Biomedical subjects

W Li

Publications and source records attributed to W Li.

At least 811 records · Page 45Linked to original sources

PubMed1998

[FTIR study on the normal and tumor gastrointestinal tissues].

Series of cancer tissues and corresponding normal tissues of gastrointestinal tract (stomach, colon, esophagus) were studied by FTIR technique and the results showed the analogy of the spectra for the cancer tissues, while the spectra of normal tissues can be classified into three kinds. The secondary structures of protein were obtained by using deconvolution and curve-fitting techniques with Amide I bands and the results showed that the contents of alpha -helix and beta-sheet are different between the normal and cancer tissues.

Connective Tissue↗

PubMed1998

In vivo three-dimensional MR microscopy of mice with chronic relapsing experimental autoimmune encephalomyelitis after treatment with insulin-like growth factor-I.

PURPOSE: The purpose of this study was to determine the ability of three-dimensional in vivo MR microscopy to depict the treatment effects of insulin-like growth factor-I (IGF-I) in SJL mice with chronic relapsing experimental autoimmune encephalomyelitis (crEAE). METHODS: The experiments were performed at 4.7-T on 10 crEAE mice and on one set of control animals. Five crEAE mice were treated with IGF-I and five were treated with a placebo. RESULTS: In the crEAE mice treated with the placebo, in vivo MR microscopy showed areas of abnormal signal throughout the cerebrum, brain stem, and cerebellum. These findings were not present in either the IGF-I-treated mice or the normal control animals. The diffuse alterations in signal intensity in the placebo-treated crEAE mice were not identified on histologic sections of the same areas. CONCLUSION: Differences between the IGF-I- and placebo-treated groups may reflect changes in stabilization or permeability of cell membranes and/or of the blood-brain barrier, although other alternative contrast mechanisms could be playing a role. In vivo MR microscopy depicted changes resulting from treatment of crEAE with IGF-I.

Animals↗

PubMed1998

[The genetic analysis of amplitude of progesterone secretion in serial blood collection from tail in min sows].

28 adult Min sows (3-7 parities), represented 9 sire families were selected to conduct the continual blood collection from the tail for two hours at the interval of ten minutes between 9:00-11:00 in the morning on the sixth day after the last mating. The content of progesterone was assayed by radio-immunological assay. The amplitudes of the secretion of progesterone were generated by the procedure of HORM fft. exe complied by the author. It is difficult to get a reasonable conclusion from the content analysis, because the content varied widely between different points of blood collection in single sow. After Fourier conversion, the heritability of amplitude for the fifth partial wave was high (h2 = 0.932); middle for the basal, 2nd and 3rd partial waves; lower for others. This supported the idea that prolific sows had more active secretion of progesterone in luteal cells. The coexist of low and high heritability components in amplitude of progesterone secretion showed that the activities of progesterone secretion were influenced by both the genetics and environments. The genetic correlation of basal, 1st, 5th partial waves were negative, the others were positive, with the litter size and litter size alive. It showed that the reproductive performance of Min pigs were not expressed perfectly, as the balance of secretion of progesterone did not reach the best. The genetic correlation of all partial waves with duration of estrus was negative which supported that the progesterone suppresses the estrus. The genetic correlation of 3rd, 5th partial waves were stronger than others, but selection for 5th partial wave would make high responses than the 3rd wave, because the genetic correlation of 3rd partial wave with litter size and litter size alive was positive, therefore, the more appropriats method of selection for progesterone was adopted, the better results would be achieved in the improvement of indirect selection response for litter size.

Animals↗

PubMed1998

Expression of apoptosis regulatory genes by retinal pericytes after rapid glucose reduction.

PURPOSE: Retinal capillary pericytes underwent apoptosis in vitro after they had been exposed long-term to high levels of glucose followed by a rapid glucose reduction. The present work was designed to study the expression of bcl-2 family members and apoptosis regulatory genes and to determine the status of oxidative stress induced by high concentrations of glucose in this in vitro apoptosis model. METHODS: Pericytes were grown in normal or high glucose concentrations (5, 20, 30, and 40 mM) for 10 days and then exposed to a rapid reduction of glucose to 0.5 mM or 5 mM. Pericyte cell death was evaluated by determining the loss of cell viability and the fragmentation of DNA using agarose gel electrophoresis. In parallel, the quantitative reverse transcription-polymerase chain reaction technique was used to determine the expression of bcl-2, bax, p53, and glutathione peroxidase (GSH-Px) genes. The intracellular level of glutathione (GSH) and the DNA fragmentation were determined simultaneously for pericytes treated with or without exogenous GSH monoethylester. Retinal capillary endothelial cells, experiencing the same glucose variation, were studied as a comparison. RESULTS: For pericytes, downregulation of bcl-2 was observed as early as 24 hours after rapid glucose reduction, whereas DNA fragmentation was not detectable at that time. After 72 hours, a decreased protein ratio of Bcl-2 to Bax was concomitant to evident loss of pericyte viability. During the period of high glucose and the following glucose reduction, p53 expression essentially was unchanged. Decreased levels of GSH induced by high concentrations of glucose (>30 mM) became further depleted when the glucose levels were rapidly reduced. Addition of GSH monoethylester to the medium restored the level of GSH in pericytes and prevented pericyte apoptosis induced by glucose variation. Moreover, the mRNA levels of GSH-Px were significantly elevated. By contrast, with the same glucose reduction endothelial cells did not undergo apoptosis. Their mRNA levels of bcl-2, bax, and GSH-Px essentially were unchanged. CONCLUSIONS: High levels of glucose and rapid reduction of glucose modulate the expression of bcl-2 family genes in retinal pericytes. Upregulation of GSH-Px and depletion of GSH indicate a reparative process of accelerated elimination of reactive oxygen species following rapid glucose reduction. These findings indicate that the aggravated oxidative stress and the weakened antioxidant defense induced by the combined effects of high levels of glucose and subsequent rapid glucose reduction cause pericyte apoptosis. Prevention of DNA fragmentation of pericytes by exogenous GSH further supports this notion. Because endothelial cells did not show similar pathologic changes, this proposed mechanism seems to be specific to pericytes.

Animals↗

PubMed1998

Synergistic action of tiazofurin and genistein in human ovarian carcinoma cells.

Tiazofurin, an oncolytic drug, reduces PI kinase activity and arrests chiefly in S phase. Genistein, an inhibitor of PIP kinase, tyrosine kinase, and topoisomerase-II, induces arrest in G2 and/or early M phase in most carcinoma cells. Both tiazofurin and genistein reduce second messenger IP3 concentration in ovarian carcinoma cells. Because genistein and tiazofurin attack different enzymic targets and arrest the cell cycle at different phases, we tested the hypothesis that tiazofurin might be synergistic with genistein. Human ovarian carcinoma OVCAR-5 cells were grown in flasks in monolayers. In growth inhibition assay for tiazofurin and genistein the IC50s were 26 and 18 microM, respectively, and in clonogenic assays the LC50s were 17 and 4 microM, respectively. Various combinations of the two drugs were tested. The best protocol took into consideration that tiazofurin decreased GTP concentration in cells by 50% at 12 h after administration. Tiazofurin (20 microM) and genistein (20 microM) as single agents reduced cell counts to 60% and 50%, respectively. The predicted value, as a sum of the effect of two drugs, would have been 30% of controls. However, genistein added 12 h after tiazofurin decreased cell counts to 8%, showing synergistic action of the two drugs for growth inhibition. Similar results were observed in the clonogenic assays, which also revealed synergistic cytotoxicity. The protocol yielding synergism might be of value in the clinical treatment of human ovarian carcinoma.

Antineoplastic Agents↗

PubMed1998

[Identification and prediction of responders to a therapy. A model and its preliminary application to hypertension].

The effect of a given treatment for a given disease may be estimated from randomized controlled clinical trials, expressed as a single treatment effect averaged over the trial population. However, in recent years there has been an increasing willingness to individualize therapeutic decisions. The method we report here identifies responders by assessing the individual probability of an event, according to the treatment group. We used a treatment-stratified Cox regression model including interaction between treatment and patient's covariates, with common regression coefficients for treated and untreated, except for the special case of a prognostic variable which has an interaction with treatment. Further, we used a discriminate function based on the final model, representing the absolute individual therapeutic effect, to identify the patients to be treated according to a given threshold of clinical efficacy. The model was explored on the INDANA database (which pools individual patient data from clinical trials of anti-hypertensive drug intervention). Data on 36,444 patients, from five randomized controlled trials were included. The results show the relationship between the proportion of avoided events among the avoidable ones, and the proportion of patients treated who were responders, as a function of the threshold of absolute benefit defining responders. The confidence intervals of the absolute therapeutic benefit for each individual were calculated, by using the Monte Carlo simulation method. A comparison of the survival of treated and controlled individuals, in both subgroups of responders and non responders, illustrated the relevance of the model. We conclude that the tools for predicting individual therapeutic benefit do exist. It will be important to assess the reproducibility of these results in other models or in other populations before widespread application. It will be necessary to have a properly computerized environment and to train doctors to use these tools.

Decision Support Techniques↗

PubMed1998

Human Bub1: a putative spindle checkpoint kinase closely linked to cell proliferation.

Eukaryotic cells have evolved a mechanism that delays the onset of anaphase until chromosomes are properly positioned on the spindle. To understand the molecular basis of such surveillance mechanism in human cells, we have cloned a full-length cDNA encoding a putative mitotic checkpoint kinase termed hBub1. Sequence comparison reveals that hBub1 is a structurally conserved protein, sharing 23% amino acid residue identity with BUB1 of budding yeast. In addition, the NH2-terminal portion (161 amino acids) of hBub1 shows a significant homology to yeast MAD3, a protein also known to be involved in the mitotic checkpoint response pathway. Northern blot analyses show that the hBub1 mRNA level is abundantly expressed in tissues or cells with a high mitotic index. When Dami cells undergo terminal differentiation after treatment with phorbol ester, hBub1 expression in this cell line is down-regulated rapidly. The hBub1 protein level is low in G1 and remains relatively constant in S, G2, and M phases. Immunofluorescence analysis shows that hBub1 protein colocalizes with a centromere-kinetochore antigen CREST in interphase, mitotic prophase, and nocodazole-treated cells. Antibody electroporation experiments show that hBub1 is an important component of the spindle checkpoint pathway. Furthermore, fluorescence in situ hybridization analysis maps the hBub1 gene to chromosome 2q12-13. Our studies suggest that hBub1 expression is restricted to proliferating cells and appears to be involved in regulating cell cycle progression. The molecular cloning of hBub1 cDNA will facilitate the study of its role in spindle checkpoint control as well as its potential role in certain genetic disorders.

Amino Acid Sequence↗

PubMed1997

Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival.

BACKGROUND: Failure of costimulatory molecule-deficient donor dendritic cells (DCs) to induce indefinite allograft acceptance may be a result of the 'late" up-regulation of these molecules on the DCs after interaction with host T cells. Ligation of CD40 on antigen-presenting cells by its cognate ligand CD40L is thought to induce expression of CD80 (B7-1) and CD86 (B7-2). We examined the influence of anti-CD40L monoclonal antibody (mAb) on the capacity of donor-derived DC progenitors to induce long-term allograft survival. METHODS: High purity DC progenitors were grown from B10 (H2b) mouse bone marrow in granulocyte-macrophage colony-stimulating factor and transforming growth factor beta1 (TGFbeta1). Mature DC were propagated in granulocyte-macrophage colony-stimulating factor and interleukin-4. Their phenotype was characterized by flow cytometric analysis and their function by mixed leukocyte reactivity. Anti-donor cytotoxic T lymphocyte activity in grafts and spleens of vascularized heart allograft recipients was also assessed. RESULTS: The TGFbeta3-cultured cells were (1) DEC 205-positive, MHC class II-positive, CD80dim, CD86dim, and CD40dim, (2) poor stimulators of naive allogeneic T-cell proliferation, and (3) able to prolong significantly B10 cardiac allograft survival in C3H (H2k) recipients when given (2 x 10[6] i.v.) 7 days before organ transplantation (median survival time [MST] 26 days vs. 12 days in controls, and 5 days in interleukin-4 DC-treated animals). Their allostimulatory activity was further diminished by addition of anti-CD40L mAb at the start of the mixed leukocyte cultures. Anti-CD40L mAb alone (250 microg/mouse, i.p.; day -7) did not prolong cardiac graft survival (MST 12 days). In contrast, TGFbeta-cultured DCs + anti-CD40L mAb extended graft survival to a MST of 77 days, and inhibited substantially the anti-donor cytotoxic T lymphocyte activity of graft-infiltrating cells and host spleen cells assessed 8 days after transplant. CONCLUSIONS: The CD40-CD40L pathway appears important in regulation of allogeneic DC-T-cell functional interaction in vivo; its blockade increases markedly the potential of costimulatory molecule-deficient DCs of donor origin to induce long-lasting allograft survival.

Animals↗

PubMed1997

Three-dimensional cryoelectron microscopic reconstruction of the 2.25-MDa homomultimeric phosphoenolpyruvate synthase from Staphylothermus marinus.

The phosphoenolpyruvate synthase (EC 2.7.9.2) of the hyperthermophilic archaeon Staphylothermus marinus forms a 2.25 MDa homomultimeric complex of 24 subunits. Here, computational analysis of low-dose cryoelectron micrographs was used to ascertain that the major rotational symmetry axes were 2-fold, 3-fold, and 4-fold. These symmetry considerations were used to perform a three-dimensional reconstruction to a spatial resolution of 4 nm. This assembly has an octahedral architecture with a solvent accessible interior and with ill-defined yet seemingly flexible appendages on the periphery. This macromolecular assembly is unusually large in mass compared with most other known globular proteins--especially other identified phosphoenolpyruvate synthases which are usually dimeric--and its elaborate quaternary arrangement might represent an adaptation to its extreme environment.

Archaeal Proteins↗

PubMed1997

Footprinting of yeast DNA topoisomerase II lysyl side chains involved in substrate binding and interdomainal interactions.

Footprinting of yeast DNA topoisomerase II and its NH2- and COOH-terminal truncation derivatives was carried out to map the locations of lysyl side chains that are involved in enzyme-DNA interaction, in the binding of ATP, or in interaction between domains of the same enzyme molecule. Several conclusions were drawn based on these measurements and the crystal structures of a 92-kDa fragment of the yeast enzyme and a 43-kDa fragment of Escherichia coli gyrase B-subunit. First, the footprinting results support the model previously inferred from the 92-kDa fragment crystal structure that the main site of DNA binding is comprised of a pair of semicircular grooves. Second, the binding of a nonhydrolyzable ATP analog to the yeast enzyme appears to affect citraconylation at a minimum of six lysines in the ATPase domain of each polypeptide. Two of these lysines are probably involved in contacting the nucleotide directly, and one probably becomes buried when the two ATPase domains of a dimeric enzyme come into contact upon ATP binding; for the others, changes in lysine reactivity appear to reflect allosteric changes following ATP binding. Third, from a comparison of the footprint of the intact enzyme and those of the truncated polypeptides comprised of either the NH2- or the COOH-terminal half of the intact polypeptide, it appears that there are few contacts between the NH2- and COOH-terminal half of yeast DNA topoisomerase II.

Adenylyl Imidodiphosphate↗

PubMed1997

Augmentation of dendritic cells in murine organ donors by Flt3 ligand alters the balance between transplant tolerance and immunity.

Treatment of mice with the recently cloned hemopoietic growth factor Flt3 ligand (FL; 10 microg/day for 10 days) resulted in a large increase in myeloid lineage cells within the liver. While the number of nonparenchymal cells (NPC) harvested from liver increased about 9-fold, a 90-fold increase was observed in the proportion of CD11c+ dendritic cells (DC) recovered from NPC following overnight (18-h) culture in granulocyte-macrophage CSF. In contrast, only a 50% increase was seen in CD11c+ cells within heart single cell suspensions and in the number of DC obtained from hearts after 18-h culture. Liver NPC and heart cell suspensions freshly isolated from 10-day FL-treated animals exhibited increased T cell allostimulatory capacity compared with controls. Overnight cultured DC from livers of FL-treated animals expressed both higher levels of costimulatory molecules (CD80 and CD86) and allostimulatory activity than those from controls. Heart-derived DC also displayed enhanced stimulatory capacity. Pretreatment of organ donors with FL for either 5 or 10 days before transplant of organs to normal recipients abrogated the spontaneous liver allograft acceptance normally observed and resulted in delayed or acute graft rejection (median survival times, 40 and 12 days, respectively). Heart rejection was significantly accelerated by pretreatment of donors with FL for 5 or 10 days (median survival times, 8 and 7 days, respectively, vs 12 days in controls). These novel findings reveal the potent immunologic adjuvant properties of FL in vivo. They also show that substantial augmentation of the number of potential allostimulatory cells in donor organs before transplantation favors rejection rather than tolerance induction.

Animals↗

PubMed1997

Localization and activity of lysyl oxidase within nuclei of fibrogenic cells.

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Amino Acid Sequence↗

PubMed1997

ECG-gated three-dimensional intravascular ultrasound: feasibility and reproducibility of the automated analysis of coronary lumen and atherosclerotic plaque dimensions in humans.

BACKGROUND: Automated systems for the quantitative analysis of three-dimensional (3D) sets of intravascular ultrasound (IVUS) images have been developed to reduce the time required to perform volumetric analyses; however, 3D image reconstruction by these nongated systems is frequently hampered by cyclic artifacts. METHODS AND RESULTS: We used an ECG-gated 3D IVUS image acquisition workstation and a dedicated pullback device in atherosclerotic coronary segments of 30 patients to evaluate (1) the feasibility of this approach of image acquisition, (2) the reproducibility of an automated contour detection algorithm in measuring lumen, external elastic membrane, and plaque+media cross-sectional areas (CSAs) and volumes and the cross-sectional and volumetric plaque+media burden, and (3) the agreement between the automated area measurements and the results of manual tracing. The gated image acquisition took 3.9+/-1.5 minutes. The length of the segments analyzed was 9.6 to 40.0 mm, with 2.3+/-1.5 side branches per segment. The minimum lumen CSA measured 6.4+/-1.7 mm2, and the maximum and average CSA plaque+media burden measured 60.5+/-10.2% and 46.5+/-9.9%, respectively. The automated contour-detection required 34.3+/-7.3 minutes per segment. The differences between these measurements and manual tracing did not exceed 1.6% (SD<6.8%). Intraobserver and interobserver differences in area measurements (n=3421; r=.97 to.99) were <1.6% (SD<7.2%); intraobserver and interobserver differences in volumetric measurements (n=30; r=.99) were <0.4% (SD<3.2%). CONCLUSIONS: ECG-gated acquisition of 3D IVUS image sets is feasible and permits the application of automated contour detection to provide reproducible measurements of the lumen and atherosclerotic plaque CSA and volume in a relatively short analysis time.

Adult↗

PubMed1997

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor.

The SH3-SH3-SH3-SH2 adapter protein Nck links receptor tyrosine kinases, such as EGF and PDGF receptors, to downstream signaling pathways, among which p21cdc42/rac-activated kinase cascade, Sos-activated Ras signaling and the human Wiskott-Aldrich Syndrome protein (WASp)-mediated actin cytoskeleton changes, have been implicated. In EGF stimulated cells, Nck co-immunoprecipitates with a number of phosphotyrosine proteins including the EGF receptor (Li et al., 1992 Mol. Cell. Biol. 12: 5824-2833). To identify the phosphotyrosine protein(s) that directly interacts with Nck and to distinguish it from indirectly associated proteins, preexisting phosphoytrosine protein complexes in the cell lysate were dissociated by heat and SDS prior to the test for binding to Nck. We found that Nck does not directly bind to EGF receptor, instead it binds via its SH2 domain to a 62 kDa phosphotyrosine protein. We present evidence demonstrating that the Nck-bound p62 is related to the previously identified GTPase-activating protein (GAP)-associated phosphotyrosine protein p62. (1) The Nck-bound and the GAP-bound p62 proteins co-migrate with each other in SDS-PAGE. (2) SH2 domains from Nck and GAP compete for binding to p62 in vitro. (3) Purified GST-Nck-SH2 binds directly to the GAP-associated p62. Under these conditions, SH2 domains from PLCgamma, PI-3 kinase, SHC, and Grb2 did not bind p62. (4) Tryptic phosphopeptide maps of the Nck- and the GAP-associated p62 proteins are identical. However, Nck and GAP do not co-immunoprecipitate with each other and apparently bind to different pools of p62. This study suggests that the GAP-associated p62 acts as an SH2 domain docking protein and mediates the interaction between Nck and EGF receptor in response to EGF stimulation.

3T3 Cells↗

PubMed1997

Comparisons of the conformational biases imposed by trans-2,3-methanomethionine and alpha-methylmethionine.

A comparative study of four peptidomimetics of the sequence Phe-Met-Arg-Phe-amide (FMRFa) was performed to compare the conformational bias caused by trans-2,3-methanomethionine and alpha-methylmethionine stereoisomers. The specific compounds studied were {(2S,3S)-cyclo-M} RFa, F{(2R,3R)-cyclo-M} RFa, F{(S)-alpha-Mem} RFa, and F{(R)-alpha-MeM} RFa. Molecular simulations based on CHARMm 22 indicate that gamma-turn, inverse gamma-turn, and alpha-helical conformations about the cyclo-M residue are accessible to the two F{cyclo-M} RFa stereoisomers. Similar calculations for F{(S)-alpha-MeM} RFa, and F{(R)-alpha-MeM}RFa indicate that the alpha-methylamino acids tend to favor alpha-helical conformations. The nmr data is presented for the four peptidomimetics. Most informative were the rotating frame nuclear Overhauser effect cross peaks between the NH protons proximal to the methionine surrogates, and the C beta hydrogens. Overall, these nmr data indicate F{(2S,3S)-cyclo-M} RFa and F{(2R,3R)-cyclo-M} RFa preferentially adopt inverse gamma-turn and gamma-turn conformations, respectively, whereas F{(S)-alpha-MeM} RFa and F{(R)-alpha-MeM} RFa tend to form partial left- and right-handed helical structures (although energy differences between the two turn structures, and between the two helical structures are likely to be small). It is suggested that the wider NH-C alpha-CO angle of cyclopropane amino acids and their more severe steric requirements around the C beta carbons force the peptidomimetic N- and C-termini into the same region of conformational space. This favors C7 turns in the cyclopropane amino acid series relative to the less constrained alpha-methyl derivatives.

FMRFamide↗

PubMed1997

Identification of serine 643 of protein kinase C-delta as an important autophosphorylation site for its enzymatic activity.

To investigate the role of serine/threonine autophosphorylation of protein kinase C-delta (PKC-delta), we mutated serine 643 of PKC-delta to an alanine residue (PKC-deltaS643A). Two different expression vectors containing PKC-deltaS643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells. In vitro autophosphorylation assays demonstrated 65-83% reduction in autophosphorylation of PKC-deltaS643A in comparison to wild type PKC-delta (PKC-deltaWT). The enzymatic activity of PKC-deltaS643A mutant as measured by phosphorylating the PKC-delta pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PKC-deltaWT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-deltaS643A when compared with PKC-deltaWT, further substantiating that serine 643 is phosphorylated in vivo. Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-deltaS643A were not altered in comparison to PKC-deltaWT, indicating that mutagenesis did not affect the structural integrity of the mutant protein. 12-O-Tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells overexpressing PKC-deltaS643A mutant protein was impaired in comparison to that of PKC-deltaWT transfectant. Taken together, our results demonstrate that serine 643 of PKC-delta is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function.

Amino Acid Sequence↗

PubMed1997

Modulation of cell cycle control by vitamin D3 and its analogue, EB1089, in human breast cancer cells.

Examination of a panel of ER positive breast cancer cell lines showed that they were differentially growth inhibited by vitamin D3 and its analogue EB1089. EB1089 treatment of the breast cancer cell lines MCF-7 E, BT20, T47D, and ZR75 demonstrated a correlation between a reduction in Cdk2 kinase activity towards phosphorylation of histone H1 and a decrease in DNA synthesis, while no modulation of Cdk2 activity was observed in the vitamin D3 and EB1089 resistant cell line MCF-7 L. This was accompanied by a time dependent decrease in the percentage of S phase cells in the responsive lines. Characterization of the expression levels of Cdk2 and its related cell cycle proteins in MCF-7 E cells showed that after EB1089 treatment, there was a concentration and time dependent up-regulation of p21 as well as a decrease in cyclin A proteins. Paradoxically, cyclin E levels were increased as a function of treatment. Analysis of cyclin-Cdk2-Cdki complex formation showed that in EB1089 treated MCF-7 E cells, Cdk2, cyclin A and cyclin E immunoprecipitates contained an increased abundance of p21. In contrast to MCF-7 E cells, increases in both p21 and p27 as well as their complex formation with Cdk2 were observed in BT20 and ZR75 cells. These findings indicate that up-regulation of p21 as well as p27 in some cell types may account for the inactivation of Cdk2 activity and a G1 block of the cell cycle following EB1089 treatment.

Antineoplastic Agents↗

PubMed1997

An NADPH:FAD oxidoreductase from the valanimycin producer, Streptomyces viridifaciens. Cloning, analysis, and overexpression.

The valanimycin producer Streptomyces viridifaciens contains a two-component enzyme system that catalyzes the oxidation of isobutylamine to isobutylhydroxylamine. One component of this enzyme system is isobutylamine hydroxylase, and the other component is a flavin reductase. The gene (vlmR) encoding the flavin reductase required by isobutylamine hydroxylase has been cloned from S. viridifaciens by chromosome walking. The gene codes for a protein of 194 amino acids with a calculated mass of 21,265 Da and a calculated pI of 10.2. Overexpression of the vlmR gene in Escherichia coli as an N-terminal His-tag derivative yielded a soluble protein that was purified to homogeneity. Removal of the N-terminal His-tag from the overexpressed protein by thrombin cleavage also produced a soluble protein. Both forms of the protein exhibited a high degree of flavin reductase activity, and the thrombin-cleaved form functioned in combination with isobutylamine hydroxylase to catalyze the conversion of isobutylamine to isobutylhydroxylamine. Kinetic data indicate that the overexpressed protein utilizes FAD and NADPH in preference to FMN, riboflavin, and NADH. The deduced amino acid sequence of the VlmR protein exhibited similarity to several other flavin reductases that may constitute a new family of flavin reductases.

Amino Acid Sequence↗