Biomedical subjects

Y Xiong

Publications and source records attributed to Y Xiong.

At least 145 records · Page 8Linked to original sources

PubMed1996

Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.

Cyclin-dependent kinases 4 and 6 are complexed with many small cellular proteins in vivo. We have isolated cDNA sequences, INK4d, encoding a 19-kDa protein that is associated with CDK6 in several hematopoietic cell lines. p19 shares equal similarity and a common ancestor with other identified inhibitors of the p16/INK4 family. p19 interacts with and inhibits the activity of both CDK4 and CDK6 and exhibits no detectable interaction with the other known CDKs. p19 protein is present in both cell nuclei and cytoplasm. The p19 gene has been mapped to chromosome 19p13.2, and the level of its mRNA expression varies widely between different tissues. In contrast to p21 and p27 whose interaction with CDK subunits is dependent on or stimulated by the cyclin subunit, the interaction of p19 and p18 with CDK6 is hindered by the cyclin protein. Binary cyclin D1-p18/p19 or cyclin D1-CDK6 complexes are highly stable and cannot be dissociated by excess amounts of cyclin D1 or p19/p18 proteins, suggesting that p16 inhibitors and D cyclins may interact with CDKs 4 and 6 in a competing or potentially mutually exclusive manner.

Amino Acid Sequence↗

PubMed1996

Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.

Amino Acid Sequence↗

PubMed1996

P/O ratios reassessed: mitochondrial P/O ratios consistently exceed 1.5 with succinate and 2.5 with NAD-linked substrates.

The efficiency of ATP synthesis coupled to cell respiration, commonly referred to as the P/O ratio, has been the subject of extensive studies for more that 50 years. The general conclusion from these studies is that respiring mitochondria can convert external ADP to ATP at a maximal P/O ratio of 3 for NAD-linked substrates and 2 for succinate. However, in recent years the validity of these "integral" values has been questioned on both mechanistic and thermodynamic grounds, and a mechanistic P/O ratio of 2.5 for NAD-linked substrates and 1.5 for succinate have been concluded on the basis of experiments with isolated mitochondria. These values have been widely adopted in the scientific literature, including several recent textbooks. In this paper we report that under optimal conditions with respect to preparation and assay procedures, the P/O ratios obtained with isolated rat liver mitochondria consistently exceed 2.5 with NAD-linked substrates and 1.5 with succinate. These results, although not excluding "nonintegral" P/O ratios due to various energy-dissipating side reactions, warrant caution in accepting the reported lower values and, in general, in referring to mechanistic considerations unless the underlying molecular mechanisms are understood.

Adenosine Diphosphate↗

PubMed1996

Thapsigargin induces rapid, transient growth inhibition and c-fos expression followed by sustained growth stimulation in mouse keratinocyte cultures.

Although the sesquiterpene lactone thapsigargin has been shown to possess hyperplastic and tumor-promoting activities when applied topically to mouse skin in vivo, the cellular mechanism(s) which underlie these effects are unclear. We show here that thapsigargin treatment of Primary mouse epidermal keratinocytes increased intracellular free Ca2+ concentration (Cai) in a concentration-dependent manner. Thapsigargin induced a rapid, transient elevation in keratinocyte Cai, in part due to the release of Ca2+ from intracellular stores. This response was followed by a sustained elevation in Ca2+, resulting entirely from calcium influx. Thapsigargin elicited a biphasic effect on keratinocyte DNA synthesis: a rapid inhibitory effect (50-60% inhibition at 4-8 h), followed by a very marked and sustained elevation. Prolonged treatment of keratinocytes with thapsigargin at relatively high concentrations resulted in cytotoxicity (inhibition of neutral red uptake). The rapid antiproliferative effect of thapsigargin was not associated with cytotoxicity, as determined by either neutral red uptake or by trypan blue exclusion, and was not blocked by pretreatment with Ro 31-7349, a selective inhibitor of protein kinase C. The rapid antiproliferative effect of thapsigargin was associated with rapid, transient activation of keratinocyte c-fos expression and rapid inhibition of total protein synthesis. Taken together, these findings raise the possibility that the hyperplastic and tumor-promoting activities of thapsigargin on epidermis in vivo result from direct keratinocyte growth stimulation as a consequence of a prolonged elevation in levels of Cai.

Animals↗

PubMed1996

Alteration of cell cycle kinase complexes in human papillomavirus E6- and E7-expressing fibroblasts precedes neoplastic transformation.

Expression of viral oncoproteins results in the loss of cell cycle checkpoint control and the accumulation of chromosomal abnormalities. Expression of both human papillomavirus type 16 oncoproteins, E6 and E7, in normal human fibroblasts completely dissociates p21 and proliferating cell nuclear antigen from the quarternary cyclin-cyclin-dependent kinase (CDK) complexes present in normal cells, causes disruption of the cyclin D-CDK4 complex and replacement with a CDK4-p16 complex, and leaves binary complexes of cyclin B1-CDC2 and cyclin A-CDK2 intact. These results are identical to those observed in fully transformed cells. The expression of the individual oncoproteins dramatically affects the association of proliferating cell nuclear antigen into the complexes while leaving the total cellular levels unaltered. Expression of low-risk human papillomavirus has no effect on cyclin complexes. These findings provide evidence for the gross alteration of cyclin-CDK complexes in preneoplastic cells and links this alteration to the loss of genomic stability.

Carrier Proteins↗

PubMed1996

A role for a p21-E2F interaction during senescence arrest of normal human fibroblasts.

The family of E2F transcription factors forms different multiprotein complexes with cell cycle regulatory proteins to control the expression of genes important in cell proliferation. In this study, we identified four distinct E2F complexes present in aged and senescent normal, human diploid fibroblasts. Two appeared to be identical to the previously described G1-specific p130 and Rb-E2F complexes present in young G0-arrested cells. The other two were novel E2F complexes that contained the cyclin-dependent kinase inhibitor p21 (cip1/WAF1/Sdi1/CAP20/PIC1) complexed with Rb/CDK2/cyclin E or with the Rb-related p107/CDK2/ cyclin D. These p21-E2F complexes, while present in young G1 cells at very low levels, were elevated in senescent cells. The p21 containing E2F complexes were not detected during the S-phase in young cells. The DNA-binding stability of the p21 complexes was approximately 10 times greater than the stability of any other E2F complex or uncomplexed E2F. Addition of purified p21 protein to the S-phase-specific cyclin A/ CDK2-p107-E2F complex from young cells dissociated cyclin A and CDK2 from p107/E2F, suggesting an additional novel function for p21. Finally, expression of p21 specifically inhibited transcription from an E2F-dependent promoter but had no effect on a mutant E2 promoter. In addition to its inhibition of CDK enzymes and proliferating cell nuclear antigen function in DNA replication, these studies reveal a novel mechanism by which p21 mediates growth arrest: direct interaction with E2F complexes and negative regulation of E2F transcription factor activity.

CDC2-CDC28 Kinases↗

PubMed1996

[Immunohistochemical detection of antigen of human cytomegalovirus in hepatocyte].

The expression plasmid of human cytomegalovirus (HCMV) 52kd glycoprotein was induced and amplified, and the recombinant protein was extracted. The rabbit antiserum to the recombinant protein was prepared. The antiserum-reacted to the antigen of HCMV AD165, and the titer of antiserum was 1 : 3200 by using 125I-Protein A. Using Avidin-Biotinperoxidase complex technique (ABC method), we detected the HCMV 52 kd glycoprotein in the hapatic slides of 9 cases. Among them, one case (an infant died of congenital heart disease) was held as a negative control. The other 8 cases (patients with clinically diagnosed cytomegalic inclusion disease) had viral hepatitis histopathologically. Of these 8 cases, 7 had positive signals but found in the nucleus and cytomembrane of the hepatocytes, no positive signal was found in the hepatocytes of the control. The result showed that HCMV 52kd glycoprotein was located in the nucleus and on the membrane of the infected hepatocyte. The antibody prepared with the antigen of gene engineering could find the virus antigen and represent the virus gene state which was active or silent. ABC method is not only simple, sensitive and specific, but also effective in localization. It is of importance to clinical diagnosis, differential diagnosis and prognostication.

Cytomegalovirus↗

PubMed1996

[Restriction fragment length polymorphism assays of clinical isolated human cytomegalovirus strain genomic DNA].

Ten clinical isolated strains of Human cytomegalovirus (HCMV) were obtained from 73 urine specimens of different people. Isolations from urine were carried out in human embryolung fibroblasts. Viral isolates were passaged four times. HCMV DNAs of laboratory strain AD169 and 10 clinical isolated strains were extracted with Hirt method, digested with each of the restriction enzymes EcoRI, Hind II. Comparison of restriction fragment length polymorphism (RFLP) of AD169 and isolated strains were made by hybridizing digested DNA with 32P labeled with HCMV Hind II cloned subgenomic fragments as the probe (pCM1035, pCM1015). pCM1035 is located in the joining region between the long(L) and short (S) unique sequences of the virus (L-S junction) pCM1015 is located in the terminal sequences of the virus. The results showed the genomic high degree of homology existed among all strains and the variable restriction site was in the L-S junction and terminal portion. The RFLP patterns of the clinical isolates which did not have relation in epidemiology were different, but the patterns of clinical isolates related in epidemiology were quite similar. Polymorphism frequently occurred in this case of EcoRI digested fragment hybridized with the probe of pCM1035. Southern hybridization of HCMV isolations is useful to researches into the molecular epidemiology and pathogenesis of HCMV infection.

Adult↗

PubMed1996

[Kidney reinforcing and yang supporting action of cistanche deserticola Y. C. Ma before and after preparation].

The weights of seminal vesicle and prostate gland of castrated young rats were significantly increased by administration of alcoholsoluble extract from decoction of Cistanche deserticola. The weights of testes, seminal vesicle and prostate gland in mice and rats were also increased by the extract. The phagocytic function of intra-abdominal macrophage in mice was activated by decoction of Cistanche deserticola. The results showed no statistical differences between crude and prepared drugs. The maximum oral tolerance for mouse was 40 g/kg.

Animals↗

PubMed1996

Endogenous inhibitors of nitric oxide synthesis and lipid peroxidation in hyperlipidemic rabbits.

AIM: To examine whether the elevation of endogenous NG, NG-dimethylarginine (DMA) content is related to lipid peroxidation in the high lipid-fed rabbit. METHODS: In high lipid diet-fed rabbits, concentrations of serum cholesterol, triglyceride, malondialdehyde (MDA), and DMA were measured, and endothelium-dependent relaxation to acetylcholine (ACh) was tested. RESULTS: After 6-wk on a high lipid-diet, the levels of serum total cholesterol, triglyceride, MDA, and DMA were increased vs those in control group (MDA was 2.88 +/- s 0.20 vs 1.54 +/- 0.13 nmol.L-1, P < 0.01 and DMA was 1.51 +/- 0.07 vs 0.75 +/- 0.13 mumol.L-1, P < 0.01), while the endothelium-dependent vasodilation in the isolated thoracic aorta was impaired (the maximal response to ACh was 45.59 +/- 3.10 vs 76.93 +/- 5.68%). Supplementation with vitamin E decreased MDA and DMA content and improved the endothelium-dependent vasodilation. CONCLUSIONS: An increase in serum concentration of DMA may be secondary to the elevation of lipid peroxides in the hyperlipidemic rabbit.

Animals↗

PubMed1996

[Influence of temperature on the chemical constituents and pharmacological effects of semen Cassiae].

The influence of temperature on the chemical constituents and pharmacological effects of seeds of Cassia tora was examined. As the baking temperature was raised, the contents of free chrysophanol increased. The contents of antihepatotoxic constituents in the samples baked at different temperatures were compared. They decreased as the temperature rose. The pharmacological results basically accorded with the contents of the constituents.

Alanine Transaminase↗

PubMed1995

Molecular cloning of CDK7-associated human MAT1, a cyclin-dependent kinase-activating kinase (CAK) assembly factor.

Mammalian CDK7 is a protein kinase identified as the catalytic subunit of cyclin-dependent kinase (CDK)-activating kinase and as an essential component of the transcription factor TFIIH that is involved in transcription initiation and DNA repair. We have identified in human cells a number of CDK7-associated cellular proteins that appear to fall into two classes based on their relative [35S] metabolic labeling intensity. One class of proteins present in CDK7 immunocomplexes as a minor fraction contains components of the TFIIH transcription complex such as p62 and p89ERCC3, whereas the other fraction contains four polypeptides (p35, p37Cyclin H, p75, and p95) that are stoichiometrically associated with CDK7. Whereas the levels of association of p35, p37Cyclin H, and p75 with CDK7 remain unchanged between density-arrested and proliferating Ewing sarcoma EW-1 cells, the association of p95 with CDK7 was significantly decreased as cells reached confluency. Through a large-scale immunopurification of CDK7 complexes and protein microsequencing, we have isolated a cDNA that encodes p35 and have shown that it is the human homologue of Mat1 that is involved in the assembly of CAK. MAT1 contains a highly conserved C3HC4 motif at its NH2 terminus, a characteristic feature shared among RING finger proteins. The human MAT1 gene expresses a single 1.6-kb transcript, the steady-state level of which, like CDK7 and cyclin H, varies significantly in different cell lines and in different terminally differentiated tissues.

Amino Acid Sequence↗

PubMed1995

Transforming growth factor beta activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site.

Transforming growth factor beta (TGF-beta) causes growth arrest in the G1 phase in many cell types. One probable pathway for this growth inhibition is through the TGF-beta-mediated up-regulation of the cyclin-dependent kinase (CDK) inhibitor p15INK4B, which specifically inhibits the enzymatic activities of CDK4 and CDK6. An active cyclin D-CDK4/6 complex is required for pRb phosphorylation to allow the cell cycle to progress from G1 to S phase. To study the molecular mechanism of the p15INK4B induction by TGF-beta, we isolated a 780-base pair promoter sequence of the human p15 gene and inserted this fragment upstream of a luciferase reporter gene. When this construct was transiently transfected into HaCaT cells, luciferase activity was induced more than 10-fold upon TGF-beta treatment, indicating that the induction of p15INK4B expression by TGF-beta is partly exerted at the transcription level. Promoter deletion analysis revealed that the sequence from -110 to -40 relative to the transcription start site is capable of conferring the 10-fold induction by TGF-beta. Within this region there are three Sp1 consensus sites. Mutation of one of these sites, GGGGCGGAG, substantially reduced both the induction by TGF-beta and the basal promoter activity, whereas mutations in the other two Sp1 sites and the spacer sequences had little effect. In addition, gel mobility shift assay indicates that the transcription factors Sp1 and Sp3 bind to this Sp1 site. Taken together, these data suggest that a specific Sp1 consensus site is involved in the mediation of TGF-beta induction as well as the basal promoter activity of the p15 gene and that Sp1 and Sp3 transcription factors might be involved in this regulation.

Base Sequence↗

PubMed1995

Deletion of cyclin-dependent kinase 4 inhibitor genes P15 and P16 in non-Hodgkin's lymphoma.

B-cell non-Hodgkin's lymphoma (NHL) is a heterogeneous lymphoid malignancy consisting of several histologic types. Alterations in proto-oncogenes caused by reciprocal chromosome translocations have been implicated in the etiology of specific histologic groups. In this study, we examined the contribution of the cell cycle inhibitor genes P15, P16, and P18 to pathogenesis in a large panel of 209 cytogenetically characterized B-cell NHL tumors representing varied histologic groups. We identified the homozygous deletion of P15 and P16 genes in 13 tumors from 12 patients, all belonging to diffuse large-cell histology; 10 had this diagnosis made on presentation, 1 had transformed from small lymphocytic lymphoma, and 1 had transformed from Hodgkin's disease. Tumor-specific point mutations were not identified in the coding regions of these genes. Cytogenetically, chromosome 9p was normal in all but one tumor. On the other hand, eight tumors hemizygous for 9p by cytogenetic analysis showed wild-type configuration of these genes. Our study, therefore, indicates that deletion of P15 and P16 occurs in about 15% of diffuse large-cell NHL and is not usually detected by cytogenetic analysis. P18 was wild-type in all tumors including the 13 tumors hemizygous for 1p.

Aged↗

PubMed1995

Both p16 and p21 families of cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin-dependent kinases by the CDK-activating kinase.

Phosphorylation of cyclin-dependent kinases (CDKs) by the CDK-activating kinase is required for the activation of CDK enzymes. Members of two families of CDK inhibitors, p16/p18 and p21/p27, become physically associated with and inhibit the activity of CDKs in response to a variety of growth-modulating signals. Here, we show that the representative members of both families of CDK inhibitors, p21waf1,cip1, p27kip1, and p18, can prevent the phosphorylation of their CDK partners, CDK2 and CDK6, by CDK-activating kinase. No direct interaction between CDK-activating kinase and the CDK inhibitors could be detected, suggesting that binding of these CDK inhibitors to CDK subunits renders CDK inaccessible to the CDK-activating kinase phosphorylation. These findings suggest that a general mechanism of CDK inhibitor function is to block the phosphorylation of CDK enzymes by CDK-activating kinase.

Base Sequence↗

PubMed1995

The alternatively spliced type II corticotropin-releasing factor receptor, stably expressed in LLCPK-1 cells, is not well coupled to the G protein(s).

Two alternatively spliced corticotropin-releasing factor receptor (CRF-R) cDNAs, type I and type II, were recently isolated from a human cDNA library. The two cDNAs are identical except that the type II cDNA encodes an additional 29 amino acid inserted in the first putative cytoplasmic loop. Since the first cytoplasmic loop is highly conserved in all the members of the hCRF receptor family we have examined whether the presence of the 29 amino acid cassette in CRF-RII influences G protein coupling in LLCPK-1 cells stably expressing the type I and type II hCRF receptors. Whether measured in intact cells or in membrane preparations, LLCPK-1 cells stably expressing CRF-RII have a 4-5 fold lower binding affinity. Maximal CRF-stimulated cAMP accumulation in LLCPK-1 cells stably expressing CRF-RI was 10-15-fold higher than that in LLCPK-1 cells expressing CRF-RII. The EC50 for CRF-stimulated cAMP accumulation in hCRF-RI-expressing cells was in the range of 0.5 +/- 0.2 nM. In contrast, the EC50 for CRF-stimulated cAMP accumulation in hCRF-RII expressing cells was 7.7 +/- 0.2 nM. hCRF increased phosphoinositide turnover in LLCPK-1 cells stably expressing CRF-RI but not in those expressing CRF-RII; this effect required hCRF concentrations of 100 nM and higher. In membrane preparations, GTP-gamma-S inhibited hCRF binding to CRF-RI and shifted the binding Kd from 4.5 nM to 16.7 nM. Conversely, GTP-gamma-S did not influence hCRF binding to CRF-RII in broken cell membranes. Additionally, CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RI was potentiated by GTP, whereas CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RII was insensitive to GTP. These data indicate that CRF-RII is not well coupled to the G protein. Since the only difference between the CRF-RII and CRF-RI is the insert in the first putative cytoplasmic loop, these data indicate that the first cytoplasmic loop plays a crucial role in hCRF receptor coupling to the G protein.

Alternative Splicing↗

PubMed1995

Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism.

The transforming growth factor beta s (TGF-beta s) are a group of multifunctional growth factors which inhibit cell cycle progression in many cell types. The TGF-beta-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-beta on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of small proteins, p21 (WAF1, Cip1), p27Kip1, p16, and p15INK4B, that physically associate with cyclins, cyclin-dependent kinases, or cyclin-Cdk complexes. p21 has been previously shown to be transcriptionally induced by DNA damage through p53 as a mediator. We demonstrate that TGF-beta also causes a rapid transcriptional induction of p21, suggesting that p21 can respond to both intracellular and extracellular signals for cell cycle arrest. In contrast to DNA damage, however, induction of p21 by TGF-beta is not dependent on wild-type p53. The cell line studied in these experiments, HaCaT, contains two mutant alleles of p53, which are unable to activate transcription from the p21 promoter when overexpressed. In addition, TGF-beta and p53 act through distinct elements in the p21 promoter. Taken together, these findings suggest that TGF-beta can induce p21 through a p53-independent pathway. Previous findings have implicated p27Kip1 and p15INK2B as effectors mediating the TGF-beta growth inhibitory effect. These results demonstrate that a single extracellular antiproliferative signal, TGF-beta, can act through multiple signaling pathways to elicit a growth arrest response.

Amino Acid Sequence↗

PubMed1995

A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning.

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Amides↗