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Y W Qian

Publications and source records attributed to Y W Qian.

At least 19 recordsLinked to original sources
PubMed2001

Spatial heterogeneity of calcium transient alternans during the early phase of myocardial ischemia in the blood-perfused rabbit heart.

BACKGROUND: Optical mapping of cytosolic calcium transients in intact mammalian hearts is now possible using long-wavelength [Ca(2+)](i) indicators. We propose that beat-to-beat [Ca(2+)](i) transient alternans during ischemia may lead to spatial and temporal heterogeneity of calcium-activated membrane currents. METHODS AND RESULTS: To test this hypothesis, isolated rabbit hearts were loaded with the fluorescent [Ca(2+)](i) indicator, rhod-2 AM, and imaged at 300 frames/sec during blood-perfused ischemic trials. High-quality [Ca(2+)](i) transients were recorded in each of 8 hearts.[Ca(2+)](i) transient alternans was never present in control records but occurred in each of the hearts during ischemia, with onset after 2 to 4 minutes. Alternans was confined to circumscribed regions of the heart surface 5 to 15 mm across. Multiple regions of alternans were found in most hearts, and regions that were out of phase with one another were found in 6 hearts. Quantitative maps of alternans were constructed by calculating an alternans ratio. This ratio behaved as a continuous variable that reached a maximum value in the center of the regions with alternans. CONCLUSIONS: These results demonstrate marked spatial heterogeneity of the [Ca(2+)](i) transient during the early phase of ischemia, which could produce electrical instability and arrhythmias in large mammalian hearts.

Animals↗
PubMed2001

Cell cycle transitions in early Xenopus development.

Xenopus oocytes and embryos undergo two major maternally controlled cell-cycle transitions: oocyte maturation and the mid-blastula transition (MBT). During maturation, the essential order of events in the cell cycle is perturbed in that the M phases of Meiosis I and II occur consecutively without an intervening S phase. Use of U0126, a new potent inhibitor of MAPK kinase (MEK), shows that MAPK activation is essential to inhibit the anaphase-promoting complex and cyclin B degradation at the MI/MII transition. If MAPK is inactivated, cyclin B is degraded, S phase commences and meiotic spindles do not form. These events are restored in U0126-treated oocytes by a constitutively active form of the protein kinase p90Rsk. Thus all actions of MAPK during maturation are mediated solely by activation of p90Rsk. At the MBT, commencing with the 13th cleavage division, there are profound changes in the cell cycle. MBT events such as maternal cyclin E degradation and sensitivity to apoptosis are regulated by a developmental timer insensitive to inhibition of DNA, RNA or protein synthesis. Other events, such as zygotic transcription and the DNA replication checkpoint, are controlled by the nuclear:cytoplasmic ratio. Lengthening of the cell cycle at the MBT is caused by increased Tyr15 phosphorylation of Cdc2 resulting from degradation of the maternal phosphatase Cdc25A and continued expression of maternal Wee1. Ionizing radiation causes activation of a checkpoint mediating apoptosis when administered before but not after the MBT. Resistance to apoptosis is associated with increased p27Xic1, the relative fraction of Bcl-2 or Bax in pro- versus anti-apoptotic complexes, and the activity of the protein kinase Akt.

Animals↗
PubMed2001

The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes.

In the Xenopus oocyte system mitogen treatment triggers the G(2)/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21(Cip1) had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.

Animals↗
PubMed2000

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk).

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.

Animals↗
PubMed1999

Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1.

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a "trigger" kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.

Amino Acid Sequence↗
PubMed1998

Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase Plx1.

The Xenopus polo-like kinase 1 (Plx1) is essential during mitosis for the activation of Cdc25C, for spindle assembly, and for cyclin B degradation. Polo-like kinases from various organisms are activated by phosphorylation by an unidentified protein kinase. A protein kinase, polo-like kinase kinase 1 or xPlkk1, that phosphorylates and activates Plx1 in vitro was purified to near homogeneity and cloned. Phosphopeptide mapping of Plx1 phosphorylated in vitro by recombinant xPlkk1 or in progesterone-treated oocytes indicates that xPlkk1 may activate Plx1 in vivo. The xPlkk1 protein itself was also activated by phosphorylation on serine and threonine residues, and the kinetics of activation of xPlkk1 in vivo closely paralleled the activation of Plx1. Moreover, microinjection of xPlkk1 into Xenopus oocytes accelerated the timing of activation of Plx1 and the transition from G2 to M phase of the cell cycle. These results define a protein kinase cascade that regulates several events of mitosis.

Adenosine Triphosphate↗
PubMed1998

Activated polo-like kinase Plx1 is required at multiple points during mitosis in Xenopus laevis.

Entry into mitosis depends upon activation of the dual-specificity phosphatase Cdc25C, which dephosphorylates and activates the cyclin B-Cdc2 complex. Previous work has shown that the Xenopus polo-like kinase Plx1 can phosphorylate and activate Cdc25C in vitro. In the work presented here, we demonstrate that Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25C. Microinjection of wild-type Plx1 into Xenopus oocytes accelerated the rate of activation of Cdc25C and cyclin B-Cdc2. Conversely, microinjection of either an antibody against Plx1 or kinase-dead Plx1 significantly inhibited the activation of Cdc25C and cyclin B-Cdc2. This effect could be reversed by injection of active Cdc25C, indicating that Plx1 is upstream of Cdc25C. However, injection of Cdc25C, which directly activates cyclin B-Cdc2, also caused activation of Plx1, suggesting that a positive feedback loop exists in the Plx1 activation pathway. Other experiments show that injection of Plx1 antibody into early embryos, which do not require Cdc25C for the activation of cyclin B-Cdc2, resulted in an arrest of cleavage that was associated with monopolar spindles. These results demonstrate that in Xenopus laevis, Plx1 plays important roles both in the activation of Cdc25C at the initiation of mitosis and in spindle assembly at late stages of mitosis.

Animals↗
PubMed1995

Dual retinoblastoma-binding proteins with properties related to a negative regulator of ras in yeast.

The retinoblastoma protein (Rb) interacts with multiple cellular proteins that mediate its cellular function. We have identified nine polypeptides that bind to the T-binding domains of Rb using an Rb affinity resin. RbAp48 and RbAp46 are quantitatively the major Rb-associated proteins purified by this approach. RbAp48 was characterized previously and was found to be related to MSI1, a negative regulator of Ras in the yeast Saccharomyces cerevisiae. Here we report the cloning and characterization of RbAp46. RbAp46 shares 89.4% amino acid identity with RbAp48. The internal WD repeats, which are found in a growing number of eukaryotic proteins, are conserved between RbAp46 and RbAp48. Like RbAp48, RbAp46 forms a complex with Rb both in vitro and in vivo and suppresses the heat-shock sensitivity of the yeast RAS2Val-19 strains. We have also isolated the murine cDNA homologs of RbAp48 and RbAp46. Although both mRNA can be detected in all mouse tissues, their mRNA levels vary dramatically between different tissues. No significant differences were observed in the expression patterns of these genes in most tissues except thymus, testis, and ovary/uterus, in which 2-fold differences were observed. Interestingly, the mouse and human RbAp48 amino acid sequences are completely identical, and the mouse and human RbAp46 differ only by one conserved amino acid substitution. These results suggest that RbAp48 and RbAp46 may have shared as well as unique functions in the regulation of cell proliferation and differentiation.

Amino Acid Sequence↗
PubMed1994

Identification of discrete structural domains in the retinoblastoma protein. Amino-terminal domain is required for its oligomerization.

To characterize the protein product of the retinoblastoma tumor suppressor gene biochemically, a recombinant human protein was produced in an Escherichia coli expression system. The full-length protein, p110RB, and an amino-terminal truncated form, p56RB, were expressed and purified to near homogeneity by conventional chromatographic procedures. To probe the structural organization of the retinoblastoma protein the purified proteins were subjected to partial proteolysis by trypsin, chymotrypsin, and subtilisin. Four discrete structural domains were revealed in p110RB by this method. Two of these structural domains, found in both p56RB and p110RB, were mapped to the carboxyl-terminal half of the protein and corresponded to the SV40 large T binding domains defined previously by genetic methods. In addition two distinct domains in the amino-terminal half of the protein were also defined. A potential role for these newly defined amino-terminal domains was uncovered upon analysis of the purified proteins by nondenaturing polyacrylamide gel electrophoresis. p110RB revealed multiple bands by this method, suggesting the formation of oligomeric structures by the protein, while this property was not observed for p56RB. Electron microscopy of p110RB revealed linearly extended, macromolecular structures, further supporting the formation of homologous higher order structures by the full-length retinoblastoma protein. Analysis of the interactions between retinoblastoma protein molecules using the yeast two-hybrid system confirmed that the retinoblastoma protein could self-associate and that this association was mediated by interactions between the amino- and carboxyl-terminal ends of the protein. These observations suggest that the retinoblastoma protein contains multiple structural domains with the amino-terminal domains being required for oligomerization of the full-length protein.

Amino Acid Sequence↗
PubMed1993

A retinoblastoma-binding protein related to a negative regulator of Ras in yeast.

The growth suppression function of the retinoblastoma protein (Rb) is though to be mediated by Rb binding to cellular proteins. p48 is one of the major proteins that binds to a putative functional domain at the carboxy terminus of the Rb protein. Here we report the isolation of a full-length complementary DNA (RbAp48) encoding p48. Complex formation between p48 and Rb occurs in vitro and in vivo, and apparently involves direct interaction between the proteins. Like Rb, p48 is a ubiquitously expressed nuclear protein. RbAp48 share sequence homology with MSI1, a negative regulator of the Ras-cyclic AMP pathway in the yeast Saccharomyces cerevisiae. Furthermore, like MSI1, human RbAp48 suppresses the heat-shock sensitivity of the yeast ira1 strains and RAS2Val19 strains. Interaction with p48 may be one of the mechanisms for suppression of growth mediated by Rb.

Amino Acid Sequence↗
PubMed1992

The retinoblastoma protein region required for interaction with the E2F transcription factor includes the T/E1A binding and carboxy-terminal sequences.

Recent experiments in understanding the mechanism of the retinoblastoma protein (RB) function have revealed the existence of several cellular proteins that are complexed with RB. One of these cellular proteins is the E2F transcription factor, which was originally identified due to its inducibility by E1A during an adenovirus infection. The E2F recognition sequence is found in the promoters of several cellular genes involved in growth control, including several oncogenes. In this report, we provide evidence that the interaction of E2F and RB is mediated through a region on RB where viral oncogenes such as SV40 T antigen and adenovirus E1A bind and where tumorigenic mutations also cluster. Additional carboxy-terminal sequences are also required for the interaction with E2F. These observations provide evidence for a direct connection between tumor suppressor function and the gene expression program leading to cellular growth regulation.

Adenovirus E1A Proteins↗
PubMed1992

Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor.

The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.

Amino Acid Sequence↗
PubMed1991

The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.

The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells. Injection of either protein early in G1 inhibits progression into S phase. Co-injection of anti-RB antibodies antagonizes this effect. Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase. These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity. Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.

Animals↗
PubMed1990

Expression of the human retinoblastoma gene product pp110RB in insect cells using the baculovirus system.

The product of the retinoblastoma susceptibility gene (RB) was overproduced in cultured insect cells using the baculovirus expression system. Upon insertion of the cloned human RB complementary DNA sequence into the viral genome downstream of the promoter of the polyhedrin gene, full-length RB protein with an apparent molecular weight of 110,000 was expressed in the insect cells. This protein was found to be phosphorylated, located in the nuclei of the infected cells, and immunologically indistinguishable from pp110RB of human cells as assayed by several anti-RB antibodies. Following cell disruption and a one-step immunoaffinity chromatographic purification, 6-12 mg of soluble pp110RB with approximately 95% purity were obtained per liter of infected suspension culture. Characterization of the two known biochemical properties of RB protein showed that this purified protein from insect cells behaved similarly to the authentic human pp110RB. First, it bound to DNA, and second, it could form a specific complex with SV40 T antigen in vitro. Prompt translocation of the protein from cytoplasm to nucleus after microinjection further indicated that the purified RB protein may be active. The availability of soluble, intact, and presumably active pp110RB in large quantity represents a significant advance for studying the biochemical and biophysical properties of the RB gene product as well as its potential biological function in cancer suppression.

Animals↗
PubMed1990

Studies on natural and modified peptide Trichosanthes trypsin inhibitors.

A peptide trypsin inhibitor was isolated and purified from the roots of Trichosanthes kirilowii (a Chinese medical herb) by using immobilized anhydro-trypsin affinity chromatography and HPLC C18 column reverse chromatography. It contains two major components, both consisting of 27 amino acid residues with three pairs of disulfide bonds. The sequence determination indicated that the difference between them is only in the ninth position, being Gln and Lys, respectively. The peptide bond of the inhibitor reactive site Arg-Ile (3-4) is easy to cleave at low pH by trypsin, resulting in a modified inhibitor. It might be the smallest naturally occurring protein inhibitor so far known. The modification reaction of the Trichosanthes inhibitor with trypsin is similar to the catalytic enzyme-substrate reaction. The dissociation constant of the modified inhibitor with trypsin is around fourfold that of the natural inhibitor.

Amino Acid Sequence↗