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J Qin

Publications and source records attributed to J Qin.

At least 109 records · Page 6Linked to original sources
PubMed1998

Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2.

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

3T3 Cells↗
PubMed1998

Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5.

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.

Animals↗
PubMed1998

Prostaglandin E2 integrates the effects of fluid distension and glucocorticoid on lung maturation.

Both glucocorticoids and alveolar fluid distension affect the rate of fetal lung maturation, possibly representing a common cellular pathway. In an explant culture, there is a spontaneous increase in triglyceride incorporation into saturated phosphatidylcholine over time. This mechanism is stimulated by prostaglandin (PG) E2, blocked by both bumetanide and indomethacin, and overridden by exogenous PGE2. Type II cells synthesized and produced PGE2 between days 16 and 21 postconception, increasing fourfold between days 19 and 21. Fetal rat lung fibroblasts released triglyceride in response to PGE2, increasing 10- to 14-fold between days 19 and 21 postconception; phloretin (1 x 10(-5) M) completely blocked this effect of PGE2 on triglyceride release. Dexamethasone stimulated both type II cell PGE2 synthesis (threefold) and fibroblast triglyceride release in response to PGE2 (60%) by day 20 cells. Stretching type II cells also increased PGE2 synthesis (approximately 100% at 1, 2, and 3 h vs. static cultures). Recombination of [3H]triglyceride-labeled fibroblasts with type II cells in an organotypic culture resulted in progressive incorporation of label into saturated phosphatidylcholine by type II cells. This process was also blocked by the addition of indomethacin and overridden by exogenous PGE2. These data suggest that the combined effects of alveolar fluid dilatation and glucocorticoids may coordinate the timely transfer of triglyceride from fibroblasts to type II cells for augmented surfactant production through their effects on PGE2 production and action as term approaches.

Animals↗
PubMed1998

[Chemical constituents of Tetrastigma hemsleyanum Diels. et Gilg].

OBJECTIVE: To investigate the chemical constituents of Tetrastigma hemsleyanum. METHOD: Compounds were separated by rechromatography on silica gel from the chloroform solubles of its ethanol extracts, and the structures were determined by spectral analysis and chemical evidence. RESULT: Three compounds were isolated and elucidated as 6'-O-benzoyldaucosterol, daucosterol and beta-sitosterol. CONCLUSION: All the three compounds were isolated from this plant for the first time, of which 6'-O-benzoyldaucosterol is a new natural product.

Drugs, Chinese Herbal↗
PubMed1998

[The changes of erythrocyte deformability and cardial function in coronary heart disease with various degrees of coronary stenosis].

According to the extent of coronary maximal stenosis, 29 cases of coronary heart disease (CHD) were classified into two groups: (I) obvious stenosis (OS, narrowing > or = 50% in luminal diameter, n = 15); (II) mild stenosis (MS, marrowing < 50% in luminal diameter, n = 14). The results showed that erythrocyte deformability index (DI), left ventricular ejection fraction (EF%) and fractional shortening (FS%) in the CHD patients were decreased as compared with those in the control group (P < 0.05 or 0.01), and the DI, EF and FS in the OS group were significantly lower than those in the MS group (P < 0.05). These suggest that DI, EF and FS may be of predictable value in identifying CHD patients with or without significant coronary stenosis.

Adult↗
PubMed1998

Effects of mechanical forces on lung-specific gene expression.

Fetal breathing movements (FBM) are necessary for fetal lung growth and maturation. The authors analyzed fetal rat lungs cultured with or without lung distension and tracheal ligation, and examined the effects of mechanical stretch on a human pulmonary epithelial cell line (NCI-H441) that shows regulated expression of surfactant proteins (SP-A, SP-B). Cells were grown on silastic membranes and mounted in a Flexercell Strain Unit. Cyclic deformation simulating FBM was achieved by applying a vacuum of 22 kPa (5%-15% radial deformation) at 50 cycles per minute for 2 to 24 hours. Results indicate that static distension for as little as 4 hours decreased steady-state SP-A and SP-B mRNA levels in whole lung (n = 5-6, P < .01). In contrast, cyclic stretching of H441 cells for 24 hours increased SP-B and SP-A expression 2- to 4-fold over controls. Cyclic deformation also significantly enhanced 3H-choline incorporation into saturated phosphatidylcholine. Dynamic mechanodeformation may be a critical stimulus for fetal lung development.

Animals↗
PubMed1997

A strategy for rapid, high-confidence protein identification.

A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of > 0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications--further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA).

Cross-Linking Reagents↗
PubMed1997

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family.

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8-10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2-3 mol of P per mol. However, the catalytic domain expressed in a baculovirus-insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

Acanthamoeba↗
PubMed1997

Phosphorylation of the human La antigen on serine 366 can regulate recycling of RNA polymerase III transcription complexes.

The human La antigen is an RNA-binding protein that facilitates transcriptional termination and reinitiation by RNA polymerase III. Native La protein fractionates into transcriptionally active and inactive forms that are unphosphorylated and phosphorylated at serine 366, respectively, as determined by enzymatic and mass spectrometric analyses. Serine 366 comprises a casein kinase II phosphorylation site that resides within a conserved region in the La proteins from several species. RNA synthesis from isolated transcription complexes is inhibited by casein kinase II-mediated phosphorylation of La serine 366 and is reversible by dephosphorylation. This work demonstrates a novel mechanism of transcriptional control at the level of recycling of stable transcription complexes.

Autoantigens↗
PubMed1997

Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase.

Adenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C1 and C2, which are homologous to each other and are together responsible for catalytic activity. Recombinant C1 and C2 domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C2 domains alone also manifest reduced levels of forskolin-stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C2 catalytic domain to residues 871-1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC2-delta 4. Crystals belong to space group P4n2(1)2, where n = 1 or 3; a = b = 81.3, and c = 180.5 A; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash-frozen crystals diffract anisotropically to 2.2 A along the c* direction and to 2.8 A along the a* and b* directions using synchrotron radiation.

Adenylyl Cyclases↗
PubMed1997

Nuclear magnetic resonance assignment and secondary structure of an ankyrin-like repeat-bearing protein: myotrophin.

Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 11B amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two- and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly 15N-labeled or uniformly 15N/13C-labeled protein to obtain the 1H, 15N, and 13C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOEs, NH exchange data, 3JHN alpha coupling constants, and chemical shifts of 1H alpha, 13C alpha, and 13 C beta. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.

Amino Acid Sequence↗
PubMed1997

Children's religious knowledge: implications for understanding satanic ritual abuse allegations.

OBJECTIVES: The goals of the present study were to examine the extent of children's religious, especially satanic, knowledge and to understand the influence of children's age, religious training, family, and media exposure on that knowledge. METHODS: Using a structured interview, 48 3- to 16-year-old children were questioned about their knowledge of: (a) religion and religious worship; (b) religion-related symbols and pictures; and (c) movies, music, and television shows with religious and horror themes. RESULTS: Although few children evinced direct knowledge of ritual abuse, many revealed general knowledge of satanism and satanic worship. With age, children's religious knowledge increased and became more sophisticated. Increased exposure to nonsatanic horror media was associated with more nonreligious knowledge that could be considered precursory to satanic knowledge, and increased exposure to satanic media was associated with more knowledge related to satanism. CONCLUSIONS: Our results suggest that children do not generally possess sufficient knowledge of satanic ritual abuse to make up false allegations on their own. However, many children have knowledge of satanism as well as nonreligious knowledge of violence, death, and illegal activities. It is possible that such knowledge could prompt an investigation of satanic ritual abuse or possibly serve as a starting point from which an allegation is erected.

Adolescent↗
PubMed1997

Expression of c-myc in human colonic tissue in response to beta-carotene supplementation.

Dietary supplementation with beta-carotene at 30 mg/day results in an increased serum trans-retinoic acid concentration in patients with a prior colonic polyp. In a number of human cell lines, trans-retinoic acid upregulates c-myc mRNA expression in colonic mucosa by reverse transcription polymerase chain reaction and correlated the results with serum concentrations of all-trans- (ATRA), 13-cis-(13-cRA), and total retinoic acid. Serum and colonic biopsy samples were obtained before and 90 days after administration of a placebo (n = 7) or 30 mg of beta-carotene (n = 5) daily. An increase in c-myc expression after supplementation was observed in 6 of 12 subjects, but 5 of these 6 subjects had decreased total serum retinoic acid concentration and 4 had decreased ATRA concentration. In addition, five of the six subjects with increased c-myc expression had received a placebo. Conversely, c-myc expression was increased in only two of five paired samples from subjects whose total serum retinoic acid concentration increased during the 90-day supplementation period. We conclude that c-myc expression is not correlated with ATRA, 13-cRA, or total retinoic acid concentration in vivo and that increased serum retinoic acid secondary to increased tissue beta-carotene is not sufficient to activate c-myc transcription.

Aged↗
PubMed1997

Beta-carotene isomers in human serum, breast milk and buccal mucosa cells after continuous oral doses of all-trans and 9-cis beta-carotene.

The concentrations of all-trans beta-carotene (tBC) and 9-cis beta-carotene (9cBC) isomers in serum, breast milk and buccal mucosa cells were determined after continuous oral doses as a simple, non-invasive method to determine whether differences in tissue uptake are important determinants of serum responses. Twelve healthy lactating women were recruited for a nonresidential study. On d 1, blood samples were obtained from fasting subjects for baseline concentrations of beta-carotene isomers. Over a 1-wk period, subjects were given either seven doses of a placebo (n = 4) or seven doses of naturally occurring BC (n = 8) derived from Dunaliella bardawil (64 mg tBC, 69 mg 9cBC). Subjects were instructed to consume a single beta-carotene dose along with a meal containing adequate fat each day for 1 wk. On d 2, 3, 5 and 8, blood samples and breast milk were collected from fasting subjects. On d 1 and 8, buccal mucosa cells were collected. Samples were analyzed for carotenoids by HPLC. In the experimental group, the mean serum concentration of tBC significantly increased to seven times the baseline level by the end of the supplementation period (P < 0.0001). The serum concentration of 9cBC significantly increased to three times the baseline level by the end of the supplementation period (P < 0.0001). The changes in milk and buccal mucosa cells levels of tBC and 9cBC followed a pattern similar to that for serum, showing significant increases at the end of the supplementation period. In the control group, the serum, milk and buccal mucosa cell concentrations of BC isomers did not change. This study confirms the previously reported differences in the serum response curves of tBC and 9cBC and provides evidence that there is no difference in tissue uptake of tBC and 9cBC.

Administration, Oral↗
PubMed1997

Ingestion by men of a combined dose of beta-carotene and lycopene does not affect the absorption of beta-carotene but improves that of lycopene.

A double-blind study was conducted in 10 healthy men to investigate serum beta-carotene and lycopene responses after ingestion of individual and combined doses of beta-carotene (BC) and lycopene. On each dosing day, a baseline blood sample was drawn, followed by an oral dose of 0.11 mmol (60 mg) of either all-trans BC or all-trans lycopene or by a combined oral dose of 0.11 mmol each. Subjects were tested with each of the three doses. The dose type was randomized. Blood (10 mL) was drawn at 1, 3, 5, 7, 9, 12 and 24 h after dosing. At 2 and 4 wk after the first dose, the protocol was repeated with the other doses. After ingestion of the BC dose, serum BC concentrations significantly decreased from baseline at 1 and 3 h followed by a continuous increase from baseline that was significant at 12 and 24 h (P < 0.01). Serum lycopene concentrations significantly increased from baseline at 5 h after the lycopene dose (P < 0.008) and returned to baseline thereafter. Ingestion of a combined dose of BC and lycopene resulted in a significant increase in serum concentrations of both BC and lycopene at 24 h (P < 0.05). The 24-h area under the curve (AUC) for BC was not different when BC was ingested alone or with lycopene, whereas the 24-h AUC for lycopene was significantly greater when lycopene was ingested with BC than when ingested alone (P < 0.05). Our data suggest that ingestion of a combined dose of BC and lycopene has little effect on the absorption of BC but improves that of lycopene in men.

Administration, Oral↗
PubMed1997

[Clinical study of eliminating dampness and removing blood stasis in treating coronary heart disease--the summary about serial study of blood stasis due to dampness].

OBJECTIVE: To elucidate the relationship between Dampness and blood stasis in patients with coronary heart disease (CHD). METHODS: One hundred and twenty-eight CHD patients with Phlegm-Dampness Syndrome (PDS) and 69 with blood stasis syndrome (BSS) were chosen and treated by eliminating Dampness and removing blood stasis. RESULTS: (1) CHD-PDS and -BSS possessed the similar changes on hemorheology, oxygen free radical, blood lipids and vascular active substance. (2) Both of these two methods could alleviate angina (effective rate was 88.3%, 89.9%), improve ischemic S-T segment changes in ECG obviously and reduce consumption of nitroglycerin significantly (P < 0.01), improve abnormal hemorheology obviously (P < 0.001), raise PGI2, SOD obviously (P < 0.01) and lowered TXA2, MDA, ANP, ET and blood lipids significantly (P < 0.001). (3) The two methods could regulate imbalanced state of PGI2/TXA2,SOD/MDA. CONCLUSIONS: CHD-PDS and -BSS had similar pathologic basis or pathologic changes, could eliminate the Dampness also had possessed similar pharmacologic or pharmacodynamic action of removing blood stasis. According to this, the viewpoint of "blood stasis due to Dampness" might be proved from clinical aspect.

Adult↗