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Five new cytotoxic triterpenoid saponins from the roots of Symplocos chinensis.

Five new triterpenoid saponins, named symplocososides G-K, were isolated from the roots of Symplocos chinensis. Their structures were elucidated by spectral and chemical methods as symplocososide G, 3beta-O-{[beta- D-glucopyranosyl(1-->2)][alpha-L-arabinofuranosyl(1-->4)]-beta-D-(3-O-acetyl)-glucuronopyranosyl}-21beta- O-[(2 Z)-3,7-dimethyl-2,6-octadienoyl]-22 alpha-O-(2-methylbutanoyl)-R1-barrigenol, symplocososide H, 3beta-O-{[beta-D-glucopyranosyl(1-->2)][alpha-L-arabinofuranosyl(1-->4)]- beta-D-(3-O-acetyl)-glucuronopyranosyl}-21beta-O-[(2E)3,7-dimethyl-2,6-octadienoyl]-22alpha-O-(2-methylbutanoyl)-R1-barrigenol, symplocososide I, 3beta-O-{[beta-D-glucopyranosyl(1-->2)][ alpha-L-arabinofuranosyl(1-->4)]-beta-D-(3- O-acetyl-6-O-methyl)-glucuronopyranosyl}-21beta-O-[(2 Z)3,7-dimethyl-2,6-octadienoyl]-22alpha-O-(2-methylbutanoyl)-R1-barrigenol, symplocososide J, 3 beta-O-{[ beta-D-glucopyranosyl(1-->2)][alpha-L-arabinofuranosyl(1-->4)]-beta-D-(3- O-acetyl)-glucuronopyranosyl}-21beta-O-[(2 Z)3,7-dimethyl-2,6-octadienoyl]-22alpha-O-benzoyl-R1-barrigenol, and symplocososide K, 3beta-O-{[beta-D-glucopyranosyl (1-->2)][alpha-L-arabinofuranosyl(1-->4)]- beta-D-(3-O-acetyl-6-O-methyl)-glucuronopyranosyl}-21beta-O-[(2Z)3,7-dimethyl-2,6-octadienoyl]-22alpha-O-benzoyl-R1-barrigenol. Symplocososides G-K showed significant cytotoxicity against cancer cell lines KB, HCT-8, Bel-7402, BGC-823 and A549 with IC50 values ranging from 0.82 microM to 5.09 microM, except for symplocososide I against cancer cell lines KB, BGC-823, A549 and symplocososide K against cancer cell line BGC-823 with IC50 values >10.00 microM.

Antineoplastic Agents, Phytogenic↗

Dual roles of Nur77 in selective regulation of apoptosis and cell cycle by TPA and ATRA in gastric cancer cells.

Nur77 is an orphan receptor. Although Nur77 affects cell proliferation and apoptosis through its capability of binding to a variety of response elements and regulating their transactivation activities, the intrinsic function of Nur77 is not yet fully understood; in particular, its regulation of apoptosis and proliferation has been characterized as cell type-dependent and agent context-dependent. In this study, Nur77 can be seen to regulate apoptosis via its expression and translocation, rather than its transactivation activity in gastric cancer cells. Nur77 was constitutively expressed in BGC-823 cells. The tetradecanoylphorbol-1,3-acetate (TPA) treatment not only resulted in up-regulation of the Nur77 mRNA level, but also led to translocation of Nur77 protein from the nucleus to the mitochondria, and caused the release of cytochrome c. This TPA-induced translocation of Nur77 was in association with the initiation of apoptosis in gastric cancer cells. Although all-trans retinoic acid (ATRA) could not induce apoptosis in BGC-823 cells due to failure of stimulating Nur77 translocation, expression of Nur77 in the nucleus was required for cell growth inhibition by ATRA. Transfection of antisense Nur77 receptor into BGC-823 cells resulted in resistance of cell growth against ATRA inhibition, and the cells were still arrested in the S phase. Furthermore, the action of Nur77 in TPA-induced apoptosis was mediated through a protein kinase C signaling pathway, while mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways were responsible for the regulation of Nur77 mRNA expression. Taken together, the data revealed the dual functioning mechanisms of Nur77 in gastric cancer cells in response to TPA and ATRA.

Apoptosis↗

Primary Bartholin gland carcinoma: a report of seven cases.

This study reviews our experience with 7 patients with primary Bartholin gland cancer (BGC) treated at the Queensland Gynaecological Cancer Centre (QCGC) and compares this with previously published data. A retrospective clinicopathologic review of all patients with primary BGC treated at QCGC from 1988 to 2000 was performed. Of the 7 patients treated, all underwent primary surgery and 5 of the 7 patients received radiotherapy postoperatively. All patients presented with a local swelling or a lump. Two had associated discharge and 2 had associated pain. Of the 7 patients, 2, 3 and 2 respectively were classified as having Stage IB, II or III disease. Five of the 7 patients had squamous cell carcinoma (SCC), one had adenoid-cystic carcinoma and 1 had a small-cell neuroendocrine cancer of the Bartholin gland. None of the patients with SCC developed recurrent disease. The patient with adenoid-cystic carcinoma experienced local recurrences at 4 years and again at 5 years and 3 months. Nine years after primary treatment she was diagnosed with pulmonary metastases. The patient with small-cell neuroendocrine cancer of the Bartholin gland was considered tumour-free after operation. Thorough imaging, including a CT scan of her chest, abdomen and pelvis showed no evidence of disease. She died 1 year and three months after diagnosis from disseminated pulmonary disease. We present the first report of small cell neuroendocrine cancer of the Bartholin gland. Therapeutic principles in the management of vulval cancer at other sites appear to be appropriate for management of BGC.

Adult↗

The problem of accurate initial diagnosis of Bartholin's gland carcinoma resulting in delayed treatment and aggressive course of the disease.

Carcinoma of the Bartholin's gland is a rare lesion accounting for only 5% of all vulvar cancers. Initial diagnosis of Bartholin's gland cancer (BGC) and recurrence after primary radical surgery and adjuvant radiotherapy poses a challenge in the treatment of BGC. This case report presents a particularly aggressive course of BGC. Spontaneous rupture of the femoral artery in the postoperative period was observed. The length of the survival period from the moment of diagnosis amounted to less than 16 months. Aspects of initial diagnosis problem and treatment options in groin recurrence of vulvar carcinoma and vascular complications are discussed.

Bartholin's Glands↗

Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a morphological and molecular approach.

Currently, there is no consensus concerning the geographic distribution and extent of endemism in Antarctic cyanobacteria. In this paper we describe the phenotypic and genotypic diversity of cyanobacteria in a field microbial mat sample from Lake Fryxell and in an artificial cold-adapted sample cultured in a benthic gradient chamber (BGC) by using an inoculum from the same mat. Light microscopy and molecular tools, including 16S rRNA gene clone libraries, denaturing gradient gel electrophoresis, and sequencing, were used. For the first time in the study of cyanobacterial diversity of environmental samples, internal transcribed spacer (ITS) sequences were retrieved and analyzed to complement the information obtained from the 16S rRNA gene. Microscopy allowed eight morphotypes to be identified, only one of which is likely to be an Antarctic endemic morphotype. Molecular analysis, however, revealed an entirely different pattern. A much higher number of phylotypes (15 phylotypes) was found, but no sequences from Nodularia and Hydrocoryne, as observed by microscopy, were retrieved. The 16S rRNA gene sequences determined in this study were distributed in 11 phylogenetic lineages, 3 of which were exclusively Antarctic and 2 of which were novel. Collectively, these Antarctic sequences together with all the other polar sequences were distributed in 22 lineages, 9 of which were exclusively Antarctic, including the 2 novel lineages observed in this study. The cultured BGC mat had lower diversity than the field mat. However, the two samples shared three morphotypes and three phylotypes. Moreover, the BGC mat allowed enrichment of one additional phylotype. ITS sequence analysis revealed a complex signal that was difficult to interpret. Finally, this study provided evidence of molecular diversity of cyanobacteria in Antarctica that is much greater than the diversity currently known based on traditional microscopic analysis. Furthermore, Antarctic endemic species were more abundant than was estimated on the basis of morphological features. Decisive arguments concerning the global geographic distribution of cyanobacteria should therefore incorporate data obtained with the molecular tools described here.

Acclimatization↗

Metagenomic insights and biosynthetic potential of Candidatus Entotheonella symbiont associated with Halichondria marine sponges.

Korea, being surrounded by the sea, provides a rich habitat for marine sponges, which have been a prolific source of bioactive natural products. Although a diverse array of structurally novel natural products has been isolated from Korean marine sponges, their biosynthetic origins remain largely unknown. To explore the biosynthetic potential of Korean marine sponges, we conducted metagenomic analyses of sponges inhabiting the East Sea of Korea. This analysis revealed a symbiotic association of Candidatus Entotheonella bacteria with Halichondria sponges. Here, we report a new chemically rich Entotheonella variant, which we named Ca. Entotheonella halido. Remarkably, this symbiont makes up 69% of the microbial community in the sponge Halichondira dokdoensis. Genome-resolved metagenomics enabled us to obtain a high-quality Ca. E. halido genome, which represents the largest (12 Mb) and highest quality among previously reported Entotheonella genomes. We also identified the biosynthetic gene cluster (BGC) of the known sponge-derived Halicylindramides from the Ca. E. halido genome, enabling us to determine their biosynthetic origin. This new symbiotic association expands the host diversity and biosynthetic potential of metabolically talented bacterial genus Ca. Entotheonella symbionts.IMPORTANCEOur study reports the discovery of a new bacterial symbiont Ca. Entotheonella halido associated with the Korean marine sponge Halichondria dokdoensis. Using genome-resolved metagenomics, we recovered a high-quality Ca. E. halido MAG (Metagenome-Assembled Genome), which represents the largest and most complete Ca. Entotheonella MAG reported to date. Pangenome and BGC network analyses revealed a remarkably high BGC diversity within the Ca. Entotheonella pangenome, with almost no overlapping BGCs between different MAGs. The cryptic and genetically unique BGCs present in the Ca. Entotheonella pangenome represents a promising source of new bioactive natural products.

Animals↗

Efficient isolation and long-term viability of bovine small preantral follicles in vitro.

A comparison of isolation techniques for small preantral follicles (30-70 microm) from bovine ovaries using a mechanical method with a grating device or collagenase treatment was performed. The mean number (157.0) of intact follicles per ovary isolated by the mechanical method was significantly greater (P < 0.05) than that (26.0) of follicles isolated by the enzymatic method. Isolated morphologically normal follicles (MNF) were cultured for up to 30 d either in control cultures (non-coculture) or in coculture with bovine ovary mesenchymal cells (BOM), fetal bovine skin fibroblasts (FBF), and/or bovine granulosa cells (BGC). In control cultures, most of the follicles degenerated and only a few MNF (1.2%) were present after 30 d in culture. In contrast, the cocultures with BOM, FBF, and BGC resulted in 50.7, 46.6, and 21.4% viable MNF, respectively. Trypan blue and Hoechst 33258 staining were used for a quick and sensitive assessment of oocyte and granulosa cell viability during follicle isolation and culture in vitro. After 30 d, percentages of viable follicles in coculture with BOM (18.6%) and FBF (17.1%) were significantly greater than those of follicles in the control cultures (0%) or in coculture with BGC (10.0%). There was a gradual increase in the average diameter of the MNF during culture. The mean diameter of the follicles increased by 15.4 and 30.0% in coculture with BOM and FBF, respectively, by day 30. In conclusion, small bovine preantral follicles were efficiently isolated using a mechanical method that utilizes a grating device, and could be maintained for up to 30 d in the presence of mesenchymal cell cocultures such as BOM and FBF. This in vitro culture system that supports long-term survival of bovine preantral follicles should be beneficial for studying follicle growth and development.

Animals↗

Alterations in bone regenerative capacity after low level gamma irradiation. A quantitative study.

In the present study the influence of single 2.5 and 5 Gy doses of irradiation on the regenerative capacity of mature bone tissue has been investigated. To the knowledge of the present authors, no quantitative analysis of bone repair after low doses of irradiation has been presented previously. The experimental model used was the Bone Growth Chamber (BGC), which is a porous implant made of titanium. Each one of twenty animals was irradiated with 2.5 or 5 Gy to one tibial metaphysis. Directly after irradiation each animal had BGC:s inserted bilaterally into the tibial metaphyses. Thus, each animal served as its own control. Four weeks after irradiation the BGC:s were removed and the newly formed bone was collected from the implant pores and was analyzed by microradiography and quantified by microdensitometry and histology. It was found that 2.5 Gy irradiation led to no statistically significant alteration in bone formation as compared to non-irradiated controls. At the 5 Gy dose level, however, there was a significant reduction of bone formation as compared to non-irradiated controls.

Animals↗

Degradation of retinoid X receptor alpha by TPA through proteasome pathway in gastric cancer cells.

AIM: To investigate and determine the mechanism and signal pathway of tetradecanoylphorbol-1, 3-acetate (TPA) in degradation of RXRalpha. METHODS: Gastric cancer cell line, BGC-823 was used in the experiments. The expression level of RXRalpha protein was detected by Western blot. Nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation. Localization and translocation of RXRalpha were observed under laser-scanning confocal microscope through labeling specific anti-RXRalpha antibody and corresponding immunofluorescent antibody as secondary antibody. Different inhibitors were used as required. RESULTS: In BGC-823 cells, RXRalpha was expressed in the nucleus. When cells were treated with TPA, expression of RXRalpha was repressed in a time-dependent and TPA-concentration-dependent manner. Meanwhile, translocation of RXRalpha from the nucleus to the cytoplasm occurred, also in a time-dependent manner. When cells were pre-incubated with proteasome inhibitor MG132 for 3 hrs, followed by TPA for another 12 hrs, TPA-induced RXRalpha degradation was inhibited. Further observation of RXRalpha translocation in the presence of MG132 showed that MG-132 could block TPA-induced RXRalpha redistribution. Conversely, when RXRalpha translocation was inhibited by LMB, an inhibitor for blocking protein export from the nucleus, TPA could not repress expression of RXRalpha. CONCLUSION: TPA could induce the degradation of RXRalpha protein in BGC-823 cells, and this degradation is time- and TPA-concentration-dependent. Furthermore, the degradation of RXRalpha by TPA is via a proteasome pathway and associated with RXRalphatranslocation from the nucleus to the cytoplasm.

Carcinogens↗

[Cytocompatibility of three substituted materials of human beings' hard tissue].

OBJECTIVE: To evaluate the osteocompatibility of three substituted materials of human beings' hard tissue (hydroxyapatite, bioglass ceramic and Ti-6Al-4V) and a new experimental method for evaluating the osteocompatibility of replaced materials of hard tissues in vitro. METHODS: The isolated osteoblasts from parietal and frontal bones of Sparague Dawley rats from 1 day to 3 days were used to estimate osteocompatibility of three substituted materials in vitro. The cells were inoculated onto specimens of HA, BGC and Ti-6Al-4V for 1 day, 3, 5 and 7 days, then the morphology and proliferating rates of cells on different materials were tested. RESULTS: Rat osteoblasts could attach, spread and proliferate on three materials as normal cells, and proliferating rates of cells on HA and BGC surface were as the same as normal, but that on Ti-6Al-4V was lower than the normal cells. CONCLUSION: The osteocompatibility of HA and BGC are better than that of Ti-6Al-4V and rat osteoblast culture system can be used to evaluate the osteocompatibility of substituted materials in vitro.

Alloys↗

[In vitro studies about the effects of implant materials on calmodulin, introcellular calcium of rat osteoblasts].

OBJECTIVE: 1. To evaluate the effects of the four implant materials, hydroxyl poly calcium sodium phosphate (HPA), hydroxyapatite (HA), bioglass ceramics (BGC) and titanium (Ti) on the calmodulin (CaM) content of osteoblasts, the concentration of intracellular free calcium and the ATPases activity of cell membrane of rat osteoblasts cultured in vitro; 2. To reveal the biological compatibility of the four implant materials and the index to estimate the biological compatibility condition. METHODS: The powders of HPA, HA, BGC and Ti were sterilized and made into extracted solutions with 199 culture medium. The isolated osteoblasts, which were taken from three-day-old sparague Dawley rats' calvaria, were incubated in 199 culture medium also. Then the osteoblasts were mixed and incubated into the four kinds of extracted solutions of the implant materials. After three days of co-incubation, the cells were taken out, and the cellular calmodulin content were measured with radiobiological analysis. The concentration of intracellular free calcium were under fluorescent analysis, and the ATPases activities of cell membrane were estimated with colorimetric analysis. RESULTS: 1. Calmodulin content of the osteoblasts co-incubated with all the implant materials decreased, and moreover, only that of the Ti group decreased more obviously than that of the Ti and HPA group (P < 0.05). 2. The concentration of intracellular free calcium of all the co-incubated osteoblasts were as normal as that of the control group. 3. Ca(2+)-ATPases activities of osteoblasts co-incubated with HPA, HA, BGC increased (P < 0.05), and Na+/K(+)-ATPases activities of the cells co-incubated with HPA, HA were strenghtened (P < 0.05). CONCLUSION: The indexes of cellular calmodulin content, concentration of intracellular free calcium and ATPases activity of cell membrane can reflect the biological compatibility of implant materials, and they may be used as indexes of estimating biological compatibility condition.

Animals↗

[In vitro studies about the effects of implanted materials on osteocalcin and alkaline phosphatase of rat osteoblasts].

OBJECTIVE: To evaluate the effects of the four implant materials, hydroxyl poly calcium sodium phosphate (HPA), hydroxyapatite (HA), bioglass ceramics (BGC) and titanium (Ti) on osteocalcin secreting amount and alkaline phosphatase activity of rat osteoblasts cultured in vitro, and expose the biological compatibility conditions of the implant materials as well as the index to estimate cytocompatibility. METHODS: The powders of HPA, HA, BGC and Ti were sterilized and made into extracted solutions with 199 culture medium, and the isolated osteoblasts which were taken from three-day-old Sprague-Dawley rats' calvaria, were incubated in 199 culture medium too. Then the osteoblasts were mixed and incubated into the four kinds of extracted solutions of the implant materials. After eight days of co-incubation, the cells were taken out to measure alkaline phosphatase activity with velocity analysis, and the extracellular liquid was used to estimate osteocalcin secreting amount with radioisotope analysis. RESULTS: 1. Osteocalcin secreting amount of the osteoblasts co-incubated with HPA, HA, BGC were as normal as the control, while that of other cells co-incubated with titanium was reduced (P < 0.05). 2. Alkaline phosphatase activities of all the co-incubated osteoblasts were as normal as the control. CONCLUSION: The osteocalcin secreting amount and alkaline phosphatase activity can reflect the biological compatibility of implant materials, and they may be used as indexes to evaluate biological compatibility condition of implant materials.

Alkaline Phosphatase↗

Effects of DNA methylation inhibitor on down-regulation of ribonuclease inhibitor expression in cancer cell lines.

BACKGROUND & OBJECTIVE: Human ribonuclease inhibitor (RI) could effectively block angiogenin-induced angiogenesis, and inhibit growth of transplant solid tumors in animals. However, its exact molecular mechanism of antitumor has not been totally ascertained. Many tumor suppressor genes occur loss of expression by aberrant methylation in promoter region. Demethylation, treated with methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR),can restore the gene expression. To further explore functions of RI, and investigate relationship between RI and tumorigenesis, the study was designed to explore effects of 5-Aza-CdR on expression of RI in cancer cell lines. METHODS: Human breast cancer cell line MCF-7, human gastric cancer cell line BGC-823, human prostate cancer cell line DU-145, and human colon cancer cell line HT-29 were treated with 5-Aza-CdR. Expression of RI was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, and immunocytochemistry. RESULTS: Expression of RI significantly elevated by 5-Aza-CdR at both mRNA and protein level in MCF-7, BGC-823, and DU-145 cells (P< 0.01). Compared with control cells, mRNA levels of RI in MCF-7, BGC-823, and DU-145 cells treated with 5-Aza-CdR were increased by percentages of 37.2%, 46.0%, and 32.4%, respectively; protein levels of RI were increased by percentages of 26.4%, 20.9%, and 24.4%, respectively; but no obvious change observed in HT-29 cells. CONCLUSION: RI gene may be involved in tumorigenesis of gastric, prostate, and breast cancer.

Azacitidine↗

[The killing action of a Chinese herbal mixture on human tumor cells in vitro and its effect on cell-cycle].

Previous studies had shown that injection of Chinese herbal mixture (ICHM) had antitumor and anti-metastatic actions on various transplantable animal tumors. In the present study, the authors observed the obvious killing actions of ICHM on human gastric cancer cell line BGC-823 and breast cancer cell line MCF-7 in vitro. The IC50 were 0.02 g crude drug/ml and 0.018 g crude drug/ml respectively. The study of the effect of ICHM on the cell-cycle of BGC-823 cell showed that ICHM decreased the number of cells in S phase and increased the number of cells in G1 phase significantly. The results indicated that the BGC-823 cells was blocked on S phase by ICHM. Meanwhile, ICHM reduced 3H-TdR incorporation into DNA, the cpm value was much lower than that of the control.

Antineoplastic Agents, Phytogenic↗

Right anterior oblique first-pass radionuclide ejection fractions: effects of temporal smoothing and various background corrections.

Thirty-seven patients undergoing contrast left ventriculography were studied by first-pass radionuclide angiography (FPRA) in the right anterior oblique view. Ejection fraction (LVEF) was calculated from FPRA using (a) a spatially and temporally varying background correction (BGC) based on a matrix of activity in lung and left atrium and (b) BGC with temporal fluctuation but with no allowance for spatial variations. The two methods were performed on both raw and temporally smoothed data. All four LVEFs correlated well with contrast LVEF (r = 0.90 - 0.94). Absolute values differed significantly from contrast values except for the method using the spatially and temporally varying BGC on smoothed data, which provided the closest overall agreement at all levels of LVEFs, despite occasional large individual variations. The same method on raw data overestimated low LVEFs and the method applying only temporal fluctuation in background underestimated high LVWFs. Allowance for spatial and temporal variations in background is therefore important when first-pass radionuclide angiography is performed in the RAO view.

Adult↗

[Protein kinase inhibitor staurosporine enhances cytotoxicity of antitumor drugs to cancer cells].

Treated with low dosage (5 ng.ml-1) of staurosporine for 18 h, human embryo lung 2BS cells were blocked at the G1/S boundary, but human gastric carcinoma BGC-823 cells still kept their cell cycle. In comparison with IC50 of 2BS and BGC-823 cells treated with cell cycle phase specific antitumor drugs adriamycin, Ara-C and BLM A5 alone or combined with staurosporine 5 ng.ml-1, the IC50 values increased from 0.325 microgram.ml-1, 5 micrograms.ml-1 and 6.5 micrograms.ml-1 to 0.45 microgram.ml-1, 10 micrograms.ml-1 and 6.5 micrograms.ml-1, respectively in 2BS cells; but decreased from 0.325 microgram.ml-1, 25 micrograms.ml-1 and 1.1 micrograms.ml-1 to 0.07 microgram.ml-1, 6.25 micrograms.ml-1 and 0.4 microgram.ml-1, respectively in BGC-823 cells. These results suggest that combination of staurosporine 5 ng.ml-1 with antitumor drugs showed different effects on tumor cells and normal cells. With the GSH fluorescent probe mBCL, we found that GSH contents increased in 2BS cells treated with staurosporine 5 ng.ml-1.

Antibiotics, Antineoplastic↗

[Changes of intracellular calcium, calmodulin in normal and tumor cells traggered by staurosporine].

Treated with low dosage staurosporine, the normal cell 2 BS were arrested in G 1 phase, but tumor cell BGC-823 were not. We measured the intracellular calcium, calmodulin, Ca(2+)-activited calmodulin (Ca(2+)-CaM) contents of single cells according to cell cycle with microphotometry. Our results showed that treatment of 5 ng/ml staurosporine for 18 h caused the changes of calcium and calmodulin. In 2 BS cells, CaM leval decreased in G 1 and S phase. In each cell cycle phase of BGC-823 cells, there was no changes in CaM level, but Ca(2+)-CaM level increased. The causes of that staurosporine blocked 2 BS cells at G 1 phase but not effected on cell cycle progression in BGC-823 cells may be the results of staurosporine decreased the contents of CaM at G 1 phase and inhibited the phosphoralation of p107 in 2 BS cells.

Calcium↗