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Biomedical subjects

K Guan

Publications and source records attributed to K Guan.

At least 19 recordsLinked to original sources
PubMed2003

Lumbar spinal fusion with a mineralized collagen matrix and rhBMP-2 in a rabbit model.

STUDY DESIGN: A new mineralized collagen matrix combined with or without growth factor was used for the posterolateral spinal fusion in the rabbit lumbar spine. OBJECTIVES: The availability of a new osteoconductive matrix with or without recombinant osteoinductive growth factors offers a possible alternative to the use of autogenous bone for grafting indications. This study evaluated the bone-forming activity of the biomimetic matrix: nano-hydroxyapatite/collagen/polylactic acid (nHAC/PLA) combined without or with recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rabbit posterolateral spinal fusion. SUMMARY OF BACKGROUND DATA: Many bone grafting materials such as titanium alloy, ceramics, and polymers were used to repair bony defects. However, each has specific disadvantaged. The permanent implantation still has possibility to be eroded in vivo, which is caused by late breakdown and abscess formation. The acidic outcome of polymer biodegradation was also negatively affected in the later-stage results of bone repair. It needed a promising material for an alternative to the use of autogenous bone for grafting indications. MATERIALS AND METHODS: Sixty-four rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC/PLA composite group (nHAC/PLA), autologous iliac crest bone mixed with nHAC/PLA composite group (ACB + nHAC/PLA), nHAC/PLA composite with recombinant human BMP-2 group (nHAC/PLA + rhBMP-2). The lumbar intertransverse process fusions were assessed by manual palpation, radiographic, histologic, and mechanical strength, and scanning electronic microscopy (SEM) in a 10-week observation. RESULTS: Optimal formulations of the ACB + nHAC/PLA and nHAC/PLA + rhBMP-2 groups were shown to perform similar to ACB in both the fusion ratio and mechanical strength in the 6 and 10 weeks after surgery. From the microstructure analysis of the samples, there was no negative effect when the compound implanted this composite with autogenous iliac crest, and there was also new bone-like tissue formation implanted this composite without combined rhBMP-2 early at the second week after surgery. CONCLUSIONS: This study shows the effective results of nHAC/PLA in rabbit posterolateral spinal fusion combined with rhBMP-2. It is an alternative method to autograft by compounding this osteoconductive matrix with growth factors.

Animals↗
PubMed2001

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling.

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.

Animals↗
PubMed2001

Expression and cellular distribution of alpha(v)integrins in beta(1)integrin-deficient embryonic stem cell-derived cardiac cells.

beta(1)integrin-deficient (beta(1)-/-) ES cells showed increased differentiation of cardiac cells characterized by reduced adhesion and high beating frequency. Whereas in whole embryoid body outgrowths of beta(1)-/- cells maximum levels of alpha(v), beta(3)and beta(5)integrin mRNA were delayed and transiently upregulated, in cardiac clusters isolated from beta(1)-/- cells, only beta(3)integrin mRNA levels were enhanced in comparison to wild-type (wt) cells. To answer the question, whether alpha(v)and beta(3)integrins may compensate, at least partially, the loss of beta(1)integrin function during cardiac differentiation, the distribution of alpha(v)and beta(3)integrins in beta(1)-/- and wt pacemaker-like cardiac cells was analyzed. A different distribution of alpha(v)and beta(3)integrins in beta(1)-/- v wt cardiac cells was found. In wt cardiac cells, beta(1)integrin was localized in specialized subsarcolemmal regions, in particular, at focal contacts and costameres, but alpha(v)integrin was diffusely distributed. In contrast, in beta(1)-/- cardiac cells, alpha(v)integrin was preponderantly localized at cell membranes, focal contacts and costameres. beta(3)integrin displayed a diffuse pattern both in wt and in beta(1)-/- pacemaker-like cells at early differentiation stages, whereas at terminal stages, beta(3)was colocalized with sarcomeres in wt, but not in beta(1)-/- pacemaker-like cells. Quantitative immunofluorescence analysis revealed increased alpha(v)and beta(3)integrin levels in beta(1)-/- pacemaker-like cardiac cells. Our results led us to conclude that altered cellular distribution of alpha(v)integrin and upregulation of beta(3)integrin correlate with growth and survival of beta(1)-/- cardiac pacemaker-like cells at an early developmental state. However, alpha(v)and beta(3)integrins cannot functionally compensate the loss of beta(1)integrin during terminal differentiation of cardiac cells implicating that cardiomyocytes require specific beta(1)integrin functions for cardiac specialization.

Animals↗
PubMed2001

Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells.

Embryonic stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However. the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the differentiation of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal differentiation paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific differentiation protocols by lineage selection of neuronal cells have been established using growth and extracellular matrix factors. After formation of cells of the three primary germ layers, mesodermal differentiation was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast growth factor, followed by differentiation induction by neuronal differentiation factors. Further application of survival-promoting factors such as neurotrophic factors and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable: (1) for studying commitment and neuronal specification in vitro, (2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.

Animals↗
PubMed2001

Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects.

Primary cultures or established cell lines of vertebrates are commonly used to analyse the mutagenic, embryotoxic or teratogenic potential of environmental factors, drugs and xenobiotics in vitro. However, these cellular systems do not include developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach has been offered by permanent lines of pluripotent stem cells of embryonic origin, such as embryonic carcinoma (EC), embryonic stem (ES) and embryonic germ (EG) cells. The undifferentiated stem cell lines are characterized by nearly unlimited self-renewal capacity and have been shown to differentiate in vitro into cells of all three primary germ layers. Pluripotent embryonic stem cell lines recapitulate cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation, data which are summarized in this review. In addition, recent studies are presented which investigated mutagenic, cytotoxic and embryotoxic effects of chemical substances using in vitro systems of pluripotent embryonic stem cells. Furthermore, an outlook is given on future molecular technologies using embryonic stem cells in developmental toxicology and embryotoxicology.

Animal Testing Alternatives↗
PubMed2001

Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.

Here, we describe the generation of viable and dopamine-producing neurons derived from pluripotent mouse embryonic stem cells. Neurotrophic factors in combination with survival-promoting factors, such as interleukin-1beta, glial cell line-derived neurotrophic factor, neurturin, transforming growth factor-beta(3) and dibutyryl-cyclic AMP, significantly enhanced Nurr1 and tyrosine hydroxylase (TH) mRNA levels, whereas En-1, mash-1 and dopamine-2-receptor mRNA levels were not upregulated. In parallel, mRNA levels of the anti-apoptotic gene bcl-2 were found to be upregulated at terminal stages. Double immunofluorescence analysis revealed increased numbers of TH- and dopamine transporter-, but not gamma-aminobutyric acid- and serotonin-positive neurons in relation to synaptophysin-labeled cells by survival-promoting factors. Moreover, high-performance liquid chromatography analysis showed detectable levels of intracellular dopamine. We conclude that survival-promoting factors enhance differentiation, survival and maintenance of dopaminergic neurons derived from embryonic stem cells.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2001

p53CP is p51/p63, the third member of the p53 gene family: partial purification and characterization.

The p53 tumor suppressor is a transcription factor that upon activation by DNA-damaging agents induces growth arrest or apoptosis mainly through transactivation and transrepression of its downstream target genes. Two additional p53 family members, p73 and p51/p63, were recently identified and characterized. Although the three family members share some similarities in transcription activation and apoptosis induction, each of them appears to play a distinct role in development and tumor suppression. We have previously identified a nuclear protein, p53CP (p53 competing protein), that is not p53 but binds to the p53 consensus sequence. Here we report the partial purification of p53CP from HeLa cells by ammonium sulfate precipitation, followed by a series of chromatography steps through heparin-agarose, Mono S ion exchange and DNA affinity columns, coupled with a gel shift assay. Although p53CP activity is readily detectable in HeLa cells by gel shift assay, only a trace amount of p53CP protein was partially purified, which was not sufficient for direct protein sequencing. Using a monoclonal antibody (4A4) specific for all p51/p63 isoforms or a polyclonal antibody (N-18) recognizing the N-terminus-containing p51/p63 isoforms we detected a significant enrichment of p51/p63 protein in p53CP-containing fractions following each step of purification. Significantly, p51/p63 was detected only in the DNA affinity column fractions that contain p53CP activity. Thus, p53CP appears to be p51/p63, the third member of the p53 gene family.

Blotting, Western↗
PubMed2001

Lithium influences differentiation and tissue-specific gene expression of mouse embryonic stem (ES) cells in vitro.

The effects of lithium chloride (LiCl) on differentiation of mouse embryonic stem (ES) cells were investigated in order to evaluate the ES cell test (EST) used in a European Union validation study for screening of embryotoxic agents in vitro. We show that LiCl inhibited concentration-dependently the differentiation of ES cells into cardiac and myogenic cells. Whereas the inhibition of cardiac differentiation by high concentrations of LiCl was obvious at day 5 + 5, decreased skeletal muscle cell differentiation was observed only at day 5 + 8. Semi-quantitative RT-PCR analyses revealed significantly lower levels of mRNA encoding cardiac-specific alpha-myosin heavy chain and skeletal muscle-specific myoD. By morphological investigation, an influence of lithium on neuronal differentiation was not evident. However, mRNA levels of genes encoding synaptophysin and the 160 kDa neurofilament protein were increased by high LiCl concentrations, whereas mRNA levels of mash-1 and Engrailed-1 were decreased, suggesting a specific influence of lithium on neuronal differentiation. Furthermore, LiCl treatment resulted in a slight, but non-significant increase of beta-catenin levels in ES cell-derived embryoid bodies. Our results demonstrate that the ES cell test, EST may be suitable to detect inhibitory effects of test compounds especially on cardiac differentiation, whereas effects on neuronal cells would not be detected. Therefore, we propose that morphological analyses of cardiac differentiation alone are insufficient to detect embryotoxic effects. The assay of other cell lineages at different developmental stages, and expression analyses of tissue-specific genes should also be employed.

Animals↗
PubMed2000

Stimulation of the mitogen-activated protein kinase cascade and tyrosine phosphorylation of the epidermal growth factor receptor by hepatopoietin.

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor, which specifically stimulates proliferation of cultured primary hepatocytes in vitro and liver regeneration after liver partial hepatectomy in vivo. Recently, the identification of the mitogenic effect of HPO on hepatoma cell lines and the existence of HPO-specific receptors indicate that HPO acts via its specific cell surface receptor. However, the molecular mechanism of HPO action is not fully elucidated. In this report, we examined the signal transduction events induced by HPO in hepatoma cell line (HepG2). Our results demonstrated that HPO induces phosphorylation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase (MAPK) in a rapid and transient manner. HPO stimulates tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Furthermore, we observed that both MAPK activation and the mitogenic effect of HPO on HepG2 cells were completely blocked by AG1478, a specific inhibitor of EGFR tyrosine kinase activity. However, the effects of HPO were not antagonized by an EGFR-blocking antibody, mAb528, which blocks the interaction between epidermal growth factor and EGFR, indicating that stimulation of tyrosine phosphorylation of EGFR by HPO was not mediated by epidermal growth factor. In contrast, genistein, a general tyrosine kinase inhibitor, significantly attenuated the tyrosine phosphorylation of EGFR in response to HPO. In conclusion, our results suggest that tyrosine phosphorylation of EGFR may play a critical role in MAPK activation and mitogenic stimulation by HPO.

Enzyme Inhibitors↗
PubMed2000

Establishment of SSEA-1- and Oct-4-expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth.

Here, we demonstrate long-term cultivation of alkaline phosphatase-positive rat embryonic stem-like (RES) cell lines. RES cells were characterized by their typical growth in highly compacted cell clusters, which were found to be sensitive against enzymatic dissociation. RES cells expressed stage-specific embryonic antigen-1 (SSEA-1) and transcription factor Oct-4, but Oct-4 mRNA was detected at lower levels compared to mouse ES cells. Once established to tissue culture, RES cells were able to grow in the absence of feeder cells under clonal conditions. Cytokines of the interleukin-6 family known to maintain the undifferentiated state of mouse ES cells were comparatively analyzed for their capacity to maintain the undifferentiated growth of two cell lines, RES-1 and RES-15, in a clonal assay. Rat ciliary neurotrophic factor (rCNTF), human oncostatin M (hOSM), and interleukin-6 and soluble interleukin-6 receptor (IL-6/sIL-6R) were found to support clonal growth of RES cells, but the cytokines did not reach the efficiency of the colony forming ability of leukemia inhibitory factor (LIF). When RES-1 and RES-15 cells were cultivated without feeder cells, SSEA-1 expression was maintained after clonal growth in the presence of LIF and LIF + rCNTF, respectively. Oct-4 mRNA was significantly detected in RES-15 cells when cultivated in the absence of feeder cells in media substituted by LIF and/or IL-6/sIL-6R, as well as without cytokines. In summary, rat embryonic stem-like cell lines could be established from rat blastocysts and were able to proliferate as undifferentiated alkaline phosphatase-positive cells. Embryonal stem cell properties, such as SSEA-1 and Oct-4 expression, were maintained by members of the IL-6 family of cytokines, but most significantly by LIF.

Alkaline Phosphatase↗
PubMed2000

MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1.

The bone-specific transcription factor, Cbfa1, regulates expression of the osteocalcin (OCN) gene and is essential for bone formation. However, little is known about the mechanisms regulating Cbfa1 activity. This work examines the role of the MAPK pathway in regulating Cbfa1-dependent transcription. Stimulation of MAPK by transfecting a constitutively active form of MEK1, MEK(SP), into MC3T3-E1 preosteoblast cells increased endogenous OCN mRNA, while a dominant negative mutant, MEK(DN), was inhibitory. MEK(SP) also stimulated activity of a 147-base pair minimal OCN promoter, and this stimulation required an intact copy of OSE2, the DNA binding site for Cbfa1. Effects of MEK(SP) were specific to Cbfa1-positive osteoblast-like cells. A purified His-tagged Cbfa1 fusion protein was directly phosphorylated by activated recombinant MAPK in vitro. Furthermore, (32)P metabolic labeling studies demonstrated that MEK(SP) clearly enhanced phosphorylation of Cbfa1 in intact cells, while MEK(DN) decreased phosphorylation. The specific MEK1/MEK2 inhibitor, PD98059, inhibited extracellular matrix-dependent up-regulation of the OCN promoter, indicating that the MAPK pathway and, presumably, Cbfa1 phosphorylation are also required for responsiveness of osteoblasts to extracellular matrix signals. This study is the first demonstration that Cbfa1 is controlled by MAPKs and suggests that this pathway has an important role in the control of osteoblast-specific gene expression.

Animals↗
PubMed2000

PTGF-beta, a type beta transforming growth factor (TGF-beta) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF-beta signaling pathway.

Identification and characterization of p53 target genes would lead to a better understanding of p53 functions and p53-mediated signaling pathways. Two putative p53 binding sites were identified in the promoter of a gene encoding PTGF-beta, a type beta transforming growth factor (TGF-beta) superfamily member. Gel shift assay showed that p53 bound to both sites. Luciferase-coupled transactivation assay revealed that the gene promoter was activated in a p53 dose- as well as p53 binding site-dependent manner by wild-type p53 but not by several p53 mutants. The p53 binding and transactivation of the PTGF-beta promoter was enhanced by etoposide, a p53 activator, and was largely blocked by a dominant negative p53 mutant. Furthermore, expression of endogenous PTGF-beta was remarkably induced by etoposide in p53-positive, but not in p53-negative, cell lines. Finally, the conditioned medium collected from PTGF-beta-overexpressing cells, but not from the control cells, suppressed tumor cell growth. Growth suppression was not, however, seen in cells that lack functional TGF-beta receptors or Smad4, suggesting that PTGF-beta acts through the TGF-beta signaling pathway. Thus, PTGF-beta, a secretory protein, is a p53 target that could mediate p53-induced growth suppression in autocrinal as well as paracrinal fashions. The finding made a vertical connection between p53 and TGF-beta signaling pathways in controlling cell growth and implied a potential important role of p53 in inflammation regulation via PTGF-beta.

Binding Sites↗
PubMed2000

Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4.

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta(1), BMP-2 and -4). ES cell differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation could be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(1), whereas BMP-2 or -4 induced chondrogenic differentiation. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate.

Aggrecans↗
PubMed1999

Transcriptional activation of the human glutathione peroxidase promoter by p53.

Glutathione peroxidase (GPX) is a primary antioxidant enzyme that scavenges hydrogen peroxide or organic hydroperoxides. We have recently found that GPX is induced by etoposide, a topoisomerase II inhibitor and a p53 activator. In a search for a cis-element that confers potential p53 regulation of GPX, we identified a p53 binding site in the promoter of the GPX gene. This site bound to purified p53 as well as p53 in nuclear extract activated by etoposide. A luciferase reporter driven by a 262-base pair GPX promoter fragment was transcriptionally activated by wild type p53 in a p53 binding site-dependent manner. The same reporter was also activated in a p53 binding site-independent manner by several p53 mutants. The p53 binding and transactivation of the GPX promoter were enhanced by etoposide in p53-positive U2-OS cells. Etoposide-induced transactivation was blocked by a dominant negative p53 mutant, indicating that endogenous wild type p53, upon activation by etoposide, transactivated the GPX promoter. Furthermore, expression of endogenous GPX was induced significantly at both mRNA and enzyme activity levels by etoposide in U2-OS cells but not in p53-negative Saos-2 cells. This is the first report demonstrating that GPX is a novel p53 target gene. The finding links the p53 tumor suppressor to an antioxidant enzyme and will facilitate study of the p53 signaling pathway and antioxidant enzyme regulation.

Base Sequence↗
PubMed1999

Transcriptional activation of the human S100A2 promoter by wild-type p53.

S100A2, a calcium binding protein of the EF-hand family, was recently identified to be inducible by etoposide, a p53 activator. A potential p53 binding site was identified in the promoter of the S100A2 gene, which binds to purified p53 as well as p53 in nuclear extract activated by etoposide. Transactivation assays using the promoter driven luciferase reporters revealed that the S100A2 promoter was transcriptionally activated by wild-type p53, but not by p53 mutants, in a dose-dependent as well as a p53 binding site-dependent manner. The p53-induced transactivation of the S100A2 promoter was enhanced by etoposide and blocked by a dominant negative p53 mutant. Furthermore, endogenous S100A2 mRNA expression is induced by etoposide in p53 positive, but not in p53 negative cells. Thus, p53 appears to positively regulate S100A2 expression.

Binding Sites↗
PubMed1999

Modulation of sarcomere organization during embryonic stem cell-derived cardiomyocyte differentiation.

Myofibrillogenesis - sarcomeres - mouse embryonic stem cells - cardiomyocytes - beta1 integrin Mouse embryonic stem (ES) cells, when cultivated as embryoid bodies, differentiate in vitro into cardiomyocytes of ventricle-, atrium- and pacemaker-like cell types characterized by developmentally controlled expression of cardiac-specific genes, structural proteins and ion channels. Using this model system, we show here, (I) that during cardiac myofibrillogenesis sarcomeric proteins are organized in a developmentally regulated manner following the order: titin (Z-disk), alpha-actinin, myomesin, titin (M-band), myosin heavy chain, alpha-actin, cardiac troponin T and M-protein, recapitulating the sarcomeric organization in the chicken embryonal heart in vivo. Our data support the view that the formation of I-Z-I complexes is developmentally delayed with respect to A-band assembly. We show (2) that the process of cardiogenic differentiation in vitro is influenced by medium components: Using a culture medium supplemented with glucose, amino acids, vitamins and selenium ions, we were able to increase the efficiency of cardiac differentiation of wild-type, as well as of beta1 integrin-deficient (beta1-/-) ES cells, and to improve the degree of organization of sarcomeric structures in wild-type and in beta1-/- cardiac cells. The data demonstrate the plasticity of cardiogenesis during the differentiation of wild-type and of genetically modified ES cells.

Adaptation, Physiological↗