) Biochemistry 33, 773-779]. To identify side chains in this peptide that potentially contribute hydrogen bonding or catalytic determinants for the RNA-dependent step of the aminoacylation reaction, peptide functional side chains that are conserved among sequenced alanine enzymes (Asp, Asn, Arg, Glu, Gln, and Tyr) were individually replaced. Of the 21 mutant proteins so generated, one was identified that was not viable even though it accumulated in vivo. This Asp235-->Ala mutant enzyme is defective in the rate of transfer of the activated amino acid to the 3'-end of the RNA substrate. The conserved Asp235 is at the beginning of motif 3. By comparison with the crystal structure of the related class II yeast aspartate tRNA synthetase complexed with tRNA(Asp) (Cavarelli et al., 1993), we suggest that D235 is not in direct contact with acceptor helix base pairs such as G3:U70. Instead, we propose that D235 contributes to transfer-step interactions at the 3'-end of alanine tRNA. Because D235 in alanine tRNA synthetase is at the beginning of one of the conserved motifs that define class II tRNA synthetases, this region of the structure may in general be important for the transfer step.

Alanine-tRNA Ligase↗
PubMed1993

Levonorgestrel levels in serum and uterine flushings in women bearing LNG-Cu-IUD.

Radioimmunoassay was employed to measure levonorgestrel levels in serum and uterine flushings in women using intrauterine device releasing both copper ions and levonorgestrel (LNG-Cu-IUD) after one-year insertion. In the meanwhile physical characteristics of cervical mucus have been observed in women using LNG-Cu-IUD and compared with those using Tcu-220c-IUD. The mean concentrations of levonorgestrel in serum and uterine flushings (5 ml) were 84.53 pg/ml and 266.93 pg/ml respectively. In LNG-Cu-IUD group, the cervical mucus lacking in ferny crystals had a viscosity significantly higher than that in Tcu-220c-IUD group, suggesting that the local high concentration of levonorgestrel changed the environment of the uterine cavity and the cervical mucus status.

Adult↗
PubMed1992

RNA tetraloops as minimalist substrates for aminoacylation.

Previous work established that seven-base-pair hairpin microhelices with sequences based on the acceptor stems of alanine, glycine, methionine, and histidine tRNAs can be aminoacylated specifically with their cognate amino acids. To obtain "minimalist" substrates with fewer base pairs, we took advantage of the high thermodynamic stability of RNA tetraloop motifs that are found in ribosomal RNAs. We show here that rationally designed RNA tetraloops with as few as four base pairs are substrates for aminoacylation. Major nucleotide determinants for recognition by the class II synthetases were incorporated into each of the respective tetraloop substrates, resulting in specific aminoacylation by the alanine, glycine, and histidine tRNA synthetases. An analysis of the kinetics of aminoacylation shows that, for the alanine system, the majority of the transition-state stabilization provided by the synthetase-tRNA interaction is reproduced by the interaction of the synthetase with nucleotides in its minimalist tetraloop substrate. In an extension of this work, we also observed specific aminoacylation with the class I methionine tRNA synthetase of RNA tetraloops based on sequences in the acceptor stem of methionine tRNA. Thus, the results demonstrate four different examples where specific aminoacylation is directed by sequences/structures contained in less than half of a turn of an RNA helix.

Acylation↗
PubMed1992

Overlapping nucleotide determinants for specific aminoacylation of RNA microhelices.

A seven-base pair microhelix that recapitulates a glycine transfer RNA (tRNA) acceptor helix can be specifically aminoacylated with glycine. A single base pair and the single-stranded discriminator base near the attachment site are essential for aminoacylation. These nucleotide sequence elements, and those in microhelices that can be charged with histidine and alanine, occur in the same positions and therefore overlap. Studies on a systematic set of sequence variants showed that no microhelix could be charged with more than one amino acid. Also, none of the three cognate aminoacyl-tRNA synthetases (aaRSs) gave a detectable amount of aminoacylation of the CCA trinucleotide that is common to the 3' ends of all tRNAs, showing that the specific acceptor stem nucleotide bases confer aminoacylation. An analysis of the relative contributions of these microhelices to overall tRNA recognition indicated that their interaction with aaRSs constitutes a substantial part of the recognition of the whole tRNAs.

Base Sequence↗
PubMed1991

Aminoacylation of alanine minihelices. "Discriminator" base modulates transition state of single turnover reaction.

RNA hairpin minihelices that recreate the acceptor-T psi C stem of Escherichia coli alanine tRNA are charged specifically with alanine, provided that they encode the critical G3:U70 base pair that is the major determinant for the identity of an alanine tRNA. These model substrates were used to investigate the role in charging of N73, the unpaired nucleotide that is just three positions removed from the amino acid attachment site and which is sometimes referred to as the "discriminator" base. Previous work showed that, while substrates which encode G3:U70 are all charged by alanine tRNA synthetase regardless of the base at position 73, catalytic efficiency is substantially higher with substrates that have the wild-type A73. To identify a specific step in aminoacylation that is affected by substitutions of A73, we studied the single turnover charging of A73, U73, C73, and G73 minihelices, using preformed, enzyme-bound alanyl adenylate and saturating concentrations of the respective minihelices. Compared to the A73 substrate, the transfer of activated amino acid to bound RNA is sharply reduced for the substituted N73 minihelices. The low efficiency of transfer is not due to an abortive reaction in which the adenylate or a transiently charged RNA is hydrolyzed. Instead, under the conditions used, the active adenylate remains on the enzyme for extended periods and simply reacts slowly with bound N73 RNA. The results suggest that the nature of the discriminator base is a critical determinant of the transition state for the reaction of bound alanyl adenylate with RNA on the surface of the enzyme.

Acylation↗
PubMed1991

Studies on the antifertility components from Marsdenia koi.

By random screening test, Marsdenia koi was found to have antifertility activity on SD rat. From MeOH extracts of this plant two steroidal glycosides, marsdekoiside A and B, were isolated, and their structures were elucidated on the basis of spectral evidence and by comparison of the hydrolysis products with the authentic samples. Both are newly identified compounds, and marsdekoiside A has good antifertility activity.

Animals↗
PubMed1991

Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg).

The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights. This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported. Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered. During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated.

Affinity Labels↗
PubMed1990

A nucleotide that enhances the charging of RNA minihelix sequence variants with alanine.

We showed earlier that a single G3.U70 base pair within the amino acid acceptor helix is a major determinant of the identity of tRNA(Ala). In addition, we demonstrated that an RNA hairpin minihelix that recreates the 12 base pair acceptor-T psi C stem of tRNA(Ala) is also aminoacylated in a G3.U70-dependent manner. Determinants for efficient aminoacylation at pH 7.5 have been further investigated with minihelix substrates that have sequence variations at 3.70 and other locations. Although a U,U mismatch and other 3.70 nucleotide alternatives to G.U were recently proposed by others as also important for alanine acceptance, neither that mismatch nor any of four other 3.70 nucleotide combinations confer aminoacylation in vitro with alanine, even with substrate levels of enzyme. In contrast, permutations of the so-called discriminator nucleotide N73 (at position 73) strongly modulate, but do not block, aminoacylation of those substrates that encode G3.U70. In particular, the efficiency of G3.U70-dependent aminoacylation with alanine is strongly enhanced by having the wild-type A73. The effect of N73 alone can explain most of the difference in aminoacylation efficiency of a G3.U70-containing tRNA and a minihelix substrate whose sequences vary significantly from their tRNA(Ala) counterparts. Comparison with earlier work suggests that the substantial modulating effect of N73 is partly or completely obscured when N73 tRNA variants are expressed as amber suppressors in vivo.

Alanine-tRNA Ligase↗
PubMed1990

[Epidemiological survey on patients with advanced filariasis in Shandong Province with bancroftian filariasis basically eliminated].

This paper reports on the incidence of advanced filariasis after basic elimination of bancroftian filariasis since 1983 in Shandong Province. Investigation was carried out in a population of 166,776 between 1984 and 1988 in 252 villages of Teng Xian and other 5 counties/cities, the erstwhile highly endemic areas. A total of 1,038 filariasis patients were found with an average incidence of 0.6%. Among them, 383 were with elephantiasis, 357 with chyluria and 298 with hydrocele. 902 cases (86.9%) who suffered the disease before the elimination of filariasis and 136 cases (13.1%) after it, 125 (91.9%) being chyluria cases. The oldest of the 1,038 cases was 86 years of age and the youngest, 6 years of age. The course of duration as 8-74 years in elephantiasis cases, 82.8% of them (317 cases) had previously lymphangitis and/or lymphadenitis. In the past three years 16.4% (52 cases) suffered from prolonged or intermittent acute lymphangitis and/or lymphadenitis. The results of this survey indicated that, after the basic elimination of filariasis in Shandong Province together with thorough clearance of infection source, elephantiasis and hydrocele persisted while new cases of chyluria continued to develop. Therefore, in such areas more emphasis should be put on the treatment of clinical patients. New patients should be surveyed and old patients be treated actively so as to reach the goal of eradicating filariasis.

Adolescent↗
PubMed1989

Limited tryptic digestion of leucyl-tRNA synthetase and characterization of its active fragment.

Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) from E. coli underwent limited proteolysis by trypsin which cut off 6K peptide and converted the intact LeuRS into a 96K fragment. The truncated enzyme retained the PPi exchange activity with the same kinetic parameters as those of native LeuRS but lost the tRNALeu charging, binding and other tRNALeu-related activities. N-terminus analysis showed that the 6K peptide was located at the C-terminus of Leu-RS. This small part played a crucial role in tRNALeu binding. Our results suggest that the two activities, PPi exchange and tRNA charging are independent of each other and correspond to different structural regions of LeuRS. The C-terminal region might be the tRNALeu binding site of LeuRS.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Arginyl-tRNA synthetase from Escherichia coli, purification by affinity chromatography, properties, and steady-state kinetics.

A Blue Sephadex G-150 affinity column adsorbs the arginyl-tRNA synthetase of Escherichia coli K12 and purifies it with high efficiency. The relatively low enzyme content was conveniently purified by DEAE-cellulose chromatography, affinity chromatography, and fast protein liquid chromatography to a preparation with high activity capable of catalyzing the esterification of about 23,000 nmol of arginine to the cognate tRNA per milligram of enzyme within 1 min, at 37 degrees C, pH 7.4. The turnover number is about 27 s-1. The purification was about 1200-fold, and the overall yield was more than 30%. The enzyme has a single polypeptide chain of about Mr 70,000 and binds arginine and tRNA with 1:1 stoichiometry. For the aminoacylation reaction, the Km values at pH 7.4, 37 degrees C, for various substrates were determined: 12 microM, 0.9 mM, and 2.5 microM for arginine, ATP, and tRNA, respectively. The Km value for cognate tRNA is higher than those of most of the aminoacyl-tRNA synthetase systems so far reported. The ATP-PPi exchange reaction proceeds only in the presence of arginine-specific tRNA. The Km values of the exchange at pH 7.2, 37 degrees C, are 0.11 mM, 2.9 mM, and 0.5 mM for arginine, ATP, and PPi, respectively, with a turnover number of 40 s-1. The pH dependence shows that the reaction is favored toward slightly acidic conditions where the aminoacylation is relatively depressed.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Affinity labelling of E. coli leucyl-tRNA synthetase with 3'-oxidized tRNA(Leu).

The E. coli leucyl-tRNA synthetase (E.C. 6.1.1.4) was specifically labelled with 3'-oxidized tRNA(Leu) (tRNA(oxLeu)). The procedure involves a Schiff's base formation and its subsequent reduction by sodium cyanoborohydride. Stoichiometric inactivation of aminoacylation was achieved with the incorporation of 1 mol of tRNA(oxLeu) per mol LeuRS. On the other hand, the amino acid activation activity of LeuRS-tRNA(ox) complex was partially inhibited. After extensive digestion of the complex by pancreatic ribonuclease, the amino acid activation activity was fully recovered, while the aminoacylation activity was not restored at all.

Affinity Labels↗
PubMed1984

Fidelity of DNA replication under conditions used for oligodeoxynucleotide-directed mutagenesis.

The fidelity of DNA replication in vitro by DNA polymerase I (large subfragment) of Escherichia coli has been measured by the standard bioassay: single-stranded phi X174 DNA (plus strand) containing an amber codon was primed with a synthetic oligodeoxynucleotide, replicated and the frequency of point mutations formed in the synthetic minus strand of the resultant double-stranded DNA determined from the number of revertant phage produced in a spheroplast assay. Since the assay depends crucially on the frequency of expression of the mutations in the heteroduplex, and this can vary for a variety of reasons, parallel control experiments were performed using a primer that covered the amber codon but contained the same mismatch that occurred during replication. The frequency of expression of these mutations was found to vary from 40 to 100% in fully ligated heteroduplexes, depending upon the age and batch of spheroplasts used. The variation probably reflects the viability of the post-replicative mismatch repair enzymes in the spheroplasts used for transfection. Far lower frequencies of expression were found under conditions of poor replication. Accurate data and rate laws for fidelity are obtained only when the bioassay is normalized for the variation in the expression frequency. There is active proofreading by the 3'-5'-exonuclease activity of the polymerase of a misincorporation resulting from a dGTP:T mismatch. The contribution of proofreading to fidelity is low: accuracy is enhanced by a factor of less than 7 at the concentrations of dNTPs in vivo. The lower accuracy of Pol I than Pol III is due mainly to poorer proofreading, which is manifested in a lower "cost" of replication: only 0.7 to 1.7% of the dNTPs are turned over to dNMPs during replication compared with 6 to 13% for Pol III. The error rates measured for Pol I under conditions used for oligodeoxynucleotide-directed mutagenesis are sufficiently low that extraneous errors should not be induced when the concentrations of dNTPs are balanced. However, even higher fidelity will be obtained using the lowest concentrations of dNTPs consistent with efficient replication (approximately 20 microM). Highly unbalanced concentrations as used in pulsed labelling should be avoided.

Bacteriophage phi X 174↗