Inhibition Of Colony Growth Research Articles

Contamination by uranium mine drainages affects fungal growth and interactions between fungal species and strains

The presence of aquatic hyphomycetes has been reported for several heavy metal-contaminated waters. Tolerance probably is one adaptation to coping with heavy metals. To help clarify this issue strains of two species of aquatic hyphomycetes (Tricladium splendens Ingold and Varicosporium elodeae Kegel) were isolated from a reference stream and a stream contaminated with heavy metals and grown on malt extract agar prepared with reference and contaminated water to characterize colony morphology, growth rate, growth inhibition and interaction among species and strains. In V. elodeae the morphology of colonies differed between strains. Colony diameter increased linearly over time with growth rates being lower for strains isolated from contaminated than from reference streams (mostly for V. elodeae). Strains from the contaminated stream grew faster in medium prepared with contaminated water than in medium prepared with reference water, while for strains from the reference stream there was no significant difference in growth rates on the two media. In interacting isolates radial growth toward the opposing colony was generally lower than toward the dish edge. Percentage growth inhibition was higher for isolates in intraspecific interactions (13–37%) than in interspecific interactions (3–27%). However differences in growth inhibition experienced by interacting isolates were observed only in three cases out of 16. The difference between the percentage inhibition caused and experienced by a given isolate was highest in interactions involving isolates with distinct growth rates. Our results suggest that strains from the reference stream tolerate heavy metals while strains from the contaminated stream seem to be adapted to contaminated waters. We hypothesize that in natural environments fungal species-specific limits of tolerance to metal contamination might determine an abrupt or gradual response of the original fungal community to mine pollution giving origin to a poorer fungal community dominated by adapted strains with distinct functional efficiency.

The relationship between cellular and calcium responses of Aspergillus awamori to external influences

The cellular and Ca2+ responses to physiological stimuli of different nature were studied in the experiments with the strain Aspergillus awamori 66A containing recombinant aequorin, a Ca2+-dependent photosensitive protein. The relationship between the cellular response registered by changes in the development of the mycelial fungus (colony growth, hyphal branching, and the rate of spore formation) and the level and duration of calcium flares in the cytosol was assessed. The physical or chemical stimuli (mechanical effect, osmotic shock) inducing short-time calcium flares in the cytosol did not influence significantly the development of A. awamori grown in liquid or on solid nutrient media. The action on the 24-h A. awamori culture of the Ca2+-selective ionophore (A23187) inducing long-term changes in calcium homeostasis caused disorders in the fungus development and morphology (hyperbranching of mycelial hyphae, formation of spherical cells, and inhibition of colony growth and spore formation). Thus, it was established that the development of cellular response in the micromycete correlated with the duration of the calcium flare in the cytosol.

Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine

Ten strains of Pseudomonas aeruginosa (PN1 ˜ PN10) isolated from rhizosphere of chir-pine were tested for their plant growth promontory properties and antagonistic activities against Macrophomina phaseolina in vitro and in vivo. P. aeruginosa PN1 produced siderophore, IAA, cyanogen and solubilized phosphorus, besides producing chitinase and β-1,3-glucanase. In dual culture, P. aeruginosa PN1 caused 69% colony growth inhibition. However, cell free culture filtrate also posed inhibitory effect but to a lesser extent. After 90 days, P. aeruginosa PN1 increased plant growth and biomass in pots trial containing M. phaseolina-infested soil. PN1 showed the strong chemotaxis toward root exudates resulting in effective root colonization. Moreover, increased population in rhizosphere of these bacteria was also recorded after 90 days of treatment. Thus, chemotactic fluorescent P. aeruginosa PN1 exhibited strong antagonistic property against M. phaseolina, suppressed the disease and improved plant growth of the seedlings of chir-pine proving potential biocontrol agent.

Dose-Response Relationship between Norepinephrine and Erythropoiesis: Evidence for a Critical Threshold

Severe traumatic injury elicits a neuroendocrine response that activates the sympathetic nervous system. Our previous work suggests that norepinephrine (NE) influences the bone marrow (BM) erythropoietic response. However, the dose-response relationship between NE and erythropoiesis remains unclear. Two days following chemical sympathectomy with 6-hydroxydopamine (6-OHDA) or injection with saline vehicle (SHAM), male Sprague-Dawley rats were infused continuously with either saline (NS) or increasing doses of NE for 5 d via osmotic pumps. Erythropoiesis was assessed by growth of erythroid progenitor colonies (BFU-E and CFU-E for early and late progenitors, respectively). Following chemical sympathectomy with 6-OHDA, both BFU-E and CFU-E growth is inhibited (42%∗ and 43%∗ versus 100% SHAM, ∗P < 0.05). SHAM rats with continuous infusion of exogenous NE show a clear dose-response inhibition of both BFU-E and CFU-E colony growth. In the 6-OHDA rats, continuous infusion of NE restored BFU-E and CFU-E growth at 10(-8) g/h and 10(-9) g/h, respectively. Erythroid precursor colony growth is inhibited in sympathectomized rats. In addition, supraphysiologic doses of exogenous NE inhibit normal erythropoiesis in a dose-dependent fashion. Following chemical sympathectomy with 6-OHDA, exogenous NE restores erythropoiesis in a narrow window. Therefore, NE has a complex interaction within the BM and the elevation of NE following traumatic injury impacts BM erythropoietic function.

Abstract B161: U3-1287 (AMG 888), a fully human anti-HER3 mAb, demonstrates preclinical efficacy in HER2+ and HER2− breast cancer models

Abstract Background: HER3 is required for proliferation in HER2 amplified (HER2+) breast cancer cell lines, but may also be important in the HER2 negative (HER2−) setting. Though HER3 lacks kinase activity, it is a scaffold for PI3K signaling for the HER family and other receptor kinases via heterodimeric interactions. In preclinical models, activation of HER3 is a resistance mechanism to current HER family inhibitors. We report the efficacy of U3-1287 (AMG 888) in HER2+ and HER2− preclinical breast cancer models. We also evaluate human breast tumor samples for detectable levels of pHER3 and pAKT. Methods: Levels of pHER3, pERK and pAKT were determined by western blotting. To determine the inhibition of HER3 oncogenic signaling, HER2+ (SKBR3, MDA-MB-453 and HCC1569) and HER2− (MDA-MB-175 VII) breast cancer cells were treated with 10 µg/ml of U3-1287 (AMG 888), cetuximab, c2C4, trastuzumab, lapatinib (250 to 1500 nM) or controls prior to heregulin (HRG) stimulation. To determine the activity on cell proliferation in the presence of 0.4% FBS, breast cancer cell lines were incubated with 10 µg/ml of U3-1287 (AMG 888), cetuximab, c2C4, trastuzumab, lapatinib (50 to 1500 nM) or control for 1 hour prior to HRG stimulation. After 4 days, the growth of treated cells was measured with alamarBlue™. To determine the activity on anchorage-independent growth, breast cancer cells were treated with 2 to 5 g/ml U3-1287 (AMG 888), anti-HER antibodies, 500 nM lapatinib, or control. Tumor cell colonies formed in the absence or presence of HRG for 6 to 10 days and were stained with MTT for 4 to 6 hours and quantified. Results: Treatment of breast cancer cell lines with U3-1287 (AMG 888) resulted in an inhibition of pHER3 and pAKT. In cell proliferation assays, U3- 1287 (AMG 888) reduced heregulin-stimulated SkBR-3 proliferation up to 40% (p&amp;lt;0.05) as a single agent, and up to 80% (p&amp;lt;0.05) when combined with other HER inhibitors. Additive activity was observed in MDA-MB 175 VII cells when U3-1287 (AMG 888) was combined with standard of care chemotherapeutics (gemcitabine and cisplatin) (p&amp;lt;0.05 vs either single agent alone). U3-1287 (AMG 888) did not inhibit colony growth in the ZR- 75-30 model, but inhibits colony growth by 50%, 25%, 85% in the SkBr-3, MDA-MB453 and MDA-MB175VII breast cancer models, respectively, as a single agent (p&amp;lt;0.05) and up to 95% when combined with other HER inhibitors (p&amp;lt;0.05). 40%, 35% of the HER2+ and 10%, 35% of the HER2− human tumor samples had detectable pHER3 and pAKT, respectively. Conclusions: U3-1287 (AMG 888) inhibits proximal and distal HER3 oncogenic signaling in breast cell lines in vitro. Breast cancer cells are sensitive to U3-1287 (AMG 888) treatment as single agent and in combination with anti-HER agents. The preclinical data together with detectable levels of pHER3 in patient samples provide evidence for the potential clinical application of U3-1287 (AMG 888) in HER2+ and HER2− breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B161.

Abstract B171: U3-1287 (AMG 888), a fully human anti-HER3 mAb, demonstrates in vitro and in vivo efficacy in NSCLC models

Abstract Background: EGFR TKIs have demonstrated activity in NSCLC, however most patients are resistant or acquire resistance to therapy. HER3 is a member of the EGFR family of receptors and is considered a target for NSCLC treatment. Though HER3 lacks kinase activity, it is a scaffold for PI3K signaling for the HER family via heterodimeric interactions. In preclinical models, activation of HER3 is a resistance mechanism (de novo and acquired) to current HER inhibitors. Herein, we report the activity of U3-1287 (AMG 888) in preclinical models of human NSCLC. We also evaluate human NSCLC tumor specimens for detectable pHER3, pAKT and HRG, the ligand for HER3. Methods: To determine the inhibition of HER3 oncogenic signaling, A549 and Calu-3 NSCLC cells were treated with 100 nM of U3-1287 (AMG 888), panitumumab, lapatinib or control for 1 hour. To determine in vitro efficacy on anchorage independent growth, A549 cells were treated with 10 g/mL U3-1287 (AMG 888), other HER family Abs or control mAb in serum containing medium. Tumor cell colonies formed in the absence of exogenous ligand for 10 days and were stained with MTT and quantified using a Scanalyzer HTS camera imaging system. To determine in vivo efficacy, mice bearing ∼200 mm3 A549 or Calu-3 NSCLC xenografts were treated 2x/week with anti-HER family inhibitors or control. A549 xenograft tumors were analyzed for the inhibition of pHER3 byWestern blotting. The anti-tumor effect of U3-1287 (AMG 888) with EGFR inhibitors was tested in the Calu-3 NSCLC xenograft model. Levels of pHER3, pAKT and HRG in 20 NSCLC patient specimens were determined by Western blotting. Results: Treatment with U3-1287 (AMG 888) resulted in inhibition of pHER3 in both NSCLC cell lines. pAKT was inhibited in the A549 cell line with U3-1287 (AMG 888) as a single agent and in A549 and Calu-3 cells to a greater extent when combined with another HER family inhibitor. U3-1287 (AMG 888) inhibited colony growth by 50% (p&amp;lt;0.001) in the A549 cell line and resulted in tumor stasis in the A549 NSCLC xenograft model vs IgG control or other HER mAbs (p&amp;lt;0.05). U3-1287 (AMG 888) treatment resulted in an inhibition of Calu-3 xenografts as a single agent or when combined with HER family inhibitors compared to control treated mice (p&amp;lt;0.05). Combinations with panitumumab resulted in tumor growth inhibition that was greater than either single agent alone in Calu-3 xenografts (p&amp;lt;0.001). 75%, 65%, 90% of the NSCLC tumor specimens had detectable pHER3, pAKT and HRG, respectively. Conclusions: U3-1287 (AMG 888) inhibits proximal and distal HER3 oncogenic signaling and cell growth in NSCLC cell lines in vitro and in vivo. NSCLC xenografts are sensitive to U3-1287 (AMG 888) treatment as single agent or in combination with the anti-EGFR agents. The preclinical data together with the detectable levels of activated HER3 in patient samples provide evidence for the potential clinical application of U3-1287 (AMG 888) in NSCLC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B171.

Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor–related protein 1 (LRP1) on megakaryocytes

AbstractPlatelet factor 4 (PF4) is a negative regulator of megakaryopoiesis, but its mechanism of action had not been addressed. Low-density lipoprotein (LDL) receptor–related protein-1 (LRP1) has been shown to mediate endothelial cell responses to PF4 and so we tested this receptor's importance in PF4's role in megakaryopoiesis. We found that LRP1 is absent from megakaryocyte-erythrocyte progenitor cells, is maximally present on large, polyploidy megakaryocytes, and near absent on platelets. Blocking LRP1 with either receptor-associated protein (RAP), an antagonist of LDL family member receptors, or specific anti-LRP1 antibodies reversed the inhibition of megakaryocyte colony growth by PF4. In addition, using shRNA to reduce LRP1 expression was able to restore megakaryocyte colony formation in bone marrow isolated from human PF4-overexpressing mice (hPF4High). Further, shRNA knockdown of LRP1 expression was able to limit the effects of PF4 on megakaryopoiesis. Finally, infusion of RAP into hPF4High mice was able to increase baseline platelet counts without affecting other lineages, suggesting that this mechanism is important in vivo. These studies extend our understanding of PF4's negative paracrine effect in megakaryopoiesis and its potential clinical implications as well as provide insights into the biology of LRP1, which is transiently expressed during megakaryopoiesis.

CTEN/tensin 4 expression induces sensitivity to paclitaxel in prostate cancer

Recently, we established paclitaxel-resistant prostate cancer cell lines (PC-3-TxR and DU145-TxR). To determine the mechanisms of paclitaxel resistance in PC-3-TxR cells, we compared the gene expression profiles between PC-3 and PC-3-TxR cells. Our results indicated that expression of the C-terminal tensin like protein (CTEN, tensin 4) gene was down-regulated by 10-fold in PC-3-TxR cells. We investigated the possibility that CTEN overexpression restores paclitaxel sensitivity. We investigated how knockdown and overexpression of CTEN in androgen-independent cell lines affect paclitaxel sensitivity by colony formation assay and growth inhibition assay. To determine the mechanisms by which CTEN affects paclitaxel sensitivity, we investigated the relationships between CTEN and F-actin or epidermal growth factor receptor (EGFR) in PC-3 cells. We also examined the association between expression of CTEN and grade of prostate cancer by immunohistochemistry using tissue microarray analysis. Down-regulation of CTEN, which is located in the cytoskeleton, played an important role in paclitaxel resistance in PC-3-TxR cells. Knockdown of CTEN expression in PC-3 cells induced paclitaxel resistance. Overexpression of CTEN in PC-3-TxR and DU145-TxR cells restored paclitaxel sensitivity. CTEN expression was inversely correlated with F-actin and EGFR expression. Then knockdown of actin and EGFR in PC-3-TxR cells recovered paclitaxel sensitivity, indicating that CTEN down-regulation mediates paclitaxel resistance through elevation of EGFR and actin expression. Moreover, CTEN expression was inversely correlated with Gleason score. These results strongly suggested that CTEN plays an important role in paclitaxel sensitivity and that CTEN expression level may be a prognostic predictive factor for PCa patients.

The Plant Hormone Abscisic Acid Stimulates the Proliferation of Human Hemopoietic Progenitors through the Second Messenger Cyclic ADP-Ribose

Abscisic acid (ABA) is a hormone involved in pivotal physiological functions in higher plants, such as response to abiotic stress and control of seed dormancy and germination. Recently, ABA was demonstrated to be autocrinally produced by human granulocytes, beta pancreatic cells, and mesenchymal stem cells (MSC) and to stimulate cell-specific functions through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). Here we show that ABA expands human uncommitted hemopoietic progenitors (HP) in vitro, through a cADPR-mediated increase of the intracellular calcium concentration ([Ca(2+)](i)). Incubation of CD34(+) cells with micromolar ABA also induces transcriptional effects, which include NF-kappaB nuclear translocation and transcription of genes encoding for several cytokines. Human MSC stimulated with a lymphocyte-conditioned medium produce and release ABA at concentrations sufficient to exert growth-stimulatory effects on co-cultured CD34(+) cells, as demonstrated by the inhibition of colony growth in the presence of an anti-ABA monoclonal antibody. These results provide a remarkable example of conservation of a stress hormone and of its second messenger from plants to humans and identify ABA as a new hemopoietic growth factor involved in the cross-talk between HP and MSC.

The prolyl 3-hydroxylases P3H2 and P3H3 are novel targets for epigenetic silencing in breast cancer

Expression of P3H2 (Leprel1) and P3H3 (Leprel2) but not P3H1 (Leprecan) is down-regulated in breast cancer by aberrant CpG methylation in the 5′ regulatory sequences of each gene. Methylation of P3H2 appears specific to breast cancer as no methylation was detected in a range of cell lines from other epithelial cancers or from primary brain tumours or malignant melanoma. Methylation in P3H2, but not P3H3, was strongly associated with oestrogen-receptor-positive breast cancers, whereas methylation in P3H3 was associated with higher tumour grade and Nottingham Prognostic Index. Ectopic expression of P3H2 and P3H3 in cell lines with silencing of the endogenous gene results in suppression of colony growth. This is the first demonstration of epigenetic inactivation of prolyl hydroxylases in human cancer, implying that this gene family represents a novel class of tumour suppressors. The restriction of silencing in P3H2 to breast carcinomas, and its association with oestrogen-receptor-positive cases, suggests that P3H2 may be a breast-cancer-specific tumour suppressor.

EphA3 Kinase Is Constitutively Activated in Chronic Myeloid Leukaemia during Progression to Accelerated and Blast Crisis and It Could Represent a New Molecular Target.

Eph receptors tyrosine kinases are involved in many key developmental processes. Ephs are overexpressed or mutated in solid tumours suggesting their possible role in oncogenesis. The majority of the genes and processes involved in CML progression are still unknown. The aim of this study was to investigate the role of EphA3 in CML progression and to explore the possibility to selectively target EphA3 with a monoclonal antibody. The expression of EphA3 and ligands were analyzed by RQ-PCR in 92 samples from 76 CML patients and in 38 healthy controls. 56 patients were in chronic phase (CP) enrolled in TOPS (Cortes, EHA, 2008) studies, 10 in accelerated phase (AP) and 10 in blast crisis (BC). In 5 patients and 10 healthy subjects EphA3 was studied also in enriched CD34+ population. Protein expression and localization were examined using Western Blot and immunofluorescence using specific antibodies. EphA3 expression in CD34+ cells were analyzed by FACS. The effects of EphA3 overexpression were studied by transfecting EphA3 plasmid in 293T e COS cells. In addition, sequencing of EphA3 TK domain was performed in 45 EphA3+ patients. BM, PB and CD34+ cells from CP CML patients expressed very low levels of EphA3, similar to normal controls with a median of 2−Δ ΔCt of 0,0018. By contrast, during the advanced phases of disease (AP and BC) we found high transcript levels (mean of 2−Δ ΔCt 43 and 67 respectively). Moreover, purified CD34+ CML cells presented significantly higher levels as compared to the unfractioned sample (p=0,001). RT-PCR showed no increase of the expression of the ligands Ephrin A2,A3,A4,A5 and B2, all of them able to bind to the receptor EphA3. WB and immunofluorescence confirmed the presence of phosphorylated protein in EphA3 in AP and BC CML cells. Western Blot and immunofluorescence confirmed the presence and the high level of phophorylation of EphA3 protein in AP and BC, but not in CP cells. EphA3 transfection into normal cells results in loss of adhesion, cell rounding and increased proliferation. Incubation of CML cells or transfected cells with a specific antibody able to block the EphA3 receptor induced a significant reduction of proliferation (31±4% of reduction in primary cells and 45±9% in transfected cells), it reduced colony growth (median value of 34,2 vs 76,5) and mainly, it changes the adhesion properties. The antibody did not induced any significant change of proliferation or colony growth in CP cells and normal controls. Finally, no kinase domain mutations were found in EphA3 overexpressing cells. In conclusion, our experimental data have shown that EphA3 was abnormally expressed in cells derived from advanced stage CML. The ectopic expression of EphA3, as demonstrated in our experimental system is responsible for increased proliferation and loss of cell adhesion. The inhibition of EphA3 induced by a specific antibody results in proliferation and colony growth inhibition. Although preliminary, our findings suggest that EphA3 may represent a potential candidate for a molecular therapy in advanced phase of chronic myeloid leukaemia. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

Anti-AML Activity of Combined Epigenetic Therapy with Novel DNMT1 Inhibitors SGI-110 or SGI-1036 and Histone Deacetylase Inhibitor Panobinostat.

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p15 and p16. DNA methyltransferase (DNMT) inhibitors 5-azacytidine and 5-aza-2‘-deoxycytidine demethylate the CpG dinucleotide islands in or near gene promoters, leading to derepression of TSGs in AML. SGI-110 (S110) (Cancer Res. 2007; 67:6400) and SGI-1036 (SuperGen, Inc.) are novel, DNMT inhibitors, which also deplete DNMT1 levels. SGI-110 is a dinucleotide containing 5-aza-2‘-deoxycytidine and SGI-1036 is a non-nucleoside heterocycle. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces trimethylation of histone H3 on lysine (K)-27. We recently reported that treatment with the pan-HDAC inhibitor panobinostat (LBH589, Novartis Pharmaceutical Corp) acetylates and inhibits the ATP binding and chaperone function of hsp90, as well as depletes the levels of EZH2, Suz12 and EED in cultured and primary AML cells (Mol Cancer Ther. 2006; 5:3096). Within the PRC2 complex, EZH2 was shown to interact with and modulate the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, which affects their binding to the EZH2-targeted gene promoters. In the present studies we determined the effects of SGI-110 or SGI-1036 and LBH589 on the PRC2 proteins EZH2 and SUZ12, and DNMT1, in the cultured (HL-60, OCI-AML3 and K562) and primary AML cells. Treatment with SGI-110 (0.5 to 2.0 μM) or SGI-1036 (0.5 and 1.0 μM) for 24 hours depleted protein levels of DNMT1 and EZH2 in the cultured and primary AML cells. SGI-110 and SGI-1036 promoted proteasomal degradation of DNMT1 and EZH2 since co-treatment with bortezomib significantly restored DNMT1 and EZH2 levels in the AML cells. Following treatment with SGI-110 or SGI-1036, bisulfite modification and methylation specific PCR demonstrated increase in unmethylated promoter DNA of p15 and JunB. This was associated with induction of the mRNA and protein levels of p15 and JunB, as well as caused inhibition of cell cycle progression (% of cells increased in G1 and increased in S phase) and colony growth in the soft agar. Treatment with 1.0 μM of SGI-110 or SGI-1036 also induced PARP cleavage activity of caspases and induced morphologic evidence of apoptosis in the AML cells. Co-treatment with 10 to 50 nM panobinostat enhanced SGI-110 or SGI-1036 mediated depletion of DNMT1 and EZH2, with more de-repression of the p15 and JunB and significant increase in apoptosis of AML cells. Collectively, these findings indicate that, SGI-110 and SGI-1036 deplete DNMT1 and EZH2 levels, as well as exert potent anti-AML activity. Additionally, combined epigenetic therapy consisting of SGI-110 or SGI-1036 in combination with panobinostat may represent a promising novel treatment of AML.

Effectiveness and molecular characterization of Burkholderia spinosa, a prospective biocontrol agent for controlling postharvest diseases of banana

Prospective antagonists for controlling Colletotrichum musae were isolated from the fructosphere of a range of dessert and cooking type banana varieties grown in Sri Lanka. Out of the isolated microorganisms, 44 bacterial and 31 fungal isolates showed more than 50% C. musae colony growth inhibition in vitro. Among 32 bacterial isolates which showed inhibition zones against C. musae under in vitro conditions, four isolates were highly effective in controlling banana anthracnose, crown rot and blossom end rot in vivo when artificially infected banana (variety Kolikuttu) were dipped in an antagonist suspension (10 8 cfu/ml) containing Tween 20 (0.02% v/v) for 5 min. Of these four, Burkholderia spinosa was used for further studies as a prospective biocontrol agent/biopesiticide. A suspension of B. spinosa (10 5 cfu/ml) containing Tween 20 (0.02% v/v) was effective in controlling anthracnose and blossom end rot of a range of dessert banana varieties (87–95% and 81–82% disease reductions, respectively), while a concentration of 10 4 cfu/ml with Tween 20 (0.02% v/v) was sufficient to have a 86–98% control of crown rot. B. spinosa showed 45–73% colony growth inhibition of C. gloeosporioides, Botryodiplodia theobromae and Thielaviopsis paradoxa isolates causing anthracnose and stem end rot of avocado, mango and pineapple in vitro. Significant in vivo control of these diseases in avocado, mango and pineapple was also achieved with antagonist concentrations ranging from 10 4 to 10 6 cfu/ml (28% reduction of mango anthracnose by 10 5 cfu/ml, 38% reduction of avocado anthracnose by 10 4 cfu/ml, 17.5% reduction of mango stem end rot by 10 5 cfu/ml, 14% reduction of avocado stem end rot by 10 6 cfu/ml and 34% reduction of pineapple stem end rot by 10 5 cfu/ml). Molecular analysis of recA gene by PCR-RFLP revealed a unique genomic identity for B. spinosa which discriminated it from human pathogenic isolates of Burkholderia. No antagonists were present in edible parts of banana treated with the most effective postharvest treatment as determined by the present study. Based on these findings, it is concluded that B. spinosa which is an indigenous antagonist is a promising candidate to be used in biological control of postharvest diseases of banana.

Alkalinisation does not enhance the antimicrobial properties of local anaesthetic solutions

ABSTRACTBackground: The purpose of the study was to examine the previously reported finding that the addition of bicarbonate to lignocaine enhanced the antimicrobial effect of the local anaesthetic agent on a range of bacteria implicated in epidural infections and to determine if this would also hold true for bupivacaine.Methods: Bupivacaine at a concentration of 0.25% and lignocaine at a concentration of 1% with and without an alkaline buffer were inoculated with suspensions of Staphylococcus epidermidis, Staphylococcus aureus or Escherichia coli. The mixtures were plated on 2% blood agar plates immediately (T = 0) and after 24 hours at room temperature (T = 24), and numbers of colonies were recorded.The difference in the number of colonies between T = 0 and T = 24 was expressed as a fraction of the T=0 value, giving a percentage inhibition of colony growth. Statistical testing was performed using Kruskal-Wallis non-parametric One Way Analysis of Variance (ANOVA) followed by multiple comparisons of mean ...

Epigenetic Inactivation of the Canonical Wnt Antagonist SRY-Box Containing Gene 17 in Colorectal Cancer

SRY-box containing gene 17 (Sox17) is a member of the high mobility group (HMG) transcription factor superfamily, which plays critical roles in the regulation of development and stem/precursor cell function, at least partly through repression of Wnt pathway activity. Modulators controlling aberrant Wnt signaling activation are frequently disrupted in human cancers through complementary effects of epigenetic and genetic changes. Our recent global analysis of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that SOX17 gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. Here, we report that CpG island methylation-dependent silencing of SOX17 occurs in 100% of CRC cell lines, 86% of colorectal adenomas, 100% of stage I and II CRC, 89% of stage III CRC, 89% of primary esophageal cancer, and 50% of non-small cell lung cancer. Overexpression of SOX17 in HCT116 CRC cells inhibits colony growth and beta-catenin/T-cell factor-dependent transcription. Structure-based deletion analysis further shows the presence of a Wnt signaling repression domain in the SOX17 HMG box. Together, our studies suggest that SOX17 is a negative modulator of canonical Wnt signaling, and that SOX17 silencing due to promoter hypermethylation is an early event during tumorigenesis and may contribute to aberrant activation of Wnt signaling in CRC.

In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH − groups and F − ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HA < FHA < FA. None of the biomaterials induced mutagenic effects compared with the positive control ( N-methyl- N′-nitro- N-nitrosoguanidine), and DNA breakage was probably the reason for the inhibition of cell division in V79 cell colonies.

Colony induction and growth inhibition in Desmodesmus quadrispina (Chlorococcales) by allelochemicals released from the filamentous alga Uronema confervicolum (Ulotrichales)

In biofilms, the competition between microorganisms for light, nutrients and space is extreme. Moreover, planktonic algae can be considered as competitors insofar as they decrease the available light for the benthic algae. One of the strategies employed by microorganisms to eliminate competitors is the release of inhibiting compounds, a process known as allelopathy. Here we demonstrate that a benthic/epiphytic alga, Uronema confervicolum, produces allelopathic compounds that induce oxidative stress and growth inhibition in the planktonic Desmodesmus quadrispina. Some of these compounds can also trigger the formation of colony in D. quadrispina. As colonies have higher sedimentation rates than unicells, their induction by U. confervicolum might decrease shading. This study is the first report of colony induction in the context of alga-alga interaction. Our results also suggest the implication of mitogen-activated protein (MAP) kinases in the transduction of the signal leading to the formation of reactive oxygen species in the cells. A comparison with allelochemicals from another planktonic green alga, Monoraphidium aff. dybowski, emphasizes the specificity of colony induction by U. confervicolum, in contrast with oxidative stress which is induced by several compounds. The reciprocal production of inhibiting compounds by D. quadrispina makes this interaction an interesting example of co-evolution between two microorganisms belonging to different compartments of the ecosystem.

Glioma Pathogenesis-Related Protein 1 Exerts Tumor Suppressor Activities through Proapoptotic Reactive Oxygen Species–c-Jun–NH2 Kinase Signaling

Glioma pathogenesis-related protein 1 (GLIPR1), a novel p53 target gene, is down-regulated by methylation in prostate cancer and has p53-dependent and -independent proapoptotic activities in tumor cells. These properties suggest an important tumor suppressor role for GLIPR1, yet direct genetic evidence of a tumor suppressor function for GLIPR1 is lacking and the molecular mechanism(s), through which GLIPR1 exerts its tumor suppressor functions, has not been shown. Here, we report that the expression of GLIPR1 is significantly reduced in human prostate tumor tissues compared with adjacent normal prostate tissues and in multiple human cancer cell lines. Overexpression of GLIPR1 in cancer cells leads to suppression of colony growth and induction of apoptosis. Mice with an inactivated Glipr1 gene had significantly shorter tumor-free survival times than either Glipr1(+/+) or Glipr1(+/-) mice in both p53(+/+) and p53(+/-) genetic backgrounds, owing to their development of a unique array of malignant tumors. Mechanistic analysis indicated that GLIPR1 up-regulation increases the production of reactive oxygen species (ROS) leading to apoptosis through activation of the c-Jun-NH(2) kinase (JNK) signaling cascade. Thus, our results identify GLIPR1 as a proapoptotic tumor suppressor acting through the ROS-JNK pathway and support the therapeutic potential for this protein.

Combined Effects of a Pan-Aurora Kinase Inhibitor MK-0457 and Dasatinib Against T315I Mutant Form of BCR-ABL: In Vitro and In Vivo Studies.

MK-0457 is a pan-Aurora kinase inhibitor that has firstly shown clinical activity in BCR-ABL positive leukemia patients harboring the T315I mutation. Combining MK-0457 with ABL kinase inhibitors may provide several advantages, including enhanced efficacy and the potential to reduce the emergence of new resistant mutations. In the present study, we investigated the combined effects of MK-0457 and dasatinib in T315I mutant-expressing cells. Co-treatment with MK-0457and dasatinib caused significantly more inhibition of colony growth of primary leukemia cells and BaF3 cells expressing T315I BCR-ABL than either drug alone. In contrast, we did not observe the enhanced effects of MK-0457 and imatinib in T315I BCR-ABL-expressing cells. Treatment with 5 μM of MK-0457 for 48 hrs induced apoptosis in T315I BCR-ABL BaF3 cells, whereas, exposure to combination with 1.0 μM of MK-0457 and 50 nM of dasatinib exerted enhanced apoptotic effect. Combined treatment with MK-0457 and dasatinib also associated with more PARP cleavage, which is due to increased activation of caspase-3 and caspase-9 during apoptosis. Following co-treatment with MK-0457 and dasatinib caused more attenuation of the level of phospho-Stat5 and the downstream signal transducer, including Bcl-XL, Mcl-1, and cyclin D1. We also observed that combined treatment with MK-0457 and dasatinib enhanced the activation of p38MAP kinase and MAPKAP Kinase-2 (MK2). Pretreatment with p38MAP kinase or MP2 siRNA in T315I BCR-ABL BaF3 cells reduced the induction of apoptosis after MK-0457 and dasatinib exposure. These results suggest that p38MAP kinase and MK2 have a critical role for the induction of apoptosis after MK-0457 and dasatinib exposure. To assess the mechanism of combination effect between MK-0457 and dasatinib on T315I BCR-ABL-expressing cells, we used RNA interference to determine whether reduction of SRC-family kinases affected the growth inhibition. BaF3 cells expressing T315I BCR-ABL pretreated with Lyn or Hck siRNA showed enhanced growth inhibition with MK-0457. These results demonstrate that the enhanced growth inhibition by MK-0457 and dasatinib in T315I-expressing cells may be mediated by Lyn and Hck. To assess the in vivo efficacy of MK-0457 and dasatinib, athymic nude mice were injected i.v. with BaF3 cells expressing T315I mutant form of BCR-ABL. 24 hrs after injection, the mice were divided four groups (5 mice per group), with each group receiving either vehicle, MK-0457 (30mg/kg b.i.d.; ip for 5 days), dasatinib (10mg/kg q.d.; po for five days), MK-0457 (30mg/kg b.i.d.; ip for 5 days) + dasatinib (10mg/kg q.d.; po for five days). The treatment was repeated in every 3 weeks. The vehicle or dasatinib-treated mice died of a condition resembling acute leukemia by 28 days; the MK-0457 only-treated mice survived more than 56 days, and the combination of MK-0457 + dasatinib -treated mice survived more than 70 days. Histopathologic analysis of vehicle or dasatinib-treated mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, histopathologic analysis of organs from MK-0457 plus dasatinib-treated mice demonstrated normal tissue architecture and no evidence of residual leukemia. Taken together, the present study shows that the combination of MK-0457 and dasatinib exhibits a desirable therapeutic index that can reduce the in vivo growth of T315I mutant form of BCR-ABL-expressing cells in an efficacious manner.

Primary Cell Experiments with TG101348, a JAK2-Selective Inhibitor, in the Presence of Myeloproliferative Disorder-Associated JAK2V617F, MPLW515L/K, and JAK2 Exon 12 Mutations.

Introduction: Several groups are developing orally bioavailable small molecule JAK2 inhibitors as an approach to targeted therapy of myeloproliferative disorders. We previously reported that one such inhibitor (TG101209) potently inhibits JAK2V617F in vitro, and was effective in reducing tumor growth in a mouse xenograft model (Leukemia, 2007 Aug;21(8):1658–68). Here, we expand our analysis using TG101348, a highly selective JAK2 inhibitor currently in preclinical development, using cell lines and primary cells harboring JAK2V617F, MPLW515L/K, or JAK2 exon 12 mutations.Methods: Cell proliferation was assessed using the XTT assay. For primary cell experiments, PBMCs were plated in methylcellulose medium at 0, 300nM, 600nM, and 1200nM TG101348, both with and without erythropoietin (Epo). Hematopoietic colonies were scored after 12–16 days culture, and harvested for genotyping.Results: TG101348 inhibits growth of human erythroleukemia (HEL) and Ba/F3-V617F cells (IC50=300nM and 580nM, respectively), as well as Ba/F3-(MPL)W515L cells (IC50=620nM). JAK2:JAK3 selectivity of TG101348 was demonstrated by its 10-fold more potent inhibition of CHRF (JAK2T875N) as compared to CMK (JAK3A572V) cell proliferation. TG101348's inhibitory effect on ex vivo hematopoietic colony growth was studied in 12 MPD patients (JAK2V617F=9, MPLW515L/K=1, JAK2 exon 12 mutation [N542-E543del]=1, no mutation=1), and 2 healthy controls (Table).Conclusions: (1) TG101348 inhibits colony growth from MPD patients more potently than healthy controls, reflecting its potential therapeutic window. (2) Endogenous colony growth is inhibited at nanomolar concentrations in all cases, with some alleles showing greater sensitivity than others (MPLW515K = exon 12 mutation > JAK2V617F). The corresponding Epo-supported erythroid colonies are less potently inhibited, reflecting the generally lower prevalence of JAK2 mutations in such colonies. (3) EEC growth in the absence of known JAK2 or MPLW515 mutations is also inhibited, indicating a similar dependence on dysregulated JAK-STAT signaling in such cases. (4) The ability of TG101348 to selectively suppress mutation-positive hematopoietic colonies will be discussed.Inhibitor effect on ex vivo hematopoietic colony growthPaientMutationIC50 (nM)% Mutation-positive coloniesEpoNo EpoEpoNo Epo (EEC)Normal 1None>1200n/an/an/aNormal 2None>1200n/an/an.aPV1V617F600<300540PV2V617F6003003350PV3V617F600–1200300–600970PV4V617F300–600600015PV5V617F600–120030080100PV6V617F600300–600719PV7V617F600–12006003236PV8V617F600–1200<3004537PV9V617F600600–12002012PV10Exon 12 (N542–E543del)900<300960PMF1W515K300–600<30010033PMF2None>1200300n/an/a