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Sample records for agents hairy cell

  1. 75 FR 14391 - Diseases Associated With Exposure to Certain Herbicide Agents (Hairy Cell Leukemia and Other...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-25

    ... Cell Leukemia and Other Chronic B Cell Leukemias, Parkinson's Disease and Ischemic Heart Disease... between exposure to herbicides and the subsequent development of hairy cell leukemia and other chronic B- cell leukemias, Parkinson's disease, and ischemic heart disease. The intended effect of this...

  2. 75 FR 54496 - Diseases Associated With Exposure to Certain Herbicide Agents (Hairy Cell Leukemia and Other...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-08

    ... Federal Register (75 FR 53202), an amendment to 38 CFR 3.309 to add hairy cell leukemia and other chronic B-cell leukemias, Parkinson's disease and ischemic heart disease to the list of diseases subject to... Cell Leukemia and Other Chronic B-Cell Leukemias, Parkinson's Disease and Ischemic Heart...

  3. Hairy Cell Leukemia Treatment Option Overview

    MedlinePlus

    ... ALL Treatment Childhood AML Treatment Research Hairy Cell Leukemia Treatment (PDQ®)–Patient Version General Information About Hairy Cell Leukemia Go to Health Professional Version Key Points Hairy ...

  4. General Information About Hairy Cell Leukemia

    MedlinePlus

    ... Hairy Cell Leukemia Treatment (PDQ®)–Patient Version General Information About Hairy Cell Leukemia Go to Health Professional ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  5. [Lymphoid myelofibrosis or hairy cell leukemia].

    PubMed

    Lovisetto, P; Pellegrino, P; Tallone, M V; Biarese, V; La Rosa, G F

    1977-05-26

    By lymphoid myelofibrosis or hairy cell leukaemia or tricholeukaemia is meant an unusual haemopathic condition known only for the past few years. It is characterized pathognomonically by the presence of lymphocyte type cells with villous extroflexions, hence the name "hairy cell". Clinically the disease presents as an involutive myelopathy associated with splenomegaly, generally without any particular lymph gland involvement. The attention of students today is concentrated on the nature of the hairy cells; while some are inclined to admit their monocyte or histiocyte derivation, others consider that they derive from B lymphocytes. Therapeutically, almost everybody agrees that splenectomy is the only valid step. A case of H.C.L., which was typical from the clinical and laboratory viewpoints is reported. It is probable that certain haemopathic pictures once classified among atypical leucoses and lymphomas, would today be more correctly classed as hairy cell leukaemia. PMID:327348

  6. Eliminating Hairy Cell Leukemia Minimal Residual Disease

    Cancer.gov

    In this trial, patients with hairy cell leukemia who have disease-related symptoms that require treatment will be randomly assigned to receive cladribine with either concurrent rituximab or rituximab at least 6 months after completing cladribine therapy.

  7. Immunotoxin Therapy for Relapsed Hairy Cell Leukemia

    Cancer.gov

    In this trial, patients with hairy cell leukemia who have relapsed multiple times or not responded to prior chemotherapy will be treated with an experimental immunotoxin called moxetumomab pasudotox given intravenously on days 1, 3, and 5 of 28-day cycles

  8. Hairy cell leukemia: clinical features and therapeutic advances.

    PubMed

    Lembersky, B C; Golomb, H M

    1987-01-01

    Hairy cell leukemia (HCL) is a rare chronic lymphoproliferative disorder which has been extensively studied over the past decade. Much has been learned regarding the diagnosis, natural history, biology, and treatment of this unique neoplasm. The disease most commonly affects middle aged men and characteristic clinical features include splenomegaly, cytopenias, and usually the presence in the peripheral blood of distinctive 'hairy cells' with irregular cytoplasmic projections. Diagnosis can usually be confirmed by bone marrow biopsy. Although the natural history can be extremely variable among patients, complications are usually referable to the cytopenias, with anemia and infection being most frequent. In addition to pyogenic infections, patients are susceptible to unusual organisms including atypical mycobacterium, legionella, and fungi. The requirement of red blood cell transfusion, severe granulocytopenia or thrombocytopenia, frequent infections, or painful splenomegaly are all indications for treatment. Splenectomy is the standard initial treatment of choice. However, in the past few years there have been exciting major advances in the therapeutic modalities for HCL. Recombinant alpha-interferon is highly effective, with beneficial responses occurring in close to 90% of patients. The Food and Drug Administration has recently approved the use of interferon for HCL. This represents the first time a biological response modifier has been approved for the treatment of human disease. In addition, preliminary results with the adenosine deaminase inhibitor, 2'deoxycoformycin (dcf), have been encouraging. Further clinical trials are required in order to determine the optimal sequential treatment strategy for HCL. The exact mechanisms of action of both interferon and dcf in HCL remain to be elucidated. A better understanding of the unusual features of the hairy cell and the underlying biological effect of these two agents in HCL may have important applications in other

  9. Treating Multiply Relapsed or Refractory Hairy Cell Leukemia

    Cancer.gov

    In this trial, patients with hairy cell leukemia who have not responded or relapsed after initial chemotherapy will be randomly assigned to receive rituximab combined with either pentostatin or bendamustine.

  10. A Unique Hairy Cell Leukemia Variant.

    PubMed

    Jian, Charles; Hsia, Cyrus C

    2016-01-01

    A 65-year-old woman presented with easy bruising, left upper quadrant pain, decreased appetite, and weight loss. She had splenomegaly and lymphocytosis (lymphocyte count of 11.6 × 10(9)/l), with remarkably abnormal appearing morphology. Her hemoglobin and platelet counts were normal. Peripheral blood flow cytometry revealed a monoclonal B-cell population expressing CD11c, CD25, CD19, CD20, and CD103. An initial diagnosis of hairy cell leukemia (HCL) was made, and the patient was treated with a standard 5-day course of cladribine. However, her lymphocytosis improved transiently, with a relapse 4 months later. There was no improvement in her splenomegaly. An HCL variant (HCL-v) was considered based on her resistance to treatment with a purine nucleoside analog. A subsequent splenectomy improved symptoms. Two years after, the patient suffered a relapse and underwent 6 cycles of CHOP-R (cyclophosphamide, hydroxydaunomycin, oncovin, prednisone, and rituximab), achieving partial remission. While under observation, she progressed with lymphocytosis 6 months later and was treated with pentostatin. There was no significant improvement in her disease, and she died 8 weeks following treatment initiation. HCL-v is a clinically more aggressive mature B-cell lymphoma than HCL with worse splenomegaly, higher lymphocyte counts, and resistance to typical HCL therapy with purine nucleoside analogs. Early recognition of HCL-v in the history, physical examination, and investigations with morphology and flow cytometry is key to patient management. Further, as in our case of HCL-v, cell morphology can be distinctly atypical, with large nucleoli and extremely convoluted nuclei. The distinction between HCL and HCL-v is important as HCL-v patients require more aggressive therapy and closer follow-up. PMID:27462230

  11. A Unique Hairy Cell Leukemia Variant

    PubMed Central

    Jian, Charles; Hsia, Cyrus C.

    2016-01-01

    A 65-year-old woman presented with easy bruising, left upper quadrant pain, decreased appetite, and weight loss. She had splenomegaly and lymphocytosis (lymphocyte count of 11.6 × 109/l), with remarkably abnormal appearing morphology. Her hemoglobin and platelet counts were normal. Peripheral blood flow cytometry revealed a monoclonal B-cell population expressing CD11c, CD25, CD19, CD20, and CD103. An initial diagnosis of hairy cell leukemia (HCL) was made, and the patient was treated with a standard 5-day course of cladribine. However, her lymphocytosis improved transiently, with a relapse 4 months later. There was no improvement in her splenomegaly. An HCL variant (HCL-v) was considered based on her resistance to treatment with a purine nucleoside analog. A subsequent splenectomy improved symptoms. Two years after, the patient suffered a relapse and underwent 6 cycles of CHOP-R (cyclophosphamide, hydroxydaunomycin, oncovin, prednisone, and rituximab), achieving partial remission. While under observation, she progressed with lymphocytosis 6 months later and was treated with pentostatin. There was no significant improvement in her disease, and she died 8 weeks following treatment initiation. HCL-v is a clinically more aggressive mature B-cell lymphoma than HCL with worse splenomegaly, higher lymphocyte counts, and resistance to typical HCL therapy with purine nucleoside analogs. Early recognition of HCL-v in the history, physical examination, and investigations with morphology and flow cytometry is key to patient management. Further, as in our case of HCL-v, cell morphology can be distinctly atypical, with large nucleoli and extremely convoluted nuclei. The distinction between HCL and HCL-v is important as HCL-v patients require more aggressive therapy and closer follow-up. PMID:27462230

  12. The importance of the tissue microenvironment in hairy cell leukemia.

    PubMed

    Sivina, Mariela; Burger, Jan A

    2015-12-01

    Hairy cell leukemia (HCL) cells engage in complex cellular and molecular interactions with accessory cells, matrix proteins, and various cytokines in the bone marrow and spleen, collectively referred to as the tissue microenvironment. Chemokine receptors and adhesion molecules are critical players for homing and retention within these microenvironments. Engagement of B cell antigen receptors and CD40 on HCL cells promote survival and proliferation. In this chapter, we summarize the current knowledge about the cellular and molecular interactions between HCL cells and their supportive tissue microenvironment, and provide insight into new therapeutic approaches targeting B cell receptor signaling in HCL. PMID:26614899

  13. Skeletal complications in hairy cell leukemia: diagnosis and therapy.

    PubMed

    Lembersky, B C; Ratain, M J; Golomb, H M

    1988-08-01

    We identified eight patients with skeletal complications associated with hairy cell leukemia (HCL). The median time from diagnosis of HCL to the diagnosis of skeletal complications was 20 months (range, 0 to 93). All patients complained of pain and all but one lesion were located in the axial skeleton, primarily the proximal femur. Lytic lesions were seen on radiographic examination in all but one patient, and one patient additionally had multiple osteoporotic vertebral compression fractures. Radionuclide technetium bone scan was abnormal in all patients examined. Although the peripheral blood counts were variable (only two patients had a leukemic phase of the disease), all patients examined had a hypercellular bone marrow biopsy with hairy cells comprising at least 90% of the hematopoietic elements. The skeletal abnormalities responded well to local radiation therapy. Seven patients were begun on systemic therapy with interferon alpha-2b after the diagnosis of the skeletal lesion. Four of five evaluable patients had a partial hematological response and a substantial improvement in the degree of hairy cell infiltration of the bone marrow. None of these patients has had a recurrence of skeletal complications at a median follow-up time of 29 months. One patient failed to respond hematologically and developed additional bone lesions after 1 year of treatment. Another patient developed a new skeletal lesion 3 months after the cessation of interferon therapy at which time the bone marrow was essentially packed with hairy cells. This retrospective study indicates that bone involvement is a rare complication of HCL and is associated with a high tumor burden in the bone marrow. In addition to local radiation therapy, systemic treatment with interferon should be considered. PMID:3411340

  14. Radiation exposure as a possible etiologic factor in hairy cell leukemia (leukemic reticuloendotheliosis)

    SciTech Connect

    Stewart, D.J.; Keating, M.J.

    1980-10-01

    The frequency of prior occupational, accidental, or therapeutic radiation exposure was significantly higher for hairy cell leukemia patients than for a control group of solid tumor patients. Hairy cell leukemia patients were also more frequently involved in occupations at high risk of radiation exposure such as chemist, engineer, physicist, and health care facility worker. The observation that the incidence of thyroid disorders among hairy cell leukemia patients was also unusually high was interpreted as further indirect evidence of excessive radiation exposure. It appears that radiation exposure may be an important contributing factor in the development of some cases of hairy cell leukemia.

  15. Food-grade chemical and biological agents permeabilize red beet hairy roots, assisting the release of betalaines.

    PubMed

    Thimmaraju, R; Bhagyalakshmi, N; Narayan, M S; Ravishankar, G A

    2003-01-01

    Hairy root cultures of red beet, Beta vulgaris L., were permeabilized under the functions of food-grade chemical and biological agents cetyl trimethylammonium bromide (CTAB), Triton X-100, Tween-80, Lactobacillus helveticus, Saccharomyces cereviseae, and Candida utilis, as well as cell fractions of L. helveticus, for the recovery of betalaines with or without oxygen stress. Tween-80 (0.15%), Triton X-100 (0.2%), and CTAB (0.05%), in combination with oxygen stress, released 45%, 70%, and 90% pigment into the medium, respectively, with significantly lesser levels in agitated cultures receiving similar treatments. The release was rapid (1 h) in CTAB treatment with a much slower release in Tween-80. CTAB (0.002%) was found to be also useful in effluxing betalaines (80%) from hairy roots grown in a bubble column reactor. Viability of permeabilized hairy roots, tested on agar medium, was not affected by any level of CTAB treatment and was significantly retarded at higher levels of Triton X-100 and Tween-80. An altogether new approach of pigment release using biological agents such as live cells of food-grade microbes was used where C. utilis, L. helveticus, and S. cereviseae released 60%, 85%, and 54% betalaines, respectively, in 24 h, though lower level treatments also released similar levels of pigment by 48 h. Dried whole cell powder of L. helveticus, its total insoluble carbohydrate, and free lipid fractions released 10%, 0%, and 85% pigment, respectively. An extended study with a bubble column reactor using the free lipid fraction of L. helveticus showed 50% and 84% pigment release in 8 and 12 h, respectively, exhibiting good viability when plated on agar medium. Even in the bioreactor, replenishment of medium 8 h after treatment with free lipid of L. helveticus allowed regrowth of hairy roots. The high level of pigment release recorded here, using CTAB or lipid of L. helveticus, appears useful for developing processes for in situ recovery of betalaines. The live

  16. Bone marrow and splenic histology in hairy cell leukaemia.

    PubMed

    Wotherspoon, Andrew; Attygalle, Ayoma; Mendes, Larissa Sena Teixeira

    2015-12-01

    Hairy cell leukaemia is a rare chronic neoplastic B-cell lymphoproliferation that characteristically involves blood, bone marrow and spleen with liver, lymph node and skin less commonly involved. Histologically, the cells have a characteristic appearance with pale/clear cytoplasm and round or reniform nuclei. In the spleen, the infiltrate involves the red pulp and is frequently associated with areas of haemorrhage (blood lakes). The cells stain for B-cell related antigens as well as with antibodies against tartrate-resistant acid phosphatase, DBA44 (CD72), CD11c, CD25, CD103, CD123, cyclin D1 and annexin A1. Mutation of BRAF -V600E is present and antibody to the mutant protein can be used as a specific marker. Bone marrow biopsy is essential in the initial assessment of disease as the bone marrow may be inaspirable or unrepresentative of degree of marrow infiltration as a result of the tumour associated fibrosis preventing aspiration of the tumour cell component. Bone marrow biopsy is important in the assessment of therapy response but in this context staining for CD11c and Annexin A1 is not helpful as they are also markers of myeloid lineage and identification of low level infiltration may be obscured. In this context staining for CD20 may be used in conjunction with morphological assessment and staining of serial sections for cyclin D1 and DBA44 to identify subtle residual infiltration. Staining for CD79a and CD19 is not recommended as these antibodies will identify plasma cells and can lead to over-estimation of disease. Staining for CD20 should not be used in patients following with anti-CD20 based treatments. Down regulation of cyclin D1 and CD25 has been reported in patients following BRAF inhibitor therapy and assessment of these antigens should not be used in this context. Histologically, hairy cell leukaemia needs to be distinguished from other B-cell lymphoproliferations associated with splenomegaly including splenic marginal zone lymphoma, splenic

  17. Epidemiology and environmental risk in hairy cell leukemia.

    PubMed

    Tadmor, Tamar; Polliack, Aaron

    2015-12-01

    Hairy cell leukaemia (HCL) is an orphan subtype of leukaemia which constitutes less than 2% of all leukaemia's, with an incidence of less than 1 per 100,000 persons per annum. Median age at presentation is 55 years and it is 3-4 times more frequent in males. It is also more frequently encountered in whites and less in Asians, Africans and Arabs. The epidemiologic data are multi-factorial and influenced by ethnicity and geographical factors. Other reported associations relate to some environmental exposures and possible occupational factors. Smoking appears to have an inverse correlation with the development of hairy cell leukaemia, while farming and exposure to pesticides, petroleum products, diesel and ionizing radiation have also been reported to be associated with an increased risk. National and international collaborative efforts are needed in order to undertake more extensive studies involving larger patient cohorts, aiming to determine the role of occupational and environmental risk factors in the development of this rare form of chronic leukaemia.

  18. [2 cases of hairy cell leukemia].

    PubMed

    Moda, S; Ceriani, A; Caretta, E

    1981-01-01

    Two cases of tricholeukaemia are reported. In both, onset and course of the disease were dominated by infectious episodes of varying gravity. Diagnosis was based on the finding in the blood and bone marrow of mononucleate cells containing the characteristic cytoplasmatic projections, best recognised in the phase contrast microscope, and on the cytochemical finding of intense positivity of the acid phosphatase reaction, that a very serious septic state starting from a dental abscess was possible in a patients by associating infusions of paps of leucocyte concentrates with massive target antibiotic therapy. Splenectomy carried out in the same patient led to an increase in the number of circulating platelets and leukocytes. After operation, two episodes of cutaneous inflammation presented by the same patient were less serious than similar previous infectious episodes.

  19. [The presence of an endogenous peroxidase activity in hairy cell leukemia cells].

    PubMed

    Reyes, F; Gourdin, M F; Farcet, J P; Dreyfus, B; Breton-Gorius, J

    1977-02-01

    Mononuclear cells from hairy cell leukemia have been studied in three cases by ultrastructural immunocytochemistry. Cells have fairly detectable surface immunoglobulins, without monoclonal distribution however. In addition these cells have a peroxidatic activity which is revealed in the perinuclear space and strands of endoplasmic reticulum. PMID:404081

  20. Hairy cell leukemia: enzyme-histochemical and ultrastructural investigation of one case.

    PubMed

    Pilotti, S; Carbone, A; Lombardi, L; Tavolato, C; Rilke, F

    1978-10-31

    The investigation was carried out on blood smears, bone marrow aspirates, one lymph node biopsy, and the surgically removed spleen of a 53-year-old man with hairy cell leukemia. In the blood smears stained with May-Grünwald-Giemsa, 60 to 70% of the hairy cells contained tubular inclusions that corresponded to the ribosome-lamella complexes demonstrated at electron microscopy. In blood smears, imprints and cryostatic sections of the lymph node and of the spleen, hairy cells revealed tartrate-resistant acid phosphatase, beta-glucuronidase and adenosine-triphosphatase activity. In the spleen neutral esterase and alkaline phosphatase demonstrated the numerical increase of the histiocytes, which ultrastructurally displayed phagocytic activity. The presence in the spleen of pseudosinuses lined by hairy cells was confirmed by electron microscopy as well as by cytoenzymology.

  1. CD27-positive hairy cell leukemia-Japanese variant.

    PubMed

    Tabata, Rie; Tabata, Chiharu; Iwama, Hideaki; Yasumizu, Ryoji; Kojima, Masaru

    2016-03-01

    We report a very rare case of a 45-year-old Japanese male patient with hairy cell leukemia-Japanese variant (HCL-JV) expressing CD27. The patient showed a high number of abnormal peripheral lymphocytes, thrombocytopenia, and severe splenomegaly but no lymphadenopathy. Histology of the resected spleen showed small-sized lymphoma cells diffusely infiltrating the red pulp without follicle formation. By immunohistochemistry, lymphoma cells were negative for CD3, CD5, CD8, CD10, CD34, cyclin-D1, and annexin A1 but positive for CD20 and BCL2. BRAF V600E mutation was not observed. Bone marrow aspirate showed preserved normal hematopoietic cells with invasion of lymphoma cells in an interstitial pattern without obvious nodules. The cells had abundant pale cytoplasm and round nuclei with inconspicuous nucleoli. After natural drying, the cells had unevenly distributed microvilli. Flow cytometric analysis demonstrated positivity for CD11a, CD11c, CD19, CD20, CD22, CD27, surface IgG, and λ but not for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD21, CD23, CD25, CD30, CD34, CD38, CD43, CD56, CD57, CD103, IgD, IgM, and κ. Monoclonal expansion of B cells was confirmed by an immunoglobulin heavy chain (IgH) rearrangement band as demonstrated by Southern blot hybridization. The lymphoma cells had unevenly distributed long, large, and broad-based microvilli, which resembled splenic diffuse red pulp small B cell lymphoma (SDRPL) cells. CD27 expression is extremely rare in HCL-JV, but the young age of the patient and high peripheral WBC counts were similar to HCL-JV, which suggests, in this case, an intermediate disease between SDRPL and HCL-JV. PMID:26868143

  2. Synchronous gastric and ampullary adenocarcinomas in a hairy cell leukemia patient treated with pentostatin eight years prior.

    PubMed

    Senatore, Frank J; Dasanu, Constantin A

    2016-06-01

    Hairy cell leukemia patients are at increased risk for second malignancies, including both solid and lymphoid neoplasms. Along with other factors, multiple immune defects present in hairy cell leukemia likely contribute to subsequent carcinogenesis. We report herein a case of synchronous high-grade gastric and ampullary adenocarcinomas in a patient with a history of hairy cell leukemia treated eight years prior with pentostatin. We include a review of immune alterations induced by both hairy cell leukemia and its therapies, and link them with the occurrence of second cancers in these patients. PMID:25712625

  3. Hairy cell leukaemia and occupational exposure to benzene.

    PubMed Central

    Clavel, J; Conso, F; Limasset, J C; Mandereau, L; Roche, P; Flandrin, G; Hémon, D

    1996-01-01

    OBJECTIVES: The role of occupational exposures in hairy cell leukaemia (HCL) was investigated through a multicentre, hospital based, case-control study. This paper analyses the role of exposure to benzene in HCL. METHODS: A population of 226 male cases of HCL and 425 matched controls were included in the study. Benzene exposure was evaluated by expert review of the detailed data on occupational exposures generated by case-control interviews. RESULTS: No association was found between HCL and employment in a job exposed to benzene (odds ratio (OR) 0.9 (95% confidence interval (95% CI) 0.6-1.3)). The sample included 125 subjects, 34 cases (15%), and 91 controls (21%) who had been exposed to benzene, as individually assessed by the experts, for at least one hour a month during one of their jobs. Benzene exposure was not associated with a risk of HCL (OR 0.8 (0.5-1.2)). No trend towards an increase in OR was detected for increasing exposures, the percentage of work time involving exposure to > 1 ppm, or the duration of exposure. No findings suggested a particular risk period, when the OR associated with the time since first or last exposure, or since the end of exposure, were examined. CONCLUSIONS: In conclusion, with the low exposures prevalent in the sample, the study did not show any association between benzene exposure and HCL. PMID:8983464

  4. Persistent Legionnaire's disease in an adult with hairy cell leukemia successfully treated with prolonged levofloxacin therapy.

    PubMed

    Cunha, Burke A; Munoz-Gomez, Sigridh; Gran, Arthur; Raza, Muhammad; Irshad, Nadia

    2015-01-01

    Legionnaire's disease (LD) manifests most commonly as an atypical community acquired pneumonia (CAP) with systemic extrapulmonary manifestations. Disorders associated with impaired cell mediated immunity (CMI) are particularly predisposed to LD. Hairy cell leukemia (HCL) is a rare B-cell lymphoproliferative leukemia associated with decreased CMI. LD has only rarely been reported in HCL. We present a most interesting case of persistent LD in a elderly male with HCL who required prolonged antibiotic therapy.

  5. Phenotype study of fresh and cultured hairy cells with the use of immunologic markers and electron microscopy.

    PubMed

    Divine, M; Farcet, J P; Gourdin, M F; Tabilio, A; Vasconcelos, A; Andre, C; Jouault, H; Bouguet, J; Reyes, F

    1984-08-01

    The phenotype of fresh and cultured leukemic cells from patients with hairy cell leukemia was studied using a panel of monoclonal antibodies in addition to the detection of peroxidase activity under electron microscopy. In fresh samples, the leukemic cells from 11 patients displayed predominantly a B phenotype, as judged by their reactivity with the B1 monoclonal antibody and surface immunoglobulin expression. Ultrastructural peroxidase activity, characteristic of hairy cells, was observed in all cases studied. When hairy cells were cultured in the presence of phytohemagglutinin and irradiated T cells, their phenotype converted from surface Ig+, B1+, OKT3-, OKT11- to surface Ig-, B1+, OKT3-, OKT11+. In contrast, the peroxidase activity remained unchanged. Some hairy cells were also OKM1+, but no conclusion could be made about the MO2 antigen, a more specific marker of monocytes. The variability of the phenotype in vivo and in vitro indicates that reliable markers are required for identifying hairy cells. When studied together, the staining by B1 monoclonal antibody and the ultrastructural detection of peroxidase, enable the identification of hairy cells with certainty. PMID:6378279

  6. Incidental Detection of Hairy Cell Leukaemia with Herpes Simplex Virus (HSV) Related Lip Ulcer Mimicking Carcinoma.

    PubMed

    Agrawal, Pallavi; Bhartiya, Richa; Singh, Ran Vijoy Narayan

    2016-08-01

    Hairy cell leukemia is a chronic lympho-proliferative disease. It is indolent but progressive in nature. It arises from B-cell lineage. We report an incidentally detected case of Hairy Cell Leukaemia (HCL) in a 55-year-old male patient with Herpes simplex virus (HSV) - related lip ulcer mimicking squamous cell carcinoma. Clinically the patient presented with lip ulceration without pain. He was found to have moderate hepatosplenomegaly and pancytopenia on general examination. Bone marrow aspiration and flow cytometric immunophenotyping revealed HCL. The oral lesion resolved after antiviral therapy. The intriguing possibility of a combined pathogenesis for the two disorders is considered, as HCL is known to be associated with immunosuppression, second malignancies and the production of cytokines promoting epithelial growth. PMID:27656454

  7. Leukemic meningitis in a patient with hairy cell leukemia. A case report

    SciTech Connect

    Wolfe, D.W.; Scopelliti, J.A.; Boselli, B.D.

    1984-09-15

    Central nervous system involvement has not previously been described in patients with hairy cell leukemia (HCL). A patient is reported who presented with meningeal involvement as his initial symptom of HCL. Diagnosis was established by morphologic and cytochemical studies of his cerebrospinal fluid (CSF) and bone marrow. Treatment with whole-brain irradiation and intrathecal chemotherapy was successful in clearing leukemic cells from the CSF with resolution of symptoms.

  8. Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture.

    PubMed

    Farcet, J P; Gourdin, M F; Testa, U; Andre, C; Jouault, H; Reyes, F

    1983-01-01

    Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes. PMID:6601222

  9. Modeling hairy root tissue growth in in vitro environments using an agent-based, structured growth model.

    PubMed

    Lenk, Felix; Sürmann, Almuth; Oberthür, Patrick; Schneider, Mandy; Steingroewer, Juliane; Bley, Thomas

    2014-06-01

    An agent-based model for simulating the in vitro growth of Beta vulgaris hairy root cultures is described. The model fitting is based on experimental results and can be used as a virtual experimentator for root networks. It is implemented in the JAVA language and is designed to be easily modified to describe the growth of diverse biological root networks. The basic principles of the model are outlined, with descriptions of all of the relevant algorithms using the ODD protocol, and a case study is presented in which it is used to simulate the development of hairy root cultures of beetroot (Beta vulgaris) in a Petri dish. The model can predict various properties of the developing network, including the total root length, branching point distribution, segment distribution and secondary metabolite accumulation. It thus provides valuable information that can be used when optimizing cultivation parameters (e.g., medium composition) and the cultivation environment (e.g., the cultivation temperature) as well as how constructional parameters change the morphology of the root network. An image recognition solution was used to acquire experimental data that were used when fitting the model and to evaluate the agreement between the simulated results and practical experiments. Overall, the case study simulation closely reproduced experimental results for the cultures grown under equivalent conditions to those assumed in the simulation. A 3D-visualization solution was created to display the simulated results relating to the state of the root network and its environment (e.g., oxygen and nutrient levels). PMID:24218303

  10. Synthesis of a peroxidase activity by the cells of hairy cell leukemia: a study by ultrastructural cytochemistry.

    PubMed

    Reyes, F; Gourdin, M F; Farcet, J P; Dreyfus, B; Breton-Gorius, J

    1978-09-01

    The nature of cells present in the blood, marrow, and spleen of patients with hairy cell leukemia is largely debated. These cells have been tentatively categorized on the basis of either monocytic or lymphocytic markers, and the accumulating data points to the fact that they share some characteristics of both cell types. Although hairy cells are known to lack myeloperoxidase-positive granules, present in normal human monocytes, we investigated the possible presence of other peroxidase activities differing from the granule-bound myeloperoxidase. The study was carried out with several methods based on the incubation of fixed and unfixed cells in the presence of diaminobenzidine and hydrogen peroxide. A peroxidase activity was found in hairy cells, located always in the endoplasmic reticulum but not in the Golgi apparatus or in any granule. By its cytochemical characteristics it appears to be closely related to that of tissue macrophages, activated blood monocytes, and other nonlymphocytic hematopoietic cells. This peroxidase is not found in lymphocytes with B or T phenotypes. PMID:678670

  11. Hairy Cell Leukemia Presenting with Duodenal Involvement Duodenum: A Case Report

    PubMed Central

    Sen, Parijat; Shaaban, Hamid; Modi, Tejas; Kumar, Abhishek; Guron, Gunwant

    2015-01-01

    Context: A rare case of adult hairy cell leukemia (HCL) with duodenal involvement is presented. Case Report: The patient was a 48-year-old man, who had a history of hairy cell leukemia. Three days after completion of 2-chlorodeoxyadenosine (CDA) chemotherapy, the patient started experiencing abdominal pain. An extensive gastroenterological workup culminated in the patient getting an esophagogastroduodenoscopy (EGD) that revealed duodenal inflammation and biopsies were taken. The duodenal biopsy was positive for chronic inflammatory infiltrate, primarily consisting of atypical lymphocytes and plasma cells with tartrate-resistant acid phosphatase (TRAP) positivity, and hence a diagnosis of duodenal involvement with HCL was made. Repeat bone marrow biopsy done 2 weeks after finishing chemotherapy revealed residual disease. At the 3-month follow-up, the patient was asymptomatic with a normocellular marrow and no residual disease. Repeat abdomen computerized tomography (CT) scan at completion of therapy showed resolution of duodenal thickening and spleen size of 12 cm. Currently, patient is in clinical remission for 6 years with 4-6 monthly follow-up visits and continues to do well. Conclusion: This case is presented to highlight the first case report of HCL with duodenal involvement that was successfully treated with CDA. PMID:26199927

  12. Sensory Cells of the Fish Ear: A Hairy Enigma

    NASA Technical Reports Server (NTRS)

    Popper, A. N.; Saidel, W. M.

    1995-01-01

    Analysis of the structure of the ears in teleost fishes has led to the tentative suggestion that otolithic endorgans may function differently, in different species. Recently, evidence has demonstrated different 'types' of sensory hair cells can be found in the ears of teleost fishes, and individual hair cell types are found in discrete regions of individual sensory, epithelia. The presence of multiple hair cell types in fishes provides strong support to the hypothesis of regional differences in the responses of individual otolithic sensory epithelia. The finding of hair cell types in fishes that closely resemble those found in amniote vestibular endorgans also suggests that hair cell heterogeneity arose earlier in the evolution of the vertebrate ear than previously thought.

  13. Rapid response to 2'-deoxycoformycin in advanced hairy cell leukemia after failure of interferons alpha and gamma.

    PubMed

    Lembersky, B C; Ratain, M J; Westbrook, C; Golomb, H M

    1988-01-01

    A patient with advanced hairy cell leukemia initially had a short-lived minor response to interferon alpha therapy and failed to respond to interferon gamma. Subsequent treatment with 2'-deoxycoformycin (dCF) administered biweekly for 12 wk resulted in a complete hematological remission which has continued for 16 months without additional therapy. PMID:3128105

  14. The microenvironment in hairy cell leukemia: pathways and potential therapeutic targets

    PubMed Central

    Burger, Jan A.; Sivina, Mariela; Ravandi, Farhad

    2014-01-01

    Hairy cell leukemia (HCL) cells accumulate and proliferate in the spleen and the bone marrow. In these tissue compartments, HCL cells interact with accessory cells, matrix proteins, and various cyctokines, collectively referred to as the ‘microenvironment.’ Surface receptors expressed on HCL cells and respective stromal ligands are critical for this cross-talk between HCL cells and the microenvironment. Chemokine receptors, adhesion molecules (integrins, CD44), the B cell antigen receptor (BCR), and CD40, expressed on the HCL cells, are likely to be critical for homing, retention, survival, and expansion of the neoplastic B cells. Some of these pathways are now targeted in first clinical trials in other mature B-cell malignancies. We summarize key aspects of the cellular and molecular interactions between HCL cells and their microenvironment. Also, we outline future prospects for therapeutic targeting of the microenvironment in HCL, focusing on CXCR4 and kinase inhibitors (Syk, Btk, phosphatidylinositol 3-kinase [PI3K]) that target B cell receptor signaling. PMID:21438839

  15. Durability of responses to interferon alfa-2b in advanced hairy cell leukemia.

    PubMed

    Ratain, M J; Golomb, H M; Bardawil, R G; Vardiman, J W; Westbrook, C A; Kaminer, L S; Lembersky, B C; Bitter, M A; Daly, K

    1987-03-01

    Previous studies have demonstrated that significant hematologic improvement occurs in the majority of patients with hairy cell leukemia (HCL) treated with partially purified or recombinant interferon (IFN). Fifty-three patients received IFN alfa-2b for at least 3 months in a dose of 2 X 10(6) U/m2 subcutaneously thrice weekly. Of the 49 patients evaluable for response (at least 6 months of IFN therapy), there were ten complete responses and 29 partial responses for a total response rate of 80%. The peripheral blood counts and bone marrow continued to improve over the course of a full year of therapy. IFN was well tolerated, with no patients discontinuing therapy because of toxicity. Transient myelosuppression occurred in most patients during the first 1 to 2 months of therapy, occasionally precipitating a transfusion requirement. After IFN treatment was discontinued, there was a marked decrease in normal marrow elements and a relative increase in marrow hairy cells. This was associated with a transient increase in normal elements in the peripheral blood. Only one of 24 patients followed after receiving IFN for a median of 8.5 months (range, 3 to 16 months) has required further therapy. We conclude that low-dose IFN alfa-2b is highly effective in advanced HCL; responding patients should be treated for at least 1 year. The decision to initiate a second course of IFN therapy should be based primarily on peripheral blood counts and the clinical status of the patient rather than on the bone marrow. PMID:3814819

  16. Occupational exposures, animal exposure and smoking as risk factors for hairy cell leukaemia evaluated in a case-control study.

    PubMed Central

    Nordström, M.; Hardell, L.; Magnuson, A.; Hagberg, H.; Rask-Andersen, A.

    1998-01-01

    To evaluate occupational exposures as risk factors for hairy cell leukaemia (HCL), a population-based case-control study on 121 male HCL patients and 484 controls matched for age and sex was conducted. Elevated odds ratio (OR) was found for exposure to farm animals in general: OR 2.0, 95% confidence interval (CI) 1.2-3.2. The ORs were elevated for exposure to cattle, horse, hog, poultry and sheep. Exposure to herbicides (OR 2.9, CI 1.4-5.9), insecticides (OR 2.0, CI 1.1-3.5), fungicides (OR 3.8, CI 1.4-9.9) and impregnating agents (OR 2.4, CI 1.3-4.6) also showed increased risk. Certain findings suggested that recall bias may have affected the results for farm animals, herbicides and insecticides. Exposure to organic solvents yielded elevated risk (OR 1.5, CI 0.99-2.3), as did exposure to exhaust fumes (OR 2.1, CI 1.3-3.3). In an additional multivariate model, the ORs remained elevated for all these exposures with the exception of insecticides. We found a reduced risk for smokers with OR 0.6 (CI 0.4-1.1) because of an effect among non-farmers. PMID:9667691

  17. Trisomy 12 in chronic lymphocytic leukemia and hairy cell leukemia: a cytogenetic and interphase cytogenetic study.

    PubMed

    Cuneo, A; Bigoni, R; Balboni, M; Carli, M G; Piva, N; Fagioli, F; Latorraca, A; Wlodarska, I; van den Berghe, H; Castoldi, G

    1994-09-01

    Fluorescent in situ hybridization (FISH) with a chromosome 12-specific pericentromeric probe was performed in 42 patients with B-cell chronic lymphocytic leukemia (CLL) and in 10 patients with hairy cell leukemia (HCL). In all cases, a normal karyotype in more than 10 metaphase cells was obtained by conventional chromosome study. FISH documented that 6/42 patients with CLL in fact had trisomy 12 in 15-49% interphase cells. Sequential FISH studies were performed in 2 cases, showing an increase of percentage of trisomic cells over a 2-month to 4-year period. Two out of 10 patients with HCL, one of whom had morphologic features consistent with a diagnosis of HCL variant, showed 5.5 and 10% interphase nuclei with three fluorescent signals, a finding suggestive of the presence of trisomy 12. Combined immunophenotyping and FISH staining in these patients with HCL documented that trisomic cells were CD11c-positive, CD13-negative, and CD2-negative. We conclude that FISH is a sensitive technique allowing for the detection of trisomy 12 in a fraction of cytogenetically normal patients affected with CLL and HCL. PMID:7858495

  18. High prevalence of MAP2K1 mutations in variant and IGHV4-34-expressing hairy-cell leukemias.

    PubMed

    Waterfall, Joshua J; Arons, Evgeny; Walker, Robert L; Pineda, Marbin; Roth, Laura; Killian, J Keith; Abaan, Ogan D; Davis, Sean R; Kreitman, Robert J; Meltzer, Paul S

    2014-01-01

    To understand the genetic mechanisms driving variant and IGHV4-34-expressing hairy-cell leukemias, we performed whole-exome sequencing of leukemia samples from ten affected individuals, including six with matched normal samples. We identified activating mutations in the MAP2K1 gene (encoding MEK1) in 5 of these 10 samples and in 10 of 21 samples in a validation set (overall frequency of 15/31), suggesting potential new strategies for treating individuals with these diseases.

  19. Inhibition of tumor cell proliferation and induction of apoptosis in human lung carcinoma 95-D cells by a new sesquiterpene from hairy root cultures of Artemisia annua.

    PubMed

    Zhai, D-D; Supaibulwatana, K; Zhong, J-J

    2010-09-01

    Artemisia annua is a rich source of many bioactive substances, and in our recent work, a new sesquiterpene, (Z)-7-acetoxy-methyl-11-methyl-3-methylene-dodeca-1,6,10-triene (AMDT), was isolated and identified from hairy roots culture of A. annua, and its bioactivity was characterized in this work. AMDT showed moderate cytotoxic activities against the human tumor cell lines of HO8910 (ovary), 95-D (lung), QGY (liver) and HeLa (cervix) by MTT assay, whose IC(50) values were ranged within 52.44-73.3 microM. As lung cancer is the No. 1 killer of global cancer patients, our interest is to investigate the ability of AMDT in inducing apoptosis of 95-D tumor cells. The 95-D cell growth was inhibited by AMDT, and the flow cytometry analysis showed its cell cycle was arrested in the G1 phase. The apoptotic rate of the cells increased in a dose-dependent manner. AMDT lowered the mitochondrial membrane potential and increased the expression of caspase-9 and -3. These results revealed that AMDT could efficiently induce 95-D cell apoptosis through mitochondrial dependent pathway, and it may be a potential chemotherapeutic agent. PMID:20362422

  20. CD11c gene expression in hairy cell leukemia is dependent upon activation of the proto-oncogenes ras and junD.

    PubMed

    Nicolaou, Fotini; Teodoridis, Jens M; Park, Heiyoung; Georgakis, Alexander; Farokhzad, Omid C; Böttinger, Erwin P; Da Silva, Nicolas; Rousselot, Philippe; Chomienne, Christine; Ferenczi, Katalin; Arnaout, M Amin; Shelley, C Simon

    2003-05-15

    Hairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the beta2 integrin CD11c. In order to determine the cause of CD11c gene expression in HCL the CD11c gene promoter was characterized. Transfection of the CD11c promoter linked to a luciferase reporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11c promoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reduced CD11c promoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11c promoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11c promoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11c promoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11c gene characteristic of HCL is dependent upon activation of the proto-oncogenes ras and junD.

  1. Production of podophyllotoxin using cross-species coculture of Linum flavum hairy roots and Podophyllum hexandrum cell suspensions.

    PubMed

    Lin, Han-wei; Kwok, Kian H; Doran, Pauline M

    2003-01-01

    Novel cross-species coculture systems using Linum flavum hairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin. The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors. In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P. hexandrum suspensions. Because roots of L. flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P. hexandrum to provide an in situ supply of coniferin. Compared with P. hexandrum suspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P. hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively. The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture. Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.

  2. BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimens.

    PubMed

    Thomas, Carla; Amanuel, Benhur; Finlayson, Jill; Grieu-Iacopetta, Fabienne; Spagnolo, Dominic V; Erber, Wendy N

    2015-06-01

    Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.

  3. High prevalence of MAP2K1 mutations in variant and IGHV4-34-expressing hairy-cell leukemias.

    PubMed

    Waterfall, Joshua J; Arons, Evgeny; Walker, Robert L; Pineda, Marbin; Roth, Laura; Killian, J Keith; Abaan, Ogan D; Davis, Sean R; Kreitman, Robert J; Meltzer, Paul S

    2014-01-01

    To understand the genetic mechanisms driving variant and IGHV4-34-expressing hairy-cell leukemias, we performed whole-exome sequencing of leukemia samples from ten affected individuals, including six with matched normal samples. We identified activating mutations in the MAP2K1 gene (encoding MEK1) in 5 of these 10 samples and in 10 of 21 samples in a validation set (overall frequency of 15/31), suggesting potential new strategies for treating individuals with these diseases. PMID:24241536

  4. Central Role of Protein Kinase Cε in Constitutive Activation of ERK1/2 and Rac1 in the Malignant Cells of Hairy Cell Leukemia

    PubMed Central

    Slupsky, Joseph R.; Kamiguti, Aura S.; Harris, Robert J.; Cawley, John C.; Zuzel, Mirko

    2007-01-01

    We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCε in the constitutive activation of Rac1 and ERK in HCs. PKCε and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCε was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCε plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia. PMID:17255340

  5. BRAF inhibitors reverse the unique molecular signature and phenotype of hairy cell leukemia and exert potent antileukemic activity

    PubMed Central

    Pettirossi, Valentina; Santi, Alessia; Imperi, Elisa; Russo, Guido; Pucciarini, Alessandra; Bigerna, Barbara; Schiavoni, Gianluca; Fortini, Elisabetta; Spanhol-Rosseto, Ariele; Sportoletti, Paolo; Mannucci, Roberta; Martelli, Maria Paola; Klein-Hitpass, Ludger; Falini, Brunangelo

    2015-01-01

    Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF–mitogen-activated protein kinase kinase (MEK)–extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells’ hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and MEK inhibitors in HCL. PMID:25480661

  6. Black hairy tongue syndrome

    PubMed Central

    Gurvits, Grigoriy E; Tan, Amy

    2014-01-01

    Black hairy tongue (BHT) is a benign medical condition characterized by elongated filiform lingual papillae with typical carpet-like appearance of the dorsum of the tongue. Its prevalence varies geographically, typically ranging from 0.6% to 11.3%. Known predisposing factors include smoking, excessive coffee/black tea consumption, poor oral hygiene, trigeminal neuralgia, general debilitation, xerostomia, and medication use. Clinical presentation varies but is typically asymptomatic, although aesthetic concerns are common. Differential diagnosis includes pseudo-BHT, acanthosis nigricans, oral hairy leukoplakia, pigmented fungiform papillae of the tongue, and congenital melanocytic/melanotic nevi/macules. Clinical diagnosis relies on visual observation, detailed history taking, and occasionally microscopic evaluation. Treatment involves identification and discontinuation of the offending agent, modifications of chronic predisposing factors, patient’s re-assurance to the benign nature of the condition, and maintenance of adequate oral hygiene with gentle debridement to promote desquamation. Complications of BHT (burning mouth syndrome, halitosis, nausea, gagging, dysgeusia) typically respond to therapy. Prognosis is excellent with treatment of underlying medical conditions. BHT remains an important medical condition which may result in additional burden on the patient and health care system and requires appropriate prevention, recognition and treatment. PMID:25152586

  7. Black hairy tongue syndrome.

    PubMed

    Gurvits, Grigoriy E; Tan, Amy

    2014-08-21

    Black hairy tongue (BHT) is a benign medical condition characterized by elongated filiform lingual papillae with typical carpet-like appearance of the dorsum of the tongue. Its prevalence varies geographically, typically ranging from 0.6% to 11.3%. Known predisposing factors include smoking, excessive coffee/black tea consumption, poor oral hygiene, trigeminal neuralgia, general debilitation, xerostomia, and medication use. Clinical presentation varies but is typically asymptomatic, although aesthetic concerns are common. Differential diagnosis includes pseudo-BHT, acanthosis nigricans, oral hairy leukoplakia, pigmented fungiform papillae of the tongue, and congenital melanocytic/melanotic nevi/macules. Clinical diagnosis relies on visual observation, detailed history taking, and occasionally microscopic evaluation. Treatment involves identification and discontinuation of the offending agent, modifications of chronic predisposing factors, patient's re-assurance to the benign nature of the condition, and maintenance of adequate oral hygiene with gentle debridement to promote desquamation. Complications of BHT (burning mouth syndrome, halitosis, nausea, gagging, dysgeusia) typically respond to therapy. Prognosis is excellent with treatment of underlying medical conditions. BHT remains an important medical condition which may result in additional burden on the patient and health care system and requires appropriate prevention, recognition and treatment.

  8. Variant B Cell Receptor Isotype Functions Differ in Hairy Cell Leukemia with Mutated BRAF and IGHV Genes

    PubMed Central

    Weston-Bell, Nicola J.; Forconi, Francesco; Kluin-Nelemans, Hanneke C.; Sahota, Surinder S.

    2014-01-01

    A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD−ve mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with s

  9. The BTK Inhibitor Ibrutinib (PCI-32765) Blocks Hairy Cell Leukaemia Survival, Proliferation and BCR Signalling: A New Therapeutic Approach

    PubMed Central

    Sivina, Mariela; Kreitman, Robert J.; Arons, Evgeny; Ravandi, Farhad; Burger, Jan A.

    2014-01-01

    B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins (A, G, and M) and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL. PMID:24697238

  10. Hairy cell leukemia associated with large granular lymphocyte leukemia: immunologic and genomic study, effect of interferon treatment.

    PubMed

    Marolleau, J P; Henni, T; Gaulard, P; Le Couedic, J P; Gourdin, M F; Divine, M; Katz, A; Tulliez, M; Goossens, M; Reyes, F

    1988-08-01

    The authors describe a patient who presented an association of hairy cell leukemia (HCL) and large granular lymphocyte (LGL) leukemia. An eventual relationship between these two rare entities is analyzed. Hairy cells (HCs) were present in the blood, bone marrow, and spleen. An excess of LGLs was found only in the blood and bone marrow. After splenectomy the patient received an alpha 2-interferon (alpha 2-IFN) treatment. The HCs surface phenotype was mu+delta+kappa+, CD20+, and CD25+. The LGLs consisted in CD3+, CD8+, HNK1+, WT31+ T lymphocytes. These were absent in the spleen. alpha 2-IFN treatment resulted in the disappearance of the HCs in the blood and bone marrow, whereas the LGLs remained unchanged. Before alpha 2-IFN treatment, peripheral blood cells, predominantly LGLs, exerted low cytotoxicity that increased up to a normal level after treatment. Using Southern blotting the authors studied the rearrangements of the T-cell receptor beta--chain (C beta) and gamma-chain (J gamma) genes and immunoglobulin heavy (JH)- and light (C kappa, C lambda)- chain genes. An unique JH and C kappa gene rearrangement was found in the blood and spleen, whereas C beta and J gamma gene rearrangements were present in the blood, not in the spleen. Under alpha 2-IFN treatment, the JH gene rearrangement fainted dramatically, in contrast to that of the C beta gene. The study of messenger RNA (mRNA) of the T cell receptor alpha and beta chains evidenced the 1.3-kilobase (kb) and 1.6-kb bands in the blood and their absence in the spleen. The patient was human T-cell leukemia virus (HTLV)-II negative by Southern analysis of blood and spleen cells. It is concluded that the LGL expansion was clonal and not reactive to the HCL. Although the authors cannot definitely exclude that both HC and LGL proliferations stem in a common leukemic precursor, their findings support an association of the two entities. PMID:2840988

  11. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    PubMed Central

    2012-01-01

    Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens. PMID:22559055

  12. β-catenin signalling in dermal papilla cells leads to a hairy situation.

    PubMed

    Gemayel, Rita; Chenette, Emily J

    2016-08-01

    Dermal papilla (DP) are specialised mesenchymal cells that activate the formation of new hair follicles. In this issue of The FEBS Journal, Zhang and colleagues show that enhancing the β-catenin signalling pathway in DP cells allows faster and denser hair growth, providing a potential target for hair loss treatments and for improving hair regeneration techniques. PMID:27450439

  13. A comprehensive immunophenotypic marker analysis of hairy cell leukemia in paraffin-embedded bone marrow trephine biopsies--a tissue microarray study.

    PubMed

    Tóth-Lipták, Judit; Piukovics, Klára; Borbényi, Zita; Demeter, Judit; Bagdi, Enikő; Krenács, László

    2015-01-01

    Hairy cell leukemia (HCL) is an uncommon B cell lymphoproliferation characterized by a unique immunophenotype. Due to low number of circulating neoplastic cells and 'dry tap' aspiration, the diagnosis is often based on BM trephine biopsy. We have performed a consecutive immunohistochemical analysis to evaluate diagnostic usefulness of various HCL markers (CD11c, CD25, CD68, CD103, CD123, CD200, annexin A1, cyclin D1, DBA.44, HBME-1, phospho-ERK1/2, TRAP, and T-bet) currently available against fixation resistant epitopes. We analyzed tissue microarrays consisting of samples gained from 73 small B-cell lymphoma cases, including hairy cell leukemia (HCL) (n = 32), HCL variant (HCL-v) (n = 4), B-cell chronic lymphocytic leukemia (B-CLL) (n = 11), lymphoplasmacytic lymphoma (LPL) (n = 3), mantle cell lymphoma (MCL) (n = 10), splenic diffuse red pulp small B cell lymphoma (SDRPL) (n = 2), splenic B cell marginal zone lymphoma (SMZL) (n = 8), and splenic B cell lymphoma/leukemia, unclassifiable (SBCL) (n = 3) cases. The HCL cases were 100% positive for all but 2 (DBA.44 and CD123) of these markers. Annexin A1 showed 100% specificity and accuracy, which was followed by CD123, pERK, CD103, HBME-1, CD11c, CD25, CD68, cyclin D1, CD200, T-bet, DBA.44, and TRAP, in decreasing order. In conclusion, our results reassured the high specificity of annexin A1 and pERK, as well as the diagnostic value of standard HCL markers of CD11c, CD25, CD103, and CD123 also in paraffin-embedded BM samples. Additional markers, including HBME-1, cyclin D1, CD200, and T-bet also represent valuable tools in the differential diagnosis of HCL and its mimics.

  14. 75 FR 53202 - Diseases Associated With Exposure to Certain Herbicide Agents (Hairy Cell Leukemia and Other...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-31

    ... number.) SUPPLEMENTARY INFORMATION: On March 25, 2010, VA published in the Federal Register (75 FR 14391... between myocardial oxygen supply and demand.'' 75 FR 14393; See Harrison's Principles of Internal Medicine...; it typically occurs when there is an imbalance between myocardial oxygen supply and demand.'' 75...

  15. Hairy Cell Leukemia Presenting with Isolated Skeletal Involvement Successfully Treated by Radiation Therapy and Cladribine: A Case Report and Review of the Literature

    PubMed Central

    Yonal-Hindilerden, Ipek; Hindilerden, Fehmi; Bulut-Dereli, Sanem; Yıldız, Eren; Dogan, Ibrahim Oner; Nalcaci, Meliha

    2015-01-01

    We describe an unusual case of hairy cell leukemia (HCL) in a 55-year-old male presenting with isolated skeletal disease as the initial manifestation without abnormal peripheral blood counts, bone marrow involvement, or splenomegaly. To the best of our knowledge, there have been only two previous reports of a similar case. The patient presented with pain in the right femur. Anteroposterior radiographs of both femurs revealed mixed lytic-sclerotic lesions. PET scan showed multiple metastatic lesions on axial skeleton, pelvis, and both femurs. Histopathological examination of the bone biopsy revealed an infiltrate of HCL. Localized radiation therapy to both proximal femurs and subsequently 4 weeks later, a 7-day course of 0.1 mg/kg/day cladribine provided complete remission with relief of symptoms and resolution of bone lesions. We addressed the manifestations and management of HCL patients with skeletal involvement. PMID:26788382

  16. Hairy AdS solitons

    NASA Astrophysics Data System (ADS)

    Anabalón, Andrés; Astefanesei, Dumitru; Choque, David

    2016-11-01

    We construct exact hairy AdS soliton solutions in Einstein-dilaton gravity theory. We examine their thermodynamic properties and discuss the role of these solutions for the existence of first order phase transitions for hairy black holes. The negative energy density associated to hairy AdS solitons can be interpreted as the Casimir energy that is generated in the dual filed theory when the fermions are antiperiodic on the compact coordinate.

  17. Remediation of textile azo dye acid red 114 by hairy roots of Ipomoea carnea Jacq. and assessment of degraded dye toxicity with human keratinocyte cell line.

    PubMed

    Jha, Pamela; Jobby, Renitta; Desai, N S

    2016-07-01

    Bioremediation has proven to be the most desirable and cost effective method to counter textile dye pollution. Hairy roots (HRs) of Ipomoea carnea J. were tested for decolourization of 25 textile azo dyes, out of which >90% decolourization was observed in 15 dyes. A diazo dye, Acid Red 114 was decolourized to >98% and hence, was chosen as the model dye. A significant increase in the activities of oxidoreductive enzymes was observed during decolourization of AR114. The phytodegradation of AR114 was confirmed by HPLC, UV-vis and FTIR spectroscopy. The possible metabolites were identified by GCMS as 4- aminobenzene sulfonic acid 2-methylaniline and 4- aminophenyl 4-ethyl benzene sulfonate and a probable pathway for the biodegradation of AR114 has been proposed. The nontoxic nature of the metabolites and toxicity of AR114 was confirmed by cytotoxicity tests on human keratinocyte cell line (HaCaT). When HaCaT cells were treated separately with 150 μg mL(-1) of AR114 and metabolites, MTT assay showed 50% and ≈100% viability respectively. Furthermore, flow cytometry data showed that, as compared to control, the cells in G2-M and death phase increased by 2.4 and 3.6 folds respectively on treatment with AR114 but remained unaltered in cells treated with metabolites.

  18. Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion

    PubMed Central

    Weston-Bell, Nicola J.; Tapper, Will; Gibson, Jane; Bryant, Dean; Moreno, Yurany; John, Melford; Ennis, Sarah; Kluin-Nelemans, Hanneke C.; Collins, Andrew R.; Sahota, Surinder S.

    2016-01-01

    In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease. PMID:26871591

  19. Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model.

    PubMed

    Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B; Bailey-Serres, Julia; Brady, Siobhan M

    2014-10-01

    Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

  20. Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN

    PubMed Central

    Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.

    2014-01-01

    Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

  1. Aspects of hairy black holes

    SciTech Connect

    Anabalón, Andrés; Astefanesei, Dumitru

    2015-03-26

    We review the existence of exact hairy black holes in asymptotically flat, anti-de Sitter and de Sitter space-times. We briefly discuss the issue of stability and the charging of the black holes with a Maxwell field.

  2. Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression.

    PubMed

    Saha, Tusar T; Shin, Sang Woon; Dou, Wei; Roy, Sourav; Zhao, Bo; Hou, Yuan; Wang, Xue-Li; Zou, Zhen; Girke, Thomas; Raikhel, Alexander S

    2016-02-01

    The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box-like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met. PMID:26744312

  3. [Induction of polyploid in hairy roots of Nicotiana tabacum and its plant regeneration].

    PubMed

    Hou, Lili; Shi, Heping; Yu, Wu; Tsang, Po Keung Eric; Chow, Cheuk Fai Stephen

    2014-04-01

    By genetic transformation with Agrobacterum rhizogenes and artificial chromosome doubling techniques, we studied the induction of hairy roots and their polyploidization, and subsequent plant regeneration and nicotine determination to enhance the content of nicotine in Nicotiana tabacum. The results show that hairy roots could be induced from the basal surface of leaf explants of N. tabacum 8 days after inoculation with Agrobacterium rhizogenes ATCC15834. The percentage of the rooting leaf explants was 100% 15 days after inoculation. The hairy roots could grow rapidly and autonomously on solid or liquid phytohormones-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and paper electrophoresis of opines from N. tabacum hairy roots. The highest rate of polyploidy induction, more than 64.71%, was obtained after treatment of hairy roots with 0.1% colchicine for 36 h. The optimum medium for plant regeneration from polyploid hairy roots was MS+2.0 mg/L 6-BA +0.2 mg/L NAA. Compared with the control diploid plants, the hairy roots-regenerated plants had weak apical dominance, more axillary buds and more narrow leaves; whereas the polyploid hairy root-regenerated plants had thicker stems, shorter internodes and the colour, width and thickness of leaves were significantly higher than that of the control. Observation of the number of chromosomes in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 96 (4n = 96) chromosomes. Pot-grown experiments showed compared to the control, the flowering was delayed by 21 days in diploid hairy roots-regenerated plants and polyploid hairy root-regenerated plants. GC-MS detection shows that the content of nicotine in polyploid plants was about 6.90 and 4.57 times the control and the diploid hairy roots-regenerated plants, respectively. PMID:25195248

  4. Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression

    PubMed Central

    Saha, Tusar T.; Shin, Sang Woon; Dou, Wei; Roy, Sourav; Zhao, Bo; Hou, Yuan; Wang, Xue-Li; Zou, Zhen; Girke, Thomas; Raikhel, Alexander S.

    2016-01-01

    The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box–like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met. PMID:26744312

  5. Requisite analytic and diagnostic performance characteristics for the clinical detection of BRAF V600E in hairy cell leukemia: a comparison of 2 allele-specific PCR assays.

    PubMed

    Brown, Noah A; Weigelin, Helmut C; Bailey, Nathanael; Laliberte, Julie; Elenitoba-Johnson, Kojo S J; Lim, Megan S; Betz, Bryan L

    2015-09-01

    Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assay-myT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.

  6. Medical History, Lifestyle, and Occupational Risk Factors for Hairy Cell Leukemia: The InterLymph Non-Hodgkin Lymphoma Subtypes Project

    PubMed Central

    Slager, Susan L.; Hughes, Ann Maree; Smith, Alex; Glimelius, Bengt; Habermann, Thomas M.; Berndt, Sonja I.; Staines, Anthony; Norman, Aaron D.; Cerhan, James R.; Sampson, Joshua N.; Morton, Lindsay M.; Clavel, Jacqueline

    2014-01-01

    Background Little is known about the etiology of hairy cell leukemia (HCL), a rare B-cell lymphoproliferative disorder with marked male predominance. Our aim was to identify key risk factors for HCL. Methods A pooled analysis of individual-level data for 154 histologically confirmed HCL cases and 8834 controls from five case–control studies, conducted in Europe and Australia, was undertaken. Age-, race and/or ethnicity-, sex-, and study-adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated using unconditional logistic regression. Results The usual patterns for age and sex in HCL were observed, with a median age of 55 years and sex ratio of 3.7 males to females. Cigarette smoking was inversely associated with HCL (OR = 0.51, 95% CI = 0.37 to 0.71) with dose–response relationships observed for duration, frequency, and lifetime cigarette smoking (P trend < .001). In contrast, occupation as a farmer was positively associated with HCL (OR = 2.34, 95% CI = 1.36 to 4.01), with a dose–response relationship observed for duration (OR = 1.82, 95% CI = 0.85 to 3.88 for ≤10 years vs never; and OR = 2.98, 95% CI = 1.50 to 5.93 for >10 years vs never; P trend = .025). Adult height was also positively associated with HCL (OR = 2.69, 95% CI = 1.39 to 5.29 for upper vs lower quartile of height). The observed associations remained consistent in multivariate analysis. Conclusions Our observations of an increased risk of HCL from farming exposures and decreased risk from smoking exposures, independent of one another, support a multifactorial origin and an etiological specificity of HCL compared with other non-Hodgkin lymphoma subtypes. The positive association with height is a novel finding that needs replication. PMID:25174032

  7. Concentrations of organochlorines related to titers to Epstein-Barr virus early antigen IgG as risk factors for hairy cell leukemia.

    PubMed Central

    Nordström, M; Hardell, L; Lindström, G; Wingfors, H; Hardell, K; Linde, A

    2000-01-01

    Hairy cell leukemia (HCL) is a rare chronic B-cell malignancy that, according to modern classifications, is a subgroup of non-Hodgkin lymphomas (NHLs). A rapid increase in incidence of NHL has been reported in many countries. The reasons for this increase are largely unknown, but exposure to organochlorines has been suggested as a risk factor. Epstein-Barr virus is a human herpesvirus that has been associated with certain subgroups of NHL. In this study, we measured lipid adjusted blood concentrations (in nanogram per gram) of 36 congeners of polychlorinated biphenyls (PCBs), p, p'-dichlorodiphenyldichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), and four subgroups of chlordanes (trans-nonachlor, cis-nonachlor, MC6, and oxychlordane) in incident cases of HCL and controls from the general population. We obtained results on organochlorines and antibodies for 54 cases and 54 controls. Titers of antibodies to the Epstein-Barr early antigen and Epstein-Barr nuclear antigen, measured as P107, were correlated to concentrations of organochlorines to evaluate the possibility of an interaction between these factors in the pathogenesis of HCL. We found no significant difference in lipid-adjusted blood concentrations of total PCBs, p,p'-DDE, HCB, or the sum of the chlordanes between cases and controls. Titers of antibodies to Epstein-Barr early antigen IgG [Greater and equal to] 40 were correlated to an increased risk for HCL. This risk was further increased in those with a level above the median value of p,p'-DDE, HCB, or the sum of the chlordanes, suggesting an interaction between Epstein-Barr virus and a higher concentration of these chemicals. We also found increased risk for the sum of immunotoxic PCB group. PMID:10811571

  8. Podophyllotoxin and 6-methoxy podophyllotoxin Production in Hairy Root Cultures of Liunm mucronatum ssp. mucronatum

    PubMed Central

    Samadi, Afsaneh; Jafari, Morad; Nejhad, Nasim Mohammad; Hossenian, Farah

    2014-01-01

    Aim: Two bacterial strains of Agrobacterium rhizogenes, A13 and 9534 were evaluated for induction of transformed hairy roots in Linum mucronatum ssp. mucronatum, a high value medicinal plant. Materials and Methods: The hairy roots were successfully initiated, through infecting the hypocotyl and root explants and the A13 strain performed a high transformation frequency for hairy roots induction. Transgenic status of hairy roots was confirmed by polymerase chain reaction (PCR) analysis of the rol genes. Growth kinetics of transgenic roots induced by two strains indicated a similar pattern of growth, with maximum growth occurring between 42 to 56 days. The lignan contents in hairy roots were analyzed using high-performance liquid chromatography (HPLC) method. Results: Transformed cultures showed significant differences (P < 0.05) in lignan content. The highest amount of Podophyllotoxin (PTOX, 5.78 mg/g DW) and 6-methoxy podophyllotoxin (MPTOX, 49.19 mg/g DW) was found in transformed lines induced by strain A13, which was four times higher than those of non-transformed roots. The results showed that hairy root cultures of L. mucronatum are rich sources of MPTOX. Conclusion: hairy root cultures from L. mucronatum can be used as a useful system for scale-up producing MPTOX and precursors for the production of antitumor agents in substitution with PTOX by considering the appropriate optimizations in future studies. PMID:24914281

  9. Air entrainment in hairy surfaces

    NASA Astrophysics Data System (ADS)

    Nasto, Alice; Regli, Marianne; Brun, P.-T.; Alvarado, José; Clanet, Christophe; Hosoi, A. E.

    2016-07-01

    Motivated by diving semiaquatic mammals, we investigate the mechanism of dynamic air entrainment in hairy surfaces submerged in liquid. Hairy surfaces are cast out of polydimethylsiloxane elastomer and plunged into a fluid bath at different velocities. Experimentally, we find that the amount of air entrained is greater than what is expected for smooth surfaces. Theoretically, we show that the hairy surface can be considered as a porous medium and we describe the air entrainment via a competition between the hydrostatic forcing and the viscous resistance in the pores. A phase diagram that includes data from our experiments and biological data from diving semiaquatic mammals is included to place the model system in a biological context and predict the regime for which the animal is protected by a plastron of air.

  10. The Droplet Digital PCR: A New Valid Molecular Approach for the Assessment of B-RAF V600E Mutation in Hairy Cell Leukemia

    PubMed Central

    Guerrini, Francesca; Paolicchi, Matteo; Ghio, Francesco; Ciabatti, Elena; Grassi, Susanna; Salehzadeh, Serena; Ercolano, Giacomo; Metelli, Maria R.; Del Re, Marzia; Iovino, Lorenzo; Petrini, Iacopo; Carulli, Giovanni; Cecconi, Nadia; Rousseau, Martina; Cervetti, Giulia; Galimberti, Sara

    2016-01-01

    Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where the B-RAF V600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of the B-RAF V600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds. This retrospective study enrolled 47 patients observed at the Hematology Unit of the University of Pisa, Italy, from January 2005 to January 2014: 27 patients were affected by “classic” HCL, two by the variant HCL (vHCL), and 18 by splenic marginal zone lymphoma (SMZL). The aim of the study was to compare dd-PCR to “classic” quantitative PCR (QT-PCR) in terms of sensitivity and specificity and to demonstrate its possible use in HCL. Results showed that: (1) the sensitivity of dd-PCR is about half a logarithm superior to QT-PCR (5 × 10-5 vs. 2.5 × 10-4), (2) the specificity of the dd-PCR is comparable to QT-PCR (no patient with marginal splenic lymphoma or HCL variant resulted mutated), (3) its high sensitivity would allow to use dd-PCR in the monitoring of MRD. At the end of treatment, among patients in complete remission, 33% were still MRD-positive by dd-PCR versus 28% by QT-PCR versus 11% by the evaluation of the B-cell clonality, after 12 months, dd-PCR was comparable to QT-PCR and both detected the B-RAF mutation in 15% of cases defined as MRD-negative by IgH rearrangement. Moreover, (4) the feasibility and the costs of dd-PCR are comparable to those of QT-PCR. In conclusion, our study supports the introduction of dd-PCR in the scenario of HCL, also during the follow-up. PMID:27790140

  11. Photoacoustic cell for ultrasound contrast agent characterization.

    PubMed

    Alippi, A; Bettucci, A; Biagioni, A; D'Orazio, A; Germano, M; Passeri, D

    2010-10-01

    Photoacoustics has emerged as a tool for the study of liquid gel suspension behavior and has been recently employed in a number of new biomedical applications. In this paper, a photoacoustic sensor is presented which was designed and realized for analyzing photothermal signals from solutions filled with microbubbles, commonly used as ultrasound contrast agents in echographic imaging techniques. It is a closed cell device, where photothermal volume variation of an aqueous solution produces the periodic deflection of a thin membrane closing the cell at the end of a short pipe. The cell then acts as a Helmholtz resonator, where the displacement of the membrane is measured through a laser probe interferometer, whereas photoacoustic signal is generated by a laser chopped light beam impinging onto the solution through a glass window. Particularly, the microbubble shell has been modeled through an effective surface tension parameter, which has been then evaluated from experimental data through the shift of the resonance frequencies of the photoacoustic sensor. This shift of the resonance frequencies of the photoacoustic sensor caused by microbubble solutions is high enough for making such a cell a reliable tool for testing ultrasound contrast agent, particularly for bubble shell characterization. PMID:21034110

  12. Phase I Trial of Anti-CD22 Recombinant Immunotoxin Moxetumomab Pasudotox (CAT-8015 or HA22) in Patients With Hairy Cell Leukemia

    PubMed Central

    Kreitman, Robert J.; Tallman, Martin S.; Robak, Tadeusz; Coutre, Steven; Wilson, Wyndham H.; Stetler-Stevenson, Maryalice; FitzGerald, David J.; Lechleider, Robert; Pastan, Ira

    2012-01-01

    Purpose To conduct a phase I dose-escalation trial assessing safety and response of recombinant immunotoxin moxetumomab pasudotox (CAT-8015, HA22) in chemotherapy-resistant hairy cell leukemia (HCL). Patients and Methods Eligible patients had relapsed/refractory HCL after ≥ two prior therapies and required treatment because of abnormal blood counts. Patients received moxetumomab pasudotox 5 to 50 μg/kg every other day for three doses (QOD ×3), with up to 16 cycles repeating at ≥ 4-week intervals if patients did not experience disease progression or develop neutralizing antibodies. Results Twenty-eight patients were enrolled, including three patients each at 5, 10, 20, and 30 μg/kg, four patients at 40 μg/kg, and 12 patients at 50 μg/kg QOD ×3 for one to 16 cycles each (median, four cycles). Dose-limiting toxicity was not observed. Two patients had transient laboratory abnormalities consistent with grade 2 hemolytic uremic syndrome with peak creatinine of 1.53 to 1.66 mg/dL and platelet nadir of 106,000 to 120,000/μL. Drug-related toxicities in 25% to 64% of the 28 patients included (in decreasing frequency) grade 1 to 2 hypoalbuminemia, aminotransferase elevations, edema, headache, hypotension, nausea, and fatigue. Of 26 patients evaluable for immunogenicity, 10 patients (38%) made antibodies neutralizing more than 75% of the cytotoxicity of 1,000 ng/mL of immunotoxin, but this immunogenicity was rare (5%) after cycle 1. The overall response rate was 86%, with responses observed at all dose levels, and 13 patients (46%) achieved complete remission (CR). Only 1 CR lasted less than 1 year, with the median disease-free survival time not yet reached at 26 months. Conclusion Moxetumomab pasudotox at doses up to 50 μg/kg QOD ×3 has activity in relapsed/refractory HCL and has a safety profile that supports further clinical development for treatment of this disease. PMID:22355053

  13. Stable, Electroinactive Wetting Agent For Fuel Cells

    NASA Technical Reports Server (NTRS)

    Prakash, Surya G.; Olah, George A.; Narayanan, Sekharipuram R.; Surampudi, Subbarao; Halpert, Gerald

    1994-01-01

    Straight-chain perfluorooctanesulfonic acid (C8 acid) identified as innocuous and stable wetting agent for use with polytetrafluoroethylene-containing electrodes in liquid-feed direct-oxidation fuel cells suggested for use in vehicles and portable power supplies. C8 acid in small concentrations in aqueous liquid solutions of methanol, trimethoxymethane, dimethoxymethane, and trioxane enables oxidation of these substances by use of commercially available electrodes of type designed originally for use with gases. This function specific to C8 acid molecule and not achieved by other related perfluorolkanesulfonic acids.

  14. [Induction of polyploid hairy roots and its plant regeneration in Pogostemon cablin].

    PubMed

    Shi, Heping; Yu, Wu; Zhang, Guopeng; Tsang, Pokeung Eric; Chow, Cheuk Fai Stephen

    2014-08-01

    Abstract: In order to enhance the content of secondary metabolites patchouli alcohol in Pogostemon cablin, we induced polyploid hairy roots and their plant regeneration, and determined the content of patchouli alcohol through artificial chromosome doubling with colchicine. The highest rate of polyploidy induction was more than 40% when hairy roots were treated with 0.05% colchicine for 36 h. The obtained polyploid hairy roots formed adventitious shoots when cultured in an MS medium with 6-BA 0.2 mg/L and NAA 0.1 mg/L for 60 d. Compared with the control diploid plants, the polyploid hairy root-regenerated plants of P. cablin had more developed root systems, thicker stems, shorter internodes and longer, wider and thicker leaves. Observation of the chromosome number in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 128 (4n = 128) chromosomes. The leaves contained around twice as many stomatal guard cells and chloroplasts as the controls, but the stomatal density declined with increasing ploidy. The stomatal density in diploid plants was around 1.67 times of that in polyploid plants. GC-MS analysis shows that the content of patchouli alcholol in the hairy root-derived polyploid plants was about 4.25 mg/g dry weight, which was 2.3 times of that in diploid plants. The present study demonstrates that polyploidization of hairy roots can stimulate the content of patchouli alcholol in medicinal plant of P. cablin. PMID:25507476

  15. [Induction of polyploid hairy roots and its plant regeneration in Pogostemon cablin].

    PubMed

    Shi, Heping; Yu, Wu; Zhang, Guopeng; Tsang, Pokeung Eric; Chow, Cheuk Fai Stephen

    2014-08-01

    Abstract: In order to enhance the content of secondary metabolites patchouli alcohol in Pogostemon cablin, we induced polyploid hairy roots and their plant regeneration, and determined the content of patchouli alcohol through artificial chromosome doubling with colchicine. The highest rate of polyploidy induction was more than 40% when hairy roots were treated with 0.05% colchicine for 36 h. The obtained polyploid hairy roots formed adventitious shoots when cultured in an MS medium with 6-BA 0.2 mg/L and NAA 0.1 mg/L for 60 d. Compared with the control diploid plants, the polyploid hairy root-regenerated plants of P. cablin had more developed root systems, thicker stems, shorter internodes and longer, wider and thicker leaves. Observation of the chromosome number in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 128 (4n = 128) chromosomes. The leaves contained around twice as many stomatal guard cells and chloroplasts as the controls, but the stomatal density declined with increasing ploidy. The stomatal density in diploid plants was around 1.67 times of that in polyploid plants. GC-MS analysis shows that the content of patchouli alcholol in the hairy root-derived polyploid plants was about 4.25 mg/g dry weight, which was 2.3 times of that in diploid plants. The present study demonstrates that polyploidization of hairy roots can stimulate the content of patchouli alcholol in medicinal plant of P. cablin. PMID:25423753

  16. Polyprenols in hairy roots of Coluria geoides.

    PubMed

    Skorupińska-Tudek, K; Hung, V S; Olszowska, O; Furmanowa, M; Chojnacki, T; Swiezewska, E

    2000-12-01

    Long-chain polyisoprenoid alcohols built from several up to more than 100 isoprenoid units are common constituents of all living organisms. They were found mostly in plants, bacteria, yeasts and mammalian cells. In vitro hairy root culture of Coluria geoides was obtained from plants transformed with Agrobacterium rhizogenes. Growth was optimal at 0.75% (w/v) glucose and at 22 degrees C. Dry samples of roots were extracted and lipid content was analysed by HPLC. According to our estimation, polyprenols are accumulated in roots of C. geoides cultivated in vitro as a mixture of several prenologues with the dominating prenol composed of 16 isoprenoid units. The content of polyprenols in tissue was approx. 300 microg/g of dry weight.

  17. Modelling of robotic work cells using agent based-approach

    NASA Astrophysics Data System (ADS)

    Sękala, A.; Banaś, W.; Gwiazda, A.; Monica, Z.; Kost, G.; Hryniewicz, P.

    2016-08-01

    In the case of modern manufacturing systems the requirements, both according the scope and according characteristics of technical procedures are dynamically changing. This results in production system organization inability to keep up with changes in a market demand. Accordingly, there is a need for new design methods, characterized, on the one hand with a high efficiency and on the other with the adequate level of the generated organizational solutions. One of the tools that could be used for this purpose is the concept of agent systems. These systems are the tools of artificial intelligence. They allow assigning to agents the proper domains of procedures and knowledge so that they represent in a self-organizing system of an agent environment, components of a real system. The agent-based system for modelling robotic work cell should be designed taking into consideration many limitations considered with the characteristic of this production unit. It is possible to distinguish some grouped of structural components that constitute such a system. This confirms the structural complexity of a work cell as a specific production system. So it is necessary to develop agents depicting various aspects of the work cell structure. The main groups of agents that are used to model a robotic work cell should at least include next pattern representatives: machine tool agents, auxiliary equipment agents, robots agents, transport equipment agents, organizational agents as well as data and knowledge bases agents. In this way it is possible to create the holarchy of the agent-based system.

  18. Hyoscyamine biosynthesis in Datura stramonium hairy root in vitro systems with different ploidy levels.

    PubMed

    Pavlov, A; Berkov, S; Weber, J; Bley, Th

    2009-05-01

    Hyoscyamine biosynthesis in Datura stramonium hairy roots with different ploidy levels was investigated. For the first time, we report that hairy roots undergo endoreduplication and therefore consist mainly of cells with doupled sets of chromosomes of primary plant tissues, used for Agrobacterium transformation. The alkaloid profiles of hairy roots obtained from diploid and tetraploid plants were similar in terms of the major compounds, but they differed significantly with respect to the minor compounds (here defined as those that accounted for <1% of the total ion current of the alkaloid mixture in gas chromatography-mass spectrometric analyses). Significant differences in the effects of the main nutrients on the growth of the hairy roots obtained from diploid and tetraploid plants and their hyoscyamine contents were observed. The maximal yield of hyoscyamine (177 mg/L) was obtained when hairy roots from tetraploid plants were cultivated in Murashige-Skoog nutrient medium supplemented with 6% sucrose. Time courses of utilization of the main nutrients in the medium during cultivation of D. stramonium hairy root cultures are also presented.

  19. Iron Oxide as an MRI Contrast Agent for Cell Tracking

    PubMed Central

    Korchinski, Daniel J.; Taha, May; Yang, Runze; Nathoo, Nabeela; Dunn, Jeff F.

    2015-01-01

    Iron oxide contrast agents have been combined with magnetic resonance imaging for cell tracking. In this review, we discuss coating properties and provide an overview of ex vivo and in vivo labeling of different cell types, including stem cells, red blood cells, and monocytes/macrophages. Furthermore, we provide examples of applications of cell tracking with iron contrast agents in stroke, multiple sclerosis, cancer, arteriovenous malformations, and aortic and cerebral aneurysms. Attempts at quantifying iron oxide concentrations and other vascular properties are examined. We advise on designing studies using iron contrast agents including methods for validation. PMID:26483609

  20. Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance

    PubMed Central

    Häkkinen, Suvi T.; Moyano, Elisabeth; Cusidó, Rosa M.; Oksman-Caldentey, Kirsi-Marja

    2016-01-01

    Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds – commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity. PMID:27746806

  1. Organic acid complexation, heavy metal distribution and the effect of ATPase inhibition in hairy roots of hyperaccumulator plant species.

    PubMed

    Boominathan, Rengasamy; Doran, Pauline M

    2003-03-01

    Heavy metal uptake and distribution were investigated in hairy roots of the Cd hyperaccumulator, Thlaspi caerulescens, and the Ni hyperaccumulator, Alyssum bertolonii. Hairy roots of both species contained high constitutive levels of citric, malic and malonic acids. After treatment with 20 ppm Cd or 25 ppm Ni, about 13% of the total Cd in T. caerulescens roots and 28% of the total Ni in A. bertolonii were associated with organic acids. T. caerulescens and A. bertolonii hairy roots remained healthy and grew well at high concentrations of Cd and Ni, respectively, whereas hairy roots of the non-hyperaccumulator, Nicotiana tabacum, did not. Most of the Cd in T. caerulescens and N. tabacum roots was localised in the cell walls. In contrast, 85-95% of the Ni in A. bertolonii and N. tabacum was associated with the symplasm. Growth of T. caerulescens and A. bertolonii hairy roots was severely reduced in the presence of diethylstilbestrol (DES), an inhibitor of plasma membrane H(+)-ATPase. Treatment with DES increased the concentration of Cd in the symplasm of T. caerulescens about 6-fold with retention of root viability, whereas viability and Ni transport across the plasma membrane were both reduced in A. bertolonii. These results suggest that the mechanisms of Cd tolerance and hyperaccumulation in T. caerulescens hairy roots are capable of withstanding the effects of plasma membrane depolarisation, whereas Ni tolerance and hyperaccumulation in A. bertolonii hairy roots are not. PMID:12568742

  2. Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots.

    PubMed Central

    Diaz, C. L.; Logman, TJJ.; Stam, H. C.; Kijne, J. W.

    1995-01-01

    Introduction of the pea (Pisum sativum L.) lectin (PSL) gene into white clover (Trifolium repens L.) hairy roots facilitates nodulation by the nitrogen-fixing bacterium Rhizobium leguminosarum biovar viciae, which normally nodulates pea and not white clover (C.L. Diaz, L.S. Melchers, P.J.J. Hooykaas, B.J.J. Lugtenberg, and J.W. Kijne [1989] Nature 338: 579-581). Here, we show that PSL is functionally expressed in transgenic white clover hairy roots transformed with the PSL gene. PSL could be isolated from these roots by affinity chromatography. Immunoanalysis of PSL showed the presence of polypeptides corresponding to the PSL precursor and its [beta] subunits. In addition, we developed a highly sensitive localization technique based on specific binding of a glycan moiety of rat IgE to PSL. Similar to the situation in pea roots, PSL appeared to be localized on the external cell surface of elongated epidermal cells and on the tips of emerging and growing root hairs of transgenic white clover hairy roots. PSL was not observed on normal white clover roots and on hairy roots without the PSL gene. These results show that (a) in transgenic white clover hairy roots, PSL is correctly processed and targeted to root cells susceptible to rhizobial infection, and (b) like in pea roots, PSL is surface bound with at least one of its two sugar-binding sites available for (rhizobial) ligands. PMID:12228660

  3. Curcumin: a promising agent targeting cancer stem cells.

    PubMed

    Zang, Shufei; Liu, Tao; Shi, Junping; Qiao, Liang

    2014-01-01

    Cancer stem cells are a subset of cells that are responsible for cancer initiation and relapse. They are generally resistant to the current anticancer agents. Successful anticancer therapy must consist of approaches that can target not only the differentiated cancer cells, but also cancer stem cells. Emerging evidence suggested that the dietary agent curcumin exerted its anti-cancer activities via targeting cancer stem cells of various origins such as those of colorectal cancer, pancreatic cancer, breast cancer, brain cancer, and head and neck cancer. In order to enhance the therapeutic potential of curcumin, this agent has been modified or used in combination with other agents in the experimental therapy for many cancers. In this mini-review, we discussed the effect of curcumin and its derivatives in eliminating cancer stem cells and the possible underlying mechanisms.

  4. Cancer stem cells targeting agents--a review.

    PubMed

    Shi, A-M; Tao, Z-Q; Li, H; Wang, Y-Q; Zhao, J

    2015-11-01

    Major current cancer strategies like surgery, radiotherapy, and chemotherapy are compromised due to major problem of recurrence, which usually lead to mortality. The widely accepted reason for this is resistance offered by cancer cells towards cancer drugs or inability of a therapeutic procedure to target real culprits viz. cancer-initiating cells (cancer stem cells). So, there is a current need of development of new agents targeting these cancer stem cells in order to overcome resistance to therapeutic procedures. The present review article is focused on new cancer cell targeting agents like salinomycin, apopotin etc and their mechanisms to target cancer stems cells will be discussed.

  5. Loss of Sertoli-germ cell adhesion determines the rapid germ cell elimination during the seasonal regression of the seminiferous epithelium of the large hairy armadillo Chaetophractus villosus.

    PubMed

    Luaces, Juan Pablo; Rossi, Luis Francisco; Sciurano, Roberta Beatriz; Rebuzzini, Paola; Merico, Valeria; Zuccotti, Maurizio; Merani, Maria Susana; Garagna, Silvia

    2014-03-01

    The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis. PMID:24451984

  6. HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls.

    PubMed

    Rahman, Laiq ur; Kouno, Hitomi; Hashiguchi, Yuya; Yamamoto, Hirobumi; Narbad, Arjan; Parr, Adrian; Walton, Nicholas; Ikenaga, Toshihiko; Kitamura, Yoshie

    2009-10-01

    As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.

  7. Viability of plant hairy roots is sustained without propagation in low sugar medium kept at ambient temperature.

    PubMed

    Nagatome; Yamamoto; Taya; Tanaka

    2000-08-01

    The effect of sucrose concentration in the medium on the growth and resumption ability to form lateral roots was investigated using the hairy roots of pak-bung and tobacco. It was found that the growth evaluated by root tip elongation of pak-bung and tobacco hairy roots was suppressed in the medium having an initial sucrose concentration of <2.5kg/m(3), and that the resumption abilities of both the hairy roots could be preserved when the hairy roots were kept at an initial sucrose concentration of 2.5kg/m(3) under ambient temperature conditions. The values of maintenance energy for pak-bung and tobacco hairy roots were determined to be 0.11 and 0.12 per day, respectively, from the total sugar consumption rates. Under the oligotrophic condition of the sucrose concentration of 2.5kg/m(3), the hairy roots were considered to exist as resting cells with maintenance metabolism, and the minimum demand for the energy source to ensure survival of the cells was met because the cells hardly multiplied and sugar consumption was not significant. In addition, long-term storage of pak-bung hairy roots in the liquid medium with 2.5kg/m(3) sucrose was performed at 25 degrees C. It was demonstrated that the hairy roots could maintain their resumption abilities without a serious loss of viability over 600 days and that the number of budding lateral roots per unit length of the main roots remained a value of 72 roots/m after the 600-day storage.

  8. Melanoma and non-melanoma skin cancers in hairy cell leukaemia: a Surveillance, Epidemiology and End Results population analysis and the 30-year experience at Memorial Sloan Kettering Cancer Center.

    PubMed

    Watts, Justin M; Kishtagari, Ashwin; Hsu, Meier; Lacouture, Mario E; Postow, Michael A; Park, Jae H; Stein, Eytan M; Teruya-Feldstein, Julie; Abdel-Wahab, Omar; Devlin, Sean M; Tallman, Martin S

    2015-10-01

    Few studies have examined melanoma and non-melanoma skin cancer (NMSC) incidence rates after a diagnosis of hairy cell leukaemia (HCL). We assessed 267 HCL patients treated at Memorial Sloan Kettering Cancer Center (MSKCC) and Surveillance, Epidemiology and End Results (SEER) data for melanoma and NMSC incidence rates after HCL. Incidence data from MSKCC patients demonstrated a 10-year combined melanoma and NMSC skin cancer rate of 11·3%, melanoma 4·4% and NMSC 6·9%. Molecular analysis of skin cancers from MSKCC patients revealed activating RAS mutations in 3/9 patients, including one patient with melanoma. Of 4750 SEER patients with HCL, 55 (1·2%) had a subsequent diagnosis of melanoma. Standardized incidence ratios (SIRs) did not show that melanoma was more common in HCL patients versus the general population (SIR 1·3, 95% CI 0·78-2·03). Analysis of SEER HCL patients diagnosed before and after 1990 (approximately before and after purine analogue therapy was introduced) showed no evidence of an increased incidence after 1990. A better understanding of any potential association between HCL and skin cancer is highly relevant given ongoing trials using BRAF inhibitors, such as vemurafenib, for relapsed HCL, as RAS-mutant skin cancers could be paradoxically activated in these patients.

  9. [Medicinal plant hairy roots generating and their applications].

    PubMed

    Zhang, Meng; Gao, Wei; Wang, Xiu-Juan

    2014-06-01

    As a kind of the plant tissue cultures, hairy root culture is characterized by rapid growth without exogenous hormones source and high yield of secondary metabolites, which attracted the attention of scholars in resent years. This work systematically summarized the research of medicinal plant hairy roots, including the mechanism, current situation of medicinal plant hairy roots, and their applications. PMID:25272822

  10. [Effects of 6-benzylaminopurine and α-naphthaleneacetic acid on growth and isoflavone contents of Pueraria phaseoloides hairy roots].

    PubMed

    He, Hanjie; Shi, Heping

    2014-10-01

    In order to study the effect of phytohormone on growth and isoflavones contents of Pueraria phaseoloides hairy roots, we cultured the hairy roots with different concentrations of 6-benzylaminopurine (6-BA) alone or in combination with α-naphthaleneacetic acid (NAA). Then we determined the effects of 6-BA alone or in combination with NAA on the growth and the contents of isoflavones compounds and levels of antioxidase activities of hairy roots by spectrophotometry. The results show that 6-BA inhibited the growth, and decreased biomass and total isoflavones compounds of P. phaseoloides hairy roots. Furthermore, the inhibition was increased with the concentrations of 6-BA. Compared with the controls, different concentrations of 6-BA in combination with NAA 2.0 mg/L could inhibit the growth of hairy roots and decrease the content of total isoflavone compounds, and also significantly enhanced the contents of soluble protein and levels of peroxidase (POD) activities, but decreased the activities of superoxide dismutase (SOD). DNA ladders detected by agarose gel electrophoresis can be observed after hairy roots of P. phaseoloides were cultured with 6-BA alone for 30 days, but can appear on the 20th day after culture with 6-BA in combination with NAA 2.0 mg/L. This result indicates that 6-BA or 6-BA in combination with NAA can both stimulate appearance of programmed cell death (PCD), and NAA may play a synergistic role on PCD. PMID:25726582

  11. Drag reduction of a hairy disk

    NASA Astrophysics Data System (ADS)

    Niu, Jun; Hu, David L.

    2011-10-01

    We investigate experimentally the hydrodynamics of a hairy disk immersed in a two-dimensional flowing soap film. Drag force is measured as a function of hair length, density, and coating area. An optimum combination of these parameters yields a drag reduction of 17%, which confirms previous numerical predictions (15%). Flow visualization indicates the primary mechanism for drag reduction is the bending, adhesion, and reinforcement of hairs trailing the disk, which reduces wake width and traps "dead water." Thus, the use of hairy coatings can substantially reduce an object's drag while negligibly increasing its weight.

  12. Enzymes and other agents that enhance cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  13. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  14. Genome-wide errant targeting by Hairy

    PubMed Central

    Kok, Kurtulus; Ay, Ahmet; Li, Li M; Arnosti, David N

    2015-01-01

    Metazoan transcriptional repressors regulate chromatin through diverse histone modifications. Contributions of individual factors to the chromatin landscape in development is difficult to establish, as global surveys reflect multiple changes in regulators. Therefore, we studied the conserved Hairy/Enhancer of Split family repressor Hairy, analyzing histone marks and gene expression in Drosophila embryos. This long-range repressor mediates histone acetylation and methylation in large blocks, with highly context-specific effects on target genes. Most strikingly, Hairy exhibits biochemical activity on many loci that are uncoupled to changes in gene expression. Rather than representing inert binding sites, as suggested for many eukaryotic factors, many regions are targeted errantly by Hairy to modify the chromatin landscape. Our findings emphasize that identification of active cis-regulatory elements must extend beyond the survey of prototypical chromatin marks. We speculate that this errant activity may provide a path for creation of new regulatory elements, facilitating the evolution of novel transcriptional circuits. DOI: http://dx.doi.org/10.7554/eLife.06394.001 PMID:26305409

  15. Cryptic variation in vulva development by cis-regulatory evolution of a HAIRY-binding site.

    PubMed

    Kienle, Simone; Sommer, Ralf J

    2013-01-01

    Robustness to mutations is a general principle of biological systems that allows for the accumulation of cryptic variation. However, little is known about robustness and cryptic variation in core developmental pathways. Here we show through gonad-ablation screens in natural isolates of Pristionchus pacificus cryptic variation in nematode vulva development. This variation is mainly caused by cis-regulatory evolution in the conserved Notch ligand apx-1/Delta and involves binding sites for the transcription factor HAIRY. In some isolates, including a Bolivian strain, absence of a HAIRY-binding site results in Ppa-apx-1 expression in the vulva precursor cell P6.p and causes gonad-independent vulva differentiation. In contrast, a Californian strain that gained a HAIRY-binding site lacks Ppa-apx-1 vulval expression and shows gonad-dependence of vulva development. Addition of this HAIRY-binding site to the Bolivian Ppa-apx-1 promoter eliminates expression in the vulva. Our findings indicate significant cis-regulatory evolution in a core developmental pathway leading to intraspecific cryptic variation.

  16. Activity of quinone alkylating agents in quinone-resistant cells.

    PubMed

    Begleiter, A; Leith, M K

    1990-05-15

    The role of the quinone group in the antitumor activity of quinone alkylating agents, such as mitomycin C and 2,5-diaziridinyl-3,5-bis(carboethoxyamino)-1,4-benzoquinone, is still uncertain. The quinone group may contribute to antitumor activity by inducing DNA strand breaks through the formation of free radicals and/or by influencing the alkylating activity of the quinone alkylators. The cytotoxic activity and DNA damage produced by the model quinone alkylating agents, benzoquinone mustard and benzoquinone dimustard, were compared in L5178Y murine lymphoblasts sensitive and resistant to the model quinone antitumor agent, hydrolyzed benzoquinone mustard. The resistant cell lines, L5178Y/HBM2 and L5178Y/HBM10, have increased concentrations of glutathione and elevated catalase, superoxide dismutase, glutathione S-transferase, and DT-diaphorase activity. L5178Y/HBM2 and L5178Y/HBM10 cells were 7.4- and 8.5-fold less sensitive to benzoquinone mustard and 1.7- and 4.3-fold less sensitive to benzoquinone dimustard, respectively, compared with sensitive cells, but showed no resistance to the non-quinone alkylating agent, aniline mustard. The formation of DNA double strand breaks by benzoquinone mustard was reduced by 2- and 8-fold in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, while double strand break formation by benzoquinone dimustard was reduced only in the L5178Y/HBM10 cells. The number of DNA-DNA cross-links produced by benzoquinone mustard was 3- and 6-fold lower, and the number produced by benzoquinone dimustard was 35% and 2-fold lower in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, compared with L5178Y parental cells. In contrast, cross-linking by aniline mustard was unchanged in sensitive and resistant cells. Dicoumarol, an inhibitor of DT-diaphorase, increased the cytotoxic activity of both benzoquinone mustard and benzoquinone dimustard in L5178Y/HBM10 cells. This study provides evidence that elevated DT-diaphorase activity in the resistant cells

  17. Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids.

    PubMed

    De Jesus-Gonzalez, L; Weathers, P J

    2003-04-01

    Hairy root cultures of diploid Artemisia annua L. (clone YUT16) grow rapidly and produce the antimalarial sesquiterpene artemisinin. Little is known about how polyploidy affects the growth of transformed hairy roots and the production of secondary metabolites. Using colchicine, we produced four stable tetraploid clones of A. annua L. from the YUT16 hairy root clone. Analysis showed major differences in growth and artemisinin production compared to the diploid clone. Tetraploid clones produced up to six times more artemisinin than the diploid parent. This study provides an initial step in increasing our understanding of the role of polyploidy in secondary metabolite production, especially in hairy roots. PMID:12789527

  18. Habitat Suitability Index Models: Hairy Woodpecker

    USGS Publications Warehouse

    Sousa, Patrick J.

    1987-01-01

    A review and synthesis of existing information were used to develop a Habitat Suitability Index (HSI) model for the hairy woodpecker (Picoides villosus). The model consolidates habitat use information into a framework appropriate for field application, and is scaled to produce an index between 0.0 (unsuitable habitat) to 1.0 (optimum habitat). HSI models are designed to be used with Habitat Evaluation Procedures previously developed by the U.S. Fish and Wildlife Service.

  19. Agent-based models in robotized manufacturing cells designing

    NASA Astrophysics Data System (ADS)

    Sekala, A.; Gwiazda, A.; Foit, K.; Banas, W.; Hryniewicz, P.; Kost, G.

    2015-11-01

    The complexity of the components, presented in robotized manufacturing workcells, causes that already at the design phase is necessary to develop models presenting various aspects of their structure and functioning. These models are simplified representation of real systems and allow to, among others, systematize knowledge about the designed manufacturing workcell. They also facilitate defining and analyzing the interrelationships between its particular components. This paper proposes the agent-based approach applied for designing robotized manufacturing cells.

  20. Other Chemotherapeutic Agents in Cutaneous T-Cell Lymphoma.

    PubMed

    Chung, Catherine G; Poligone, Brian

    2015-10-01

    Traditional chemotherapies, interleukins, phosphorylase inhibitors, and proteasome inhibitors are important therapies available to patients with cutaneous T-cell lymphoma (CTCL). Traditional chemotherapies, both in combination and as single agents, are commonly used in relapsed, refractory CTCLs that behave in an aggressive manner. Interleukins, phosphorylase inhibitors, and proteasome inhibitors are less commonly used but data support a role in patients with more refractory disease. PMID:26433850

  1. Detection of chemical agents using a novel energy cell

    NASA Astrophysics Data System (ADS)

    Shewchun, John

    2007-04-01

    The detection, classification and tracking of chemical agents (explosives) being surreptitiously smuggled into public areas, such as airports, for destructive purposes is difficult to solve by unobtrusive means. We propose the use of a novel energy cell with gas/vapor sniffing capability. Variants of such devices are routinely used by police to detect alcohol emanating from the breath of suspected impaired vehicle drivers. We have advanced this technology with the development of a Pethanol Alkaline Energy Cell which is capable of reading gaseous emissions ultimately in the parts per billion range. Our work is described in terms of detecting TATAP (acetone peroxide).

  2. Thymol derivatives from hairy roots of Arnica montana.

    PubMed

    Weremczuk-Jezyna, I; Kisiel, W; Wysokińska, H

    2006-09-01

    Five known thymol derivatives were isolated from roots of Arnica montana transformed with Agrobacterium rhizogenes LBA 9402. The compounds were characterized by spectral methods. The pattern of thymol derivatives in light-grown hairy roots was slightly different from that in dark-grown ones. This is the first report on the presence of thymol derivatives in hairy roots of the plant.

  3. Hairy root culture: bioreactor design and process intensification.

    PubMed

    Stiles, Amanda R; Liu, Chun-Zhao

    2013-01-01

    The cultivation of hairy roots for the production of secondary metabolites offers numerous advantages; hairy roots have a fast growth rate, are genetically stable, and are relatively simple to maintain in phytohormone free media. Hairy roots provide a continuous source of secondary metabolites, and are useful for the production of chemicals for pharmaceuticals, cosmetics, and food additives. In order for hairy roots to be utilized on a commercial scale, it is necessary to scale-up their production. Over the last several decades, significant research has been conducted on the cultivation of hairy roots in various types of bioreactor systems. In this review, we discuss the advantages and disadvantages of various bioreactor systems, the major factors related to large-scale bioreactor cultures, process intensification technologies and overview the mathematical models and computer-aided methods that have been utilized for bioreactor design and development.

  4. Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots.

    PubMed

    Huet, Yoann; Ekouna, Jean-Pierre Ele; Caron, Aurore; Mezreb, Katiba; Boitel-Conti, Michèle; Guerineau, François

    2014-01-01

    A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.

  5. Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots.

    PubMed

    Huet, Yoann; Ekouna, Jean-Pierre Ele; Caron, Aurore; Mezreb, Katiba; Boitel-Conti, Michèle; Guerineau, François

    2014-01-01

    A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots. PMID:24078130

  6. Cell wall proteins of Sporothrix schenckii as immunoprotective agents.

    PubMed

    Alba-Fierro, Carlos A; Pérez-Torres, Armando; López-Romero, Everardo; Cuéllar-Cruz, Mayra; Ruiz-Baca, Estela

    2014-01-01

    Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America. Cell wall (CW) proteins located on the cell surface are inducers of cellular and humoral immune responses, potential candidates for diagnosis purposes and to generate vaccines to prevent fungal infections. This mini-review emphasizes the potential use of S. schenckii CW proteins as protective and therapeutic immune response inducers against sporotrichosis. A number of pathogenic fungi display CW components that have been characterized as inducers of protective cellular and humoral immune responses against the whole pathogen from which they were originally purified. The isolation and characterization of immunodominant protein components of the CW of S. schenckii have become relevant because of their potential in the development of protective and therapeutic immune responses against sporotrichosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

  7. Hairy root cultures for secondary metabolites production.

    PubMed

    Pistelli, Laura; Giovannini, Annalisa; Ruffoni, Barbara; Bertoli, Alessandra; Pistelli, Luisa

    2010-01-01

    Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. This pathogen causes the HR disease leading to the neoplastic growth of roots that are characterized by high growth rate in hormone free media and genetic stability. HRs produce the same phytochemicals pattern of the corresponding wild type organ. High stability and productivity features allow the exploitation of HRs as valuable biotechnological tool for the production of plant secondary metabolites. In addition, several elicitation methods can be used to further enhance their accumulation in both small and large scale production. However, in the latter case, cultivation in bioreactors should be still optimized. HRs can be also utilised as biological farm for the production of recombinant proteins, hence holding additional potential for industrial use. HR technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. The present review summarizes updated aspects of the hairy root induction, genetics and metabolite production. PMID:21520711

  8. Decreased stability of DNA in cells treated with alkylating agents

    SciTech Connect

    Frankfurt, O.S. )

    1990-12-01

    A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg{sup 2+}. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl{sub 2}. Thus, the presence of phosphates and MgCl{sub 2} during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S{sub 1} nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.

  9. Effect of elicitors and precursors on azadirachtin production in hairy root culture of Azadirachta indica.

    PubMed

    Srivastava, Smita; Srivastava, A K

    2014-02-01

    The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag(+), Hg(+2), Co(+2), Cu(+2)) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l(-1) of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g(-1)). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 % v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g(-1)) with respect to the control (3.3 mg g(-1)). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l(-1) in 25 days) was found to be the highest with cholesterol (50 mg l(-1)) addition as an indirect precursor in the medium. PMID:24357500

  10. Plant hairy root cultures as plasmodium modulators of the slime mold emergent computing substrate Physarum polycephalum.

    PubMed

    Ricigliano, Vincent; Chitaman, Javed; Tong, Jingjing; Adamatzky, Andrew; Howarth, Dianella G

    2015-01-01

    Roots of the medicinal plant Valeriana officinalis are well-studied for their various biological activities. We applied genetically transformed V. officinalis root biomass to exert control of Physarum polycephalum, an amoeba-based emergent computing substrate. The plasmodial stage of the P. polycephalum life cycle constitutes a single, multinucleate cell visible by unaided eye. The plasmodium modifies its network of oscillating protoplasm in response to spatial configurations of attractants and repellents, a behavior that is interpreted as biological computation. To program the computing behavior of P. polycephalum, a diverse and sustainable library of plasmodium modulators is required. Hairy roots produced by genetic transformation with Agrobacterium rhizogenes are a metabolically stable source of bioactive compounds. Adventitious roots were induced on in vitro V. officinalis plants following infection with A. rhizogenes. A single hairy root clone was selected for massive propagation and the biomass was characterized in P. polycephalum chemotaxis, maze-solving, and electrical activity assays. The Agrobacterium-derived roots of V. officinalis elicited a positive chemotactic response and augmented maze-solving behavior. In a simple plasmodium circuit, introduction of hairy root biomass stimulated the oscillation patterns of slime mold's surface electrical activity. We propose that manipulation of P. polycephalum with the plant root culture platform can be applied to the development of slime mold microfluidic devices as well as future models for engineering the plant rhizosphere.

  11. Micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin production.

    PubMed

    Ya-ut, Pornwilai; Chareonsap, Piyarat; Sukrong, Suchada

    2011-12-01

    An efficient system was developed for the in vitro micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin (CPT) production. Shoot multiplication on leaf and node explants from germinated seeds of O. alata was successful on half-strength Murashige and Skoog medium supplemented with varying amounts of kinetin and α-naphthaleneacetic acid. Node explants grown in vitro were successfully infected by Agrobacterium rhizogenes TISTR 1450 for the establishment of hairy root culture. The amount of CPT in various parts of O. alata was analyzed by HPLC. The accumulation of CPT in transformed hairy roots was twice that in soil-grown plants (785 ± 52 and 388 ± 32 μg/g dry wt, respectively). In the presence of a polystyrene resin (Diaion HP-20) that absorbed CPT, the CPT content in the culture media increased sevenfold compared with controls (1,036 and 151 μg per 250 ml medium, respectively). These results enable the feasible production of CPT of O. alata by means of a cell culture strategy. These measures can help safeguard the plant from extinction.

  12. Plant hairy root cultures as plasmodium modulators of the slime mold emergent computing substrate Physarum polycephalum

    PubMed Central

    Ricigliano, Vincent; Chitaman, Javed; Tong, Jingjing; Adamatzky, Andrew; Howarth, Dianella G.

    2015-01-01

    Roots of the medicinal plant Valeriana officinalis are well-studied for their various biological activities. We applied genetically transformed V. officinalis root biomass to exert control of Physarum polycephalum, an amoeba-based emergent computing substrate. The plasmodial stage of the P. polycephalum life cycle constitutes a single, multinucleate cell visible by unaided eye. The plasmodium modifies its network of oscillating protoplasm in response to spatial configurations of attractants and repellents, a behavior that is interpreted as biological computation. To program the computing behavior of P. polycephalum, a diverse and sustainable library of plasmodium modulators is required. Hairy roots produced by genetic transformation with Agrobacterium rhizogenes are a metabolically stable source of bioactive compounds. Adventitious roots were induced on in vitro V. officinalis plants following infection with A. rhizogenes. A single hairy root clone was selected for massive propagation and the biomass was characterized in P. polycephalum chemotaxis, maze-solving, and electrical activity assays. The Agrobacterium-derived roots of V. officinalis elicited a positive chemotactic response and augmented maze-solving behavior. In a simple plasmodium circuit, introduction of hairy root biomass stimulated the oscillation patterns of slime mold's surface electrical activity. We propose that manipulation of P. polycephalum with the plant root culture platform can be applied to the development of slime mold microfluidic devices as well as future models for engineering the plant rhizosphere. PMID:26236301

  13. Plant hairy root cultures as plasmodium modulators of the slime mold emergent computing substrate Physarum polycephalum.

    PubMed

    Ricigliano, Vincent; Chitaman, Javed; Tong, Jingjing; Adamatzky, Andrew; Howarth, Dianella G

    2015-01-01

    Roots of the medicinal plant Valeriana officinalis are well-studied for their various biological activities. We applied genetically transformed V. officinalis root biomass to exert control of Physarum polycephalum, an amoeba-based emergent computing substrate. The plasmodial stage of the P. polycephalum life cycle constitutes a single, multinucleate cell visible by unaided eye. The plasmodium modifies its network of oscillating protoplasm in response to spatial configurations of attractants and repellents, a behavior that is interpreted as biological computation. To program the computing behavior of P. polycephalum, a diverse and sustainable library of plasmodium modulators is required. Hairy roots produced by genetic transformation with Agrobacterium rhizogenes are a metabolically stable source of bioactive compounds. Adventitious roots were induced on in vitro V. officinalis plants following infection with A. rhizogenes. A single hairy root clone was selected for massive propagation and the biomass was characterized in P. polycephalum chemotaxis, maze-solving, and electrical activity assays. The Agrobacterium-derived roots of V. officinalis elicited a positive chemotactic response and augmented maze-solving behavior. In a simple plasmodium circuit, introduction of hairy root biomass stimulated the oscillation patterns of slime mold's surface electrical activity. We propose that manipulation of P. polycephalum with the plant root culture platform can be applied to the development of slime mold microfluidic devices as well as future models for engineering the plant rhizosphere. PMID:26236301

  14. Linezolid induced black hairy tongue: a rare side effect.

    PubMed

    Aijazi, Ishma; Abdulla, Fadhil M

    2014-01-01

    Linezolid induced black hairy tongue is a rare benign reversible side effect of linezolid therapy. We report a case of a 61 year old diabetic lady who developed thrombocytopenia and black hairy discoloration of the tongue after being prescribed linezolid for foot osteomyelitis by the orthopaedic surgeon. Patient was encouraged to practice good oral dental hygiene, advised to use a soft tooth brush, regular mouth wash and baking soda containing tooth paste. The condition resolved four weeks after cessation of the antibiotic therapy.

  15. Hairy polyp of the tongue: a case report.

    PubMed

    Erdogan, Seyda; Tunali, Nurdan; Canpolat, Tuba; Tuncer, Recep

    2004-12-01

    Hairy polyps or dermoids of the oro- and nasopharynx are benign lesions containing elements of both ectodermal and mesodermal origin. Because of its rarity, we report a case of hairy polyp arising from the tongue in a 40-day-old infant. The lesion was covered by squamous epithelium and a central core of fibroadipose tissue, minor salivary glands, and cartilage. We discuss the clinicopathological features, terminology, etiology, and differential diagnosis of this condition.

  16. Exact formation of hairy planar black holes

    NASA Astrophysics Data System (ADS)

    Fan, Zhong-Ying; Chen, Bin

    2016-04-01

    We consider Einstein gravity minimally coupled to a scalar field with a given potential in general dimensions. We obtain large classes of static hairy planar black holes which are asymptotic to anti-de Sitter (AdS) space-times. In particular, for a special case μ =(n -2 )/2 , we obtain new classes of exact dynamical solutions describing black hole formation. We find there are two classes of collapse solutions. The first class of solutions describes the evolution start from AdS space-time with a naked singularity at the origin. The space-time is linearly unstable and evolves into stationary black hole states even under small perturbation. The second class of solutions describes the space-time spontaneously evolving from AdS vacua into stationary black hole states undergoing nonlinear instability. We also discuss the global properties of all these dynamical solutions.

  17. Antimalarial evaluation of the chemical constituents of hairy root culture of Bixa orellana L.

    PubMed

    Zhai, Bo; Clark, Julie; Ling, Taotao; Connelly, Michele; Medina-Bolivar, Fabricio; Rivas, Fatima

    2013-01-01

    Over 216 million malaria cases are reported annually worldwide and about a third of these cases, primarily children under the age of five years old, will not survive the infection. Despite this significant world health impact, only a limited number of therapeutic agents are currently available. The lack of scaffold diversity poses a threat in the event that multi-drug-resistant strains emerge. Terrestrial natural products have provided a major source of chemical diversity for starting materials in many FDA approved drugs over the past century. Bixa orellana L. is a popular plant used in South America for the treatment of malaria. In search of new potential therapeutic agents, the chemical constituents of a selected hairy root culture line of Bixa orellana L. were characterized utilizing NMR and mass spectrometry methods, followed by its biological evaluation against malaria strains 3D7 and K1. The crude extract and its isolated compounds demonstrated EC50 values in the micromolar range. Herein, we report our findings on the chemical constituents of Bixa orellana L. from hairy roots responsible for the observed antimalarial activity. PMID:24406786

  18. Elicitation Based Enhancement of Secondary Metabolites in Rauwolfia serpentina and Solanum khasianum Hairy Root Cultures

    PubMed Central

    Srivastava, Mrinalini; Sharma, Swati; Misra, Pratibha

    2016-01-01

    Background: Rauwolfia serpentina and Solanum khasianum are well-known medicinally important plants contained important alkaloids in their different parts. Elicitation of these alkaloids is important because of associated pharmaceutical properties. Targeted metabolites were ajmaline and ajmalicine in R. serpentina; solasodine and α-solanine in S. khasianum. Objective: Enhancement of secondary metabolites through biotic and abiotic elicitors in hairy root cultures of R. serpentina and S. khasianum. Materials and Methods: In this report, hairy root cultures of these two plants were established through Agrobacterium rhizogenes mediated transformation by optimizing various parameters as age of explants, duration of preculture, and co-cultivation period. NaCl was used as abiotic elicitors in these two plants. Cellulase from Aspergillus niger was used as biotic elicitor in S. khasianum and mannan from Saccharomyces cerevisiae was used in R. serpentina. Results: First time we have reported the effect of biotic and abiotic elicitors on the production of important metabolites in hairy root cultures of these two plants. Ajmalicine production was stimulated up to 14.8-fold at 100 mM concentration of NaCl after 1 week of treatment. Ajmaline concentration was also increased 2.9-fold at 100 mg/l dose of mannan after 1 week. Solasodine content was enhanced up to 4.0-fold and 3.6-fold at 100 mM and 200 mM NaCl, respectively, after 6 days of treatments. Conclusion: This study explored the potential of the elicitation strategy in A. rhizogenes transformed cell cultures and this potential further used for commercial production of these pharmaceutically important secondary metabolites. SUMMARY Hairy roots of Rauwolfia serpentina were subjected to salt (abiotic stress) and mannan (biotic stress) treatment for 1 week. Ajmaline and ajmalicine secondary metabolites were quantified before and after stress treatmentAjmalicine yield was enhanced up to 14.8-fold at 100 mM concentration of Na

  19. Antiretroviral Agents Effectively Block HIV Replication after Cell-to-Cell Transfer

    PubMed Central

    Permanyer, Marc; Ballana, Ester; Ruiz, Alba; Badia, Roger; Riveira-Munoz, Eva; Gonzalo, Encarna; Clotet, Bonaventura

    2012-01-01

    Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo. PMID:22696642

  20. Gentiana dinarica Beck hairy root cultures and evaluation of factors affecting growth and xanthone production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The induction and establishment of hairy root cultures of Gentiana dinarica using two strains of Agrobacterium rhizogenes (A4M70GUS and 15834/PI) is reported for the first time. Hairy roots were formed from the shoots 25 days after inoculation, and strain 15834/PI had higher induction rate of hairy ...

  1. HTLV-1 Tax protein sensitizes cells to apoptotic cell death induced by DNA damaging agents.

    PubMed

    Kao, S Y; Lemoine, F J; Mariott, S J

    2000-04-27

    Transient HTLV-1 Tax expression suppresses cellular nucleotide excision repair, and this effect correlates with Tax transactivation of the proliferating cell nuclear antigen promoter. The inability to repair DNA damage typically induces apoptotic cell death. Therefore, we investigated the effect of Tax-mediated suppression of DNA repair on apoptosis in stable Tax-expressing cells. Constitutive Tax expression reduced cellular nucleotide excision repair activity compared with parental and control cells. Tax-expressing cells were also more sensitive to apoptosis induced by DNA damaging agents than control cells. Even though Tax-expressing cells displayed reduced DNA repair, they showed increased DNA replication following UV damage. These results suggest that Tax suppresses the cell's ability to repair DNA damage and stimulates DNA replication even in the presence of damage. The inability to repair DNA damage is likely to stimulate apoptotic cell death in the majority of Tax-expressing cells while the ability to promote DNA replication may also allow the survival of a small population of cells. We propose that together these effects contribute to the monoclonal nature and low efficiency of HTLV-1 transformation.

  2. Double-layer interaction between two plates with hairy surfaces.

    PubMed

    Huang, Haohao; Ruckenstein, Eli

    2004-05-01

    In most theoretical treatments of colloidal particles with hairy surfaces, only the steric effect is taken into account. The steric force is a short-range interaction and acts only when the chains on different particles begin to interpenetrate each other. However, since the hairy chains are extended into the continuous phase, they constrain the orientation of the water molecules near the surface and, as a result, the dielectric constant in that region can become very different from that in the bulk. The low dielectric constant affects the distributions of ion concentrations and the gradient of the electric field. Therefore, the double-layer interactions between two plates with hairy surfaces cannot be calculated on the basis of the classical Gouy-Chapman theory, which involves a uniform dielectric constant in the Poisson-Boltzmann equation. A model which accounts for the difference in dielectric constants in the hairy region and outside that region is therefore proposed. The ion specificity is also taken into account by using Born's expression for the free energy of hydration of ions. The repulsive forces calculated via the Gouy-Chapman theory and via the new model are compared. The hairy region can have a long range effect on the repulsive double-layer interactions.

  3. Cell-based phenotypic screening of mast cell degranulation unveils kinetic perturbations of agents targeting phosphorylation

    PubMed Central

    Qin, Shenlu; Wang, Xumeng; Wu, Huanwen; Xiao, Peng; Cheng, Hongqiang; Zhang, Xue; Ke, Yuehai

    2016-01-01

    Mast cells play an essential role in initiating allergic diseases. The activation of mast cells are controlled by a complicated signal network of reversible phosphorylation, and finding the key regulators involved in this network has been the focus of the pharmaceutical industry. In this work, we used a method named Time-dependent cell responding profile (TCRP) to track the process of mast cell degranulation under various perturbations caused by agents targeting phosphorylation. To test the feasibility of this high-throughput cell-based phenotypic screening method, a variety of biological techniques were used. We further screened 145 inhibitors and clustered them based on the similarities of their TCRPs. Stat3 phosphorylation has been widely reported as a key step in mast cell degranulation. Interestingly, our TCRP results showed that a Stat3 inhibitor JSI124 did not inhibit degranulation like other Stat3 inhibitors, such as Stattic, clearly inhibited degranulation. Regular endpoint assays demonstrated that the distinctive TCRP of JSI124 potentially correlated with the ability to induce apoptosis. Consequently, different agents possibly have disparate functions, which can be conveniently detected by TCRP. From this perspective, our TCRP screening method is reliable and sensitive when it comes to discovering and selecting novel compounds for new drug developments. PMID:27502076

  4. Using Hairy Roots for Production of Valuable Plant Secondary Metabolites.

    PubMed

    Tian, Li

    2015-01-01

    Plants synthesize a wide variety of natural products, which are traditionally termed secondary metabolites and, more recently, coined specialized metabolites. While these chemical compounds are employed by plants for interactions with their environment, humans have long since explored and exploited plant secondary metabolites for medicinal and practical uses. Due to the tissue-specific and low-abundance accumulation of these metabolites, alternative means of production in systems other than intact plants are sought after. To this end, hairy root culture presents an excellent platform for producing valuable secondary metabolites. This chapter will focus on several major groups of secondary metabolites that are manufactured by hairy roots established from different plant species. Additionally, the methods for preservations of hairy roots will also be reviewed. PMID:25583225

  5. Hairy black holes in the general Skyrme model

    NASA Astrophysics Data System (ADS)

    Adam, C.; Kichakova, O.; Shnir, Ya.; Wereszczynski, A.

    2016-07-01

    We study the existence of hairy black holes in the generalized Einstein-Skyrme model. It is proven that in the Bogomol'nyi-Prasad-Sommerfield model limit there are no hairy black hole solutions, although the model admits gravitating (and flat space) solitons. Furthermore, we find strong evidence that a necessary condition for the existence of black holes with Skyrmionic hair is the inclusion of the Skyrme term L4. As an example, we show that there are no hairy black holes in the L2+L6+L0 model and present a new kind of black hole solutions with compact Skyrmion hair in the L4+L6+L0 model.

  6. Linezolid induced black hairy tongue: a rare side effect.

    PubMed

    Aijazi, Ishma; Abdulla, Fadhil M

    2014-01-01

    Linezolid induced black hairy tongue is a rare benign reversible side effect of linezolid therapy. We report a case of a 61 year old diabetic lady who developed thrombocytopenia and black hairy discoloration of the tongue after being prescribed linezolid for foot osteomyelitis by the orthopaedic surgeon. Patient was encouraged to practice good oral dental hygiene, advised to use a soft tooth brush, regular mouth wash and baking soda containing tooth paste. The condition resolved four weeks after cessation of the antibiotic therapy. PMID:25671958

  7. Experimental Observation of Hairy Surface Exposed in Airflow

    NASA Astrophysics Data System (ADS)

    Hasegawa, Mitsugu; Sakaue, Hirotaka

    2015-11-01

    The development of drag reduction method is important to reduce the consumption of limited energy in the field of engineering. While active method which needs external energy has received significant attention, passive method which means no external energy use has been focused. As one of the potential passive drag reduction method for offshore structure, aircraft, wind turbine, flexible hair implanted on the object surface has been studied. Here we make hairy surface. We conduct flow visualization to investigate the behavior of hairy surface exposed in wind tunnel. In the presentation, a current status of this experiment will be presented.

  8. MALDI-TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor.

    PubMed

    López, Edgar García; Ramírez, Emma Gloria Ramos; Gúzman, Octavio Gómez; Calva, Graciano Calva; Ariza-Castolo, Armando; Pérez-Vargas, Josefina; Rodríguez, Herminia Guadalupe Martínez

    2014-01-01

    Expression systems based on plant cells, tissue, and organ cultures have been investigated as an alternative for production of human therapeutic proteins in bioreactors. In this work, hairy root cultures of Brassica oleracea var. italica (broccoli) were established in an airlift with mesh bioreactor to produce isoform 1 of the human growth hormone (hGH1) as a model therapeutic protein. The hGH1 cDNA was cloned into the pCAMBIA1105.1 binary vector to induce hairy roots in hypocotyls of broccoli plantlets via Agrobacterium rhizogenes. Most of the infected plantlets (90%) developed hairy roots when inoculated before the appearance of true leaves, and keeping the emerging roots attached to hypocotyl explants during transfer to solid Schenk and Hildebrandt medium. The incorporation of the cDNA into the hairy root genome was confirmed by PCR amplification from genomic DNA. The expression and structure of the transgenic hGH1 was assessed by ELISA, western blot, and MALDITOF-MS analysis of the purified protein extracted from the biomass of hairy roots cultivated in bioreactor for 24 days. Production of hGH1 was 5.1 ± 0.42 µg/g dry weight (DW) for flask cultures, and 7.8 ± 0.3 µg/g DW for bioreactor, with productivity of 0.68 ± 0.05 and 1.5 ± 0.06 µg/g DW*days, respectively, indicating that the production of hGH1 was not affected by the growth rate, but might be affected by the culture system. These results demonstrate that hairy root cultures of broccoli have potential as an alternative expression system for production of hGH1, and might also be useful for production of other therapeutic proteins. PMID:24124083

  9. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    SciTech Connect

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells.

  10. Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.

    PubMed

    Plasencia, Anna; Soler, Marçal; Dupas, Annabelle; Ladouce, Nathalie; Silva-Martins, Guilherme; Martinez, Yves; Lapierre, Catherine; Franche, Claudine; Truchet, Isabelle; Grima-Pettenati, Jacqueline

    2016-06-01

    Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes. PMID:26579999

  11. Effect of harvest timing and leaf hairiness on fiber quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent concerns over leaf grades have generated questions of how both time of day cotton is harvested, as well as leaf hairiness levels of certain varieties, influence fiber quality. To address this, two smooth leaf varieties and two varieties with higher levels of leaf pubescence were harvested at...

  12. Hairy vetch seedbank persistence and implications for cover crop management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hairy vetch (Vicia villosa Roth) is a fast growing, winter hardy annual legume that can produce shoot biomass levels upwards of 6500 kg ha-1. This cover crop is well suited for summer annual grain rotations, as it fixes considerable amounts of nitrogen, reduces erosion through rapid ground cover, an...

  13. Hairy polyp can be lethal even when small in size.

    PubMed

    Koike, Yuhki; Uchida, Keiichi; Inoue, Mikihiro; Ohtsu, Kazuya; Tanaka, Takaaki; Otake, Kohei; Tanaka, Koji; Kusunoki, Masato

    2013-06-01

    A case of sudden cardiopulmonary arrest in a 3-month-old girl is presented. The patient had barely recovered from hypoxic encephalopathy when she presented with repeated respiratory distress. Computed tomography and endoscopic analysis revealed a shiny polyp in the lateral wall of the nasopharynx, and this polyp was suspected to be the main cause of respiratory distress. After referral to our hospital, surgical removal was performed, and the histopathological diagnosis was hairy polyp. Hairy polyp is a rare congenital benign tumor that sometimes induces respiratory distress. This polyp can potentially induce a life-threatening event. In a systematic review of 40 reported cases, polyps of ≤ 3.0 cm in diameter have a higher risk of respiratory distress than do those >3.0 cm in diameter (P = 0.01). Small hairy polyps may be lethal because of delayed diagnosis. To locate small hairy polyps, physicians should not hesitate to perform further examination because there is the possibility of oversight with only physical examination.

  14. Quantitative imaging of cell-permeable magnetic resonance contrast agents using x-ray fluorescence.

    PubMed

    Endres, Paul J; Macrenaris, Keith W; Vogt, Stefan; Allen, Matthew J; Meade, Thomas J

    2006-01-01

    The inability to transduce cellular membranes is a limitation of current magnetic resonance imaging probes used in biologic and clinical settings. This constraint confines contrast agents to extracellular and vascular regions of the body, drastically reducing their viability for investigating processes and cycles in developmental biology. Conversely, a contrast agent with the ability to permeate cell membranes could be used in visualizing cell patterning, cell fate mapping, gene therapy, and, eventually, noninvasive cancer diagnosis. Therefore, we describe the synthesis and quantitative imaging of four contrast agents with the capability to cross cell membranes in sufficient quantity for detection. Each agent is based on the conjugation of a Gd(III) chelator with a cellular transduction moiety. Specifically, we coupled Gd(III)-diethylenetriaminepentaacetic acid DTPA and Gd(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with an 8-amino acid polyarginine oligomer and an amphipathic stilbene molecule, 4-amino-4'-(N,N-dimethylamino)stilbene. The imaging modality that provided the best sensitivity and spatial resolution for direct detection of the contrast agents is synchrotron radiation x-ray fluorescence (SR-XRF). Unlike optical microscopy, SR-XRF provides two-dimensional images with resolution 10(3) better than (153)Gd gamma counting, without altering the agent by organic fluorophore conjugation. The transduction efficiency of the intracellular agents was evaluated by T(1) analysis and inductively coupled plasma mass spectrometry to determine the efficacy of each chelate-transporter combination. PMID:17150161

  15. Agent-Based Modeling of Cancer Stem Cell Driven Solid Tumor Growth.

    PubMed

    Poleszczuk, Jan; Macklin, Paul; Enderling, Heiko

    2016-01-01

    Computational modeling of tumor growth has become an invaluable tool to simulate complex cell-cell interactions and emerging population-level dynamics. Agent-based models are commonly used to describe the behavior and interaction of individual cells in different environments. Behavioral rules can be informed and calibrated by in vitro assays, and emerging population-level dynamics may be validated with both in vitro and in vivo experiments. Here, we describe the design and implementation of a lattice-based agent-based model of cancer stem cell driven tumor growth. PMID:27044046

  16. Quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapeutic agents.

    PubMed

    Wang, Wenge; Gallant, Jean-Nicolas; Katz, Sharyn I; Dolloff, Nathan G; Smith, Charles D; Abdulghani, Junaid; Allen, Joshua E; Dicker, David T; Hong, Bo; Navaraj, Arunasalam; El-Deiry, Wafik S

    2011-08-01

    Quinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. Treatment by quinacrine alone at concentrations of 10-20 mM for 1-2 d cannot kill hepatocellular carcinoma cells, such as HepG2, Hep3B, Huh7, which are also resistant to TRAIL. However, quinacrine renders these cells sensitive to treatment by TRAIL. Co-treatment of these cells with quinacrine and TRAIL induces overwhelming cell death within 3-4 h. Levels of DR5, a pro-apoptotic death receptor of TRAIL, are increased upon treatment with quinacrine, while levels of Mcl-1, an anti-apoptotic member of the Bcl-2 family, are decreased. While the synergistic effect of quinacrine with TRAIL appears to be in part independent of p53, knockdown of p53 in HepG2 cells by siRNA results in more cell death after treatment by quinacrine and TRAIL. The mechanism by which quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapies, and the potential for clinical application currently are being further explored. Lastly, quinacrine synergizes with chemotherapeutics, such as adriamycin, 5-FU, etoposide, CPT11, sorafenib, and gemcitabine, in killing hepatocellular carcinoma cells in vitro and the drug enhances the activity of sorafenib to delay tumor growth in vivo. PMID:21725212

  17. Sensitivity of human dental pulp cells to eighteen chemical agents used for endodontic treatments in dentistry.

    PubMed

    Kobayashi, Morio; Tsutsui, Takeo W; Kobayashi, Tomoko; Ohno, Maki; Higo, Yukari; Inaba, Tomohiro; Tsutsui, Takeki

    2013-01-01

    To determine the adverse effects against human dental pulp tissue, the sensitivity of human dental pulp cells (D824 cells) to 18 chemical agents used for endodontic treatments in dentistry was examined. The cytotoxicity, as determined by a decrease in colony-forming ability of cells treated with the chemical agents, increased as the concentration increased. As a quantitative measure of the cytotoxic effect, LC(50), the concentration which induces a 50% lethality, was extrapolated from the concentration-response curves. The rank of the chemical agents according to their cytotoxic effect (LC(50)) was sodium arsenite > formaldehyde > hydrogen peroxide > zinc oxide > thymol ≈ iodoform ≈ eugenol > guaiacol > ethylenediaminetetraacetic acid ≈ iodine > procaine > lidocaine ≈ chloramphenicol ≈ m-cresol > calcium hydroxide ≈ sodium hypochlorite ≈ phenol ≈ p-phenolsulfonic acid. To compare the cytotoxicity and the levels of apoptosis and mRNA expression of five genes related to the function of dental pulp tissue, D824 cells treated with the LC(50) concentrations of chemical agents were assayed by the TUNEL method and quantitative reverse transcription polymerase chain reaction analysis, respectively. The inducibility of apoptotic cells and the level of mRNA expression of the genes varied with the chemical agents, indicating that both effects occurred independent of the rank of cytotoxic effect of the chemical agents. The results not only provide information concerning cytotoxicity of various chemical agents to human dental pulp cells, but also show an insight into the diversity of the pharmacodynamic action of the chemical agents.

  18. Naso-oropharyngeal choristoma (hairy polyps): an overview and current update on presentation, management, origin and related controversies.

    PubMed

    Dutta, Mainak; Roy, Soham; Ghatak, Soumya

    2015-05-01

    This review presents a comprehensive and updated overview of bigerminal choristomas (hairy polyps) of naso-oropharynx/oral cavity, and discusses the controversies related to nosology and origin from a clinico-embryologic perspective. English-language texts of the last 25 years (January 1989-January 2014) were collected from the PubMed/MEDLINE database using the given keywords. Of the 330 records, 64 full-text articles (mostly case reports/series) were selected, incorporating clinical data from 78 patients, after screening through duplicates and the given exclusion criteria. With the available evidence, hairy polyps appear more common than generally believed, and are increasingly being recognized as an important, often-missed cause of respiratory distress and feeding difficulty in neonates and infants. Such a child without any apparent cause should be examined with flexible nasopharyngoscope to specifically look for hairy polyps which might be life-threatening, especially when small. The female preponderance as believed today has been found to be an overestimation in this review. These lesions are characteristically composed of mature ectodermal and mesodermal tissue derivatives presenting as heterotopic masses, hence termed choristoma. However, little is known about their origin, and whether they are developmental malformations or primitive teratomas is debatable. Involvement of Eustachian tube and tonsils as predominant subsites and the speculated molecular embryogenesis link hairy polyps to the development of the first and second pharyngeal arches. They are exceptionally rare in adults, but form a distinct entity in this age-group and could be explained as delayed pluripotent cell morphogenesis or focal neoplastic malformations, keeping with the present-day understandings of the expanded "teratoma family".

  19. Chronic B-Cell Leukemias and Agent Orange

    MedlinePlus

    ... survivors' benefits . Research on B-cell leukemias and herbicides The Health and Medicine Division (HMD) (formally known ... sufficient evidence of an association between exposure to herbicides and chronic lymphocytic leukemia. In 2003, VA recognized ...

  20. Modeling of gene therapy for regenerative cells using intelligent agents.

    PubMed

    Adly, Aya Sedky; Aboutabl, Amal Elsayed; Ibrahim, M Shaarawy

    2011-01-01

    Gene therapy is an exciting field that has attracted much interest since the first submission of clinical trials. Preliminary results were very encouraging and prompted many investigators and researchers. However, the ability of stem cells to differentiate into specific cell types holds immense potential for therapeutic use in gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. It is widely recognized that gain/loss of function approaches using gene therapy are essential for understanding specific genes functions, and such approaches would be particularly valuable in studies involving stem cells. A significant complexity is that the development stage of vectors and their variety are still not sufficient to be efficiently applied in stem cell therapy. The development of scalable computer systems constitutes one step toward understanding dynamics of its potential. Therefore, the primary goal of this work is to develop a computer model that will support investigations of virus' behavior and organization on regenerative tissues including genetically modified stem cells. Different simulation scenarios were implemented, and their results were encouraging compared to ex vivo experiments, where the error rate lies in the range of acceptable values in this domain of application.

  1. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells.

    PubMed

    He, Huan; Li, Dong-Wei; Yang, Li-Yun; Fu, Li; Zhu, Xun-Jin; Wong, Wai-Kwok; Jiang, Feng-Lei; Liu, Yi

    2015-01-01

    Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. PMID:26337336

  2. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells

    PubMed Central

    He, Huan; Li, Dong-Wei; Yang, Li-Yun; Fu, Li; Zhu, Xun-Jin; Wong, Wai-Kwok; Jiang, Feng-Lei; Liu, Yi

    2015-01-01

    Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. PMID:26337336

  3. [Effects of phytohormones on plant regeneration and production of flavonoids in transgenic Saussurea involucrata hairy roots].

    PubMed

    Qiao, Xianli; Jiang, Shuguang; Li, Xiaofeng; Li, Fengxia; Zhao, Dexiu

    2011-01-01

    We investigated the plant regeneration and production of flavonoids in three high-yield flavonoids transgenic Saussurea involucrata hairy roots C17, C27 and C46 by quantification of two phytohormones GA3 and IAA. The results showed that GA3 concentration at more than 1.0 mg/L could induce adventitious shoots in the hairy root lines. The highest shoot regeneration rate, about 82%, was obtained when the hairy roots C17 were cultured with 2.0 mg/L GA3. The results on HPLC and UV spectrophotometry showed that exogenous application of both GA3 and IAA increased the content of flavonoids in the hairy roots. The contents of flavonoids and apigenin in the hormone-treated hairy roots and regenerates were higher comparing with those in the untreated hairy roots and the regenerates. However, the content of flavonoids was not related to tissue weight, and was negatively related to the regeneration efficiency.

  4. Oestrous cycle of captive southern hairy-nosed wombats (Lasiorhinus latifrons) in South Australia, Australia.

    PubMed

    Finlayson, G R; Shimmin, G A; Taggart, D A; Skinner, J F; Gilmore, A; Paris, M C J

    2006-10-01

    There is limited information available on the oestrous cycle of female southern hairy-nosed wombats (Lasiorhinus latifrons). This is mainly due to an extremely poor breeding success in captivity and the difficulty in routine recapturing of these cryptic, semi-fossorial animals in the wild. The aim of this study was to characterise the oestrous cycle of this species by monitoring peripheral plasma concentrations of progesterone and oestradiol, assessing changes in vaginal cytology, pouch condition and the urogenital sinus. Eight adult female wombats were monitored during the breeding season (July-December) over 2 years (2002-2003). Samples were collected up to three times a week. Vaginal smears contained several cell types, categorised by morphology, as either superficial epithelial cells or parabasal-intermediate cells. Leucocytes were also counted. Plasma progesterone profiles showed a mean oestrous cycle length of 36.33+/-0.67 days with a peak progesterone concentration of 139.53+/-10.62nmol/L. Levels of oestradiol peaked at a mean level of 467.33+/-44.32pmol/L on average 5 days before a rise in plasma progesterone values. The proportion of epithelial cells in vaginal smears varied throughout the cycle, with a high percentage of superficial epithelial cells observed during the follicular phase. During periods when progesterone concentrations were high, a greater percentage of parabasal-intermediate cells was observed. In conclusion, this study has characterised the oestrous cycle of the southern hairy-nosed wombat and confirmed that changes in vaginal smears together with pouch and urogenital sinus details could be used to determine signs of oestrus in this species. PMID:16289971

  5. Oestrous cycle of captive southern hairy-nosed wombats (Lasiorhinus latifrons) in South Australia, Australia.

    PubMed

    Finlayson, G R; Shimmin, G A; Taggart, D A; Skinner, J F; Gilmore, A; Paris, M C J

    2006-10-01

    There is limited information available on the oestrous cycle of female southern hairy-nosed wombats (Lasiorhinus latifrons). This is mainly due to an extremely poor breeding success in captivity and the difficulty in routine recapturing of these cryptic, semi-fossorial animals in the wild. The aim of this study was to characterise the oestrous cycle of this species by monitoring peripheral plasma concentrations of progesterone and oestradiol, assessing changes in vaginal cytology, pouch condition and the urogenital sinus. Eight adult female wombats were monitored during the breeding season (July-December) over 2 years (2002-2003). Samples were collected up to three times a week. Vaginal smears contained several cell types, categorised by morphology, as either superficial epithelial cells or parabasal-intermediate cells. Leucocytes were also counted. Plasma progesterone profiles showed a mean oestrous cycle length of 36.33+/-0.67 days with a peak progesterone concentration of 139.53+/-10.62nmol/L. Levels of oestradiol peaked at a mean level of 467.33+/-44.32pmol/L on average 5 days before a rise in plasma progesterone values. The proportion of epithelial cells in vaginal smears varied throughout the cycle, with a high percentage of superficial epithelial cells observed during the follicular phase. During periods when progesterone concentrations were high, a greater percentage of parabasal-intermediate cells was observed. In conclusion, this study has characterised the oestrous cycle of the southern hairy-nosed wombat and confirmed that changes in vaginal smears together with pouch and urogenital sinus details could be used to determine signs of oestrus in this species.

  6. Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents

    SciTech Connect

    Yadav, N.; Kumar, S.; Marlowe, T.; Chaudhary, A. K.; Kumar, R.; Wang, J.; O'Malley, J.; Boland, P. M.; Jayanthi, S.; Kumar, T. K. S.; Yadava, N.; Chandra, D.

    2015-11-05

    Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency

  7. Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents

    DOE PAGES

    Yadav, N.; Kumar, S.; Marlowe, T.; Chaudhary, A. K.; Kumar, R.; Wang, J.; O'Malley, J.; Boland, P. M.; Jayanthi, S.; Kumar, T. K. S.; et al

    2015-11-05

    Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrialmore » biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS

  8. Rapid, cell-based toxicity screen of potentially therapeutic post-transcriptional gene silencing agents.

    PubMed

    Kolniak, Tiffany A; Sullivan, Jack M

    2011-05-01

    Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular co-localization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding

  9. Rapid, cell-based toxicity screen of potentially therapeutic post-transcriptional gene silencing agents.

    PubMed

    Kolniak, Tiffany A; Sullivan, Jack M

    2011-05-01

    Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular co-localization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding

  10. Combating photooxidative stress in green hairy roots of Daucus carota cultivated under light irradiation.

    PubMed

    Mukherjee, Chiranjit; Sircar, Debabrata; Chatterjee, Moniya; Das, Sampa; Mitra, Adinpunya

    2014-01-15

    The light-dependent generation of active oxygen species, which can disrupt normal metabolic process of plant, is termed as photo-oxidative stress. Plants are equipped with enzymatic and non-enzymatic antioxidative defence system to reduce the effect of such stress. Hairy root culture of Daucus carota when cultivated under continuous illumination (250 μmol m(-2)s(-1)) turned green. To know the reason behind that and photo-oxidative stress response in green hairy roots, activities of several antioxidant enzymes were measured. When compared with normal hairy roots, green hairy roots showed an enhanced superoxide dismutase (SOD) activity. Treatment with a SOD inhibitor diethyldithiocarbamate led to suppression of SOD activity in a concentration-dependent manner in green hairy roots. Interestingly, SOD-suppressed root showed three-fold enhanced caffeic acid glucoside accumulation in the soluble fraction as compared to untreated ones. While ascorbate peroxidase activity showed marginal increase in green hairy roots, a decrease in the activities of guaiacol peroxidase and catalase were observed. SDS-PAGE of crude protein profile from green hairy roots showed a distinct band, which was absent in normal hairy roots. MALDI-TOF-MS/MS analysis of the extracted protein confirmed it as the large subunit of RuBisCO. RT-PCR based expression analysis of betaine aldehyde dehydrogenase showed enhanced transcript levels in green hairy roots as compared to normal hairy roots, whereas reverse trends were observed with the transcripts accumulation for phenylalanine ammonia-lyase and chalcone synthase. These findings corroborate with the in vitro BADH activities in hairy roots, and thus indicate an important role of this stress enzyme in combating photo-oxidative stress in green hairy roots upon continuous light exposure.

  11. Hairy carbon electrodes studied by cyclic voltammetry and battery discharge testing

    NASA Technical Reports Server (NTRS)

    Chung, Deborah D. L.; Shui, Xiaoping; Frysz, Christine A.

    1993-01-01

    Hairy carbon is a new material developed by growing submicron carbon filaments on conventional carbon substrates. Typical substrate materials include carbon black, graphite powder, carbon fibers, and glassy carbon. A catalyst is used to initiate hair growth with carbonaceous gases serving as the carbon source. To study the electrochemical behavior of hairy carbons, cyclic voltammetry (CV) and discharge testing were conducted. In both cases, hairy carbon results surpassed those of the substrate material alone.

  12. Spying on Cells: Toward a Perfect Sleeper Agent.

    PubMed

    Wiens, Matthew D; Lu, Xiaocen; Campbell, Robert E

    2016-07-21

    Creative engineering of fluorescent proteins has yielded a variety of tools for visualization of biochemical events in vivo. In this issue of Cell Chemical Biology, To et al. (2016) describe a fluorogenic green fluorescent protein that is activated by caspase-3 activity and enables imaging of apoptosis in developing zebrafish embryos (To et al., 2016).

  13. Manipulating biological agents and cells in micro-scale volumes for applications in medicine

    PubMed Central

    Tasoglu, Savas; Gurkan, Umut Atakan; Wang, ShuQi

    2013-01-01

    Recent technological advances provide new tools to manipulate cells and biological agents in micro/nano-liter volumes. With precise control over small volumes, the cell microenvironment and other biological agents can be bioengineered; interactions between cells and external stimuli can be monitored; and the fundamental mechanisms such as cancer metastasis and stem cell differentiation can be elucidated. Technological advances based on the principles of electrical, magnetic, chemical, optical, acoustic, and mechanical forces lead to novel applications in point-of-care diagnostics, regenerative medicine, in vitro drug testing, cryopreservation, and cell isolation/purification. In this review, we first focus on the underlying mechanisms of emerging examples for cell manipulation in small volumes targeting applications such as tissue engineering. Then, we illustrate how these mechanisms impact the aforementioned biomedical applications, discuss the associated challenges, and provide perspectives for further development. PMID:23575660

  14. Manipulating biological agents and cells in micro-scale volumes for applications in medicine.

    PubMed

    Tasoglu, Savas; Gurkan, Umut Atakan; Wang, Shuqi; Demirci, Utkan

    2013-07-01

    Recent technological advances provide new tools to manipulate cells and biological agents in micro/nano-liter volumes. With precise control over small volumes, the cell microenvironment and other biological agents can be bioengineered; interactions between cells and external stimuli can be monitored; and the fundamental mechanisms such as cancer metastasis and stem cell differentiation can be elucidated. Technological advances based on the principles of electrical, magnetic, chemical, optical, acoustic, and mechanical forces lead to novel applications in point-of-care diagnostics, regenerative medicine, in vitro drug testing, cryopreservation, and cell isolation/purification. In this review, we first focus on the underlying mechanisms of emerging examples for cell manipulation in small volumes targeting applications such as tissue engineering. Then, we illustrate how these mechanisms impact the aforementioned biomedical applications, discuss the associated challenges, and provide perspectives for further development.

  15. Novel therapeutic agents for cutaneous T-Cell lymphoma

    PubMed Central

    2012-01-01

    Mycosis fungoides (MF) and Sezary Syndrome (SS) represent the most common subtypes of primary Cutaneous T-cell lymphoma (CTCL). Patients with advanced MF and SS have a poor prognosis leading to an interest in the development of new therapies with targeted mechanisms of action and acceptable safety profiles. In this review we focus on such novel strategies that have changed the treatment paradigm of this rare malignancy. PMID:22594538

  16. Multiwall carbon nanotubes as MRI contrast agents for tracking stem cells

    NASA Astrophysics Data System (ADS)

    Vittorio, Orazio; Duce, Suzanne L.; Pietrabissa, Andrea; Cuschieri, Alfred

    2011-03-01

    In this study we investigate the potential of multiwall carbon nanotubes (MWCNTs) with low metal impurities (2.57% iron) as magnetic resonance imaging (MRI) contrast agents. Taking into account probable aggregation at high MWCNTs concentration analysis shows that the r2 relaxivity of MWCNTs in 1% agarose gels at 19 °C is 564 ± 41 s - 1 mM - 1; this is attributed to both the presence of iron oxide impurities and also to the carbon MWCNT structure itself. Stem cells were labelled with MWCNTs to demonstrate the effectiveness of MWCNTs as MRI contrast agents for cellular MRI. The MWCNTs did not impair cell viability or proliferation. These results suggest that the MRI contrast agent properties of the MWCNTs could be used in vivo for stem cell tracking/imaging and during MWCNT-mediated targeted electro-chemotherapy of tumours.

  17. A model of hematopoietic stem cell proliferation under the influence of a chemotherapeutic agent in combination with a hematopoietic inducing agent

    PubMed Central

    2014-01-01

    Background Hematopoiesis is a complex process that encompasses both pro-mitotic and anti-mitotic stimuli. Pharmacological agents used in chemotherapy have a prominent anti-mitotic effect. The approach of inhibiting cell proliferation is rational with respect to the rapidly dividing malignant cells. However, it poses a serious problem with respect to cell proliferation of cell types required for the ‘house-keeping’ operations of the human body. One such affected system is hematopoiesis. Chemotherapy induced anemia is an undesired side effect of chemotherapy that can lead to serious complications. Patients exhibiting anemia or leukopenia during chemotherapy are frequently administered a hematopoietic inducing agent that enhances hematopoiesis. Methods In previous work, we derived a mathematical model consisting of a set of delay differential equations that was dependent on the effect of a hematopoietic inducing agent. The aim of the current work was to formulate a mathematical model that captures both the effect of a chemotherapeutic agent in combination with a hematopoietic inducing agent. Steady state solutions and stability analysis of the system of equations is performed and numerical simulations of the stem cell population are provided. Results Numerical simulations confirm that our mathematical model captures the desired result which is that the use of hematopoietic agents in conjunction with chemotherapeutic agents can decrease the negative secondary effects often experienced by patients. Conclusions The proposed model indicates that the introduction of hematopoietic inducing agents have clinical potential to offset the deleterious effects of chemotherapy treatment. Furthermore, the proposed model is relevant in that it enhances the understanding of stem cell dynamics and provides insight on the stem cell kinetics. PMID:24438084

  18. Microbial cell wall agents as an occupational hazard

    SciTech Connect

    Sigsgaard, T. . E-mail: ts@mil.au.dk; Bonefeld-Jorgensen, E.C.; Hoffmann, H.J.; Bonlokke, J.; Krueger, T.

    2005-09-01

    Organic dusts cause inflammatory reactions in the tissues exposed. The lung and the cells lining the surface of the respiratory tract are a primary target. Many receptors have been shown to react specifically on the presence of microorganisms that are ubiquitous elements in organic dusts. There is a great variability in the individual response to organic dusts. Almost 50% of Caucasians are hyporesponders to LPS exposure, and people with alpha-1-antitrypsin deficiency are hyperresponsive to organic dust exposure. The diseases resulting from organic dust exposures include asthma, allergy, hypersensitivity pneumonitis and toxic pneumonitis (organic dust toxic syndrome).This paper deals with inflammation and the subsequent mechanism of disease as it is encountered in industries with these exposures. Toxicological studies including human experimental exposures and ex vivo studies of cells are described. Cellular reactions are mediated through the attachment of, e.g. LPS and {beta} (1,3)-D-glucan to lipopolysaccharide binding protein, CD14 and Toll-like receptors. The relation between protein release and the gene activation is described. Furthermore, studies of the individual susceptibility will be reviewed.

  19. Brassica napus hairy roots and rhizobacteria for phenolic compounds removal.

    PubMed

    González, Paola S; Ontañon, Ornella M; Armendariz, Ana L; Talano, Melina A; Paisio, Cintia E; Agostini, Elizabeth

    2013-03-01

    Phenolic compounds are contaminants frequently found in water and soils. In the last years, some technologies such as phytoremediation have emerged to remediate contaminated sites. Plants alone are unable to completely degrade some pollutants; therefore, their association with rhizospheric bacteria has been proposed to increase phytoremediation potential, an approach called rhizoremediation. In this work, the ability of two rhizobacteria, Burkholderia kururiensis KP 23 and Agrobacterium rhizogenes LBA 9402, to tolerate and degrade phenolic compounds was evaluated. Both microorganisms were capable of tolerating high concentrations of phenol, 2,4-dichlorophenol (2,4-DCP), guaiacol, or pentachlorophenol (PCP), and degrading different concentrations of phenol and 2,4-DCP. Association of these bacterial strains with B. napus hairy roots, as model plant system, showed that the presence of both rhizospheric microorganisms, along with B. napus hairy roots, enhanced phenol degradation compared to B. napus hairy roots alone. These findings are interesting for future applications of these strains in phenol rhizoremediation processes, with whole plants, providing an efficient, economic, and sustainable remediation technology. PMID:22961561

  20. Water-Dispersible, Responsive, and Carbonizable Hairy Microporous Polymeric Nanospheres.

    PubMed

    Mai, Weicong; Sun, Bin; Chen, Luyi; Xu, Fei; Liu, Hao; Liang, Yeru; Fu, Ruowen; Wu, Dingcai; Matyjaszewski, Krzysztof

    2015-10-21

    Multifunctionalization of microporous polymers is highly desirable but remains a significant challenge, considering that the current microporous polymers are generally hydrophobic and nonresponsive to different environmental stimuli and difficult to be carbonized without damage of their well-defined nanomorphology. Herein, we demonstrate a facile and versatile method to fabricate water-dispersible, pH/temperature responsive and readily carbonizable hairy microporous polymeric nanospheres based on combination of the hyper-cross-linking chemistry with the surface-initiated atom transfer radical polymerization (SI-ATRP). The hyper-cross-linking creates a highly microporous core, whereas the SI-ATRP provides diverse functionalities by surface grafting of hairy functional blocks. The as-prepared materials present multifunctional properties, including sensitive response to pH/temperature, high adsorption capacity toward adsorbates from aqueous solution, and valuable transformation into well-defined microporous carbon nanospheres because of hybrid of carbonizable core and thermo-decomposable protection shell. We hope this strategy could promote the development of both functional microporous polymers and advanced hairy nanoparticles for multipurpose applications.

  1. Response to microtubule-interacting agents in primary epithelial ovarian cancer cells

    PubMed Central

    2013-01-01

    Background Ovarian cancer constitutes nearly 4% of all cancers among women and is the leading cause of death from gynecologic malignancies in the Western world. Standard first line adjuvant chemotherapy treatments include Paclitaxel (Taxol) and platinum-based agents. Taxol, epothilone B (EpoB) and discodermolide belong to a family of anti-neoplastic agents that specifically interferes with microtubules and arrests cells in the G2/M phase of the cell cycle. Despite initial success with chemotherapy treatment, many patients relapse due to chemotherapy resistance. In vitro establishment of primary ovarian cancer cells provides a powerful tool for better understanding the mechanisms of ovarian cancer resistance. We describe the generation and characterization of primary ovarian cancer cells derived from ascites fluids of patients with epithelial ovarian cancer. Methods Chemosensitivity of these cell lines to Taxol, EpoB and discodermolide was tested, and cell cycle analysis was compared to that of immortalized ovarian cancer cell lines SKOV3 and Hey. The relationship between drug resistance and αβ-tubulin and p53 status was also investigated. Results All newly generated primary cancer cells were highly sensitive to the drugs. αβ-tubulin mutation was not found in any primary cell lines tested. However, one cell line that harbors p53 mutation at residue 72 (Arg to Pro) exhibits altered cell cycle profile in response to all drug treatments. Immortalized ovarian cancer cells respond differently to EpoB treatment when compared to primary ovarian cancer cells, and p53 polymorphism suggests clinical significance in the anti-tumor response in patients. Conclusions The isolation and characterization of primary ovarian cancer cells from ovarian cancer patients’ specimens contribute to further understanding the nature of drug resistance to microtubule interacting agents (MIAs) currently used in clinical settings. PMID:23574945

  2. Tumor lysing genetically engineered T cells loaded with multi-modal imaging agents.

    PubMed

    Bhatnagar, Parijat; Alauddin, Mian; Bankson, James A; Kirui, Dickson; Seifi, Payam; Huls, Helen; Lee, Dean A; Babakhani, Aydin; Ferrari, Mauro; Li, King C; Cooper, Laurence J N

    2014-03-28

    Genetically-modified T cells expressing chimeric antigen receptors (CAR) exert anti-tumor effect by identifying tumor-associated antigen (TAA), independent of major histocompatibility complex. For maximal efficacy and safety of adoptively transferred cells, imaging their biodistribution is critical. This will determine if cells home to the tumor and assist in moderating cell dose. Here, T cells are modified to express CAR. An efficient, non-toxic process with potential for cGMP compliance is developed for loading high cell number with multi-modal (PET-MRI) contrast agents (Super Paramagnetic Iron Oxide Nanoparticles - Copper-64; SPION-(64)Cu). This can now be potentially used for (64)Cu-based whole-body PET to detect T cell accumulation region with high-sensitivity, followed by SPION-based MRI of these regions for high-resolution anatomically correlated images of T cells. CD19-specific-CAR(+)SPION(pos) T cells effectively target in vitro CD19(+) lymphoma.

  3. Tumor Lysing Genetically Engineered T Cells Loaded with Multi-Modal Imaging Agents

    NASA Astrophysics Data System (ADS)

    Bhatnagar, Parijat; Alauddin, Mian; Bankson, James A.; Kirui, Dickson; Seifi, Payam; Huls, Helen; Lee, Dean A.; Babakhani, Aydin; Ferrari, Mauro; Li, King C.; Cooper, Laurence J. N.

    2014-03-01

    Genetically-modified T cells expressing chimeric antigen receptors (CAR) exert anti-tumor effect by identifying tumor-associated antigen (TAA), independent of major histocompatibility complex. For maximal efficacy and safety of adoptively transferred cells, imaging their biodistribution is critical. This will determine if cells home to the tumor and assist in moderating cell dose. Here, T cells are modified to express CAR. An efficient, non-toxic process with potential for cGMP compliance is developed for loading high cell number with multi-modal (PET-MRI) contrast agents (Super Paramagnetic Iron Oxide Nanoparticles - Copper-64; SPION-64Cu). This can now be potentially used for 64Cu-based whole-body PET to detect T cell accumulation region with high-sensitivity, followed by SPION-based MRI of these regions for high-resolution anatomically correlated images of T cells. CD19-specific-CAR+SPIONpos T cells effectively target in vitro CD19+ lymphoma.

  4. Gels from soft hairy nanoparticles in polymeric matrices

    NASA Astrophysics Data System (ADS)

    Vlassopoulos, Dimitris

    2013-03-01

    Hairy particles represent a huge class of soft colloids with tunable interactions and properties. Advances in synthetic chemistry have enabled obtaining well-characterized such systems for specific needs. In this talk we present two model hairy soft particles with diameters of the order of tens of nanometers, star polymers and polymerically grafted spherical particles. In particular, we discuss design strategies for dispersing them in polymeric matrices and eventually creating and breaking gels. Control parameters are the matrix molar mass, the grafting density (or functionality) and the size of the grafts (or arms). The linear viscoelastic properties and slow time evolution of the gels are examined in view of the existing knowledge from colloidal gels consisting of micron-sized particles, and compared. In the case of stars we start from a concentrated glassy suspension in molecular solvent and add homopolymer at increasing concentration, and as a result of the induced osmotic pressure the stars shrink and a depletion gel is formed. For the grafted colloidal particles, they are added at low concentration to a polymer matrix, and it has been shown that under certain conditions the anisotropy of interactions gives rise to network formation. We then focus on the nonlinear rheological response and in particular the effect of shear flow in inducing a solid to liquid transition. Our studies show that the yielding process is gradual and shares many common features with that of flocculated colloidal suspensions, irrespectively of the shape of the building block of the gel. Whereas shear can melt such a gel, it cannot break it into its constituent blocks and hence fully disperse the hairy nanoparticles. On the other hand, the hairy particles are intrinsically hybrid. We show how this important feature is reflected on the heating of the gels. In that case, the mismatch of thermal expansion coefficients of core and shell appears to play a role on the particle response as it

  5. Mycoplasma Contamination of Cell Cultures: Vesicular Traffic in Bacteria and Control over Infectious Agents

    PubMed Central

    Chernov, V. M.; Chernova, O. A.; Sanchez-Vega, J. T.; Kolpakov, A. I.; Ilinskaya, O. N.

    2014-01-01

    Cell cultures are subject to contamination either with cells of other cultures or with microorganisms, including fungi, viruses, and bacteria. Mycoplasma contamination of cell cultures is of particular importance. Since cell cultures are used for the production of vaccines and physiologically active compounds, designing a system for controlling contaminants becomes topical for fundamental science and biotechnological production. The discovery of extracellular membrane vesicles in mycoplasmas makes it necessary to take into consideration the bacterial vesicular traffic in systems designed for controlling infectious agents. The extracellular vesicles of bacteria mediate the traffic of proteins and genes, participate in cell-to-cell interactions, as well as in the pathogenesis and development of resistance to antibiotics. The present review discusses the features of mycoplasmas, their extracellular vesicles, and the interaction between contaminants and eukaryotic cells. Furthermore, it provides an analysis of the problems associated with modern methods of diagnosis and eradication of mycoplasma contamination from cell cultures and prospects for their solution. PMID:25349713

  6. A high-throughput, homogeneous microplate assay for agents that kill mammalian tissue culture cells.

    PubMed

    Pierce, Michael; Wang, Chunwei; Rebentisch, Matt; Endo, Mark; Stump, Mark; Kamb, Alexander

    2003-06-01

    Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens.

  7. Imaging translucent cell bodies in the living mouse retina without contrast agents

    PubMed Central

    Guevara-Torres, A.; Williams, D. R.; Schallek, J. B.

    2015-01-01

    The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina. PMID:26114032

  8. Labeling of human mesenchymal stem cell: Comparison between paramagnetic and superparamagnetic agents

    NASA Astrophysics Data System (ADS)

    Yang, Chung-Yi; Tai, Ming-Fong; Chen, Shin-Tai; Wang, Yi-Ting; Chen, Ya-Fang; Hsiao, Jong-Kai; Wang, Jaw-Lin; Liu, Hon-Man

    2009-04-01

    Paramagnetic and superparamagnetic substances are used to trace stem cell in living organisms under magnetic resonance imaging (MRI). We compared paramagnetic and superparamagnetic substance for their labeling efficiency by using clinically widely used gadolinium chelates and iron oxide nanoparticles. Without the aid of transfection agent, human mesenchymal stem cells were labeled with each agent separately in different concentration and the optimized concentration was determined by maintaining same cell viability as unlabeled cells. Iron oxide nanoparticle labeling has a detecting threshold of 12 500 cells in vitro, while gadolinium chelates labeling could be detected for at least 50 000 cells. In life animal study, we found there is an eightfold sensitivity in cells labeled with iron oxide superparamagnetic nanoparticles; however, the magnetic susceptibility artifact would obscure the detail of adjacent anatomical structures. We conclude that labeling stem cells with superparamagnetic substance is more efficacious. However, the cells labeled by superparamagnetic nanoparticles might interfere with the interpretation of anatomical structure. These findings would be beneficial to applications of magnetic substances toward stem cell biology and tissue engineering.

  9. Effect of anti-glycolytic agents on tumour cells in vitro

    NASA Astrophysics Data System (ADS)

    Korshunov, D. A.; Kondakova, I. V.

    2016-08-01

    A metabolic change is one of the tumour hallmarks, which has recently attracted a great amount of attention. One of the main metabolic characteristics of tumour cells is a high level of glycolysis even in the presence of oxygen, known as aerobic glycolysis or the Warburg effect. The energy production is much less in a glycolysis pathway than that in a tricarboxylic acid cycle. The Warburg effect constitutes a fundamental adaptation of tumour cells to a relatively hostile environment, and supports the evolution of aggressive and metastatic phenotypes. As a result, tumour glycolysis may become an attractive target for cancer therapy. Here, we research the effect of potential anticancer agents on tumour cells in vitro. In our study, we found a high sensitivity of tumour cells to anti-glycolityc drugs. In addition, tumour cells are more resistant to the agents studied in comparison with normal cells. We also observed an atypical cooperative interaction of tumour cells in the median lethal dose of drugs. They formed the specific morphological structure of the surviving cells. This behavior is not natural for the culture of tumour cells. Perhaps this is one of the mechanisms of cells' adaptation to the aggressive environment.

  10. Suppression of alkylating agent induced cell transformation and gastric ulceration by low-dose alkylating agent pretreatment

    SciTech Connect

    Onodera, Akira; Kawai, Yuichi; Kashimura, Asako; Ogita, Fumiya; Tsutsumi, Yasuo; Itoh, Norio

    2013-06-14

    Highlights: •Low-dose MNNG pretreatment suppresses high-dose MNNG induced in vitro transformation. •Gastric ulcers induced by high-dose MNNG decreased after low-dose MNNG pretreatment. •Efficacy of low-dose MNNG related to resistance of mutation and oxidative stress. -- Abstract: Exposure to mild stress by chemicals and radiation causes DNA damage and leads to acquired stress resistance. Although the linear no-threshold (LNT) model of safety assessment assumes risk from any dose, evidence from radiological research demonstrates a conflicting hormetic phenomenon known as the hormesis effect. However, the mechanisms underlying radiation hormesis have not yet been clarified, and little is known about the effects of low doses of chemical carcinogens. We analyzed the efficacy of pretreatment with low doses of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) on the subsequent induction of cell transformation and gastric ulceration by high-dose MNNG. We used an in vitro Balb/3T3 A31-1-1 cell transformation test and monitored the formation of gastric ulcers in 5-week-old male ICR mice that were administered MNNG in drinking water. The treatment concentrations of MNNG were determined by the cell survival rate and past reports. For low-dose in vitro and in vivo experiments, MNNG was used at 0.028 μM, and 2.8 μg/mL, respectively. The frequency of cell transformation induced by 10 μm MNNG was decreased by low-dose MNNG pretreatment to levels similar to that of spontaneous transformation. In addition, reactive oxygen species (ROS) and mutation frequencies induced by 10 μm MNNG were decreased by low-dose MNNG pretreatment. Importantly, low-dose MNNG pretreatment had no effect on cell proliferation. In vivo studies showed that the number of gastric ulcers induced by 1 mg/mL MNNG decreased after low-dose MNNG pretreatment. These data indicate that low-dose pretreatment with carcinogens may play a beneficial role in the prevention of chemical toxicity

  11. Spermatogenesis is seasonal in the large hairy armadillo, Chaetophractus villosus (Dasypodidae, Xenarthra, Mammalia).

    PubMed

    Luaces, Juan P; Rossi, Luis F; Merico, Valeria; Zuccotti, Maurizio; Redi, Carlo A; Solari, Alberto J; Merani, Maria S; Garagna, Silvia

    2013-01-01

    Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus. PMID:22951275

  12. Spermatogenesis is seasonal in the large hairy armadillo, Chaetophractus villosus (Dasypodidae, Xenarthra, Mammalia).

    PubMed

    Luaces, Juan P; Rossi, Luis F; Merico, Valeria; Zuccotti, Maurizio; Redi, Carlo A; Solari, Alberto J; Merani, Maria S; Garagna, Silvia

    2013-01-01

    Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.

  13. Hairy Root as a Model System for Undergraduate Laboratory Curriculum and Research

    ERIC Educational Resources Information Center

    Keyes, Carol A.; Subramanian, Senthil; Yu, Oliver

    2009-01-01

    Hairy root transformation has been widely adapted in plant laboratories to rapidly generate transgenic roots for biochemical and molecular analysis. We present hairy root transformations as a versatile and adaptable model system for a wide variety of undergraduate laboratory courses and research. This technique is easy, efficient, and fast making…

  14. Intraocular choristoma, anterior staphyloma with ipsilateral nevus sebaceus, and congenital giant hairy nevus: a case report.

    PubMed

    Nigam, Pramod K; Sudarshan, Vijaya; Chandrakar, Ashok K; Gahine, Renuka; Krishnani, Chandani

    2011-02-01

    A 5-year-old girl presented with choristoma of the eye along with nevus sebaceus and congenital giant hairy nevus over the face. Anterior staphyloma also was present. Although choristomas have been seen occasionally occurring with nevus sebaceus, an associated ipsilateral, regional, congenital giant hairy nevus is rare.

  15. Design of a hydrogen peroxide-activatable agent that specifically targets cancer cells.

    PubMed

    Vadukoot, Anish K; AbdulSalam, Safnas F; Wunderlich, Mark; Pullen, Eboni D; Landero-Figueroa, Julio; Mulloy, James C; Merino, Eddie J

    2014-12-15

    Some cancers, like acute myeloid leukemia (AML), use reactive oxygen species to endogenously activate cell proliferation and angiogenic signaling cascades. Thus many cancers display increases in reactive oxygen like hydrogen peroxide concentrations. To translate this finding into a therapeutic strategy we designed new hydrogen peroxide-activated agents with two key molecular pharmacophores. The first pharmacophore is a peroxide-acceptor and the second is a pendant amine. The acceptor is an N-(2,5-dihydroxyphenyl)acetamide susceptible to hydrogen peroxide oxidation. We hypothesized that selectivity between AML and normal cells could be achieved by tuning the pendant amine. Synthesis and testing of fourteen compounds that differed at the pendent amine led to the identification of an agent (14) with 2μM activity against AML cancer cells and an eleven fold-lower activity in healthy CD34+ blood stem cells. Interestingly, analysis shows that upon oxidation the pendant amine cyclizes, ejecting water, with the acceptor to give a bicyclic compound capable of reacting with nucleophiles. Preliminary mechanistic investigations show that AML cells made from addition of two oncogenes (NrasG12D and MLL-AF9) increase the ROS-status, is initially an anti-oxidant as hydrogen peroxide is consumed to activate the pro-drug, and cells respond by upregulating electrophilic defense as visualized by Western blotting of KEAP1. Thus, using this chemical approach we have obtained a simple, potent, and selective ROS-activated anti-AML agent.

  16. Suprafenacine, an Indazole-Hydrazide Agent, Targets Cancer Cells Through Microtubule Destabilization

    PubMed Central

    Choi, Bo-Hwa; Chattopadhaya, Souvik; Thanh, Le Nguyen; Feng, Lin; Nguyen, Quoc Toan; Lim, Chuan Bian; Harikishore, Amaravadhi; Nanga, Ravi Prakash Reddy; Bharatham, Nagakumar; Zhao, Yan; Liu, Xuewei; Yoon, Ho Sup

    2014-01-01

    Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents (TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we report the identification of a novel cell permeable, tubulin-destabilizing molecule - 4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This led to G2/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss of mitochondrial membrane potential, JNK - mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3. Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our results suggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents. PMID:25354194

  17. In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents

    NASA Astrophysics Data System (ADS)

    Zharov, Vladimir P.; Galanzha, Ekaterina I.; Shashkov, Evgeny V.; Khlebtsov, Nicolai G.; Tuchin, Valery V.

    2006-12-01

    A new photoacoustic flow cytometry was developed for real-time detection of circulating cells, nanoparticles, and contrast agents in vivo. Its capability, integrated with photothermal and optical clearing methods, was demonstrated using a near-infrared tunable laser to characterize the in vivo kinetics of Indocyanine Green alone and single cancer cells labeled with gold nanorods and Indocyanine Green in the vasculature of the mouse ear. In vivo applications are discussed, including selective nanophotothermolysis of metastatic squamous cells, label-free detection of melanoma cells, study of pharmokinetics, and immune response to apoptotic and necrotic cells, with potential translation to humans. The threshold sensitivity is estimated as one cancer cell in the background of 107 normal blood cells.

  18. Evaluation of respiration of mitochondria in cancer cells exposed to mitochondria-targeted agents.

    PubMed

    Kluckova, Katarina; Dong, Lan-Feng; Bajzikova, Martina; Rohlena, Jakub; Neuzil, Jiri

    2015-01-01

    Respiration is one of the major functions of mitochondria, whereby these vital organelles use oxygen to produce energy. Many agents that may be of potential clinical relevance act by targeting mitochondria, where they may suppress mitochondrial respiration. It is therefore important to evaluate this process and understand how this is modulated by small molecules. Here, we describe the general methodology to assess respiration in cultured cells, followed by the evaluation of the effect of one anticancer agent targeted to mitochondria on this process, and also how to assess this in tumor tissue.

  19. Highly Adaptable Triple-Negative Breast Cancer Cells as a Functional Model for Testing Anticancer Agents

    PubMed Central

    Singh, Balraj; Shamsnia, Anna; Raythatha, Milan R.; Milligan, Ryan D.; Cady, Amanda M.; Madan, Simran; Lucci, Anthony

    2014-01-01

    A major obstacle in developing effective therapies against solid tumors stems from an inability to adequately model the rare subpopulation of panresistant cancer cells that may often drive the disease. We describe a strategy for optimally modeling highly abnormal and highly adaptable human triple-negative breast cancer cells, and evaluating therapies for their ability to eradicate such cells. To overcome the shortcomings often associated with cell culture models, we incorporated several features in our model including a selection of highly adaptable cancer cells based on their ability to survive a metabolic challenge. We have previously shown that metabolically adaptable cancer cells efficiently metastasize to multiple organs in nude mice. Here we show that the cancer cells modeled in our system feature an embryo-like gene expression and amplification of the fat mass and obesity associated gene FTO. We also provide evidence of upregulation of ZEB1 and downregulation of GRHL2 indicating increased epithelial to mesenchymal transition in metabolically adaptable cancer cells. Our results obtained with a variety of anticancer agents support the validity of the model of realistic panresistance and suggest that it could be used for developing anticancer agents that would overcome panresistance. PMID:25279830

  20. Enhancement of Cytotoxicity of Three Apoptosis-inducing Agents Against Human Oral Squamous Cell Carcinoma Cell Line by Benzoxazinotropone.

    PubMed

    Tomikoshi, Yukiko; Nomura, Maki; Okudaira, Noriyuki; Sakagami, Hiroshi; Wakabayashi, Hidetsugu

    Tumor-specificity (TS) and anti-inflammatory activity of benzo[b]cyclohept[e][1,4]oxazin-6(11H)-one, generally known as benzoxazinotropone (BOT), have been reported. In order to find a new biological activity, the combination effect of BOT and three apoptosis-inducing agents was investigated. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and five human oral normal cells (gingival fibroblasts, periodontal ligament fibroblasts, pulp cells, oral keratinocytes and primary gingival epithelial cells) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. TS was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal oral cells to the one against OSCC cell lines. Synergy was evaluated by CompuSyn software program. Expression of cleaved forms of poly ADP-ribose polymerase and caspsase-3 was evaluated by western blot analysis. BOT induced activation of caspase 3, suggesting the apoptosis induction in HSC-2 OSCC cells. BOT enhanced the cytotoxicity of doxorubicin (DXR) additively and that of curcumin and resveratrol synergistically. On the other hand, BOT did not enhance, but rather inhibit the cytotoxicity of DXR against normal keratinocytes. The present study suggests that BOT may enhance the anti-tumor activity of apoptosis-inducing agents, while reducing its cytotoxicity against normal cells. PMID:27566085

  1. Tolfenamic acid inhibits neuroblastoma cell proliferation and induces apoptosis: a novel therapeutic agent for neuroblastoma.

    PubMed

    Eslin, Don; Sankpal, Umesh T; Lee, Chris; Sutphin, Robert M; Maliakal, Pius; Currier, Erika; Sholler, Giselle; Khan, Moeez; Basha, Riyaz

    2013-05-01

    Current therapeutic options for recurrent neuroblastoma have poor outcomes that warrant the development of novel therapeutic strategies. Specificity protein (Sp) transcription factors regulate several genes involved in cell proliferation, survival, and angiogenesis. Sp1 regulates genes believed to be important determinants of the biological behavior of neuroblastoma. Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to induce the degradation of Sp proteins and may serve as a novel anti-cancer agent. The objective of this investigation was to examine the anti-cancer activity of TA using established human neuroblastoma cell lines. We tested the anti-proliferative effect of TA using SH-SY5Y, CHLA90, LA1 55n, SHEP, Be2c, CMP 13Y, and SMS KCNR cell lines. Cells were treated with TA (0/25/50/100 µM) and cell viability was measured at 24, 48, and 72 h post-treatment. Selected neuroblastoma cell lines were treated with 50 µM TA for 24 and 48 h and tested for cell apoptosis using Annexin-V staining. Caspase activity was measured with caspase 3/7 Glo kit. Cell lysates were prepared and the expression of Sp1, survivin, and c-PARP were evaluated through Western blot analysis. TA significantly inhibited the growth of neuroblastoma cells in a dose/time-dependent manner and significantly decreased Sp1 and survivin expression. Apart from cell cycle (G0/G1) arrest, TA caused significant increase in the apoptotic cell population, caspase 3/7 activity, and c-PARP expression. These results show that TA effectively inhibits neuroblastoma cell growth potentially through suppressing mitosis, Sp1, and survivin expression, and inducing apoptosis. These results show TA as a novel therapeutic agent for neuroblastoma.

  2. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    PubMed

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination. PMID:11844038

  3. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    PubMed

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination.

  4. Adiaspiromycosis in south Australian hairy-nosed wombats (Lasiorhinus latifrons).

    PubMed

    Mason, R W; Gauhwin, M

    1982-01-01

    Spherical organisms, with an average diameter of about 22 microns, were detected in the lungs of adult and pouched young hairy-nosed wombats (Lasiorhinus latifrons). Although infections of up to 640 X 10(3) organisms per cubic centimeter were detected, their presence produced only limited pathological change. In-vitro growth was obtained at 30 C but not at 37 C or 40 C. However, at the higher temperatures, typical chlamydospore spherules were produced by colonies initially grown at 30 c. This report presents the first record of adiaspiromycosis in Australia and in wombats. PMID:7201528

  5. Hairy roots are more sensitive to auxin than normal roots

    PubMed Central

    Shen, Wen Hui; Petit, Annik; Guern, Jean; Tempé, Jacques

    1988-01-01

    Responses to auxin of Lotus corniculatus root tips or protoplasts transformed by Agrobacterium rhizogenes strains 15834 and 8196 were compared to those of their normal counterparts. Three different types of experiments were performed, involving long-term, medium-term, or short-term responses to a synthetic auxin, 1-naphthaleneacetic acid. Root tip elongation, proton excretion by root tips, and transmembrane electrical potential difference of root protoplasts were measured as a function of exogenous auxin concentration. The sensitivity of hairy root tips or protoplasts to exogenous auxin was found to be 100-1000 times higher than that of untransformed material. PMID:16593928

  6. Antimicrobial polyacetylenes from Panax ginseng hairy root culture.

    PubMed

    Fukuyama, Noriaki; Shibuya, Masaaki; Orihara, Yutaka

    2012-01-01

    Two new polyacetylenes, 1-hydroxydihydropanaxacol (3) and 17-hydroxypanaxacol (4), were isolated from Panax ginseng hairy root culture, along with dihydropanaxacol (1), panaxacol (2) and ginsenoyne D (5). Highly hydroxylated compounds 1-4 were isolated from the medium and compound 5, which was a biosynthetic precursor of compound 1, was isolated from the roots. Compounds 1-4 showed antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Cryptococcus neoformans and Aspergillus fumigatus. It is suggested that P. ginseng plants release antimicrobial polyacetylenes into the surrounding soil from the roots as defense compounds.

  7. Adiaspiromycosis in south Australian hairy-nosed wombats (Lasiorhinus latifrons).

    PubMed

    Mason, R W; Gauhwin, M

    1982-01-01

    Spherical organisms, with an average diameter of about 22 microns, were detected in the lungs of adult and pouched young hairy-nosed wombats (Lasiorhinus latifrons). Although infections of up to 640 X 10(3) organisms per cubic centimeter were detected, their presence produced only limited pathological change. In-vitro growth was obtained at 30 C but not at 37 C or 40 C. However, at the higher temperatures, typical chlamydospore spherules were produced by colonies initially grown at 30 c. This report presents the first record of adiaspiromycosis in Australia and in wombats.

  8. Canonical energy and hairy AdS black holes

    NASA Astrophysics Data System (ADS)

    Hyun, Seungjoon; Park, Sang-A.; Yi, Sang-Heon

    2016-08-01

    We propose the modified version of the canonical energy which was introduced originally by Hollands and Wald. Our construction depends only on the Euler-Lagrange expression of the system and thus is independent of the ambiguity in the Lagrangian. After some comments on our construction, we briefly mention on the relevance of our construction to the boundary information metric in the context of the AdS/CFT correspondence. We also study the stability of three-dimensional hairy extremal black holes by using our construction.

  9. Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy.

    PubMed

    Zhou, Jiehua; Rossi, John J

    2014-06-17

    One hundred years ago, Dr. Paul Ehrlich popularized the "magic bullet" concept for cancer therapy in which an ideal therapeutic agent would only kill the specific tumor cells it targeted. Since then, "targeted therapy" that specifically targets the molecular defects responsible for a patient's condition has become a long-standing goal for treating human disease. However, safe and efficient drug delivery during the treatment of cancer and infectious disease remains a major challenge for clinical translation and the development of new therapies. The advent of SELEX technology has inspired many groundbreaking studies that successfully adapted cell-specific aptamers for targeted delivery of active drug substances in both in vitro and in vivo models. By covalently linking or physically functionalizing the cell-specific aptamers with therapeutic agents, such as siRNA, microRNA, chemotherapeutics or toxins, or delivery vehicles, such as organic or inorganic nanocarriers, the targeted cells and tissues can be specifically recognized and the therapeutic compounds internalized, thereby improving the local concentration of the drug and its therapeutic efficacy. Currently, many cell-type-specific aptamers have been developed that can target distinct diseases or tissues in a cell-type-specific manner. In this review, we discuss recent advances in the use of cell-specific aptamers for targeted disease therapy, as well as conjugation strategies and challenges.

  10. The microtubule stabilizing agent discodermolide is a potent inducer of accelerated cell senescence.

    PubMed

    Klein, Laura E; Freeze, B Scott; Smith, Amos B; Horwitz, Susan Band

    2005-03-01

    Discodermolide is a microtubule stabilizing agent that suppresses dynamic instability and blocks cells in mitosis. Selection of A549 nonsmall cell lung carcinoma cells with increasing concentrations of discodermolide yielded a clone that proliferated in 8 nM. When these cells were exposed to any concentration greater than 8 nM, replication ceased and the cells developed a flattened, enlarged, granular morphology. Accelerated senescence was demonstrated by a functional beta-galactosidase activity at pH 6. When parental A549 cells were treated with IC50-concentrations of doxorubicin, Taxol or discodermolide, the latter two drugs quickly produced aberrant mitosis. However, discodermolide, but not Taxol, also produced a large increase in senescence-associated beta-galactosidase activity and altered levels of known senescence markers. Although some of these differences between Taxol and discodermolide were dose dependent, only discodermolide produced a doxorubicin-like induction of a senescence phenotype, including a senescence-associated beta-galactosidase activity, up-regulation of PAI-1 and p66Shc, and a strong, sustained, Erk1/2 activation. This research provides insights into the mechanism of action of discodermolide and provides the first demonstration of a microtubule stabilizing agent that inhibits tumor cell growth with a powerful induction of accelerated senescence.

  11. Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy

    PubMed Central

    Zhou, Jiehua; Rossi, John J.

    2014-01-01

    One hundred years ago, Dr. Paul Ehrlich popularized the “magic bullet” concept for cancer therapy in which an ideal therapeutic agent would only kill the specific tumor cells it targeted. Since then, “targeted therapy” that specifically targets the molecular defects responsible for a patient's condition has become a long-standing goal for treating human disease. However, safe and efficient drug delivery during the treatment of cancer and infectious disease remains a major challenge for clinical translation and the development of new therapies. The advent of SELEX technology has inspired many groundbreaking studies that successfully adapted cell-specific aptamers for targeted delivery of active drug substances in both in vitro and in vivo models. By covalently linking or physically functionalizing the cell-specific aptamers with therapeutic agents, such as siRNA, microRNA, chemotherapeutics or toxins, or delivery vehicles, such as organic or inorganic nanocarriers, the targeted cells and tissues can be specifically recognized and the therapeutic compounds internalized, thereby improving the local concentration of the drug and its therapeutic efficacy. Currently, many cell-type-specific aptamers have been developed that can target distinct diseases or tissues in a cell-type-specific manner. In this review, we discuss recent advances in the use of cell-specific aptamers for targeted disease therapy, as well as conjugation strategies and challenges. PMID:24936916

  12. Gadolinium contrast agent-induced CD163+ ferroportin+ osteogenic cells in nephrogenic systemic fibrosis.

    PubMed

    Swaminathan, Sundararaman; Bose, Chhanda; Shah, Sudhir V; Hall, Kimberly A; Hiatt, Kim M

    2013-09-01

    Gadolinium-based contrast agents are linked to nephrogenic systemic fibrosis in patients with renal insufficiency. The pathology of nephrogenic systemic fibrosis is characterized by abnormal tissue repair: fibrosis and ectopic ossification. The mechanisms by which gadolinium could induce fibrosis and ossification are not known. We examined in vitro the effect of a gadolinium-based contrast agent on human peripheral blood mononuclear cells for phenotype and function relevant to the pathology of nephrogenic systemic fibrosis using immunofluorescence, flow cytometry, real-time PCR, and osteogenic assays. We also examined tissues from patients with nephrogenic systemic fibrosis, using IHC to identify the presence of cells with phenotype induced by gadolinium. Gadolinium contrast induced differentiation of human peripheral blood mononuclear cells into a unique cellular phenotype--CD163(+) cells expressing proteins involved in fibrosis and bone formation. These cells express fibroblast growth factor (FGF)23, osteoblast transcription factors Runt-related transcription factor 2, and osterix, and show an osteogenic phenotype in in vitro assays. We show in vivo the presence of CD163(+)/procollagen-1(+)/osteocalcin(+) cells in the fibrotic and calcified tissues of nephrogenic systemic fibrosis patients. Gadolinium contrast-induced CD163(+)/ferroportin(+)/FGF23(+) cells with osteogenic potential may play a role in systemic fibrosis and ectopic ossification in nephrogenic systemic fibrosis.

  13. HIV Latency-Reversing Agents Have Diverse Effects on Natural Killer Cell Function

    PubMed Central

    Garrido, Carolina; Spivak, Adam M.; Soriano-Sarabia, Natalia; Checkley, Mary Ann; Barker, Edward; Karn, Jonathan; Planelles, Vicente; Margolis, David M.

    2016-01-01

    In an effort to clear persistent HIV infection and achieve a durable therapy-free remission of HIV disease, extensive pre-clinical studies and early pilot clinical trials are underway to develop and test agents that can reverse latent HIV infection and present viral antigen to the immune system for clearance. It is, therefore, critical to understand the impact of latency-reversing agents (LRAs) on the function of immune effectors needed to clear infected cells. We assessed the impact of LRAs on the function of natural killer (NK) cells, the main effector cells of the innate immune system. We studied the effects of three histone deacetylase inhibitors [SAHA or vorinostat (VOR), romidepsin, and panobinostat (PNB)] and two protein kinase C agonists [prostratin (PROST) and ingenol] on the antiviral activity, cytotoxicity, cytokine secretion, phenotype, and viability of primary NK cells. We found that ex vivo exposure to VOR had minimal impact on all parameters assessed, while PNB caused a decrease in NK cell viability, antiviral activity, and cytotoxicity. PROST caused non-specific NK cell activation and, interestingly, improved antiviral activity. Overall, we found that LRAs can alter the function and fate of NK cells, and these effects must be carefully considered as strategies are developed to clear persistent HIV infection. PMID:27708642

  14. Downregulation of hPMC2 imparts chemotherapeutic sensitivity to alkylating agents in breast cancer cells

    PubMed Central

    Krishnamurthy, Nirmala; Liu, Lili; Xiong, Xiahui; Zhang, Junran; Montano, Monica M

    2015-01-01

    Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies. PMID:25849309

  15. Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media - Vitrification versus Slow Freezing Methods.

    PubMed

    von Bomhard, Achim; Elsässer, Alexander; Ritschl, Lucas Maximilian; Schwarz, Silke; Rotter, Nicole

    2016-01-01

    Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco's modified Eagle medium Ham's F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K+TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%).

  16. Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media – Vitrification versus Slow Freezing Methods

    PubMed Central

    von Bomhard, Achim; Elsässer, Alexander; Ritschl, Lucas Maximilian; Schwarz, Silke; Rotter, Nicole

    2016-01-01

    Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco's modified Eagle medium Ham’s F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K+TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%). PMID:26890410

  17. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-05-11

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E₂, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis.

  18. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E₂, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis. PMID:27187358

  19. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E2, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional “repair and survive, or die” hypothesis. PMID:27187358

  20. Effect of medroxyprogesterone acetate and of some antiinflammatory agents on mouse erythroleukemia cell differentiation.

    PubMed

    Supino, R; Mazzoni, A; Formelli, F

    1984-02-29

    The effects of medroxyprogesterone acetate (MPA) on differentiation were examined using mouse erythroleukemia (MEL) cells and compared with those of antiinflammatory agents. MPA at low doses (10(-6) - 10(-7)M) induced 10-15% cells to differentiate, whereas high doses (10(-4) - 10(-5)M) caused a 30% inhibition of dimethylsulfoxide (DMSO)-induced differentiation. Dexamethasone (10(-4) - 10(-8)M), a steroid antiinflammatory agent, significantly inhibited (77-70%) DMSO-induced differentiation, whereas indomethacin, aspirin, flurbiprofen and BW755c (non steroid antiinflammatory agents) at the same concentrations had no effect. If added 24 h before DMSO, the inhibitory effects of MPA and dexamethasone increased to 65% and 95%, respectively, whereas indomethacin (10(-5)M) caused only a 30% inhibition and the other drugs were inactive. None of these antiinflammatory agents affected differentiation when used without DMSO. MPA and dexamethasone inhibitory effects on DMSO-induced differentiation did not seem to be mediated through the inhibition of the synthesis of prostaglandins, since non-steroid prostaglandin inhibitors were slightly active only when added 24 h before DMSO.

  1. Celastrol induces proteasomal degradation of FANCD2 to sensitize lung cancer cells to DNA crosslinking agents.

    PubMed

    Wang, Gui-Zhen; Liu, Yong-Qiang; Cheng, Xin; Zhou, Guang-Biao

    2015-07-01

    The Fanconi anemia (FA) pathway plays a key role in interstrand crosslink (ICL) repair and maintenance of the genomic stability, while inhibition of this pathway may sensitize cancer cells to DNA ICL agents and ionizing radiation (IR). The active FA core complex acts as an E3 ligase to monoubiquitinate FANCD2, which is a functional readout of an activated FA pathway. In the present study, we aimed to identify FANCD2-targeting agents, and found that the natural compound celastrol induced degradation of FANCD2 through the ubiquitin-proteasome pathway. We demonstrated that celastrol downregulated the basal and DNA damaging agent-induced monoubiquitination of FANCD2, followed by proteolytic degradation of the substrate. Furthermore, celastrol treatment abrogated the G2 checkpoint induced by IR, and enhanced the ICL agent-induced DNA damage and inhibitory effects on lung cancer cells through depletion of FANCD2. These results indicate that celastrol is a FANCD2 inhibitor that could interfere with the monoubiquitination and protein stability of FANCD2, providing a novel opportunity to develop FA pathway inhibitor and combinational therapy for malignant neoplasms.

  2. Skin Stem Cell Hypotheses and Long Term Clone Survival – Explored Using Agent-based Modelling

    PubMed Central

    Li, X.; Upadhyay, A. K.; Bullock, A. J.; Dicolandrea, T.; Xu, J.; Binder, R. L.; Robinson, M. K.; Finlay, D. R.; Mills, K. J.; Bascom, C. C.; Kelling, C. K.; Isfort, R. J.; Haycock, J. W.; MacNeil, S.; Smallwood, R. H.

    2013-01-01

    Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation. PMID:23712735

  3. Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting.

    PubMed

    Zilbermintz, Leeor; Leonardi, William; Jeong, Sun-Young; Sjodt, Megan; McComb, Ryan; Ho, Chi-Lee C; Retterer, Cary; Gharaibeh, Dima; Zamani, Rouzbeh; Soloveva, Veronica; Bavari, Sina; Levitin, Anastasia; West, Joel; Bradley, Kenneth A; Clubb, Robert T; Cohen, Stanley N; Gupta, Vivek; Martchenko, Mikhail

    2015-08-27

    A longstanding and still-increasing threat to the effective treatment of infectious diseases is resistance to antimicrobial countermeasures. Potentially, the targeting of host proteins and pathways essential for the detrimental effects of pathogens offers an approach that may discover broad-spectrum anti-pathogen countermeasures and circumvent the effects of pathogen mutations leading to resistance. Here we report implementation of a strategy for discovering broad-spectrum host-oriented therapies against multiple pathogenic agents by multiplex screening of drugs for protection against the detrimental effects of multiple pathogens, identification of host cell pathways inhibited by the drug, and screening for effects of the agent on other pathogens exploiting the same pathway. We show that a clinically used antimalarial drug, Amodiaquine, discovered by this strategy, protects host cells against infection by multiple toxins and viruses by inhibiting host cathepsin B. Our results reveal the practicality of discovering broadly acting anti-pathogen countermeasures that target host proteins exploited by pathogens.

  4. Bax overexpression enhances cytochrome c release from mitochondria and sensitizes KATOIII gastric cancer cells to chemotherapeutic agent-induced apoptosis.

    PubMed

    Sawa, H; Kobayashi, T; Mukai, K; Zhang, W; Shiku, H

    2000-04-01

    To evaluate whether overexpression of Bax, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic agents, three stable cell lines of KATOIII transfected with Bax (KATOIII-Bax), Bcl-2 (KATOIII-Bcl-2), or control pCI-neo expression vector (KATOIII-pCI-neo) were established. The cells were treated with paclitaxel, 5-fluorouracil, or doxorubicin, and the apoptotic response was measured. Our results showed that the sensitivity of the KATOIII-Bax cells to chemotherapeutic agents was enhanced compared with that of the KATOIII-pCI-neo cells, and the KATOIII-Bcl-2 cells were more resistant to these agents. Western blotting revealed that cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells. Significant increase of cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was detected 24 h after exposure to chemotherapeutic agents, when apoptotic cells were less than 10%. The cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells at all time points examined after exposure to chemotherapeutic agents. Marked activation of caspase-3 in the KATOIII-Bax cells was observed 48 h and 72 h after exposure to chemotherapeutic agents compared with that in the KATOIII-pCI-neo cells. Consistently, zVAD-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c release from mitochondria and caspase-3 activation. These findings suggest that cytochrome c release is a major mechanism of apoptotic response and Bax overexpression sensitizes KATOIII cells to chemotherapeutic agent-induced apoptosis through enhancing the release of cytochrome c from mitochondria. PMID:10717243

  5. Surface Disinfestation of Resting Spores of Plasmodiophora brassicae Used to Infect Hairy Roots of Brassica spp.

    PubMed

    Asano, T; Kageyama, K; Hyakumachi, M

    1999-04-01

    ABSTRACT Resting spores of Plasmodiophora brassicae were surface-disinfested by treatment with 2% chloramine-T for 20 min and then with an antibiotic solution (1,000 ppm of colistin sulfate, 1,000 ppm of vancomycin hydrochloride, and 6,000 ppm of cefotaxime sodium) for 1 day. The disinfested resting spores were used to inoculate hairy roots of cabbage (Brassica oleracea var. capitata cv. Fuji Wase), Chinese cabbage (B. pekinensis cv. Musou Hakusai), turnip (B. rapa var. rapifera cv. Wase Okabu), and rape (B. napus line Dc 119). Differences among hosts in susceptibility to clubroot in hairy roots were evident. Chinese cabbage and turnip hairy roots supported the highest percentages of root hair infection (53.3 to 80%) and the greatest production of zoosporangial groups (8.5 to 32.5 per root). Moreover, gall formation was observed only on Chinese cabbage and turnip hairy roots. The morphology of zoo-sporangia, plasmodia, and resting spores in diseased hairy roots was found to be identical to that in infected intact plants by both light and scanning electron microscopy. Pathogenicity tests confirmed the infectivity of resting spores produced in hairy roots. Thus, the hairy root culture technique should prove useful as a dual culture system for P. brassicae.

  6. [Establishment of culture system of Silybum marianum hairy roots and determination of silybin].

    PubMed

    Zhang, Shu-Li; Zhang, Tian-Zhu; Yang, Shi-Hai

    2014-06-01

    This research uses six Agrobacterium rhizogenes R1601, R15384, R1000, A4, R1025 and R1 to infect silymarin explants to induce hairy roots and silibin. All of the six A. rhizogenes can induce Silybum marianum to generate hairy roots and the A. rhizogene A4 shows comparatively high infection on the plant. This research determines the condition to induce silymarin hairy roots by the factors of infection time, pre-culturing, co-culturing and pH value. The fact that MS liquid medium fits the proliferation of silymarin hairy roots is determined. Through PCR molecular identification, it can be seen that the DNA plasmids in the A. rhizogenes are successfully integrated into the genome of transformed roots. Using liquid chromatography, it is determined that the silibin content in silymarin hairy roots is 2.5 times that in the plant In this research, the silymarin hairy roots culturing system is established, which lays a foundation for the study of culturing silymarin hairy roots and producing silibin.

  7. LipoCEST and cellCEST imaging agents: opportunities and challenges.

    PubMed

    Ferrauto, Giuseppe; Delli Castelli, Daniela; Di Gregorio, Enza; Terreno, Enzo; Aime, Silvio

    2016-07-01

    From the early days of CEST agents' disclosure, it was evident that their potential for in vivo applications was strongly hampered by the intrinsic low sensitivity. Therefore, much work has been devoted to seek out suitable routes to achieve strong CEST contrast enhancement. The use of nanosized systems turned out to be a strategic choice, because a very large amount of CEST agents can be delivered at the site of interest. However, the breakthrough innovation in term of increase of sensitivity was found by designing the lipoCEST agents. The naturally inspired, liposomes vesicles, when loaded with paramagnetic lanthanide-based shift reagents, can be transformed into CEST probes. The large number of water molecules entrapped inside the inner cavity of the nanovesicles represents an enormous pool of exchanging protons for the generation of CEST contrast, whereas the presence of the shift reagent increases the separation in chemical shift of their nuclear magnetic resonance signal from that of the bulk water, thus allowing for a proper exchange regime for the activation of CEST contrast. From lipoCEST, it has been rather straightforward to evolve to cellCEST in order to exploit the cytoplasmatic water molecules as source of the CEST effect, once cells have been loaded with the proper shift reagent. The red blood cells were found to be particularly suitable for the development of the cellCEST concept. Finally, an understanding of the main determinants of the CEST effects in nanosized and cellular-sized agents has allowed the design of innovative lipoCEST/RBC aggregates for potential theranostic applications. WIREs Nanomed Nanobiotechnol 2016, 8:602-618. doi: 10.1002/wnan.1385 For further resources related to this article, please visit the WIREs website. PMID:26810631

  8. Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots.

    PubMed

    Ruiz-May, Eliel; De-la-Peña, Clelia; Galaz-Ávalos, Rosa M; Lei, Zhentian; Watson, Bonnie S; Sumner, Lloyd W; Loyola-Vargas, Víctor M

    2011-08-01

    Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.

  9. Oncolytic reovirus synergizes with chemotherapeutic agents to promote cell death in canine mammary gland tumor

    PubMed Central

    Igase, Masaya; Hwang, Chung Chew; Kambayashi, Satoshi; Kubo, Masato; Coffey, Matt; Miyama, Takako Shimokawa; Baba, Kenji; Okuda, Masaru; Noguchi, Shunsuke; Mizuno, Takuya

    2016-01-01

    The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC50) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy. PMID:26733729

  10. Hairy cellulose nanocrystalloids: a novel class of nanocellulose.

    PubMed

    van de Ven, Theo G M; Sheikhi, Amir

    2016-08-18

    Nanomaterials have secured such a promising role in today's life that imagining the modern world without them is almost impossible. A large fraction of nanomaterials is synthesized from environmentally-dangerous elements such as heavy metals, which have posed serious side-effects to ecosystems. Despite numerous advantages of synthetic nanomaterials, issues such as renewability, sustainability, biocompatibility, and cost efficiency have drawn significant attention towards natural products such as cellulose-based nanomaterials. Within the past decade, nanocelluloses, most remarkably nanocrystalline cellulose (NCC) and nanofibrillated cellulose (NFC), have successfully been used for a wide spectrum of applications spanning from nanocomposites, packaging, and mechanical and rheological property modifications, to chemical catalysis and organic templating. Yet, there has been little effort to introduce fundamentally new polysaccharide-based nanomaterials. We have been able to develop the first kind of cellulose-based nanoparticles bearing both crystalline and amorphous regions. These nanoparticles comprise a crystalline body, similar to conventional NCC, but with polymer chains protruding from both ends; therefore, these particles are called hairy cellulose nanocrystalloids (HCNC). In this article, we touch on the philosophy of HCNC synthesis, the striking superiority over existing nanocelluloses, and applications of this novel class of nanocelluloses. We hope that the emergence of hairy cellulose nanocrystalloids extends the frontiers of sustainable, green nanotechnology. PMID:27453347

  11. Effect of spermine synthase on the sensitivity of cells to anti-tumour agents.

    PubMed Central

    Ikeguchi, Yoshihiko; Mackintosh, Caroline A; McCloskey, Diane E; Pegg, Anthony E

    2003-01-01

    The role of spermine in the sensitivity of cells to various established and experimental anti-tumour agents was examined, using paired cell lines that possess or lack spermine synthase. All spermine-synthase-deficient cells had no detectable spermine, and elevated spermidine, content. Spermine content did not alter the cell growth rate. There was little or no difference in sensitivity of immortalized mouse embryonic fibroblasts to doxorubicin, etoposide, cisplatin, methylglyoxal bis(guanylhydrazone) or H(2)O(2) and only a slight increase in sensitivity to vinblastine and nocodazole. However, the absence of spermine clearly increased the sensitivity to 1,3-bis(2-chloroethyl)- N -nitrosourea, suggesting that depletion of spermine may be a useful way to increase the anti-neoplastic effects of anti-tumour agents that form chloroethyl-mediated interstrand DNA cross-links. The effects of spermine on the response to polyamine analogues (which have been proposed to be useful anti-neoplastic agents) were complex, and depended on the compound examined and on the cells tested. Sensitivity to CHENSpm ( N (1)-ethyl- N (11)-[(cycloheptyl)methyl]-4,8-diazaundecane) was substantially greater in immortalized fibroblasts that lack spermine. In contrast, BE-3-4-3 [ N (1), N (12)-bis(ethyl)spermine] and BE-3-3-3 [ N (1), N (11)-bis(ethyl)norspermine] were more active against cells that contained spermine. The presence of spermine correlated with a greater induction of spermidine/spermine- N (1)-acetyltransferase by BE-3-3-3, which is consistent with suggestions that this induction is important for the response to this drug. These findings support the concepts that different polyamine analogues have different sites of action and that CHENSpm has a different site of action from BE-3-3-3. PMID:12737625

  12. Targeting ferritin receptors for the selective delivery of imaging and therapeutic agents to breast cancer cells

    NASA Astrophysics Data System (ADS)

    Geninatti Crich, S.; Cadenazzi, M.; Lanzardo, S.; Conti, L.; Ruiu, R.; Alberti, D.; Cavallo, F.; Cutrin, J. C.; Aime, S.

    2015-04-01

    In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 μg ml-1 (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation.In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 μg ml-1 (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation. Electronic supplementary information (ESI) available: Competition studies with free apoferritin, Fig. S1; APO-FITC intracellular distribution by

  13. Hydroponics gel as a new electrolyte gelling agent for alkaline zinc-air cells

    NASA Astrophysics Data System (ADS)

    Othman, R.; Basirun, W. J.; Yahaya, A. H.; Arof, A. K.

    The viability of hydroponics gel as a new alkaline electrolyte gelling agent is investigated. Zinc-air cells are fabricated employing 12 wt.% KOH electrolyte immobilised with hydroponics gel. The cells are discharged at constant currents of 5, 50 and 100 mA. XRD and SEM analysis of the anode plates after discharge show that the failure mode is due to the formation of zinc oxide insulating layers and not due to any side reactions between the gel and the plate or the electrolyte.

  14. Devil's claw hairy root culture in flasks and in a 3-L bioreactor: bioactive metabolite accumulation and flow cytometry.

    PubMed

    Homova, Vediha; Weber, Jost; Schulze, Josef; Alipieva, Kalina; Bley, Thomas; Georgiev, Milen

    2010-01-01

    Phenylethanoids are a group of natural water-soluble compounds with high biological value, which could potentially be commercially produced by hairy root cultures. Thus, we have examined the capacity of transformed root cultures of Devil's claw (Harpagophytum procumbens) to accumulate four phenylethanoid glycosides -beta-OH-verbascoside, verbascoside, leucosceptoside A, and martynoside--in shake-flasks and a 3-L stirred tank reactor. Verbascoside was found to be the major phenylethanoid, and its maximal contents were the same (1.12 mg/g dry weight) in both kinds of culture. However, peak leucosceptoside A contents were 1.6-times higher in bioreactor cultures than in shake-flask cultures. Flow cytometry analysis revealed that G0 + G1-phase cells predominated throughout the growth of the cultures, which was in accordance with the very high proportion of quiescent cells in the transformed roots. The results provide the first demonstration of the potential utility of Devil's claw hairy roots as biofactories for producing high-value phenylethanoid glycosides. PMID:20737916

  15. Telomerase activity and telomere length in human tumor cells with acquired resistance to anticancer agents.

    PubMed

    Smith, V; Dai, F; Spitz, M; Peters, G J; Fiebig, H H; Hussain, A; Burger, A M

    2009-11-01

    Telomeres and telomerase are targets for anticancer drug development and specific inhibitors are currently under clinical investigation. However, it has been reported that standard cytotoxic agents can affect telomere length and telomerase activity suggesting that they also have of a role in drug resistance. in this study, telomere lengths and telomerase activity as well as drug efflux pump expression, glutathione (GSH) levels and polyadenosine-ribose polymerase (PARP) cleavage were assessed in a panel of human tumor cell lines made resistant to vindesine, gemcitabine and cisplatin. these included two lung cancer cell lines resistant to vindesine (LXFL 529L/Vind, LXFA 526L/Vind), a renal cancer cell line (RXF944L/Gem) and an ovarian cancer cell line (AG6000) resistant to gemcitabine, and one resistant to cisplatin (ADDP). The resistant clones were compared to their parental lines and evaluated for cross resistance to other cytotoxic agents. Several drug specific resistance patterns were found, and various complex patterns of cross resistance emerged from some cell lines, but these mechanisms of resistance could not be related to drug efflux pump expression, GSH levels or pARp cleavage. However, all displayed changes in telomerase activity and/or telomere length. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics.

  16. Effects of psoralens as anti-tumoral agents in breast cancer cells

    PubMed Central

    Panno, Maria Luisa; Giordano, Francesca

    2014-01-01

    This review examines the biological properties of coumarins, widely distributed at the highest levels in the fruit, followed by the roots, stems and leaves, by considering their beneficial effects in the prevention of some diseases and as anti-cancer agents. These compounds are well known photosensitizing drugs which have been used as pharmaceuticals for a broad number of therapeutic applications requiring cell division inhibitors. Despite this, even in the absence of ultraviolet rays they are active. The current paper mainly focuses on the effects of psoralens on human breast cancer as they are able to influence many aspects of cell behavior, such as cell growth, survival and apoptosis. In addition, analytical and pharmacological data have demonstrated that psoralens antagonize some metabolizing enzymes, affect estrogen receptor stability and counteract cell invasiveness as well as cancer drug resistance. The scientific findings summarized highlight the pleiotropic functions of phytochemical drugs, given that recently their target signals and how these are modified in the cells have been identified. The encouraging results in this field suggest that multiple modulating strategies based on coumarin drugs in combination with canonical chemotherapeutic agents or radiotherapy could be a useful approach to address the treatment of many types of cancer. PMID:25114850

  17. Suppression of STN1 enhances the cytotoxicity of chemotherapeutic agents in cancer cells by elevating DNA damage

    PubMed Central

    Zhou, Qing; Chai, Weihang

    2016-01-01

    DNA damage-inducing agents are among the most effective treatment regimens in clinical chemotherapy. However, drug resistance and severe side effects caused by these agents greatly limit their efficacy. Sensitizing malignant cells to chemotherapeutic agents has long been a goal of chemotherapy. In the present study, suppression of STN1, a gene important for safeguarding genome stability, potentiated the anticancer effect of chemotherapeutic agents in tumor cells. Using multiple cancer cells from a variety of origins, it was observed that downregulation of STN1 resulted in a significant decrease in the half maximal inhibitory concentration values of several conventional anticancer agents. When cells are treated with anticancer agents, STN1 suppression leads to a decline in colony formation and diminished anchorage-independent growth. Furthermore, it was additionally observed that STN1 knockdown augmented the levels of DNA damage caused by damage-inducing agents. The present study concluded that suppression of STN1 enhances the cytotoxicity of damage-inducing chemotherapeutic agents by increasing DNA damage in cancer cells. PMID:27446354

  18. Tumor lysing genetically engineered T cells loaded with multi-modal imaging agents.

    PubMed

    Bhatnagar, Parijat; Alauddin, Mian; Bankson, James A; Kirui, Dickson; Seifi, Payam; Huls, Helen; Lee, Dean A; Babakhani, Aydin; Ferrari, Mauro; Li, King C; Cooper, Laurence J N

    2014-01-01

    Genetically-modified T cells expressing chimeric antigen receptors (CAR) exert anti-tumor effect by identifying tumor-associated antigen (TAA), independent of major histocompatibility complex. For maximal efficacy and safety of adoptively transferred cells, imaging their biodistribution is critical. This will determine if cells home to the tumor and assist in moderating cell dose. Here, T cells are modified to express CAR. An efficient, non-toxic process with potential for cGMP compliance is developed for loading high cell number with multi-modal (PET-MRI) contrast agents (Super Paramagnetic Iron Oxide Nanoparticles - Copper-64; SPION-(64)Cu). This can now be potentially used for (64)Cu-based whole-body PET to detect T cell accumulation region with high-sensitivity, followed by SPION-based MRI of these regions for high-resolution anatomically correlated images of T cells. CD19-specific-CAR(+)SPION(pos) T cells effectively target in vitro CD19(+) lymphoma. PMID:24675806

  19. Molecularly targeted agents and immunotherapy for the treatment of head and neck squamous cell cancer (HNSCC).

    PubMed

    Azoury, SaÏd C; Gilmore, Richard C; Shukla, Vivek

    2016-06-01

    Squamous cell carcinoma is one of the most frequent tumors of the head and neck and often presents at an advanced-stage. Traditionally, treatment for head and neck squamous cell carcinoma (HNSCC) has included surgery, radiation, and chemotherapy depending on both the site and stage of disease. Although the treatment approach for local disease is often standardized, the management of recurrent and advanced disease is evolving. A better understanding of the molecular mechanisms of HNSCC has led to numerous promising investigations and the push for the development of novel therapies. Similarly, over the past several decades, growing data supports the notion that an individual's immune system can be manipulated in such a way to help eradicate cancer. The success of immunotherapeutic agents such as interleukin therapy and immune checkpoint inhibitor blockade in cancer, particularly advanced-stage melanoma, has stimulated researchers to uncover similar success stories in HNSCC. Examples of immunotherapeutics that are being studied for the treatment of HNSCC include adoptive T-cell therapy, vaccines, and immune checkpoint inhibitor proteins (e.g., anti-CTLA-4, -PD-1, -PD-L1). Molecularly targeted agents of interest include inhibitors of transmembrane growth factor receptors, angiogenesis, and PI3K/AKT/mTOR and NOTCH signaling pathways. To date, cetuximab, an epidermal growth factor receptor inhibitor, is the only targeted agent for HNSCC that was approved by the Federal Food and Drug Administration (FDA) on the basis that it improves overall survival when combined with chemotherapy or radiation. Herein, the authors provide an up-to-date review of immunotherapeutic and molecularly targeted agents for the treatment of HNSCC. PMID:27448787

  20. Identification of thiostrepton as a novel therapeutic agent that targets human colon cancer stem cells.

    PubMed

    Ju, S-Y; Huang, C-Y F; Huang, W-C; Su, Y

    2015-01-01

    Accumulating evidence shows that colorectal cancer stem cells (CRSCs) are largely responsible for the metastasis and relapse of colorectal cancer (CRC) after therapy. Hence, identifying new agents that specifically target CRSCs would help improve the effectiveness of current CRC therapies. To accelerate identification of agents targeting CRSCs, the Connectivity Map (CMap) approach was used. Among the top-ranked candidates, thiostrepton, a thiazole antibiotic, was selected for further investigation because of its known tumoricidal activity. Thiostrepton could selectively induce apoptosis in CRSC subpopulations in both parental HCT-15 and HT-29 human CRC lines as well as in EMT and chemoresistant clones derived from them. Further, we investigated its inhibitory effects on the sphere- and colony-forming capabilities of the aforementioned CRC lines. The in vitro inhibition of sphere and colony formation was associated with downregulation of various modulators of the stem cell phenotype. The combination of thiostrepton and oxaliplatin eradicated both CD44(+) HCT-15 and HT-29 cells more efficiently than either drug alone. FoxM1, an oncogenic transcription factor, was identified as a critical positive modulator of stemness and as the main target of thiostrepton in the CRC lines. This is the first report showing the selective killing of CRSCs by thiostrepton, which has been proposed to be a promising anti-neoplastic agent. On the basis of its synergism with oxaliplatin in killing CRSCs in vitro, if this activity is confirmed in vivo, thiostrepton may be a promising agent to be used clinically in combination with current chemotherapies to improve the efficacy of these regimens. PMID:26136074

  1. Osmotic Effects Induced by Pore-Forming Agent Nystatin: From Lipid Vesicles to the Cell

    PubMed Central

    Zemljič Jokhadar, Špela; Božič, Bojan; Kristanc, Luka; Gomišček, Gregor

    2016-01-01

    The responses of Chinese hamster ovary epithelial cells, caused by the pore-forming agent nystatin, were investigated using brightfield and fluorescence microscopy. Different phenomena, i.e., the detachment of cells, the formation of blebs, the occurrence of “cell-vesicles” and cell ruptures, were observed. These phenomena were compared to those discovered in giant lipid vesicles. A theoretical model, based on the osmotic effects that occur due to the size-discriminating nystatin transmembrane pores in lipid vesicles, was extended with a term that considers the conservation of the electric charge density in order to describe the cell’s behavior. The increase of the cellular volume was predicted and correlated with the observed phenomena. PMID:27788169

  2. Effect of Antimicrobial Agents on MinD Protein Oscillations in E. coli Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Kelly, Corey; Murphy, Megan; Giuliani, Maximiliano; Dutcher, John

    2011-03-01

    The pole-to-pole oscillation of the MinD proteins in E. coli determines the location of the division septum, and is integral to healthy cell division. It has been shown previously that the MinD oscillation period is approximately 40 s for healthy cells but is strongly dependant on environmental factors such as temperature, which may place stress on the cell [2,3]. We use a strain of E. coli in which the MinD proteins are tagged with green fluorescent protein (GFP), allowing fluorescence visualization of the MinD oscillation. We use high resolution total internal reflection fluorescence (TIRF) microscopy to observe the effect of exposure to antimicrobial agents on the MinD oscillation period and, more generally, to analyze the time variation of the spatial distribution of the MinD proteins within the cells. These measurements provide insight into the mechanism of antimicrobial action.

  3. Carnosine and neocuproine as neutralizing agents for copper overload-induced damages in cultured human cells.

    PubMed

    Arnal, Nathalie; de Alaniz, María J T; Marra, Carlos A

    2011-07-15

    Copper is dangerous when it is present in excess, mainly because it can participate in the Fenton reaction, which produces radical species. As a consequence of copper pollution, people are involuntarily exposed to a copper overload under sub-clinical and sub-symptomatological conditions, which may be very difficult to detect. Thus, we investigated (i) the possible use of the chelator molecules carnosine and neocuproine to prevent the Cu overload-induced damage on cellular lipids and proteins, as tested in human cell culture systems, and (ii) the differential response of these two chelating agents in relation to their protective action, and the type of copper ion involved in the process, by using two types of human cultured cells (HepG2 and A-549). Cu treatment clearly enhanced (p<0.01) the formation of protein carbonyls, thiobarbituric acid-reactive substances (TBARS) and the concentration of nitrate plus nitrites, with a concomitant decrease in cell survival, as estimated by the trypan dye exclusion test and lactate dehydrogenase leakage. Simultaneous treatment with Cu and carnosine or neocuproine indicated that carnosine is more efficient than neocuproine in protecting both types of cells from the effect of cupric ions on both the cell-associated damages and the decrease in the cellular viability. This observation was supported by the fact that carnosine is not only a complexing agent for Cu(II), but also an effective antioxidant that can dismutate superoxide radicals, scavenge hydroxyl radicals and neutralize TBARS formation. Carnosine should be investigated in more detail in order to establish its putative utility as an agent to prevent copper-associated damages in biological systems.

  4. Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death.

    PubMed

    Turunen, M J; Töyräs, J; Lammi, M J; Jurvelin, J S; Korhonen, R K

    2012-02-01

    In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent Hexabrix(TM). Samples were prepared from bovine patellae and stored in Dulbecco's Modified Eagle's Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1 MPa to 10 MPa and from 0 min to 15 min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix™, stress-relaxation tests were conducted for the samples immersed in Hexabrix™ and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix™ were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8-10 MPa contact stresses, the samples immersed in full strength Hexabrix™ showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.

  5. Efficient labeling in vitro with non-ionic gadolinium magnetic resonance imaging contrast agent and fluorescent transfection agent in bone marrow stromal cells of neonatal rats.

    PubMed

    Li, Ying-Qin; Tang, Ying; Fu, Rao; Meng, Qiu-Hua; Zhou, Xue; Ling, Ze-Min; Cheng, Xiao; Tian, Su-Wei; Wang, Guo-Jie; Liu, Xue-Guo; Zhou, Li-Hua

    2015-07-01

    Although studies have been undertaken on gadolinium labeling-based molecular imaging in magnetic resonance imaging (MRI), the use of non-ionic gadolinium in the tracking of stem cells remains uncommon. To investigate the efficiency in tracking of stem cells with non-ionic gadolinium as an MRI contrast agent, a rhodamine-conjugated fluorescent reagent was used to label bone marrow stromal cells (BMSCs) of neonatal rats in vitro, and MRI scanning was undertaken. The fluorescent-conjugated cell uptake reagents were able to deliver gadodiamide into BMSCs, and cell uptake was verified using flow cytometry. In addition, the labeled stem cells with paramagnetic contrast medium remained detectable by an MRI monitor for a minimum of 28 days. The present study suggested that this method can be applied efficiently and safely for the labeling and tracking of bone marrow stromal cells in neonatal rats.

  6. Hairy root-activation tagging: a high-throughput system for activation tagging in transformed hairy roots.

    PubMed

    Seki, Hikaru; Nishizawa, Tomoko; Tanaka, Nobukazu; Niwa, Yasuo; Yoshida, Shigeo; Muranaka, Toshiya

    2005-11-01

    Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in "transformed hairy roots". The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.

  7. Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents.

    PubMed

    Brojatsch, Jürgen; Lima, Heriberto; Palliser, Deborah; Jacobson, Lee S; Muehlbauer, Stefan M; Furtado, Raquel; Goldman, David L; Lisanti, Michael P; Chandran, Kartik

    2015-01-01

    Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis. PMID:25830414

  8. Loss of Atrx sensitizes cells to DNA damaging agents through p53-mediated death pathways.

    PubMed

    Conte, Damiano; Huh, Michael; Goodall, Emma; Delorme, Marilyne; Parks, Robin J; Picketts, David J

    2012-01-01

    Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrx(f/f) mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres) were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors. PMID:23284920

  9. Therapeutic and toxicologic evaluation of anti-lipogenic agents in cancer cells compared with non-neoplastic cells.

    PubMed

    Deepa, Perinkulam Ravi; Vandhana, Suryanarayanan; Jayanthi, Udayakumar; Krishnakumar, Subramanian

    2012-06-01

    Fatty acid synthase (FASN), a multi-enzyme complex, is involved in lipid biosynthesis. FASN is over-expressed in different types of cancers and is being widely investigated for its role in cancer progression, diagnosis and therapy. Here, three inhibitors targeting different domains of FASN--cerulenin, triclosan and orlistat--were evaluated for their anti-proliferative efficacy in ocular cancer, retinoblastoma (RB) cells and their toxicity (if any) in normal cells. FASN inhibitors were tested in cultured retinoblastoma Y79 cells, normal fibroblast (3T3) and Müller glial (MIOM1) cells. Cell viability was determined by MTT-based assay, and IC(50) (50% inhibitory concentration) of the FASN inhibitors was calculated in neoplastic and non-neoplastic cells. The IC(50) after 48 and 96 hr of incubation with the three anti-FASN agents showed that cerulenin, triclosan and orlistat inhibited retinoblastoma cell proliferation in a dose- and time-dependent manner. The cancer cells exhibited differential dose- and time-dependent response/sensitivities to cerulenin, triclosan and orlistat. The 48-hr neoplastic IC(50) dosages were, however, not toxic to the normal cells. These findings were confirmed by phase-contrast microscopic assessment of cell morphology. Therapeutic index (TI) was calculated as a ratio of the IC(50) normal cells, to the IC(50) neoplastic cells. Relative to normal MIOM1 cells, TI was 9.18 for cerulenin, while 5.32 for triclosan and 1.72 for orlistat. The TI computed relative to 3T3 cells was 28.64, 7.10 and 2.58 for cerulenin, triclosan and orlistat, respectively. DNA fragmentation analysis suggests that FASN inhibitors induced apoptotic DNA damage in retinoblastoma cells. Thus, FASN inhibition can be an effective strategy in retinoblastoma therapy. PMID:22151915

  10. Toxicity and in vitro activity of HIV-1 latency-reversing agents in primary CNS cells.

    PubMed

    Gray, Lachlan R; On, Hung; Roberts, Emma; Lu, Hao K; Moso, Michael A; Raison, Jacqueline A; Papaioannou, Catherine; Cheng, Wan-Jung; Ellett, Anne M; Jacobson, Jonathan C; Purcell, Damian F J; Wesselingh, Steve L; Gorry, Paul R; Lewin, Sharon R; Churchill, Melissa J

    2016-08-01

    Despite the success of combination antiretroviral therapy (cART), HIV persists in long lived latently infected cells in the blood and tissue, and treatment is required lifelong. Recent clinical studies have trialed latency-reversing agents (LRA) as a method to eliminate latently infected cells; however, the effects of LRA on the central nervous system (CNS), a well-known site of virus persistence on cART, are unknown. In this study, we evaluated the toxicity and potency of a panel of commonly used and well-known LRA (panobinostat, romidepsin, vorinostat, chaetocin, disulfiram, hexamethylene bisacetamide [HMBA], and JQ-1) in primary fetal astrocytes (PFA) as well as monocyte-derived macrophages as a cellular model for brain perivascular macrophages. We show that most LRA are non-toxic in these cells at therapeutic concentrations. Additionally, romidepsin, JQ-1, and panobinostat were the most potent at inducing viral transcription, with greater magnitude observed in PFA. In contrast, vorinostat, chaetocin, disulfiram, and HMBA all demonstrated little or no induction of viral transcription. Together, these data suggest that some LRA could potentially activate transcription in latently infected cells in the CNS. We recommend that future trials of LRA also examine the effects of these agents on the CNS via examination of cerebrospinal fluid.

  11. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents

    SciTech Connect

    Kaina, B.; Lohrer, H.; Karin, M.; Herrlich, P. )

    1990-04-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions.

  12. A Novel Microtubule-Disrupting Agent Induces Endoplasmic Reticular Stress-Mediated Cell Death in Human Hepatocellular Carcinoma Cells

    PubMed Central

    Ho, Chun-Te; Chang, Yu-Jia; Yang, Li-Xi; Wei, Po-Li; Liu, Tsan-Zon; Liu, Jun-Jen

    2015-01-01

    Here, we present evidence of a novel microtubule-disrupting agent, N-deacetyl-N-(chromone-2-carbonyl)-thiocolchicine (TCD), exhibiting potent antitumor activity (with IC50 values in the nanomolar range) against hepatocellular carcinoma cell lines. Cell cycle analysis revealed that TCD induced G2/M cell-cycle arrest in a dose- and time-dependent manner in both Hep-J5 and Mahlavu HCC cell lines. TCD also induced a decrease in mitochondrial membrane potential (ΔΨm) and caused DNA damage. Mechanistically, TCD activated protein kinase RNA-like endoplasmic reticular kinase and several transcription factors, including activating transcription factor (ATF) 6, ATF4, ATF3, and the CCAAT-enhancer binding protein homologous protein. These data clearly demonstrate that the antitumor activity of TCD is mechanistically linked to its capacity to trigger both intrinsic and extrinsic apoptotic cell death via endoplasmic reticular stress pathway. The potent antitumor activity of TCD was similarly demonstrated in a hepatocellular carcinoma xenograft model, where 5 and 10 mg/kg doses of TCD significantly arrested Hep-J5 and Mahlavu tumor growth. Our finding suggests that TCD is a promising therapeutic agent against hepatocellular carcinoma; further translational assessment of its clinical usage is warranted. PMID:26355599

  13. The presence of Estrogen Receptor β modulates the response of breast cancer cells to therapeutic agents.

    PubMed

    Pons, Daniel Gabriel; Torrens-Mas, Margalida; Nadal-Serrano, Mercedes; Sastre-Serra, Jorge; Roca, Pilar; Oliver, Jordi

    2015-09-01

    Breast cancer is a leading cause of death for women. The estrogen receptors (ERs) ratio is important in the maintenance of mitochondrial redox status, and higher levels of ERβ increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERβ ratio and the response to cytotoxic treatments such as cisplatin (CDDP), paclitaxel (PTX) and tamoxifen (TAM). Cell viability, apoptosis, autophagy, ROS production, mitochondrial membrane potential, mitochondrial mass and mitochondrial functionality were analyzed in MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) breast cancer cell lines. Cell viability decreased more in MCF-7 when treated with CDDP and PTX. Apoptosis was less activated after cytotoxic treatments in T47D than in MCF-7 cells. Nevertheless, autophagy was increased more in CDDP-treated MCF-7, but less in TAM-treated cells than in T47D. CDDP treatment produced a raise in mitochondrial mass in MCF-7, as well as the citochrome c oxidase (COX) and ATP synthase protein levels, however significantly reduced COX activity. In CDDP-treated cells, the overexpression of ERβ in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERβ in T47D cells promoted the opposite effects. In TAM-treated cells, ERβ-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERβ in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERβ expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.

  14. Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells.

    PubMed

    Jung, Heejun; Kim, Namyoung; Yoon, Minjung

    2016-10-01

    The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions. PMID:27546795

  15. Alkylating agents and immunotoxins exert synergistic cytotoxic activity against ovarian cancer cells. Mechanism of action.

    PubMed Central

    Lidor, Y J; O'Briant, K C; Xu, F J; Hamilton, T C; Ozols, R F; Bast, R C

    1993-01-01

    Alkylating agents can be administered in high dosage to patients with ovarian cancer using autologous bone marrow support, but drug-resistant tumor cells can still persist. Immunotoxins provide reagents that might eliminate drug resistant cells. In the present study, concurrent treatment with alkylators and immunotoxins proved superior to treatment with each agent alone. Toxin immunoconjugates prepared from different monoclonal antibodies and recombinant ricin A chain (rRTA) inhibited clonogenic growth of ovarian cancer cell lines in limiting dilution assays. When alkylating agents and toxin conjugates were used in combination, the addition of the immunotoxins to cisplatin, or to cisplatin and thiotepa, produced synergistic cytotoxic activity against the OVCA 432 and OVCAR III cell lines. Studies performed to clarify the mechanism of action showed that cisplatin and thiotepa had no influence on internalization and binding of the 317G5-rRTA immunotoxin. Intracellular uptake of [195m]Pt-cisplatin was not affected by the immunoconjugate and thiotepa. The combination of the 317G5-rRTA and thiotepa, as well as 317G5-rRTA alone, increased [195m]Pt cisplatin-DNA adduct levels. The immunotoxin alone and in combination with the alkylators decreased intracellular glutathione levels and reduced glutathione-S-transferase activity. Repair of DNA damage induced by the combination of alkylators and 317G5-rRTA was significantly reduced when compared to repair after damage with alkylators alone. These findings suggest that immunotoxins affect levels and activity of enzymes required for the prevention and repair of alkylator damage. Images PMID:8227359

  16. Harnessing the potential of hairy roots: dawn of a new era.

    PubMed

    Guillon, Stéphanie; Trémouillaux-Guiller, Jocelyne; Pati, Pratap Kumar; Rideau, Marc; Gantet, Pascal

    2006-09-01

    In the past two decades, hairy root research for the production of important secondary metabolites has received a lot of attention. The addition of knowledge to overcome the limiting culture parameters of the regulation of the metabolic pathway by specific molecules and the development of novel tools for metabolic engineering now offer new possibilities to improve the hairy root technique for the production of metabolites. Furthermore, engineering hairy roots for the production of animal proteins of therapeutic interest in confined and controlled in vitro conditions is seen as one of the exciting spin-offs of the technology. Recent progress made in the scale-up of the hairy root cultures has paved the way for industrial exploitation of this system. This review highlights some of the significant progress made in the past three years and discusses the potential implications of that research.

  17. Enhanced load-carrying capacity of hairy surfaces floating on water.

    PubMed

    Xue, Yahui; Yuan, Huijing; Su, Weidong; Shi, Yipeng; Duan, Huiling

    2014-05-01

    Water repellency of hairy surfaces depends on the geometric arrangement of these hairs and enables different applications in both nature and engineering. We investigate the mechanism and optimization of a hairy surface floating on water to obtain its maximum load-carrying capacity by the free energy and force analyses. It is demonstrated that there is an optimum cylinder spacing, as a result of the compromise between the vertical capillary force and the gravity, so that the hairy surface has both high load-carrying capacity and mechanical stability. Our analysis makes it clear that the setae on water striders' legs or some insects' wings are in such an optimized geometry. Moreover, it is shown that surface hydrophobicity can further increase the capacity of a hairy surface with thick cylinders, while the influence is negligible when the cylinders are thin. PMID:24808757

  18. Enhanced load-carrying capacity of hairy surfaces floating on water

    PubMed Central

    Xue, Yahui; Yuan, Huijing; Su, Weidong; Shi, Yipeng; Duan, Huiling

    2014-01-01

    Water repellency of hairy surfaces depends on the geometric arrangement of these hairs and enables different applications in both nature and engineering. We investigate the mechanism and optimization of a hairy surface floating on water to obtain its maximum load-carrying capacity by the free energy and force analyses. It is demonstrated that there is an optimum cylinder spacing, as a result of the compromise between the vertical capillary force and the gravity, so that the hairy surface has both high load-carrying capacity and mechanical stability. Our analysis makes it clear that the setae on water striders' legs or some insects' wings are in such an optimized geometry. Moreover, it is shown that surface hydrophobicity can further increase the capacity of a hairy surface with thick cylinders, while the influence is negligible when the cylinders are thin. PMID:24808757

  19. Short- and long-term effects of T-cell modulating agents in experimental autoimmunity.

    PubMed

    Mellergård, Johan; Havarinasab, Said; Hultman, Per

    2004-03-15

    Due to the easy and reliable induction of a disease condition with many of the features present in human autoimmunity, mercury-induced autoimmunity (mHgAI) in rodents is a favourable autoimmune model. Genetically susceptible (H-2(s)) mice develop in response to mercury (Hg) a systemic autoimmune condition with antinucleolar antibodies (ANoA) targeting the protein fibrillarin, transient polyclonal B-cell activation, hyperimmunoglobulinemia, and systemic immune-complex (IC) deposits. In order to study the short- and long-term effects of treatment with immunomodulating agents on the disease parameters in HgAI, groups of B10.S (H-2(s)) mice were given 6 mg HgCl(2)/l drinking water for 22 weeks. Three weeks initial treatment with cyclosporin A (CyA), a high dose of tacrolimus (HD tacrolimus), or anti-CD4 monoclonal antibody (a-CD4) inhibited induction of ANoA and IC deposit by Hg. This effect persisted for the subsequent 19 weeks when the mice were only treated with Hg. Initial treatment with anti-IL-4 monoclonal antibody (a-IL-4) for 3 weeks inhibited induction of IgE and IC deposits by Hg, but not ANoA. However, subsequent treatment with Hg without a-IL-4 for 19 weeks induced IC deposits. The T-cell modulating agents aggravated some of the HgAI disease parameters: a-CD4 stimulated the polyclonal B-cell activation, a-IL-4 increased the IgG antichromatin antibody response, and a low dose of tacrolimus (LD tacrolimus) enhanced the ANoA, the polyclonal B-cell activation, and the IC deposits. We conclude that a short initial treatment with a-CD4 or CyA efficiently protects against induction of systemic autoimmunity for an extended period of time. However, some of the T-cell modulating agents, especially a low dose of tacrolimus, aggravate autoimmune manifestations not only during ongoing treatment, but also after treatment with these agents has ceased.

  20. Effects of antimicrobial agents used for therapy of CNS infections on dissociated brain cell cultures.

    PubMed

    Schaad, U B; Guenin, K; Steffen, C; Herschkowtiz, N

    1988-09-01

    The prediction, measurement, and monitoring of neurologic toxicity of antibacterial agents is an exceedingly difficult matter. In this study we investigated if in vitro exposure of cultured brain cells to antibacterial drugs could predict neurotoxicity in man. Effects of antibiotics used for therapy of bacterial CNS infections on growth and differentiation in dissociated rat brain cell cultures were studied over 24 days in culture, the drugs being added from 10 to 17 days in culture, the main differentiation phase of rat CNS cells. Our results demonstrated a reversible inhibition of cerebral sulfate transferase activity (p less than 0.001 or less than 0.01) and to a lesser extent (p less than 0.001 or NS) of DNA synthesis in brain cell cultures by the highest concentrations studied of amikacin, cefuroxime, and ceftazidime which correspond to peak cerebrospinal fluid values attained by intraventricular therapy in patients. Accumulation of DNA reflects brain cell growth whereas cerebral sulfate transferase activity parallels brain cell differentiation. Our findings indicate that intraventricular therapy could be more toxic with amikacin, cefuroxime, and ceftazidime than with penicillin, chloramphenicol, or ceftriaxone. Thus, this brain cell culture model might become a supplement, complement, or even alternative technique for neurotoxicity assessment of antibiotics with proven or potential value for therapy of CNS infections.

  1. Cordycepin, a Natural Antineoplastic Agent, Induces Apoptosis of Breast Cancer Cells via Caspase-dependent Pathways.

    PubMed

    Wang, Di; Zhang, Yongfeng; Lu, Jiahui; Wang, Yang; Wang, Junyue; Meng, Qingfan; Lee, Robert J; Wang, Di; Teng, Lesheng

    2016-01-01

    Cordycepin, a major compound separated from Cordyceps sinensis, is known as a potential novel candidate for cancer therapy. Breast cancer, the most typical cancer diagnosed among women, remains a global health problem. In this study, the anti-breast cancer property of cordycepin and its underlying mechanisms was investigated. The direct effects of cordycepin on breast cancer cells both in in vitro and in vivo experiments were evaluated. Cordycepin exerted cytotoxicity in MCF-7 and MDA-MB-231 cells confirmed by reduced cell viability, inhibition of cell proliferation, enhanced lactate dehydrogenase release and reactive oxygen species accumulation, induced mitochondrial dysfunction and nuclear apoptosis in human breast cancer cells. Cordycepin increased the activation of pro-apoptotic proteins, including caspase-8, caspase-9, caspase-3 and Bax, and suppressed the expression of the anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2). The inhibition on MCF-7-xenografted tumor growth in nude mice further confirmed cordycepin's anti-breast cancer effect. These aforementioned results reveal that cordycepin induces apoptosis in human breast cancer cells via caspase-dependent pathways. The data shed light on the possibility of cordycepin being a safe agent for breast cancer treatment. PMID:26996021

  2. Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani.

    PubMed

    Lozovaya, Vera V; Lygin, Anatoliy V; Zernova, Olga V; Li, Shuxian; Hartman, Glen L; Widholm, Jack M

    2004-01-01

    Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible 'Spencer' and partially resistant 'PI567.374') to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f. sp. glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection. Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored. The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots. FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots. The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer. The de novo synthesis of isoflavones and glyceollin was confirmed by [(14)C]Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv. PI567.374 than that of Spencer. Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested. The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation. The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture. The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus. PMID:15331097

  3. Antibacterial agent triclosan suppresses RBL-2H3 mast cell function

    SciTech Connect

    Palmer, Rachel K.; Hutchinson, Lee M.; Burpee, Benjamin T.; Tupper, Emily J.; Pelletier, Jonathan H.; Kormendy, Zsolt; Hopke, Alex R.; Malay, Ethan T.; Evans, Brieana L.; Velez, Alejandro; Gosse, Julie A.

    2012-01-01

    Triclosan is a broad-spectrum antibacterial agent, which has been shown previously to alleviate human allergic skin disease. The purpose of this study was to investigate the hypothesis that the mechanism of this action of triclosan is, in part, due to effects on mast cell function. Mast cells play important roles in allergy, asthma, parasite defense, and carcinogenesis. In response to various stimuli, mast cells degranulate, releasing allergic mediators such as histamine. In order to investigate the potential anti-inflammatory effect of triclosan on mast cells, we monitored the level of degranulation in a mast cell model, rat basophilic leukemia cells, clone 2H3. Having functional homology to human mast cells, as well as a very well defined signaling pathway leading to degranulation, this cell line has been widely used to gain insight into mast-cell driven allergic disorders in humans. Using a fluorescent microplate assay, we determined that triclosan strongly dampened the release of granules from activated rat mast cells starting at 2 μM treatment, with dose-responsive suppression through 30 μM. These concentrations were found to be non-cytotoxic. The inhibition was found to persist when early signaling events (such as IgE receptor aggregation and tyrosine phosphorylation) were bypassed by using calcium ionophore stimulation, indicating that the target for triclosan in this pathway is likely downstream of the calcium signaling event. Triclosan also strongly suppressed F-actin remodeling and cell membrane ruffling, a physiological process that accompanies degranulation. Our finding that triclosan inhibits mast cell function may explain the clinical data mentioned above and supports the use of triclosan or a mechanistically similar compound as a topical treatment for allergic skin disease, such as eczema. -- Highlights: ►The effects of triclosan on mast cell function using a murine mast cell model. ►Triclosan strongly inhibits degranulation of mast cells.

  4. DNA transfer and cell killing in epidermoid cells by diagnostic ultrasound activation of contrast agent gas bodies in vitro.

    PubMed

    Miller, Douglas L; Dou, Chunyan; Song, Jianming

    2003-04-01

    DNA transfer by sonoporation and cell killing in monolayer cells were examined by contrast-aided low-power diagnostic ultrasound (US). Culture chambers with epidermoid cell monolayers were scanned at about 1 mm/s with a 1.5-MHz scan head aimed upward at the chamber in a 37 degrees C water bath. For DNA transfer tests, plasmids coding for green fluorescent protein (GFP) were added to the medium, and GFP expression was assessed by flow cytometry after 2 days. In separate tests, cell killing was determined immediately after treatment. GFP-positive cell counts were 0.4% (0.7% SD) for shams and 3.7% (1.2% SD) of cells for exposure at 2.3 MPa with 2% Optison contrast agent. The fraction of dead cells was 3.4% (1.7% SD) in shams and 28.6% (6.3% SD) in exposed chambers. Both effects increased for increasing Optison concentration and increasing peak rarefactional pressure amplitude. Contrast-aided diagnostic US has a potential therapeutic application for gene transfer, but a trade-off appears to exist with cell killing.

  5. Inhibition of cell adhesion by anti–P-selectin aptamer: a new potential therapeutic agent for sickle cell disease

    PubMed Central

    Gutsaeva, Diana R.; Parkerson, James B.; Yerigenahally, Shobha D.; Kurz, Jeffrey C.; Schaub, Robert G.; Ikuta, Tohru

    2011-01-01

    Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti–P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti–P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti–P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti–P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti–P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti–P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti–P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti–P-selectin aptamer may be useful as a novel therapeutic agent for SCD. PMID:20926770

  6. Reversible assembly of tunable nanoporous materials from "hairy" silica nanoparticles.

    PubMed

    Khabibullin, Amir; Fullwood, Emily; Kolbay, Patrick; Zharov, Ilya

    2014-10-01

    Membranes with 1-100 nm nanopores are widely used in water purification and in biotechnology, but are prone to blockage and fouling. Reversibly assembled nanoporous membranes may be advantageous due to recyclability, cleaning, and retentate recovery, as well as the ability to tune the pore size. We report the preparation and characterization of size-selective nanoporous membranes with controlled thickness, area, and pore size via reversible assembly of polymer brush-grafted ("hairy") silica nanoparticles. We describe membranes reversibly assembled from silica particles grafted with (1) polymer brushes carrying acidic and basic groups, and (2) polymer brushes carrying neutral groups. The former are stable in most organic solvents and easily disassemble in water, whereas the latter are water-stable and disassemble in organic solvents.

  7. Phytochelatin homologs induced in hairy roots of horseradish.

    PubMed

    Kubota, H; Sato, K; Yamada, T; Maitani, T

    2000-01-01

    When exposed to excess heavy metals, plants induce phytochelatins and related peptides (all designated as PCAs). Thus, when hairy roots of horseradish (Armoracia rusticana) were exposed for 3 days to cadmium (1 mM) along with reduced glutathione (2 mM), PCA induction occurred. Moreover, a new family of thiol peptides was detected as well as the previously known PCAs, as revealed by postcolumn-derivatization HPLC. Two were isolated and their structures were identified as (gamma-Glu-Cys)n-Gln (n = 3 and 4) by electrospray ionization-mass spectrometer spectra, this being confirmed by chemical synthesis of the peptides. These new analogs constitute the sixth PCA family identified to date. PMID:10680177

  8. The role of multi-agent systems in improving performance of manufacturing robotized cells

    NASA Astrophysics Data System (ADS)

    Sękala, A.; Ćwikła, G.; Kost, G.

    2015-11-01

    Present market conditions causes that modern control systems of robotized manufacturing cells should be characterized by the much greater degree of flexibility, selforganization and, above all, adaptability to emerging outer excitations. The phenomenon of information distribution is one of the most important features of modern control systems. In the paper is presented the approach, based on application of multi-agent systems, for supporting the operation of robotized manufacturing cells. The aim of this approach is to obtain the flexible response to outer excitations and preventing situations that might cause the delay of the production process. The presented paper includes description of the concept of an informatics system designed for controlling the work of production systems, including work cells. Such systems could operate independently if it would be equipped with the selforganization mechanism. It is possible in the case of the proposed multi-agent system. The implementation of the presented concept will follow the present analysis of the described concept. The advantage of the proposed concept is its hierarchical depiction that allows integrating different utilized informatics tools in one complex system. It allows preparing the final computer program.

  9. Desmoplastic small round-cell tumor: an adult with previous exposure to agent orange.

    PubMed

    Baz, Walid; El-Soueidi, Raymond; Nakhl, Fadi; Aoun, Nelly; Chin, Nena; Dhar, Meeko

    2010-06-01

    Desmoplastic small round-cell tumor is an uncommon, highly aggressive tumor with a predilection for pediatric age groups and young adults. It is very unusual in the elderly population. Although Agent Orange has been associated with soft-tissue sarcoma, an association with desmoplastic small round-cell tumor has not been reported. A 52-year-old male presented with abdominal distention, dyspnea, and a 9 kg weight loss. Prior history was significant for hepatitis C and diabetes. He was a Vietnam veteran and he admitted being exposed to Agent Orange. On physical examination, the abdomen was distended and tense. Computed tomography scan of the chest, abdomen and pelvis demonstrated extensive mediastinal and retroperitoneal adenopathy, diffuse omental masses and extensive pleural, intra-abdominal and pelvic ascites. Omental core needle biopsy was consistent with desmoplastic small round-cell tumor based on morphology and immunohistochemistry. He responded poorly to chemotherapy with high-dose cyclophosphamide, doxorubicin and vincristine and died 5 months after presentation secondary to neutropenic sepsis despite G-CSF support and antibiotics. PMID:20382635

  10. Novel Agents and Emerging Strategies for Targeting the B-Cell Receptor Pathway in CLL

    PubMed Central

    Efremov, Dimitar G.; Wiestner, Adrian; Laurenti, Luca

    2012-01-01

    Chronic lymphocytic leukemia (CLL) is a disease of malignant CD5+ B lymphocytes that are characterized by frequent expression of autoreactive B-cell receptors (BCRs) and marked dependence on microenvironmental signals for proliferation and survival. Among the latter, signals propagated through the BCR are believed to play a key role in leukemia initiation, maintenance and evolution. Drugs that can disrupt these signals have recently emerged as potential therapeutic agents in CLL and several of them are currently being evaluated in clinical trials. Particularly promising clinical responses have been obtained with inhibitors of the kinases SYK, BTK, and PI3Kδ, which function by blocking BCR signal transduction. In addition, recent studies focusing on the phosphatase PTPN22, which is involved in the pathogenesis of multiple autoimmune diseases and is markedly overexpressed in CLL cells, suggest that it may be possible in the future to develop strategies that will selectively reprogram BCR survival signals into signals that induce leukemic cell death. This review focuses on the biological basis behind these strategies and highlights some of the most promising BCR-targeting agents in ongoing preclinical and clinical studies. PMID:23170196

  11. Honokiol: a promising small molecular weight natural agent for the growth inhibition of oral squamous cell carcinoma cells.

    PubMed

    Chen, Xi-Rui; Lu, Rui; Dan, Hong-Xia; Liao, Ga; Zhou, Min; Li, Xiao-Yu; Ji, Ning

    2011-01-01

    Honokiol (HNK) is a small organic molecule purified from magnolia species and has demonstrated anticancer activities in a variety of cancer cell lines; however, its effect on oral squamous cell carcinoma (OSCC) cells is unknown. We investigated the antitumor activities of HNK on OSCC cells in vitro for the first time. The inhibitory effects of HNK on the growth and proliferation of OSCC cells were demonstrated via in vitro 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) assays, and the apoptotic cells were investigated by the observation of morphological changes and detection of DNA fragmentation via PI, TdT-mediated dUTP-biotin nick end labeling (TUNEL), and DNA ladder assays, as well as flow cytometry assay. The results showed that HNK inhibited the growth and proliferation of OSCC cells in vitro in a time and dose-dependent manner. The inhibitory effect was associated with the cell apoptosis induced by HNK, evidenced by the morphological features of apoptotic cells, TUNEL-positive cells and a degradation of chromosomal DNA into small internucleosomal fragments. The study also demonstrated here that the inhibition or apoptosis mediated by 15 microg x mL(-1) or 20 microg x mL(-1) of HNK were more stronger compared with those of 20 microg x mL(-1) 5-fluorouracil (5-Fu, the control) applied to OSCC cells, when the ratio of OSCC cell numbers were measured between the treatment of different concentrations of HNK to the 5-Fu treatment for 48 h. HNK is a promising compound that can be potentially used as a novel treatment agent for human OSCC. PMID:21449214

  12. [Induction and in vitro culture of hairy roots of Dianthus caryophyllus and its plant regeneration].

    PubMed

    Shi, Heping; Zhu, Yuanfeng; Wang, Bei; Sun, Jiangbing; Huang, Shengqin

    2014-11-01

    To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0-3.0 mg/L + NAA 0.1-0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L + NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30-40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.

  13. Metabolomic Analysis and Phenylpropanoid Biosynthesis in Hairy Root Culture of Tartary Buckwheat Cultivars

    PubMed Central

    Li, Xiaohua; Bok Kim, Yeon; Romij Uddin, Md; Kim, Sun Ju; Suzuki, Tatsuro; Park, Nam Il; Park, Sang Un

    2013-01-01

    Buckwheat, Fagopyrum tataricum Gaertn., is an important medicinal plant, which contains several phenolic compounds, including one of the highest content of rutin, a phenolic compound with anti-inflammatory properties. An experiment was conducted to investigate the level of expression of various genes in the phenylpropanoid biosynthetic pathway to analyze in vitro production of anthocyanin and phenolic compounds from hairy root cultures derived from 2 cultivars of tartary buckwheat (Hokkai T8 and T10). A total of 47 metabolites were identified by gas chromatography–time-of-flight mass spectrometry (GC-TOFMS) and subjected to principal component analysis (PCA) in order to fully distinguish between Hokkai T8 and T10 hairy roots. The expression levels of phenylpropanoid biosynthetic pathway genes, through qRT-PCR, showed higher expression for almost all the genes in T10 than T8 hairy root except for FtF3’H-2 and FtFLS-2. Rutin, quercetin, gallic acid, caffeic acid, ferulic acid, 4-hydroxybenzoic acid, and 2 anthocyanin compounds were identified in Hokkai T8 and T10 hairy roots. The concentration of rutin and anthocyanin in Hokkai T10 hairy roots of tartary buckwheat was several-fold higher compared with that obtained from Hokkai T8 hairy root. This study provides useful information on the molecular and physiological dynamic processes that are correlated with phenylpropanoid biosynthetic gene expression and phenolic compound content in F. tataricum species. PMID:23799007

  14. Atropa belladonna hairy roots: orchestration of concurrent oxidation and reduction reactions for biotransformation of carbonyl compounds.

    PubMed

    Srivastava, Vikas; Negi, Arvind Singh; Ajayakumar, P V; Khan, Shamshad A; Banerjee, Suchitra

    2012-03-01

    The biotransformation potential of a selected Atropa belladonna hairy root clone (AB-09) had been evaluated with regard to three different aromatic carbonyl compounds, i.e., 3,4,5-trimethoxybenzaldehyde (1), 3,4,5-trimethoxyacetophenone (2), and 3,4,5-trimethoxy benzoic acid (3). The results demonstrated for the first time the untapped potentials of the selected hairy root clone to perform simultaneous oxidation (34.49%) and reduction (32.68%) of 3,4,5-trimethoxy benzaldehyde (1) into 3,4,5-trimethoxy benzoic acid (3), and 3,4,5-trimethoxy benzyl alcohol (4), respectively, without any intermediate separation or addition of reagents. The same hairy root clone also demonstrated reduction (<5%) of a 3,4,5-trimethoxyacetophenone (2) into a secondary alcohol, i.e., 1-(3,4,5-trimethoxyphenyl) ethanol (5), while in the case of aromatic carboxylic acid substrate (3), no biotransformation could be obtained under the similar conditions. The current observations revealed oxidation and reduction of the formyl group of the aromatic ring, and only reduction of the carbonyl group of acetophenone through the specific hairy root clone. The concurrent oxidation and reduction reactions by the selected hairy root clone highlight the importance of this study, which, as per our observations, is the first of its kind relating the hairy root culture of A. belladonna.

  15. Elicitors from the endophytic fungus Trichoderma atroviride promote Salvia miltiorrhiza hairy root growth and tanshinone biosynthesis.

    PubMed

    Ming, Qianliang; Su, Chunyan; Zheng, Chengjian; Jia, Min; Zhang, Qiaoyan; Zhang, Hong; Rahman, Khalid; Han, Ting; Qin, Luping

    2013-12-01

    Biotic elicitors can be used to stimulate the production of secondary metabolites in plants. However, limited information is available on the effects of biotic elicitors from endophytic fungi on their host plant. Trichoderma atroviride D16 is an endophytic fungus isolated from the root of Salvia miltiorrhiza and previously reported to produce tanshinone I (T-I) and tanshinone IIA (T-IIA). Here, the effects of extract of mycelium (EM) and the polysaccharide fraction (PSF), produced by T. atroviride D16, on the growth and secondary metabolism of S. miltiorrhiza hairy roots are reported. The results indicated that both EM and PSF promoted hairy root growth and stimulated the biosynthesis of tanshinones in hairy roots. EM slightly suppressed the accumulation of phenolic acids, while PSF had no significant influence on the accumulation of these compounds. When comparing the effects of EM versus PSF, it was concluded that PSF is one of the main active constituents responsible for promoting hairy root growth, as well as stimulating biosynthesis of tanshinones in the hairy root cultures. Moreover, the transcriptional activity of genes involved in the tanshinone biosynthetic pathway increased significantly with PSF treatment. Thus, PSF from endophytic T. atroviride D16 affected the chemical composition of the host plant by influencing the expression of genes related to the secondary metabolite biosynthetic pathway. Furthermore, treatment with PSF can be effectively utilized for large-scale production of tanshinones in the S. miltiorrhiza hairy root culture system.

  16. Enhancement of rutin production in Fagopyrum tataricum hairy root cultures with its endophytic fungal elicitors.

    PubMed

    Zhao, Jianglin; Xiang, Dabing; Peng, Lianxin; Zou, Liang; Wang, Yuehua; Zhao, Gang

    2014-01-01

    Tartary buckwheat (Fagopyrum tataricum) is a potentially important source of rutin, a natural bioactive flavonoid with antihyperglycemic, antioxidative, antihypertensive, and anti-inflammatory properties. This study examines the effects of endophytic fungi on rutin production in the hairy root cultures of F. tataricum. Without obvious changes in the appearance of the hairy roots, the exogenous fungal mycelia elicitors efficiently stimulated the hairy root growth and rutin biosynthesis, and the stimulation effect was mainly dependent on the mycelia elicitor species, as well as its treatment dose. Two endophytic fungal isolates Fat9 (Fusarium oxysporum) and Fat15 (Alternaria sp.) were screened as promising candidates for promoting F. tataricum hairy root growth and rutin production. With application of polysaccharide (PS) of endophyte Fat9 (200 mg/L), and PS of endophyte Fat15 (100 mg/L) to the hairy root cultures on day 25, the rutin yield was increased to 45.9 mg/L and 47.2 mg/L, respectively. That was about 3.1- to 3.2-fold in comparison with the control level of 14.6 mg/L. Moreover, the present study revealed that the accumulation of rutin resulted from the stimulation of the phenylpropanoid pathway by mycelia PS treatments. This may be an efficient strategy for enhancing rutin production in F. tataricum hairy root culture provided with its endophytic mycelia elicitors.

  17. Cell permeable vanX inhibitors as vancomycin re-sensitizing agents.

    PubMed

    Muthyala, Ramaiah; Rastogi, Namrata; Shin, Woo Shik; Peterson, Marnie L; Sham, Yuk Yin

    2014-06-01

    VanX is an induced zinc metallo d-Ala-d-Ala dipeptidase involved in the viable remodeling of bacterial cell wall that is essential for the development of VREF. Here we report two cyclic thiohydroxamic acid-based peptide analogs that were designed, synthesized and investigated as vancomycin re-sensitizing agents. These compounds exhibit low micromolar inhibitory activity against vanX, with low cytotoxicity and were shown to increase vancomycin sensitivity against VREF. The improved pharmacological properties of these novel inhibitors over previous transition state mimics should provide an enhanced platform for designing potent vanX inhibitors for overcoming vancomycin resistance. PMID:24751446

  18. The sensitivity of yeast and yeast-like cells to new lysosomotropic agents.

    PubMed

    Krasowska, Anna; Chmielewska, Lucyna; Adamski, Ryszard; Luczyński, Jacek; Witek, Stanisław; Sigler, Karel

    2004-01-01

    The lysosomotropic action of the compounds DM-11 and DMAL-12s against Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans is species- and pH-dependent. At pH 6.0, DMAL-12s is less effective against S. cerevisiae and S. pombe but more effective against C. albicans than DM-11. At pH 8.0, DMAL-12s strongly inhibits the growth of S. cerevisiae but has only a marginal effect on the resistant C. albicans. S. pombe did not grow at pH 8.0. As shown by quinacrine accumulation, DM-11 causes a general intracellular acidification in all three species, while with DMAL-12s, the acidification is marginal. Morphological changes caused by DMAL-12s in S. cerevisiae affect the cell interior but not surface structures, while S. pombe cells exhibit a thickened and wrinkled cell wall, shrunken protoplast and "grainy" plasma membrane. A large number of blisters resembling lipid droplets were observed inside S. cerevisiae and S. pombe vacuoles. The high susceptibility of S. pombe cells to the action of DM-11 and DMAL-12s contrasts with the low sensitivity of S. pombe H+-ATPase to the agents. In our C. albicans isolate, DMAL 12s did not have an effect on cell morphology and appeared to be unable to penetrate the cells, especially at pH 8.0.

  19. Effect of Antimicrobial Agents on MinD Protein Oscillations in E. coli Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Kelly, Corey; Giuliani, Maximiliano; Dutcher, John

    2012-02-01

    The pole-to-pole oscillation of MinD proteins in E. coli cells determines the location of the division septum, and is integral to healthy cell division. It has been shown previously that the MinD oscillation period is approximately 40 s for healthy cells [1] but is strongly dependant on environmental factors such as temperature, which may place stress on the cell [2,3]. We use a strain of E. coli in which the MinD proteins are tagged with green fluorescent protein (GFP), allowing fluorescence visualization of the MinD oscillation. We use high-resolution total internal reflection fluorescence (TIRF) microscopy and a custom, temperature controlled flow cell to observe the effect of exposure to antimicrobial agents on the MinD oscillation period and, more generally, to analyze the time variation of the spatial distribution of the MinD proteins within the cells. These measurements provide insight into the mechanism of antimicrobial action. [1] Raskin, D.M.; de Boer, P. (1999) Proc. Natl. Acad. Sci. 96: 4971-4976. [2] Touhami, A.; Jericho, M; Rutenberg, A. (2006) J. Bacteriol. 188: 7661-7667. [3] Downing, B.; Rutenberg, A.; Touhami, A.; Jericho, M. (2009) PLoS ONE 4: e7285.

  20. HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress.

    PubMed

    Khanra, Kalyani; Chakraborty, Anindita; Bhattacharyya, Nandan

    2015-01-01

    The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta (polβ Δ208-304) specific for ovarian cancer. Pol β Δ208-304 has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. Polβ Δ208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H2O2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol β Δ208-304 transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

  1. Salidroside as a Novel Protective Agent to Improve Red Blood Cell Cryopreservation.

    PubMed

    Alotaibi, Noha A S; Slater, Nigel K H; Rahmoune, Hassan

    2016-01-01

    Glycerol and trehalose have been widely examined as protective agents in the cryopreservation of red blood cells (RBCs). However, the effectiveness of these reagents alone on cell viability is moderate. Here, the addition of salidroside attenuated oxidative damage of sheep RBCs prior to and post cryostorage. The supplementation of salidroside to the cryopreservation media containing 10% glycerol improved RBC survival by approximately 61.1±4.8% vs 37.9±4.6%. A smaller effect was seen in RBCs cryopreserved in 300 mM trehalose where the addition of salidroside improved survival by 7.6±0.3%. Furthermore, the addition of salidroside to cold storage solution demonstrated a significant reduction of haemolysis after 4 days for RBCs loaded with either glycerol or trehalose, compared to cells incubated without salidroside. RBCs survival was 2-fold greater following freezing in trehalose, compared with glycerol. After 10 days, salidroside enabled a lower haemolysis of 16.7±1.3% compared to 29.0±8.4% for cells incubated without salidroside. However, salidroside had no effect on RBCs which had been frozen in glycerol as the resulting haemolysis rate by day 10 was approximately 60%. Salidroside increased glutathione reductase activity and decreased lactate dehydrogenase activity. Furthermore, it led to reduced carbonylation of proteins in both glycerol and trehalose loaded cells. Finally, no effect on lipid peroxidation was found in the glycerol loaded RBCs although this was reduced in RBCs loaded with trehalose and salidroside. The present findings confirm the potential use of salidroside as a novel protective agent in cryopreservation and refrigerated storage of sheep RBCs. PMID:27631782

  2. Salidroside as a Novel Protective Agent to Improve Red Blood Cell Cryopreservation

    PubMed Central

    Alotaibi, Noha A. S.; Slater, Nigel K. H.; Rahmoune, Hassan

    2016-01-01

    Glycerol and trehalose have been widely examined as protective agents in the cryopreservation of red blood cells (RBCs). However, the effectiveness of these reagents alone on cell viability is moderate. Here, the addition of salidroside attenuated oxidative damage of sheep RBCs prior to and post cryostorage. The supplementation of salidroside to the cryopreservation media containing 10% glycerol improved RBC survival by approximately 61.1±4.8% vs 37.9±4.6%. A smaller effect was seen in RBCs cryopreserved in 300 mM trehalose where the addition of salidroside improved survival by 7.6±0.3%. Furthermore, the addition of salidroside to cold storage solution demonstrated a significant reduction of haemolysis after 4 days for RBCs loaded with either glycerol or trehalose, compared to cells incubated without salidroside. RBCs survival was 2-fold greater following freezing in trehalose, compared with glycerol. After 10 days, salidroside enabled a lower haemolysis of 16.7±1.3% compared to 29.0±8.4% for cells incubated without salidroside. However, salidroside had no effect on RBCs which had been frozen in glycerol as the resulting haemolysis rate by day 10 was approximately 60%. Salidroside increased glutathione reductase activity and decreased lactate dehydrogenase activity. Furthermore, it led to reduced carbonylation of proteins in both glycerol and trehalose loaded cells. Finally, no effect on lipid peroxidation was found in the glycerol loaded RBCs although this was reduced in RBCs loaded with trehalose and salidroside. The present findings confirm the potential use of salidroside as a novel protective agent in cryopreservation and refrigerated storage of sheep RBCs. PMID:27631782

  3. Inhibition of human telomerase enhances the effect of chemotherapeutic agents in lung cancer cells.

    PubMed

    Misawa, Masafumi; Tauchi, Tetsuzo; Sashida, Goro; Nakajima, Akihiro; Abe, Kenji; Ohyashiki, Junko H; Ohyashiki, Kazuma

    2002-11-01

    Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. Earlier studies have reported that the presence of telomerase activity in tumors of patients with non-small cell lung cancer patients correlates with a high proliferation rate and advanced pathological stage. Thus, the modification of telomerase activity may be a potential therapeutic modality for the treatment of lung and other cancers. We introduced vectors encoding dominant negative (DN)-hTERT, or wild-type (WT)-hTERT, or a control vector expressing only a drug-resistance marker, into the A549 lung cancer cell line, and assessed the biological effect of telomerase inhibition on cellular immortality. Ectopic expression of DN-hTERT resulted in complete inhibition of telomerase activity and reduction of telomere length. The entire population of telomerase-inhibited A549 cells exhibited cytoplasmic blebbling and chromatin condensation, which are features of apoptosis. In contrast, A549 cells expressing wild-type hTERT, which differs from the mutants by only two amino acids, exhibited normal morphology. Evidence for apoptosis in the telomerase-inhibited cells was provided by flow cytometric analysis with APO2.7 monoclonal antibody. We also observed enhanced induction of apoptosis by chemotherapeutic reagents, including cisplatin, docetaxel and etoposide, in DN-hTERT-expressing A549 cells, as compared with WT-hTERT-expressing cells. These results demonstrate that disruption of telomere maintenance limits the cellular lifespan of lung cancer cells, and show that the combined use of chemotherapeutic agents and telomere maintenance inhibition may be effective in the treatment of patients with non-small cell lung cancer.

  4. Effects and treatment of sarcoptic mange in southern hairy-nosed wombats (Lasiorhinus latifrons).

    PubMed

    Ruykys, Laura; Breed, Bill; Schultz, David; Taggart, David

    2013-04-01

    We examined the clinical and cellular effects of sarcoptic mange on southern hairy-nosed wombats (SHNW, Lasiorhinus latifrons) and the effectiveness of a single dose of ivermectin as a treatment for captive and wild animals. Wambats were caught at three sites in South Australia between April and August 2005 and blood and skin samples were collected. Hematology, biochemistry, and protein electrophoresis reference intervals were determined for healthy and diseased SHNW. Diseased SHNW had significantly higher white blood cell counts, neutrophils, lymphocytes, and total protein but lower red blood cell counts, hemoglobin, hematocrit, and creatinine. Microscopic investigation indicated substantial hyperplasia, hyperkeratosis, and fluid infiltration into the dermis and epidermis of diseased animals. Conclusions on the efficacy of a single dose of ivermectin were limited by low sample size (n=5, two captive and three wild SHNW) and are preliminary. However, ivermectin effectively treated mild, but not severe, mange in wild SHNW and severe mange in captive animals. This study has implications for the conservation and management of SHNW and the broader Vombatidae family. PMID:23568906

  5. Effects and treatment of sarcoptic mange in southern hairy-nosed wombats (Lasiorhinus latifrons).

    PubMed

    Ruykys, Laura; Breed, Bill; Schultz, David; Taggart, David

    2013-04-01

    We examined the clinical and cellular effects of sarcoptic mange on southern hairy-nosed wombats (SHNW, Lasiorhinus latifrons) and the effectiveness of a single dose of ivermectin as a treatment for captive and wild animals. Wambats were caught at three sites in South Australia between April and August 2005 and blood and skin samples were collected. Hematology, biochemistry, and protein electrophoresis reference intervals were determined for healthy and diseased SHNW. Diseased SHNW had significantly higher white blood cell counts, neutrophils, lymphocytes, and total protein but lower red blood cell counts, hemoglobin, hematocrit, and creatinine. Microscopic investigation indicated substantial hyperplasia, hyperkeratosis, and fluid infiltration into the dermis and epidermis of diseased animals. Conclusions on the efficacy of a single dose of ivermectin were limited by low sample size (n=5, two captive and three wild SHNW) and are preliminary. However, ivermectin effectively treated mild, but not severe, mange in wild SHNW and severe mange in captive animals. This study has implications for the conservation and management of SHNW and the broader Vombatidae family.

  6. Fluorine-19 MRI Contrast Agents for Cell Tracking and Lung Imaging

    PubMed Central

    Fox, Matthew S.; Gaudet, Jeffrey M.; Foster, Paula J.

    2015-01-01

    Fluorine-19 (19F)-based contrast agents for magnetic resonance imaging stand to revolutionize imaging-based research and clinical trials in several fields of medical intervention. First, their use in characterizing in vivo cell behavior may help bring cellular therapy closer to clinical acceptance. Second, their use in lung imaging provides novel noninvasive interrogation of the ventilated airspaces without the need for complicated, hard-to-distribute hardware. This article reviews the current state of 19F-based cell tracking and lung imaging using magnetic resonance imaging and describes the link between the methods across these fields and how they may mutually benefit from solutions to mutual problems encountered when imaging 19F-containing compounds, as well as hardware and software advancements. PMID:27042089

  7. Contribution of Cell Surface Hydrophobicity in the Resistance of Staphylococcus aureus against Antimicrobial Agents

    PubMed Central

    Lather, Puja; Mohanty, A. K.; Jha, Pankaj; Garsa, Anita Kumari

    2016-01-01

    Staphylococcus aureus is found in a wide variety of habitats, including human skin, where many strains are commensals that may be clinically significant or contaminants of food. To determine the physiological characteristics of resistant strain of Staphylococcus aureus against pediocin, a class IIa bacteriocin, a resistant strain was compared with wild type in order to investigate the contribution of hydrophobicity to this resistance. Additional clumping of resistant strain relative to wild type in light microscopy was considered as an elementary evidence of resistance attainment. A delay in log phase attainment was observed in resistant strain compared to the wild type strain. A significant increase in cell surface hydrophobicity was detected for resistant strain in both hexadecane and xylene indicating the contribution of cell surface hydrophobicity as adaptive reaction against antimicrobial agents. PMID:26966577

  8. Evaluating the toxic effect of an antimicrobial agent on single bacterial cells with optical tweezers

    PubMed Central

    Samadi, Akbar; Zhang, Chensong; Chen, Joseph; Reihani, S. N. S.; Chen, Zhigang

    2014-01-01

    We implement an optical tweezers technique to assess the effects of chemical agents on single bacterial cells. As a proof of principle, the viability of a trapped Escherichia coli bacterium is determined by monitoring its flagellar motility in the presence of varying concentrations of ethyl alcohol. We show that the “killing time” of the bacterium can be effectively identified from the correlation statistics of the positional time series recorded from the trap, while direct quantification from the time series or associated power spectra is intractable. Our results, which minimize the lethal effects of bacterial photodamage, are consistent with previous reports of ethanol toxicity that used conventional culture-based methods. This approach can be adapted to study other pairwise combinations of drugs and motile bacteria, especially to measure the response times of single cells with better precision. PMID:25657879

  9. The effect of actin disrupting agents on contact guidance of human embryonic stem cells

    PubMed Central

    Gerecht, Sharon; Bettinger, Christopher J.; Zhang, Zhitong; Borenstein, Jeffrey; Vunjak-Novakovic, Gordana; Langer, Robert

    2007-01-01

    Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography alters morpohology and proliferation of human embryonic stem cells (hESCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600 nm ridges with 600 nm spacing and 600 +/− 150 nm feature height) induced hESC alignment and elongation, mediated the organization of cytoskeletal components including actin, vimentin, and α-tubulin, and reduced proliferation. Spatial polarization of gamma tubulin complexes was also observed in response to nanotopography. Furthermore, the addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. These findings further demonstrate the importance of interplay between cytoskeleton and substrate interactions as a key modulator of morphological and proliferative cellular response in hESCs on nanotopography. PMID:17576011

  10. Neoplastic cell transformation by energetic heavy ions and its modification with chemical agents

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Tobias, C. A.

    1984-01-01

    One of the major deleterious late effects of ionizing radiation is related to the induction of neoplasms. In the present report recent experimental results on neoplastic cell transformation by heavy ions are presented, and possible means to circumvent the carcinogenic effect of space radiation are discussed. Biological effects observed in experiments involving the use of energetic heavy ions accelerated at the Bevalac suggest that many of the biological effects observed in earlier space flight experiments may be due to space radiation, particularly cosmic rays. It is found that the effect of radiation on cell transformation is dose-rate dependent. The frequency of neoplastic transformation for a given dose decreases with a decrease of dose rate of Co-60 gamma rays. It is found that various chemical agents give radiation protection, including DMSO.

  11. DNA Repair in Human Cells Exposed to Combinations of Carcinogenic Agents

    SciTech Connect

    Setlow, R. B.; Ahmed, F. E.

    1980-01-01

    Normal human and XP2 fibroblasts were treated with UV plus UV-mimetic chemicals. The UV dose used was sufficient to saturate the UV excision repair system. Excision repair after combined treatments was estimated by unscheduled DNA synthesis, BrdUrd photolysis, and the loss of sites sensitive to a UV specific endonuclease. Since the repair of damage from UV and its mimetics is coordinately controlled we expected that there would be similar rate-limiting steps in the repair of UV and chemical damage and that after a combined treatment the total amount of repair would be the same as from UV or the chemicals separately. The expectation was not fulfilled. In normal cells repair after a combined treatment was additive whereas in XP cells repair after a combined treatment was usually less than after either agent separately. The chemicals tested were AAAF, DMBA-epoxide, 4NQO, and ICR-170.

  12. Chemical warfare agent and biological toxin-induced pulmonary toxicity: could stem cells provide potential therapies?

    PubMed

    Angelini, Daniel J; Dorsey, Russell M; Willis, Kristen L; Hong, Charles; Moyer, Robert A; Oyler, Jonathan; Jensen, Neil S; Salem, Harry

    2013-01-01

    Chemical warfare agents (CWAs) as well as biological toxins present a significant inhalation injury risk to both deployed warfighters and civilian targets of terrorist attacks. Inhalation of many CWAs and biological toxins can induce severe pulmonary toxicity leading to the development of acute lung injury (ALI) as well as acute respiratory distress syndrome (ARDS). The therapeutic options currently used to treat these conditions are very limited and mortality rates remain high. Recent evidence suggests that human stem cells may provide significant therapeutic options for ALI and ARDS in the near future. The threat posed by CWAs and biological toxins for both civilian populations and military personnel is growing, thus understanding the mechanisms of toxicity and potential therapies is critical. This review will outline the pulmonary toxic effects of some of the most common CWAs and biological toxins as well as the potential role of stem cells in treating these types of toxic lung injuries.

  13. Photosensitized inactivation of infectious agents for sterilization of red blood cell concentrates and whole blood

    NASA Astrophysics Data System (ADS)

    Judy, Millard M.; Matthews, James Lester; Sogandares-Bernal, Franklin M.; Newman, Joseph T.; Chanh, Tran C.; Marengo-Rowe, Alain J.

    1992-06-01

    More than 10 million units of human blood components are transfused annually in the United States. Although donor screening and testing have greatly lowered the risk of transmission of viral and protozoan infectious agents, additional sterilization procedures which also preserve blood component function would be of significant value. Use of visible-light-range photosensitizers for sterilization of red blood cells is currently being aggressively investigated in laboratory-scale optical-mechanical systems. Both the photochemical sterilization process and the optical-mechanical system must operate without introducing significant alteration in the properties of the red cells. With successful demonstration of the efficacy and safety of these sterilization techniques, implementation in the blood bank setting will require scale-up to optical-mechanical systems capable of handling approximately 50,000 units daily in 500 - 1,000 blood banks in the United States.

  14. Antiviral activity of carbohydrate-binding agents against Nidovirales in cell culture.

    PubMed

    van der Meer, F J U M; de Haan, C A M; Schuurman, N M P; Haijema, B J; Peumans, W J; Van Damme, E J M; Delputte, P L; Balzarini, J; Egberink, H F

    2007-10-01

    Coronaviruses are important human and animal pathogens, the relevance of which increased due to the emergence of new human coronaviruses like SARS-CoV, HKU1 and NL63. Together with toroviruses, arteriviruses, and roniviruses the coronaviruses belong to the order Nidovirales. So far antivirals are hardly available to combat infections with viruses of this order. Therefore, various antiviral strategies to counter nidoviral infections are under evaluation. Lectins, which bind to N-linked oligosaccharide elements of enveloped viruses, can be considered as a conceptionally new class of virus inhibitors. These agents were recently evaluated for their antiviral activity towards a variety of enveloped viruses and were shown in most cases to inhibit virus infection at low concentrations. However, limited knowledge is available for their efficacy towards nidoviruses. In this article the application of the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Cymbidium sp. agglutinin (CA) and Urtica dioica agglutinin (UDA) as well as non-plant derived pradimicin-A (PRM-A) and cyanovirin-N (CV-N) as potential antiviral agents was evaluated. Three antiviral tests were compared based on different evaluation principles: cell viability (MTT-based colorimetric assay), number of infected cells (immunoperoxidase assay) and amount of viral protein expression (luciferase-based assay). The presence of carbohydrate-binding agents strongly inhibited coronaviruses (transmissible gastroenteritis virus, infectious bronchitis virus, feline coronaviruses serotypes I and II, mouse hepatitis virus), arteriviruses (equine arteritis virus and porcine respiratory and reproductive syndrome virus) and torovirus (equine Berne virus). Remarkably, serotype II feline coronaviruses and arteriviruses were not inhibited by PRM-A, in contrast to the other viruses tested.

  15. Cetuximab enhanced the efficacy of chemotherapeutic agent in ABCB1/P-glycoprotein-overexpressing cancer cells.

    PubMed

    Wang, Fang; Chen, Yifan; Huang, Lihua; Liu, Tao; Huang, Yue; Zhao, Jianming; Wang, Xiaokun; Yang, Ke; Ma, Shaolin; Huang, Liyan; To, Kenneth Kin Wah; Gu, Yong; Fu, Liwu

    2015-12-01

    The overexpression of ATP-binding cassette (ABC) transporters is closely associated with the development of multidrug resistance (MDR) in certain types of cancer, which represents a formidable obstacle to the successful cancer chemotherapy. Here, we investigated that cetuximab, an EGFR monoclonal antibody, reversed the chemoresistance mediated by ABCB1, ABCG2 or ABCC1. Our results showed that cetuximab significantly enhanced the cytotoxicity of ABCB1 substrate agent in ABCB1-overexpressing MDR cells but had no effect in their parental drug sensitive cells and ABCC1, ABCG2 overexpressing cells. Furthermore, cetuximab markedly increased intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABCB1-overexpressing MDR cancer cells in a concentration-dependent manner. Cetuximab stimulated the ATPase activity but did not alter the expression level of ABCB1 or block phosphorylation of AKT and ERK. Interestingly, cetuximab decreased the cell membrane fluidity which was known to decrease the function of ABCB1. Our findings advocate further clinical investigation of combination chemotherapy of cetuximab and conventional chemotherapeutic drugs in ABCB1 overexpressing cancer patients.

  16. Screening system of blocking agents of the receptor for advanced glycation endproducts in cells using fluorescence.

    PubMed

    Jung, Dong Ho; Kim, Young Sook; Kim, Jin Sook

    2012-01-01

    Activation of the receptor for advanced glycation endproducts (RAGE) triggers cellular responses implicated in the pathogenesis of diabetic complications; blockade of RAGE has been shown to inhibit the development of diabetic complications. To develop a screening system to identify novel disruptors of advanced glycation endproducts (AGE)-RAGE binding, we used an AGE-RAGE binding system in RAGE-overexpressing cells; test compounds were screened using this system. To construct human RAGE-overexpressing cells, mouse mesangial cells (MMCs) were stably transfected with the pcDNA-human RAGE (hRAGE) vector and selected under 1 mg/mL gentamicin (G418). RAGE expression in hRAGE-overexpressing MMCs was analyzed by Western blotting with specific RAGE antibody. To identify novel disruptors of AGE-RAGE binding, 50 single compounds and AGE-bovine serum albumin (BSA)-Alexa 488 (AGE-BSA labeled with Alexa 488) were treated to the hRAGE-overexpressing MMCs. Nonbinding AGE-BSA-Alexa 488 was washed and fluorescence measured by microtiter plate reader (excitation wavelength, 485 nm; emission wavelength, 528 nm). In hRAGE-overexpressing cells, only treatment with AGE-BSA-Alexa 488 significantly increased fluorescence intensity in a dose-dependent manner. Of 50 compounds tested, genistein disrupted AGE-RAGE binding in a dose-dependent manner. This AGE-RAGE binding system using AGE-BSA-Alexa 488 in hRAGE-overexpressing cells was suitable for screening of agents that disrupt AGE-hRAGE binding.

  17. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells.

    PubMed

    Bauer, Matthias R; Joerger, Andreas C; Fersht, Alan R

    2016-09-01

    The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53's oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1(MET)(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells.

  18. Cetuximab enhanced the efficacy of chemotherapeutic agent in ABCB1/P-glycoprotein-overexpressing cancer cells

    PubMed Central

    Liu, Tao; Huang, Yue; Zhao, Jianming; Wang, Xiaokun; Yang, Ke; Ma, Shaolin; Huang, Liyan; Wah To, Kenneth Kin; Gu, Yong; Fu, Liwu

    2015-01-01

    The overexpression of ATP-binding cassette (ABC) transporters is closely associated with the development of multidrug resistance (MDR) in certain types of cancer, which represents a formidable obstacle to the successful cancer chemotherapy. Here, we investigated that cetuximab, an EGFR monoclonal antibody, reversed the chemoresistance mediated by ABCB1, ABCG2 or ABCC1. Our results showed that cetuximab significantly enhanced the cytotoxicity of ABCB1 substrate agent in ABCB1-overexpressing MDR cells but had no effect in their parental drug sensitive cells and ABCC1, ABCG2 overexpressing cells. Furthermore, cetuximab markedly increased intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABCB1-overexpressing MDR cancer cells in a concentration-dependent manner. Cetuximab stimulated the ATPase activity but did not alter the expression level of ABCB1 or block phosphorylation of AKT and ERK. Interestingly, cetuximab decreased the cell membrane fluidity which was known to decrease the function of ABCB1. Our findings advocate further clinical investigation of combination chemotherapy of cetuximab and conventional chemotherapeutic drugs in ABCB1 overexpressing cancer patients. PMID:26506420

  19. Screening system of blocking agents of the receptor for advanced glycation endproducts in cells using fluorescence.

    PubMed

    Jung, Dong Ho; Kim, Young Sook; Kim, Jin Sook

    2012-01-01

    Activation of the receptor for advanced glycation endproducts (RAGE) triggers cellular responses implicated in the pathogenesis of diabetic complications; blockade of RAGE has been shown to inhibit the development of diabetic complications. To develop a screening system to identify novel disruptors of advanced glycation endproducts (AGE)-RAGE binding, we used an AGE-RAGE binding system in RAGE-overexpressing cells; test compounds were screened using this system. To construct human RAGE-overexpressing cells, mouse mesangial cells (MMCs) were stably transfected with the pcDNA-human RAGE (hRAGE) vector and selected under 1 mg/mL gentamicin (G418). RAGE expression in hRAGE-overexpressing MMCs was analyzed by Western blotting with specific RAGE antibody. To identify novel disruptors of AGE-RAGE binding, 50 single compounds and AGE-bovine serum albumin (BSA)-Alexa 488 (AGE-BSA labeled with Alexa 488) were treated to the hRAGE-overexpressing MMCs. Nonbinding AGE-BSA-Alexa 488 was washed and fluorescence measured by microtiter plate reader (excitation wavelength, 485 nm; emission wavelength, 528 nm). In hRAGE-overexpressing cells, only treatment with AGE-BSA-Alexa 488 significantly increased fluorescence intensity in a dose-dependent manner. Of 50 compounds tested, genistein disrupted AGE-RAGE binding in a dose-dependent manner. This AGE-RAGE binding system using AGE-BSA-Alexa 488 in hRAGE-overexpressing cells was suitable for screening of agents that disrupt AGE-hRAGE binding. PMID:23037172

  20. Rapid screening of potential autophagic inductor agents using mammalian cell lines.

    PubMed

    Martins, Waleska K; Severino, Divinomar; Souza, Cleidiane; Stolf, Beatriz S; Baptista, Maurício S

    2013-06-01

    Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.

  1. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells.

    PubMed

    Bauer, Matthias R; Joerger, Andreas C; Fersht, Alan R

    2016-09-01

    The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53's oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1(MET)(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells. PMID:27551077

  2. Recent progress in fungus-derived bioactive agents for targeting of signaling machinery in cancer cells

    PubMed Central

    Lin, Xiukun; Farooqi, Ammad Ahmad; Ismail, Muhammad

    2015-01-01

    It is becoming increasingly understood that tumor cells may have different mutations and dependencies on diverse intracellular signaling cascades for survival or metastatic potential. Overexpression of oncogenes, inactivation of tumor suppressor genes, genetic/epigenetic mutations, genomic instability, and loss of apoptotic cell death are some of the mechanisms that have been widely investigated in molecular oncology. We partition this multicomponent review into the most recent evidence on the anticancer activity of fungal substances obtained from in vitro and xenografted models, and these fungal substances modulate expression of oncogenic and tumor suppressor miRNAs. There are some outstanding questions regarding fungus-derived chemical-induced modulation of intracellular signaling networks in different cancer cell lines and preclinical models. Certain hints have emerged, emphasizing mechanisms via which apoptosis can be restored in TRAIL-resistant cancer cells. Reconceptualization of the knowledge obtained from these emerging areas of research will enable us to potentially identify natural agents with notable anticancer activity and minimal off-target effects. Integration of experimentally verified evidence obtained from cancer cell line gene expression with large-scale functional screening results and pharmacological sensitivity data will be helpful in identification of therapeutics with substantial efficacy. New tools and technologies will further deepen our understanding of the signaling networks that underlie the development of cancer, metastasis, and resistance to different therapeutics at both a personal and systems-wide level. PMID:25848216

  3. The methylating agent streptozotocin induces persistent telomere dysfunction in mammalian cells.

    PubMed

    Paviolo, Natalia S; Santiñaque, Federico F; Castrogiovanni, Daniel C; Folle, Gustavo A; Bolzán, Alejandro D

    2015-12-01

    We analyzed chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the methylating agent and antineoplastic/diabetogenic drug streptozotocin (STZ), to test whether it induces long-term telomere instability (by chromosome end loss and/or telomere dysfunction). Rat cells (ADIPO-P2 cell line, derived from Sprague-Dawley rat adipose cells) were treated with a single concentration of STZ (2mM). Chromosomal aberrations were analyzed 18h, 10 days, and 15 days after treatment, using PNA-FISH with a pan-telomeric probe [Cy3-(CCCTAA)3] to detect (TTAGGG)n repeats. Cytogenetic analysis revealed a higher frequency of chromosomal aberrations in STZ-exposed cultures vs. untreated cultures at each time point analyzed. The yield of induced aberrations was very similar at each time point. Induction of aberrations not involving telomere dysfunction was only observed 18h and 15 days after treatment, whereas induction of telomere dysfunction-related aberrations by STZ (mainly in the form of telomere FISH signal loss and duplications, most of them chromatid-type aberrations) was observed at each time point. Our results show that STZ induces persistent telomere instability in mammalian cells, cytogenetically manifested as telomere dysfunction-related chromosomal aberrations. Neither telomere length nor telomerase activity is related to the telomere dysfunction.

  4. Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting

    PubMed Central

    Zilbermintz, Leeor; Leonardi, William; Jeong, Sun-Young; Sjodt, Megan; McComb, Ryan; Ho, Chi-Lee C.; Retterer, Cary; Gharaibeh, Dima; Zamani, Rouzbeh; Soloveva, Veronica; Bavari, Sina; Levitin, Anastasia; West, Joel; Bradley, Kenneth A.; Clubb, Robert T.; Cohen, Stanley N.; Gupta, Vivek; Martchenko, Mikhail

    2015-01-01

    A longstanding and still-increasing threat to the effective treatment of infectious diseases is resistance to antimicrobial countermeasures. Potentially, the targeting of host proteins and pathways essential for the detrimental effects of pathogens offers an approach that may discover broad-spectrum anti-pathogen countermeasures and circumvent the effects of pathogen mutations leading to resistance. Here we report implementation of a strategy for discovering broad-spectrum host-oriented therapies against multiple pathogenic agents by multiplex screening of drugs for protection against the detrimental effects of multiple pathogens, identification of host cell pathways inhibited by the drug, and screening for effects of the agent on other pathogens exploiting the same pathway. We show that a clinically used antimalarial drug, Amodiaquine, discovered by this strategy, protects host cells against infection by multiple toxins and viruses by inhibiting host cathepsin B. Our results reveal the practicality of discovering broadly acting anti-pathogen countermeasures that target host proteins exploited by pathogens. PMID:26310922

  5. Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells

    PubMed Central

    Sharma, Bijaya; Brown, Autumn V.; Matluck, Nicole E.; Hu, Linden T.

    2015-01-01

    Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the United States. When treated early, the disease usually resolves, but when left untreated, it can result in symptoms such as arthritis and encephalopathy. Treatment of the late-stage disease may require multiple courses of antibiotic therapy. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late-stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In this study, we examined the ability of B. burgdorferi to form persisters. Killing growing cultures of B. burgdorferi with antibiotics used to treat the disease was distinctly biphasic, with a small subpopulation of surviving cells. Upon regrowth, these cells formed a new subpopulation of antibiotic-tolerant cells, indicating that these are persisters rather than resistant mutants. The level of persisters increased sharply as the culture transitioned from the exponential to stationary phase. Combinations of antibiotics did not improve killing. Daptomycin, a membrane-active bactericidal antibiotic, killed stationary-phase cells but not persisters. Mitomycin C, an anticancer agent that forms adducts with DNA, killed persisters and eradicated growing and stationary cultures of B. burgdorferi. Finally, we examined the ability of pulse dosing an antibiotic to eliminate persisters. After addition of ceftriaxone, the antibiotic was washed away, surviving persisters were allowed to resuscitate, and the antibiotic was added again. Four pulse doses of ceftriaxone killed persisters, eradicating all live bacteria in the culture. PMID:26014929

  6. Profiling of Altered Metabolomic States in Nicotiana tabacum Cells Induced by Priming Agents

    PubMed Central

    Mhlongo, Msizi I.; Steenkamp, Paul A.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Dubery, Ian A.

    2016-01-01

    Metabolomics has developed into a valuable tool for advancing our understanding of plant metabolism. Plant innate immune defenses can be activated and enhanced so that, subsequent to being pre-sensitized, plants are able to launch a stronger and faster defense response upon exposure to pathogenic microorganisms, a phenomenon known as priming. Here, three contrasting chemical activators, namely acibenzolar-S-methyl, azelaic acid and riboflavin, were used to induce a primed state in Nicotiana tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid chromatography and mass spectrometry. Multivariate data models indicated that these inducers cause time-dependent metabolic perturbations in the cultured cells and revealed biomarkers of which the levels are affected by these agents. A total of 34 metabolites were annotated from the mass spectral data and online databases. Venn diagrams were used to identify common biomarkers as well as those unique to a specific agent. Results implicate 20 cinnamic acid derivatives conjugated to (i) quinic acid (chlorogenic acids), (ii) tyramine, (iii) polyamines, or (iv) glucose as discriminatory biomarkers of priming in tobacco cells. Functional roles for most of these metabolites in plant defense responses could thus be proposed. Metabolites induced by the activators belong to the early phenylpropanoid pathway, which indicates that different stimuli can activate similar pathways but with different metabolite fingerprints. Possible linkages to phytohormone-dependent pathways at a metabolomic level were indicated in the case of cells treated with salicylic acid and methyljasmonate. The results contribute to a better understanding of the priming phenomenon and advance our knowledge of cinnamic acid derivatives as versatile defense metabolites. PMID

  7. Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells.

    PubMed

    Sharma, Bijaya; Brown, Autumn V; Matluck, Nicole E; Hu, Linden T; Lewis, Kim

    2015-08-01

    Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the United States. When treated early, the disease usually resolves, but when left untreated, it can result in symptoms such as arthritis and encephalopathy. Treatment of the late-stage disease may require multiple courses of antibiotic therapy. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late-stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In this study, we examined the ability of B. burgdorferi to form persisters. Killing growing cultures of B. burgdorferi with antibiotics used to treat the disease was distinctly biphasic, with a small subpopulation of surviving cells. Upon regrowth, these cells formed a new subpopulation of antibiotic-tolerant cells, indicating that these are persisters rather than resistant mutants. The level of persisters increased sharply as the culture transitioned from the exponential to stationary phase. Combinations of antibiotics did not improve killing. Daptomycin, a membrane-active bactericidal antibiotic, killed stationary-phase cells but not persisters. Mitomycin C, an anticancer agent that forms adducts with DNA, killed persisters and eradicated growing and stationary cultures of B. burgdorferi. Finally, we examined the ability of pulse dosing an antibiotic to eliminate persisters. After addition of ceftriaxone, the antibiotic was washed away, surviving persisters were allowed to resuscitate, and the antibiotic was added again. Four pulse doses of ceftriaxone killed persisters, eradicating all live bacteria in the culture. PMID:26014929

  8. Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells.

    PubMed

    Sharma, Bijaya; Brown, Autumn V; Matluck, Nicole E; Hu, Linden T; Lewis, Kim

    2015-08-01

    Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the United States. When treated early, the disease usually resolves, but when left untreated, it can result in symptoms such as arthritis and encephalopathy. Treatment of the late-stage disease may require multiple courses of antibiotic therapy. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late-stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In this study, we examined the ability of B. burgdorferi to form persisters. Killing growing cultures of B. burgdorferi with antibiotics used to treat the disease was distinctly biphasic, with a small subpopulation of surviving cells. Upon regrowth, these cells formed a new subpopulation of antibiotic-tolerant cells, indicating that these are persisters rather than resistant mutants. The level of persisters increased sharply as the culture transitioned from the exponential to stationary phase. Combinations of antibiotics did not improve killing. Daptomycin, a membrane-active bactericidal antibiotic, killed stationary-phase cells but not persisters. Mitomycin C, an anticancer agent that forms adducts with DNA, killed persisters and eradicated growing and stationary cultures of B. burgdorferi. Finally, we examined the ability of pulse dosing an antibiotic to eliminate persisters. After addition of ceftriaxone, the antibiotic was washed away, surviving persisters were allowed to resuscitate, and the antibiotic was added again. Four pulse doses of ceftriaxone killed persisters, eradicating all live bacteria in the culture.

  9. Ability of fourteen chemical agents used in dental practice to induce chromosome aberrations in Syrian hamster embryo cells.

    PubMed

    Hikiba, Hirohito; Watanabe, Eiko; Barrett, J Carl; Tsutsui, Takeki

    2005-01-01

    To assess the genotoxicity of 14 chemical agents used in dental practice, the ability of these agents to induce chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of chromosome aberrations were induced in SHE cells treated with 7 of 10 chemical agents used as endodontic medicaments, that is, carbol camphor, m-cresol, eugenol, guaiacol, zinc oxide, hydrogen peroxide, and formaldehyde. The other 3 chemical agents, that is, thymol, glutaraldehyde, and iodoform, did not increase the levels of chromosome aberrations. Of the 4 chemical agents that are used as an antiseptic on the oral mucosa, chromosome aberrations were induced by iodine, but not by the other 3 antiseptics, benzalkonium chloride, benzethonium chloride, and chlorhexidine. Among the 6 chemical agents exhibiting a negative response in the assay, only thymol induced chromosome aberrations in the presence of exogenous metabolic activation. Our results indicate that chemical agents having a positive response in the present study are potentially genotoxic to mammalian cells and need to be studied further in detail. PMID:15665446

  10. Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure

    PubMed Central

    2016-01-01

    Immunomodulatory drugs—agents regulating the immune response—are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 μL), short assay time (∼30 min), heightened sensitivity (∼20–30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment. PMID:27478873

  11. Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

    PubMed Central

    Gottlieb, R A; Nordberg, J; Skowronski, E; Babior, B M

    1996-01-01

    We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction. Images Fig. 5 PMID:8570610

  12. Silibinin derivatives as anti-prostate cancer agents: Synthesis and cell-based evaluations.

    PubMed

    Vue, Bao; Zhang, Sheng; Zhang, Xiaojie; Parisis, Konstantinos; Zhang, Qiang; Zheng, Shilong; Wang, Guangdi; Chen, Qiao-Hong

    2016-02-15

    This study aims to systematically explore the alkylation effect of 7-OH in silibinin and 2,3-dehydrosilibinin on the antiproliferative potency toward three prostate cancer cell lines. Eight 7-O-alkylsilibinins, eight 7-O-alkyl-2,3-dehydrosilibinins, and eight 3,7-O-dialkyl-2,3-dehydrosilibinins have been synthesized from commercially available silibinin for the in vitro cell-based evaluation. The WST-1 cell proliferation assay indicates that nineteen out of twenty-four silibinin derivatives have significantly improved antiproliferative potency when compared with silibinin. 7-O-Methylsilibinin (2) and 7-O-ethylsilibinin (3) have been identified as the most potent compounds with 98- and 123-fold enhanced potency against LNCaP human androgen-dependent prostate cancer cell line. Among 2,3-dehydrosilibinin derivatives, 7-O-methyl-2,3-dehydrosilibinin (10) and 7-O-ethyl-2,3-dehydrosilibinin (11) have been identified as the optimal compounds with the highest potency towards both androgen-dependent LNCaP and androgen-independent PC-3 prostate cancer cell lines. 7-O-Ethyl-2,3-dehydrosilibinin (11) was demonstrated to arrest PC-3 cell cycle at the G0/G1 phase and to induce PC-3 cell apoptosis. The findings in this study suggest that antiproliferative potency of silibinin and 2,3-dehydrosilibinin can be appreciably enhanced through suitable chemical modifications on the phenolic hydroxyl group at C-7 and that introduction of a chemical moiety with the potential to improve bioavailability through a linker to 7-OH in silibinin and 2,3-dehydrosilibinin would be a feasible strategy for the development of silibinin derivatives as anti-prostate cancer agents.

  13. Bone marrow mesenchymal stromal cells affect the cell cycle arrest effect of genotoxic agents on acute lymphocytic leukemia cells via p21 down-regulation.

    PubMed

    Zhang, Yiran; Hu, Kaimin; Hu, Yongxian; Liu, Lizhen; Wang, Binsheng; Huang, He

    2014-09-01

    The effect of bone marrow microenvironment on the cell cycle of acute lymphocytic leukemia (ALL) and the underlying mechanism has not been elucidated. In this study, we found that in normal condition, bone marrow mesenchymal stromal cells (BM-MSCs) had no significant effect on the cell cycle and apoptosis of ALL; in the condition when the cell cycle of ALL was blocked by genotoxic agents, BM-MSCs could increase the S-phase cell ratio and decrease the G2/M phase ratio of ALL. Besides, BM-MSCs could protect ALL cells from drug-induced apoptosis. Then, we proved that BM-MSCs affect the cell cycle arrest effect of genotoxic agents on ALL cells via p21 down-regulation. Moreover, our results indicated that activation of Wnt/β-catenin and Erk pathways might be involved in the BM-MSC-induced down-regulation of p21 in ALL cells. Targeting microenvironment-related signaling pathway may therefore be a potential novel approach for ALL therapy.

  14. Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells.

    PubMed

    Gerges, Steve; Rohde, Katharina; Fulda, Simone

    2016-05-28

    Treatment resistance in acute lymphoblastic leukemia (ALL) is often caused by defects in programmed cell death, e.g. by overexpression of Inhibitor of Apoptosis (IAP) proteins. Here, we report that small-molecule Smac mimetics (i.e. BV6, LCL161, birinapant) that neutralize x-linked IAP (XIAP), cellular IAP (cIAP)1 and cIAP2 cooperate with demethylating agents (i.e. 5-azacytidine (5AC) or 5-aza-2'-deoxycytidine (DAC)) to induce cell death in ALL cells. Molecular studies reveal that induction of cell death is preceded by BV6-mediated depletion of cIAP1 protein and involves tumor necrosis factor (TNF)α autocrine/paracrine signaling, since the TNFα-blocking antibody Enbrel significantly reduces BV6/5AC-induced cell death. While BV6/5AC cotreatment induces caspase-3 activation, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) only partly rescues ALL cells from BV6/5AC-induced cell death. This indicates that BV6/5AC cotreatment engages non-apoptotic cell death upon caspase inhibition. Indeed, genetic silencing of key components of necroptosis such as Receptor-Interacting Protein (RIP)3 or mixed lineage kinase domain-like (MLKL) in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. necrostatin-1s, GSK'872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. These findings demonstrate that in ALL cells BV6/5AC-induced cell death is mediated via both apoptotic and necroptotic pathways. Importantly, BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition. This opens new perspectives to overcome apoptosis resistance with important implications for the development of new treatment strategies

  15. Hairy root biotechnology--indicative timeline to understand missing links and future outlook.

    PubMed

    Mehrotra, Shakti; Srivastava, Vikas; Ur Rahman, Laiq; Kukreja, A K

    2015-09-01

    Agrobacterium rhizogenes-mediated hairy roots (HR) were developed in the laboratory to mimic the natural phenomenon of bacterial gene transfer and occurrence of disease syndrome. The timeline analysis revealed that during 90 s, the research expanded to the hairy root-based secondary metabolite production and different yield enhancement strategies like media optimization, up-scaling, metabolic engineering etc. An outlook indicates that much emphasis has been given to the strategies that are helpful in making this technology more practical in terms of high productivity at low cost. However, a sequential analysis of literature shows that this technique is upgraded to a biotechnology platform where different intra- and interdisciplinary work areas were established, progressed, and diverged to provide scientific benefits of various hairy root-based applications like phytoremediation, molecular farming, biotransformation, etc. In the present scenario, this biotechnology research platform includes (a) elemental research like hairy root-mediated secondary metabolite production coupled with productivity enhancement strategies and (b) HR-based functional research. The latter comprised of hairy root-based applied aspects such as generation of agro-economical traits in plants, production of high value as well as less hazardous molecules through biotransformation/farming and remediation, respectively. This review presents an indicative timeline portrayal of hairy root research reflected by a chronology of research outputs. The timeline also reveals a progressive trend in the state-of-art global advances in hairy root biotechnology. Furthermore, the review also discusses ideas to explore missing links and to deal with the challenges in future progression and prospects of research in all related fields of this important area of plant biotechnology.

  16. A cell-based screening system for anti-influenza A virus agents

    PubMed Central

    Wong, Wan Ying; Loh, Sheng Wei; Ng, Wei Lun; Tan, Ming Cheang; Yeo, Kok Siong; Looi, Chung Yeng; Maah, Mohd Jamil; Ea, Chee-Kwee

    2015-01-01

    Emerging of drug resistant influenza A virus (IAV) has been a big challenge for anti-IAV therapy. In this study, we describe a relatively easy and safe cell-based screening system for anti-IAV replication inhibitors using a non-replicative strain of IAV. A nickel (II) complex of polyhydroxybenzaldehyde N4-thiosemicarbazone (NiPT5) was recently found to exhibit anti-inflammatory activity in vivo and in vitro. NiPT5 impedes the signaling cascades that lead to the activation of NF-κB in response to different stimuli, such as LPS and TNFα. Using our cell-based screening system, we report that pretreating cells with NiPT5 protects cells from influenza A virus (IAV) and vesicular stomatitis virus (VSV) infection. Furthermore, NiPT5 inhibits replication of IAV by inhibiting transcription and translation of vRNAs of IAV. Additionally, NiPT5 reduces IAV-induced type I interferon response and cytokines production. Moreover, NiPT5 prevents activation of NF-κB, and IRF3 in response to IAV infection. These results demonstrate that NiPT5 is a potent antiviral agent that inhibits the early phase of IAV replication. PMID:25728279

  17. Hypersensitivities for acetaldehyde and other agents among cancer cells null for clinically relevant Fanconi anemia genes.

    PubMed

    Ghosh, Soma; Sur, Surojit; Yerram, Sashidhar R; Rago, Carlo; Bhunia, Anil K; Hossain, M Zulfiquer; Paun, Bogdan C; Ren, Yunzhao R; Iacobuzio-Donahue, Christine A; Azad, Nilofer A; Kern, Scott E

    2014-01-01

    Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities.

  18. Inhibition of cell-to-cell transmission of human T-cell lymphotropic virus type 1 in vitro by carbohydrate-binding agents.

    PubMed

    Balestrieri, Emanuela; Ascolani, Arianna; Igarashi, Yasuhiro; Oki, Toshikazu; Mastino, Antonio; Balzarini, Jan; Macchi, Beatrice

    2008-08-01

    Peripheral blood mononuclear cells (PBMCs) from healthy individuals can be infected by human T-lymphotropic virus type 1 (HTLV-1) upon cocultivation of the PBMCs with irradiated HTLV-1-transformed human MT-2 cells. This model system closely mimics HTLV-1 transmission through cell-to-cell contact. Carbohydrate-binding agents (CBAs) such as the alpha(1,3)/alpha(1,6)mannose-specific Hippeastrum hybrid agglutinin and the GlcNAc-specific Urtica dioica agglutinin, and also the small, nonpeptidic alpha(1,2)-mannose-specific antibiotic pradimicin A, were able to efficiently prevent cell-to-cell HTLV-1 transmission at nontoxic concentrations, as evidenced by the lack of appearance of virus-specific mRNA and of the viral protein Tax in the acceptor cells. Consistently, antivirally active doses of CBAs fully prevented HTLV-1-induced stimulation of PBMC growth. The inhibitory effects of CBAs on HTLV-1 transmission were also evident when HTLV-1-infected C5MJ cells were used in place of MT-2 cells as a virus donor cell line. The anti-HTLV-1 properties of the CBAs highlight the importance of the envelope glycans in events underlying HTLV-1 passage from cell to cell and indicate that CBAs should be further investigated for their potential to prevent HTLV-1 infection, including mother-to-child virus transmission by cell-to-cell contact through breast milk feeding. PMID:18505856

  19. Assessing the Efficacy of Nano- and Micro-Sized Magnetic Particles as Contrast Agents for MRI Cell Tracking

    PubMed Central

    Taylor, Arthur; Herrmann, Anne; Moss, Diana; Sée, Violaine; Davies, Karen; Williams, Steve R.; Murray, Patricia

    2014-01-01

    Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of labelled cells in vivo. Currently, a wide range of such contrast agents is available with sizes varying from several nanometers up to a few micrometers and consisting of single or multiple magnetic cores. Here, we evaluate the effectiveness of these different particles for labelling and imaging stem cells, using a mouse mesenchymal stem cell line to investigate intracellular uptake, retention and processing of nano- and microsized contrast agents. The effect of intracellular confinement on transverse relaxivity was measured by MRI at 7 T and in compliance with the principles of the ‘3Rs’, the suitability of the contrast agents for MR-based cell tracking in vivo was tested using a chick embryo model. We show that for all particles tested, relaxivity was markedly reduced following cellular internalisation, indicating that contrast agent relaxivity in colloidal suspension does not accurately predict performance in MR-based cell tracking studies. Using a bimodal imaging approach comprising fluorescence and MRI, we demonstrate that labelled MSC remain viable following in vivo transplantation and can be tracked effectively using MRI. Importantly, our data suggest that larger particles might confer advantages for longer-term imaging. PMID:24959883

  20. Dissecting the regulation of fructan metabolism in chicory (Cichorium intybus) hairy roots.

    PubMed

    Kusch, Ute; Greiner, Steffen; Steininger, Heike; Meyer, Alain Denis; Corbière-Divialle, Hélène; Harms, Karsten; Rausch, Thomas

    2009-01-01

    Fifteen per cent of higher plants accumulate fructans. Plant development, nutritional status and stress exposure all affect fructan metabolism, and while fructan biochemistry is well understood, knowledge of its regulation has remained fragmentary. Here, we have explored chicory (Cichorium intybus) hairy root cultures (HRCs) to study the regulation of fructan metabolism in sink tissues in response to environmental cues. In standard medium (SM), HRCs did not accumulate inulin. However, upon transfer to high-carbon (C)/low-nitrogen (N) medium, expression of sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT) was strongly induced and inulin accumulated. Upon return to SM, inulin was degraded, together with a coordinate decline of 1-SST and 1-FFT expression. In HRCs, cold-induced expression of fructan 1-exohydrolases (1-FEH I and IIa) was similar to cold induction in taproots, even in the absence of accumulated inulin. For high-C/low-N induction of 1-SST and 1-FFT, and cold induction of 1-FEH I and IIa, the signaling pathways were addressed. While 1-SST and 1-FFT induction was similarly prevented by inhibitors of Ca(2+) signaling, protein kinases and phosphatases, cold induction of 1-FEH I and IIa revealed distinct signaling pathways. In summary, this study has established chicory HRCs as a convenient experimental system with which to study the regulation of fructan active enzyme (FAZY) expression in heterotrophic cells.

  1. Renal cell carcinoma: review of novel single-agent therapeutics and combination regimens.

    PubMed

    Amato, R J

    2005-01-01

    A search of the Medline database and ASCO 2003 conference proceedings was conducted to identify clinical trials currently underway using single-agent therapy for renal cell carcinoma (RCC). Combination trials were identified using the ASCO 2003 conference proceedings. Fourteen single-agent therapies employing different mechanisms of action were identified in the published literature: imatinib mesylate (Gleevec); bevacizumab (Avastin); thalidomide (Thalomid); gefitinib (ZD1839) (Iressa); cetuximab (IMC-C225) (Erbitux); bortezomib (PS-341) (Velcade); HSPPC-96 (Oncophage); BAY 59-8862; ABT-510; G250; CCI-779; SU5416; PTK/ZK; and ABX-EGF. Six distinct fields of clinical research have emerged: monoclonal antibodies, small molecules, vaccines, second-generation taxanes, nonapeptides and immunomodulators. Five combination regimens, primarily biological response modifiers (interleukin-2 or interferon-alpha), chemotherapy- or thalidomide-based, were identified. All therapies demonstrated acceptable toxicity profiles. Clinical benefit was assessed based on each study's reported criteria: antitumor response (regression or stability) ranged from 5% to 71%. In the past several years, significant advances in the underlying biological mechanisms of RCC, particularly the role of tumor angiogenesis, have permitted the design of molecularly targeted therapeutics. Based on preliminary and limited studies, combination therapies offer the greatest clinical benefit in the management of this malignancy, although additional basic research is still warranted.

  2. Identification of anti-invasive but noncytotoxic chemotherapeutic agents using the tetrazolium dye MTT to quantitate viable cells in Matrigel.

    PubMed

    Sasaki, C Y; Passaniti, A

    1998-06-01

    Screening methods for chemotherapeutic agents usually rely on the cytotoxic properties of the drugs. However, agents that inhibit invasion may have more efficacy and cause fewer side effects. Various cellular invasion assays have been used to evaluate these types of compounds, including the modified Boyden chamber, monolayer wound models and Matrigel outgrowth assays. In this report, we have combined the use of the Matrigel outgrowth assay with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) visualization and cell viability dye to visualize invasive cells on Matrigel without magnification. Extraction of the dye's formazan byproduct allows cell viability to be assessed. Using several invasive and noninvasive cell lines, the utility of the method for various target cells was verified. Several established chemotherapeutic agents were also screened for their anti-invasive and/or cytotoxic effects when cultured on Matrigel. Our results suggest that this method may be an easy, inexpensive and nonradioactive alternative for both enumerating cells on Matrigel and screening various tumor cell lines treated with chemotherapeutic agent to look for compounds with noncytotoxic but anti-invasive properties.

  3. Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines

    PubMed Central

    Al-Asmari, Abdulrahman Khazim; Albalawi, Sulaiman Mansour; Athar, Md Tanwir; Khan, Abdul Quaiyoom; Al-Shahrani, Hamoud; Islam, Mozaffarul

    2015-01-01

    In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70–90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2–3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of

  4. Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines.

    PubMed

    Al-Asmari, Abdulrahman Khazim; Albalawi, Sulaiman Mansour; Athar, Md Tanwir; Khan, Abdul Quaiyoom; Al-Shahrani, Hamoud; Islam, Mozaffarul

    2015-01-01

    In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70-90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2-3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast

  5. Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines.

    PubMed

    Al-Asmari, Abdulrahman Khazim; Albalawi, Sulaiman Mansour; Athar, Md Tanwir; Khan, Abdul Quaiyoom; Al-Shahrani, Hamoud; Islam, Mozaffarul

    2015-01-01

    In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70-90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2-3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast

  6. Further studies on the detection of chemical agents using an alkaline energy cell

    NASA Astrophysics Data System (ADS)

    Shewchun, John

    2008-04-01

    The detection, classification and tracking of chemical agents (explosives) being surreptitiously smuggled into public areas, such as airports, for destructive purposes is difficult to solve by unobtrusive means. We propose the use of a novel Alkaline Energy Cell (AEC) with gas/vapor sniffing capability as a potential solution. Variants of such devices are routinely used by police to detect alcohol emanating from the breath of suspected impaired vehicle drivers. We reported previously at the SPIE Symposium in 2007 the details of our technology and results. We have continued to advanced this capability with the development of an AEC which is capable of detecting gaseous emissions ultimately in the parts per billion range. Our work is described in terms of detecting TATP (acetone peroxide). Other explosive materials have also been investigated and will be reported on.

  7. Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells.

    PubMed

    Ma, Wenzhe; Feng, Senling; Yao, Xiaojun; Yuan, Zhongwen; Liu, Liang; Xie, Ying

    2015-12-22

    Multidrug resistance (MDR) is the major obstacle to the successful chemotherapy treatment of many cancers. Here we found that nobiletin, a citrus methoxyflavone, significantly sensitized ABCB1 overexpressing cells A2780/T and A549/T to chemotherapeutic agents such as paclitaxel (a 433-fold reversal of MDR to PTX at 9 μM), doxorubicin (DOX), docetaxel and dounorubicin. Nobiletin profoundly inhibited ABCB1 transporter activity since it significantly increased the intracellular accumulation of DOX and Flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the mRNA and protein expression of ABCB1. Moreover, nobiletin stimulated ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, molecular docking analysis also identified favorable binding of nobiletin with the transmemberane region site 1 of homology modeled human ABCB1 transporter. Moreover, the Nrf2 protein expression and phosphorylation levels of AKT/ERK were suppressed by co-treated with nobiletin and PTX at the reversal concentrations, suggesting that inhibition of the AKT/ERK/Nrf2 pathway was associated with the sensitizing effect of nobiletin. These findings encourage further animal and clinical MDR studies with the combination therapy of nobiletin and chemotherapeutic drugs.

  8. Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells

    PubMed Central

    Ma, Wenzhe; Feng, Senling; Yao, Xiaojun; Yuan, Zhongwen; Liu, Liang; Xie, Ying

    2015-01-01

    Multidrug resistance (MDR) is the major obstacle to the successful chemotherapy treatment of many cancers. Here we found that nobiletin, a citrus methoxyflavone, significantly sensitized ABCB1 overexpressing cells A2780/T and A549/T to chemotherapeutic agents such as paclitaxel (a 433-fold reversal of MDR to PTX at 9 μM), doxorubicin (DOX), docetaxel and dounorubicin. Nobiletin profoundly inhibited ABCB1 transporter activity since it significantly increased the intracellular accumulation of DOX and Flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the mRNA and protein expression of ABCB1. Moreover, nobiletin stimulated ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, molecular docking analysis also identified favorable binding of nobiletin with the transmemberane region site 1 of homology modeled human ABCB1 transporter. Moreover, the Nrf2 protein expression and phosphorylation levels of AKT/ERK were suppressed by co-treated with nobiletin and PTX at the reversal concentrations, suggesting that inhibition of the AKT/ERK/Nrf2 pathway was associated with the sensitizing effect of nobiletin. These findings encourage further animal and clinical MDR studies with the combination therapy of nobiletin and chemotherapeutic drugs. PMID:26689156

  9. Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents.

    PubMed

    Herles, S; Olsen, S; Afflitto, J; Gaffar, A

    1994-11-01

    We developed an experimental in vitro model of dental plaque to assess the potential efficacy of antiplaque agents. The model used a chemostat, which provided a continuous source of 5 species of oral bacteria grown in an artificial "saliva-like" medium. This mixture was pumped through six flow cells, each containing two types of surfaces on which plaque formed and was subsequently measured. Formation of bacterial plaque on hydroxyapatite surfaces was assessed by measurement of the DNA and protein content of the plaque film. The amount of bacterial plaque formed on germanium surfaces was measured by attenuated total reflectance (ATR/FT-IR) spectroscopy. Plaque viability was also assessed by a fluorescent staining technique. The quantity of plaque formed on both types of surfaces gradually increased with the duration of flow (from 24 to 72 h) through the cells during a 72-hour experimental period. The flow cells were then pulsed with experimental treatment solutions for 30 s, twice daily. Parallel to results of human clinical studies, the model was capable of discriminating among water, a placebo mouthrinse, and an active antimicrobial mouthrinse formulation containing 0.03% triclosan. It therefore offers a valuable alternative to animal model testing and allows for more rapid evaluations under well-controlled experimental conditions. PMID:7983262

  10. Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents.

    PubMed

    Herles, S; Olsen, S; Afflitto, J; Gaffar, A

    1994-11-01

    We developed an experimental in vitro model of dental plaque to assess the potential efficacy of antiplaque agents. The model used a chemostat, which provided a continuous source of 5 species of oral bacteria grown in an artificial "saliva-like" medium. This mixture was pumped through six flow cells, each containing two types of surfaces on which plaque formed and was subsequently measured. Formation of bacterial plaque on hydroxyapatite surfaces was assessed by measurement of the DNA and protein content of the plaque film. The amount of bacterial plaque formed on germanium surfaces was measured by attenuated total reflectance (ATR/FT-IR) spectroscopy. Plaque viability was also assessed by a fluorescent staining technique. The quantity of plaque formed on both types of surfaces gradually increased with the duration of flow (from 24 to 72 h) through the cells during a 72-hour experimental period. The flow cells were then pulsed with experimental treatment solutions for 30 s, twice daily. Parallel to results of human clinical studies, the model was capable of discriminating among water, a placebo mouthrinse, and an active antimicrobial mouthrinse formulation containing 0.03% triclosan. It therefore offers a valuable alternative to animal model testing and allows for more rapid evaluations under well-controlled experimental conditions.

  11. Glucosinolate biosynthesis in hairy root cultures of broccoli (Brassica oleracea var. italica).

    PubMed

    Kim, Sun-Ju; Park, Woo Tae; Uddin, Md Romij; Kim, Yeon Bok; Nam, Sang-Yong; Jho, Kwang Hyun; Park, Sang Un

    2013-02-01

    Here we present previously unreported glucosinolate production by hairy root cultures of broccoli (B. oleracea var. italica). Growth media greatly influenced the growth and glucosinolate content of hairy root cultures of broccoli. Seven glucosinolates, glucoraphanin, gluconapin, glucoerucin, glucobrassicin, 4-methoxyglucobrassicin, gluconasturtiin, and neoglucobrassicin, were identified by analysis of the broccoli hairy root cultures. Both half and full strength B5 and SH media enabled the highest accumulation of glucosinolates. In most cases, the levels of glucosinolates were higher in SH and BS media. Among the 7 glucosinolates, the accumulation of neoglucobrassicin was very high, irrespective of growth medium. The neoglucobrassicin content was 7.4-fold higher in SH medium than 1/2 MS, in which its level was the lowest. The 1/2 B5 medium supported the production of the highest amounts of glucobrassicin and 4-methoxyglucobrassicin, the levels for which were 36.2- and 7.9- fold higher, respectively, than their lowest content in 1/2 MS medium. The 1/2 SH medium enabled the highest accumulation of glucoraphanin and gluconapin in the broccoli hairy root cultures, whose levels were 1.8- and 4.6-fold higher, respectively, than their lowest content in 1/2 MS medium. Our results suggest that hairy root cultures of broccoli could be a valuable alternative approach for the production of glucosinolate compounds.

  12. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC.

    PubMed

    Brijwal, Latika; Tamta, Sushma

    2015-01-01

    An efficient protocol for hairy root induction in Berberis aristata DC. was established using two different strains of Agrobacterium rhizogenes, MTCC 532 and 2364 from IMTECH (Institute of Microbial Technology), Chandigarh, India. The strain 532 was more effective than strain 2364 in hairy root induction and in vitro grown callus (61.11 ± 1.60 % transformation frequency) was found to be suitable explant in comparison to leaves (42.59 ± 0.92 % transformation frequency) and nodal segments (34.25 ± 0.92 % transformation frequency) of in vitro grown microshoots for hairy root induction. The presence of rol A and rol B genes during amplification confirmed the transgenic nature of hairy roots and transformed callus. Transformation frequency of callus was further enhanced (from 61.11 ± 1.60 % to 72.22 ± 1.60 %; when infection time was 1 h) by using acetosyringone (100 µM) during co-cultivation period (48 h) on semisolid MS (Murashige and Skoog) medium. In conclusion, this study describes the protocol for hairy root induction which could further be useful for the production of berberin and may reduce the overharvesting of this endangered species from its natural habitat.

  13. Lignan enhancement in hairy root cultures of Linum album using coniferaldehyde and methylenedioxycinnamic acid.

    PubMed

    Ahmadian Chashmi, Najmeh; Sharifi, Mohsen; Behmanesh, Mehrdad

    2016-07-01

    Feeding experiments with hairy root cultures of Linum album have established that the extracellular coniferaldehyde is a good precursor for production of two lignans: lariciresinol (LARI) and pinoresinol (PINO). The accumulation of the LARI, PINO, and podophyllotoxin (PTOX) in hairy roots were enhanced about 14.8-, 8.7-, and 1.5-fold (107.61, 8.7 and 6.42 µg g(-1) Fresh Wight), respectively, by the addition of coniferaldehyde (2 mM) to the culture media (after 24 hr). This result was correlated with an increase pinoresinol/lariciresinol reductase (PLR) expression gene and cinnamyl alcohol dehydrogenase (CAD) activity in the fed hairy roots. Adding 3,4-(methylendioxy)cinnamic acid (MDCA) precursor did not influence on the lignans accumulation, but the lignin content of the hairy roots was increased. Moreover, the expression genes of phenylalanine ammonialyase (PAL), CAD, and cinnamoyl-CoA reductase (CCR) were influenced after feeding hairy roots with MDCA.

  14. [Role of NO signal in ABA-induced phenolic acids accumulation in Salvia miltiorrhiza hairy roots].

    PubMed

    Shen, Lihong; Ren, Jiahui; Jin, Wenfang; Wang, Ruijie; Ni, Chunhong; Tong, Mengjiao; Liang, Zongsuo; Yang, Dongfeng

    2016-02-01

    To investigate roles of nitric oxide (NO) signal in accumulations of phenolic acids in abscisic.acid (ABA)-induced Salvia miltiorrhiza hairy roots, S. miltiorrhiza hairy roots were treated with different concentrations of sodium nitroprusside (SNP)-an exogenous NO donor, for 6 days, and contents of phenolic acids in the hairy roots are determined. Then with treatment of ABA and NO scavenger (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide, c-PTIO) or NO synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME), contents of phenolic acids and expression levels of three key genes involved in phenolic acids biosynthesis were detected. Phenolic acids production in S. miltiorrhiza hairy roots was most significantly improved by 100 µmoL/L SNP. Contents of RA and salvianolic acid B increased by 3 and 4 folds. ABA significantly improved transcript levels of PAL (phenylalanine ammonia lyase), TAT (tyrosine aminotransferase) and RAS (rosmarinic acid synthase), and increased phenolic acids accumulations. However, with treatments of ABA+c-PTIO or ABA+L-NAME, accumulations of phenolic acids and expression levels of the three key genes were significantly inhibited. Both NO and ABA can increase accumulations of phenolic acids in S. miltiorrhiza hairy roots. NO signal probably mediates the ABA-induced phenolic acids production. PMID:27382772

  15. Effects of epicatechin, a crosslinking agent, on human dental pulp cells cultured in collagen scaffolds

    PubMed Central

    Lim, Eun-su; Lim, Myung-Jin; Min, Kyung-San; Kwon, Young-Sun; Hwang, Yun-Chan; Yu, Mi-Kyung; Hong, Chan-Ui; Lee, Kwang-Won

    2016-01-01

    ABSTRACT Objective The purpose of this study was to investigate the biological effects of epicatechin (ECN), a crosslinking agent, on human dental pulp cells (hDPCs) cultured in collagen scaffolds. Material and Method To evaluate the effects of ECN on the proliferation of hDPCs, cell counting was performed using optical and fluorescent microscopy. Measurements of alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reactions were performed to assess odontogenic differentiation. The compressive strength and setting time of collagen scaffolds containing ECN were measured. Differential scanning calorimetry was performed to analyze the thermal behavior of collagen in the presence of ECN. Results Epicatechin increased ALP activity, mineralized nodule formation, and the mRNA expression of dentin sialophosphoprotein (DSPP), a specific odontogenic-related marker. Furthermore, ECN upregulated the expression of DSPP in hDPCs cultured in collagen scaffolds. Epicatechin activated the extracellular signal-regulated kinase (ERK) and the treatment with an ERK inhibitor (U0126) blocked the expression of DSPP. The compressive strength was increased and the setting time was shortened in a dose-dependent manner. The number of cells cultured in the ECN-treated collagen scaffolds was significantly increased compared to the cells in the untreated control group. Conclusions Our results revealed that ECN promoted the proliferation and differentiation of hDPCs. Furthermore, the differentiation was regulated by the ERK signaling pathway. Changes in mechanical properties are related to cell fate, including proliferation and differentiation. Therefore, our study suggests the ECN treatment might be desirable for dentin-pulp complex regeneration. PMID:27008260

  16. Linking a Germplasm Collection of the Cover Crop Hairy Vetch (Vicia villosa Roth) to Traits Related to Improved Nitrogen Fixation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hairy vetch is used as a leguminous cover crop throughout the United States providing important ecosystem services in agro-ecosystems (Abdul-Baki et al., 2002; Mohler and Teasdale, 1993; Puget and Drinkwater, 2001; Seo et al., 2006; Stute and Posner, 1995). Many traits found in hairy vetch have pro...

  17. 3'-Phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1) knockdown sensitizes non-small cell lung cancer cells to DNA damaging agents.

    PubMed

    Leung, Ada W Y; Dragowska, Wieslawa H; Ricaurte, Daniel; Kwok, Brian; Mathew, Veena; Roosendaal, Jeroen; Ahluwalia, Amith; Warburton, Corinna; Laskin, Janessa J; Stirling, Peter C; Qadir, Mohammed A; Bally, Marcel B

    2015-07-10

    Standard treatment for advanced non-small cell lung cancer (NSCLC) with no known driver mutation is platinum-based chemotherapy, which has a response rate of only 30-33%. Through an siRNA screen, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate PAPS, was identified as a novel platinum-sensitizing target in NSCLC cells. PAPSS1 knockdown in combination with low-dose (IC10) cisplatin reduces clonogenicity of NSCLC cells by 98.7% (p < 0.001), increases DNA damage, and induces G1/S phase cell cycle arrest and apoptosis. PAPSS1 silencing also sensitized NSCLC cells to other DNA crosslinking agents, radiation, and topoisomerase I inhibitors, but not topoisomerase II inhibitors. Chemo-sensitization was not observed in normal epithelial cells. Knocking out the PAPSS1 homolog did not sensitize yeast to cisplatin, suggesting that sulfate bioavailability for amino acid synthesis is not the cause of sensitization to DNA damaging agents. Rather, sensitization may be due to sulfation reactions involved in blocking the action of DNA damaging agents, facilitating DNA repair, promoting cancer cell survival under therapeutic stress or reducing the bioavailability of DNA damaging agents. Our study demonstrates for the first time that PAPSS1 could be targeted to improve the activity of multiple anticancer agents used to treat NSCLC. PMID:26220590

  18. 3′-Phosphoadenosine 5′-phosphosulfate synthase 1 (PAPSS1) knockdown sensitizes non-small cell lung cancer cells to DNA damaging agents

    PubMed Central

    Leung, Ada W. Y.; Dragowska, Wieslawa H.; Ricaurte, Daniel; Kwok, Brian; Mathew, Veena; Roosendaal, Jeroen; Ahluwalia, Amith; Warburton, Corinna; Laskin, Janessa J.; Stirling, Peter C.; Qadir, Mohammed A.; Bally, Marcel B.

    2015-01-01

    Standard treatment for advanced non-small cell lung cancer (NSCLC) with no known driver mutation is platinum-based chemotherapy, which has a response rate of only 30–33%. Through an siRNA screen, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate PAPS, was identified as a novel platinum-sensitizing target in NSCLC cells. PAPSS1 knockdown in combination with low-dose (IC10) cisplatin reduces clonogenicity of NSCLC cells by 98.7% (p < 0.001), increases DNA damage, and induces G1/S phase cell cycle arrest and apoptosis. PAPSS1 silencing also sensitized NSCLC cells to other DNA crosslinking agents, radiation, and topoisomerase I inhibitors, but not topoisomerase II inhibitors. Chemo-sensitization was not observed in normal epithelial cells. Knocking out the PAPSS1 homolog did not sensitize yeast to cisplatin, suggesting that sulfate bioavailability for amino acid synthesis is not the cause of sensitization to DNA damaging agents. Rather, sensitization may be due to sulfation reactions involved in blocking the action of DNA damaging agents, facilitating DNA repair, promoting cancer cell survival under therapeutic stress or reducing the bioavailability of DNA damaging agents. Our study demonstrates for the first time that PAPSS1 could be targeted to improve the activity of multiple anticancer agents used to treat NSCLC. PMID:26220590

  19. Black hairy tongue in a patient with amyotrophic lateral sclerosis

    PubMed Central

    Erriu, Matteo; Pili, Francesca Maria Giovanna; Denotti, Gloria; Garau, Valentino

    2016-01-01

    Black hairy tongue (BHT) is a condition characterized by the elongation of filiform papillae associated with a marked discoloration, from yellowish-brown to black, and a thick lingual coating. BHT is usually observed in the elderly and in patients with limited self-sufficiency, as a consequence of poor oral hygiene. In this perspective, the patients affected by amyotrophic lateral sclerosis (ALS) represent a high-risk category for the occurrence of BHT. The fast and inexorable loss of their self-sufficiency due to progressive muscle atrophy as well as the impropriate education of healthcare assistants have demonstrated to have significant reflection on the maintenance of an adequate standard of oral hygiene. This paper firstly described a case of BHT in a patient affected by ALS. A case of BHT in a patient (Caucasic, male, 63 years old) affected by ALS was described. The primary goal of the work was to teach and motivate the patient to the use of the tongue cleaner in association with the local application of chlorexidine 0.20%. Furthermore, in order to support the patient with accurate domiciliary oral hygiene, a proper training for his health-care assistant was provided. The maintenance of the oral health of ALS patient is fundamental to prevent systemic complications that could jeopardize the already fragile physical balance of these patients. The dedicated monitoring by a dentist or a dental hygienist would seem essential in order to achieve this objective. PMID:27011938

  20. Black hairy tongue in a patient with amyotrophic lateral sclerosis.

    PubMed

    Erriu, Matteo; Pili, Francesca Maria Giovanna; Denotti, Gloria; Garau, Valentino

    2016-01-01

    Black hairy tongue (BHT) is a condition characterized by the elongation of filiform papillae associated with a marked discoloration, from yellowish-brown to black, and a thick lingual coating. BHT is usually observed in the elderly and in patients with limited self-sufficiency, as a consequence of poor oral hygiene. In this perspective, the patients affected by amyotrophic lateral sclerosis (ALS) represent a high-risk category for the occurrence of BHT. The fast and inexorable loss of their self-sufficiency due to progressive muscle atrophy as well as the impropriate education of healthcare assistants have demonstrated to have significant reflection on the maintenance of an adequate standard of oral hygiene. This paper firstly described a case of BHT in a patient affected by ALS. A case of BHT in a patient (Caucasic, male, 63 years old) affected by ALS was described. The primary goal of the work was to teach and motivate the patient to the use of the tongue cleaner in association with the local application of chlorexidine 0.20%. Furthermore, in order to support the patient with accurate domiciliary oral hygiene, a proper training for his health-care assistant was provided. The maintenance of the oral health of ALS patient is fundamental to prevent systemic complications that could jeopardize the already fragile physical balance of these patients. The dedicated monitoring by a dentist or a dental hygienist would seem essential in order to achieve this objective. PMID:27011938

  1. Removal of Phenol by A. belladonna L. Hairy Root.

    PubMed

    Mazaheri, Hamide; Piri, Khosro

    2015-01-01

    Phenolic compounds that present in the several industries are harmful and dangerous for human health. In this study we have studied the potential of Atropa belladonna hairy roots in phenol removal of wastewater. The optimal conditions for the removal process were evaluated using different phenol (10-500 mg.1(-1)) and H2O2 (1-15 Mm) concentrations. In the presence of H2O2, Roots were able to remove phenol concentrations up to 500 mg.1(-1). in the wide range of pH (4-9), reaching high removal efficiency. When roots were re-used for five consecutive cycles, phenol removal efficiency decreased from 98-62%, in the last cycle. After the removal process, the solutions were obtained from the experiment were estimated for their toxicity using a test with Lactaca sativa L. seeds. Results showed that the treated solution was less toxic than the parent solution. PMID:25950155

  2. Global structure of exact scalar hairy dynamical black holes

    NASA Astrophysics Data System (ADS)

    Fan, Zhong-Ying; Chen, Bin; Lü, H.

    2016-05-01

    We study the global structure of some exact scalar hairy dynamical black holes which were constructed in Einstein gravity either minimally or non-minimally coupled to a scalar field. We find that both the apparent horizon and the local event horizon (measured in luminosity coordinate) monotonically increase with the advanced time as well as the Vaidya mass. At late advanced times, the apparent horizon approaches the event horizon and gradually becomes future outer. Correspondingly, the space-time arrives at stationary black hole states with the relaxation time inversely proportional to the 1/( n-1) power of the final black hole mass, where n is the space-time dimension. These results strongly support the solutions describing the formation of black holes with scalar hair. We also obtain new charged dynamical solutions in the non-minimal theory by introducing an Maxwell field which is non-minimally coupled to the scalar. The presence of the electric charge strongly modifies the dynamical evolution of the space-time.

  3. Critical review of topical management of oral hairy leukoplakia.

    PubMed

    Brasileiro, Cláudia B; Abreu, Mauro Henrique Ng; Mesquita, Ricardo A

    2014-07-16

    Oral hairy leukoplakia (OHL) is a disease associated with Epstein-Barr virus and human immunodeficiency virus infections. OHL is usually an asymptomatic lesion, but in some cases treatment is recommended to reestablish the normal characteristics of the tongue, to eliminate pathogenic microorganisms, to improve patient comfort and for cosmetic reasons. Proposed treatments for this condition include surgery, systemic antiviral treatment and topical management. Topical treatment is an inexpensive and safe therapy that is easy to apply, noninvasive, free of systemic adverse effects and effective over a long period of time. The aim of this study was to present a review of the literature for topical therapy for OHL. Gentian violet, retinoids, podophyllin, acyclovir and podophyllin associated with topical antiviral drugs were used to treat OHL. Reports with this focus are limited, and since 2010, no new studies have been published that discuss the efficacy of topical treatments for OHL. Podophyllin with acyclovir cream was found to be effective, causing regression of lesions with no recurrences. Additional searches are necessary to provide clinical evidence of topical management effectiveness. PMID:25032199

  4. A chronic 1 year assessment of MRI contrast agent-labelled neural stem cell transplants in stroke.

    PubMed

    Modo, M; Beech, J S; Meade, T J; Williams, S C R; Price, J

    2009-08-01

    Non-invasive identification of transplanted neural stem cells in vivo by pre-labelling with contrast agents may play an important role in the translation of cell therapy to the clinic. Understanding the impact of these labels on the cells' ability to repair is therefore vital. In rats with middle cerebral artery occlusion (MCAo), a model of stroke, the transhemispheric migration of MHP36 cells labelled with the bimodal contrast agent GRID was detected on magnetic resonance images (MRI) up to 4 weeks following transplantation. However, compared to MHP36 cells labelled with the red fluorescent dye PKH26, GRID-labelled transplants did not significantly improve behaviour, and performance was akin to non-treated animals. Likewise, the evolution of anatomical damage as assessed by serial, T(2)-weighted MRI over 1 year indicated that GRID-labelled transplants resulted in a slight increase in lesion size compared to MCAo-only animals, whereas the same, PKH26-labelled cells significantly decreased lesion size by 35%. Although GRID labelling allows the in vivo identification of transplanted cells up to 1 month after transplantation, it is likely that some is gradually degraded inside cells. The translation of cellular imaging therefore does not only require the in vitro assessment of contrast agents on cellular functions, but also requires the chronic, in vivo assessment of the label on the stem cells' ability to repair in preclinical models of neurological disease. PMID:18634886

  5. Single-agent cytarabine is insufficient for the treatment of human mantle cell lymphoma in mouse xenograft model.

    PubMed

    Klanova, M; Soukup, T; Molinsky, J; Lateckova, L; Vockova, P; Alam, M; Zivny, J; Trneny, M; Klener, P

    2016-01-01

    Mantle cell lymphoma is an aggressive type of B-cell non-Hodgkin lymphoma with adverse prognosis. It was demonstrated that alternation of CHOP and DHAP chemotherapy improved outcome of mantle cell lymphoma patients. However, which components of DHAP, cisplatin, cytarabine, or both, were responsible for the improved outcome remained unclear. To answer this question, antitumor efficacies of equally toxic doses of cytarabine, cisplatin, and three different combinations were compared in vivo using mouse xenograft models of mantle cell lymphoma. We demonstrated that cisplatin, alone or with cytarabine, is significantly superior to single-agent cytarabine in both eliminating lymphoma cells and suppressing their proliferation rate. PMID:27468882

  6. Recombinant interferon alfa-2a, an active agent in advanced cutaneous T-cell lymphomas.

    PubMed

    Bunn, P A; Ihde, D C; Foon, K A

    1987-01-01

    The cutaneous T-cell lymphomas including mycosis fungoides and the Sézary syndrome, are indolent lymphomas with early systemic dissemination. Like the indolent B-cell lymphomas, they cannot be cured by currently available systemic chemotherapy so new systemic therapies need to be developed. A study of very high-dose recombinant interferon alfa-2a was, therefore, initiated in 20 patients with advanced cutaneous T-cell lymphoma (5 in stage II, 2 in stage III and 13 in stage IV). All patients were refractory to at least 2 standard therapies, including topical nitrogen mustard (18 patients), psoralens and ultraviolet A light (12 patients), total skin electron irradiation (14 patients) and systemic chemotherapy (16 patients). Nine out of 20 patients (45%; 95% confidence interval 25-69%) had either objective partial or complete responses within 3 months of starting treatment. Maximal response, however, often did not occur for at least one year. The median duration of response was 5.5 months and all complete responses lasted more than 2 years. Response frequencies were equal at both cutaneous and extracutaneous sites and in patients with or without prior chemotherapy. Toxicity was exhibited primarily as a flu-like syndrome consisting of fever, malaise, fatigue, anorexia and weight loss which necessitated dose reductions in all patients. Transient elevations in liver function and decreases in renal function and granulocyte counts occurred in some patients. It is concluded that interferon alfa-2a is highly active against advanced cutaneous T-cell lymphomas and that it should be studied in its early stages. It should also be evaluated in combination with other biological agents and with chemotherapy.

  7. The hairy family of Burma: a four generation pedigree of congenital hypertrichosis lanuginosa.

    PubMed Central

    Bondeson, J; Miles, A E

    1996-01-01

    A Burmese family with congenital hypertrichosis lanuginosa had an eventful history in the nineteenth century. The earlier members of this family were employed at the court of Ava, but the later ones spent their lives in show business, being widely exhibited for money in the 1880s. Their extraordinary hairiness attracted much curiosity, and they were photographed several times. The hairy Burmese are the only example of a four-generation pedigree of congenital hypertrichosis lanuginosa, which is consistent with an autosomal dominant mode of inheritance. There is good evidence that, when the members of this family were hairy, their dentition was also deficient. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 (a) Figure 5 (b) PMID:8774541

  8. Hematologic and serum biochemical reference values for free-ranging northern hairy-nosed wombats.

    PubMed

    Reiss, Andrea; Portas, Timothy; Horsup, Alan

    2008-01-01

    Hematologic and serum biochemistry values were determined for 31 adult (21 male and 10 female) and four subadult male northern hairy-nosed wombats (Lasiorhinus krefftii) from the only existing population in Epping Forest National Park, Australia. Blood samples were obtained from free-ranging northern hairy-nosed wombats during trapping for population census and health and reproductive assessment in 1999. Hematologic and biochemical values were compared between adult males and adult females, and between adult and subadult wombats. Values were also compared with those previously published for southern hairy-nosed (Lasiorhinus latifrons) and common (Vombatus ursinus) wombats. The values from this study were used to create reference intervals, and they make up the first comprehensive hematologic and biochemical study for this highly endangered species. PMID:18263822

  9. Bifunctional hairy silica nanoparticles as high-performance additives for lubricant

    PubMed Central

    Sui, Tianyi; Song, Baoyu; Wen, Yu-ho; Zhang, Feng

    2016-01-01

    Bifunctional hairy silica nanoparticles (BHSNs), which are silica nanoparticles covered with alkyl and amino organic chains, were prepared as high-performance additives for lubricants. Compared with hairy silica nanoparticles covered by a single type of organic chain, binary hairy silica nanoparticles exhibit the advantages of both types of organic chains, which exhibit excellent compatibility with lubricants and adsorbability to metal surfaces. Nanoparticles with different ratios of amino and alkyl ligands were investigated. In comparison to an untreated lubricant, BHSNs reduce the friction coefficient and wear scar diameter by 40% and 60%, respectively. The wear mechanism of BHSNs was investigated, and the protective and filling effect of the nanoparticles improved because of collaboration of amino and alkyl ligands. PMID:26936117

  10. Hematologic and serum biochemical reference values for free-ranging northern hairy-nosed wombats.

    PubMed

    Reiss, Andrea; Portas, Timothy; Horsup, Alan

    2008-01-01

    Hematologic and serum biochemistry values were determined for 31 adult (21 male and 10 female) and four subadult male northern hairy-nosed wombats (Lasiorhinus krefftii) from the only existing population in Epping Forest National Park, Australia. Blood samples were obtained from free-ranging northern hairy-nosed wombats during trapping for population census and health and reproductive assessment in 1999. Hematologic and biochemical values were compared between adult males and adult females, and between adult and subadult wombats. Values were also compared with those previously published for southern hairy-nosed (Lasiorhinus latifrons) and common (Vombatus ursinus) wombats. The values from this study were used to create reference intervals, and they make up the first comprehensive hematologic and biochemical study for this highly endangered species.

  11. Novel active comb-shaped dry electrode for EEG measurement in hairy site.

    PubMed

    Huang, Yan-Jun; Wu, Chung-Yu; Wong, Alice May-Kuen; Lin, Bor-Shyh

    2015-01-01

    Electroencephalography (EEG) is an important biopotential, and has been widely applied in clinical applications. The conventional EEG electrode with conductive gels is usually used for measuring EEG. However, the use of conductive gel also encounters with the issue of drying and hardening. Recently, many dry EEG electrodes based on different conductive materials and techniques were proposed to solve the previous issue. However, measuring EEG in the hairy site is still a difficult challenge. In this study, a novel active comb-shaped dry electrode was proposed to measure EEG in hairy site. Different form other comb-shaped or spike-shaped dry electrodes, it can provide more excellent performance of avoiding the signal attenuation, phase distortion, and the reduction of common mode rejection ratio. Even under walking motion, it can effectively acquire EEG in hairy site. Finally, the experiments for alpha rhythm and steady-state visually evoked potential were also tested to validate the proposed electrode.

  12. "Hairy blobs:" microbial suspects preserved in modern and ancient extremely acid lake evaporites.

    PubMed

    Benison, Kathleen C; Jagniecki, Elliot A; Edwards, Tina B; Mormile, Melanie R; Storrie-Lombardi, Michael C

    2008-08-01

    "Hairy blobs" are unusual clumps of organic bodies and sulfate crystals that have been found in evaporite minerals grown in acid saline lakes. Here, we document modern hairy blobs in halite and gypsum from 5 modern acid saline lakes in southern Western Australia, and Permian hairy blobs trapped in halite from the mid-Permian Opeche Shale in the subsurface of North Dakota. These are among the first microbial remains described from acid saline lake environments. They give clues about the role of microorganisms in the acidity, geochemistry, and mineralogy of these extreme environments. This study also may add to the inventory of life in extreme environments and help predict possible martian life-forms and the method of preservation. PMID:18498219

  13. The shikonin derivatives and pyrrolizidine alkaloids in hairy root cultures of Lithospermum canescens (Michx.) Lehm.

    PubMed

    Pietrosiuk, A; Sykłowska-Baranek, K; Wiedenfeld, H; Wolinowska, R; Furmanowa, M; Jaroszyk, E

    2006-10-01

    Hairy root cultures of Lithospermum canescens were established using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight lines resulting from infection with A. rhizogenes ATCC 15834 demonstrated sufficient biomass increase and were submitted to further investigations. The contents of acetylshikonin (ACS) and isobutyrylshikonin (IBS) in transformed hairy roots made up ca. 10% of those observed in natural roots of L. canescens (24.35 and 14.48 mg g(-1) DW, respectively). One line, Lc1-D, produced the largest amounts of ACS (2.72 mg g(-1) DW) and IBS (0.307 mg g(-1) DW). Traces of pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots.

  14. "Hairy blobs:" microbial suspects preserved in modern and ancient extremely acid lake evaporites.

    PubMed

    Benison, Kathleen C; Jagniecki, Elliot A; Edwards, Tina B; Mormile, Melanie R; Storrie-Lombardi, Michael C

    2008-08-01

    "Hairy blobs" are unusual clumps of organic bodies and sulfate crystals that have been found in evaporite minerals grown in acid saline lakes. Here, we document modern hairy blobs in halite and gypsum from 5 modern acid saline lakes in southern Western Australia, and Permian hairy blobs trapped in halite from the mid-Permian Opeche Shale in the subsurface of North Dakota. These are among the first microbial remains described from acid saline lake environments. They give clues about the role of microorganisms in the acidity, geochemistry, and mineralogy of these extreme environments. This study also may add to the inventory of life in extreme environments and help predict possible martian life-forms and the method of preservation.

  15. Trientine, a copper-chelating agent, induced apoptosis in murine fibrosarcoma cells by activation of the p38 MAPK pathway.

    PubMed

    KADOWAKI, Shingo; ENDOH, Daiji; OKUI, Toyo; HAYASHI, Masanobu

    2009-11-01

    We have reported that treatment with trientine, Cu-chelating agent, inhibits tumor growth in a murine transplantation model using fibrosarcoma and induces apoptosis in tumor cells in vivo and in vitro. When fibrosarcoma cells were treated with 10 mM trientine, the activities of p38 MAPK in treated cells were approximately 3-4 times higher than those in untreated cells. Proportions of cells in which apoptosis was induced by trientine increased in an incubation time-dependent manner from days 2 to 6. The proportions of apoptotic cells in the cells treated with trientine and SB203580, an inhibitor of p38 MAPK, were approximately 50% in those of cells treated with trientine alone. The present results showed that the p38 MAPK pathway may play an important role in induction of apoptosis in fibrosarcoma cells by trientine.

  16. Targeted delivery of 5-fluorouracil to cholangiocarcinoma cells using folic acid as a targeting agent.

    PubMed

    Ngernyuang, Nipaporn; Seubwai, Wunchana; Daduang, Sakda; Boonsiri, Patcharee; Limpaiboon, Temduang; Daduang, Jureerut

    2016-03-01

    There are limits to the standard treatment for cholangiocarcinoma (CCA) including drug resistance and side effects. The objective of this study was to develop a new technique for carrying drugs by conjugation with gold nanoparticles and using folic acid as a targeting agent in order to increase drug sensitivity. Gold nanoparticles (AuNPs) were functionalized with 5-fluorouracil (5FU) and folic acid (FA) using polyethylene glycol (PEG) shell as a linker (AuNPs-PEG-5FU-FA). Its cytotoxicity was tested in CCA cell lines (M139 and M213) which express folic acid receptor (FA receptor). The results showed that AuNPs-PEG-5FU-FA increased the cytotoxic effects in the M139 and M213 cells by 4.76% and 7.95%, respectively compared to those treated with free 5FU+FA. It is found that the cytotoxicity of the AuNPs-PEG-5FU-FA correlates with FA receptor expression suggested the use of FA as a targeted therapy. The mechanism of cytotoxicity was mediated via mitochondrial apoptotic pathway as determined by apoptosis array. In conclusion, our findings shed some light on the use of gold nanoparticles for conjugation with potential compounds and FA as targeted therapy which contribute to the improvement of anti-cancer drug efficacy. In vivo study should be warranted for its effectiveness of stability, biosafety and side effect reduction.

  17. Targeted delivery of 5-fluorouracil to cholangiocarcinoma cells using folic acid as a targeting agent.

    PubMed

    Ngernyuang, Nipaporn; Seubwai, Wunchana; Daduang, Sakda; Boonsiri, Patcharee; Limpaiboon, Temduang; Daduang, Jureerut

    2016-03-01

    There are limits to the standard treatment for cholangiocarcinoma (CCA) including drug resistance and side effects. The objective of this study was to develop a new technique for carrying drugs by conjugation with gold nanoparticles and using folic acid as a targeting agent in order to increase drug sensitivity. Gold nanoparticles (AuNPs) were functionalized with 5-fluorouracil (5FU) and folic acid (FA) using polyethylene glycol (PEG) shell as a linker (AuNPs-PEG-5FU-FA). Its cytotoxicity was tested in CCA cell lines (M139 and M213) which express folic acid receptor (FA receptor). The results showed that AuNPs-PEG-5FU-FA increased the cytotoxic effects in the M139 and M213 cells by 4.76% and 7.95%, respectively compared to those treated with free 5FU+FA. It is found that the cytotoxicity of the AuNPs-PEG-5FU-FA correlates with FA receptor expression suggested the use of FA as a targeted therapy. The mechanism of cytotoxicity was mediated via mitochondrial apoptotic pathway as determined by apoptosis array. In conclusion, our findings shed some light on the use of gold nanoparticles for conjugation with potential compounds and FA as targeted therapy which contribute to the improvement of anti-cancer drug efficacy. In vivo study should be warranted for its effectiveness of stability, biosafety and side effect reduction. PMID:26706547

  18. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein

    PubMed Central

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  19. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

    PubMed

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  20. Pouch young removal and return to oestrus in wild southern hairy-nosed wombats (Lasiorhinus latifrons).

    PubMed

    Finlayson, G R; Taggart, D A; Shimmin, G A; White, C R; Dibben, R; Steele, V; Paris, M C J; Temple-Smith, P D

    2007-07-01

    The southern hairy-nosed wombat (Lasiorhinus latifrons) is a seasonal breeding, burrowing marsupial adapted to a semi-arid environment and the closest relative of the endangered northern hairy-nosed wombat (Lasiorhinus krefftii). Females typically give birth to one to two young every 3 years with young weaned at 360-400 days. This study examined the occurrence of polyoestry in a wild population of southern hairy-nosed wombats, and in particular the ability of this species to produce additional offspring in the same breeding season if a young was prematurely lost or removed. Pouch young were removed during the breeding seasons of 1996/1997 and 2003. No females from the 1996 (n=3)/1997 (n=3) group gave birth to a second pouch young in the same breeding season. However, two females in this group gave birth to young the following season. In contrast, all the 2003 group of females (n=6) produced a second offspring in the same breeding season after removal of pouch young (RPY). The reason for the different response to RPY between the two groups is unknown. These studies confirm that southern hairy-nosed wombats are polyoestrus in the wild and are capable of producing more than one offspring in a single breeding season. Females that failed to return to oestrus in the breeding season that pouch young were removed bred again in the following season. Rapid replacement of southern hairy-nosed wombat pouch young in the same breeding season as RPY suggests that this procedure, linked to either hand-rearing or interspecific cross-fostering, should be seriously considered as a priority conservation action to increase the population size of the critically endangered sister species, the northern hairy-nosed wombat. PMID:17023125

  1. Pouch young removal and return to oestrus in wild southern hairy-nosed wombats (Lasiorhinus latifrons).

    PubMed

    Finlayson, G R; Taggart, D A; Shimmin, G A; White, C R; Dibben, R; Steele, V; Paris, M C J; Temple-Smith, P D

    2007-07-01

    The southern hairy-nosed wombat (Lasiorhinus latifrons) is a seasonal breeding, burrowing marsupial adapted to a semi-arid environment and the closest relative of the endangered northern hairy-nosed wombat (Lasiorhinus krefftii). Females typically give birth to one to two young every 3 years with young weaned at 360-400 days. This study examined the occurrence of polyoestry in a wild population of southern hairy-nosed wombats, and in particular the ability of this species to produce additional offspring in the same breeding season if a young was prematurely lost or removed. Pouch young were removed during the breeding seasons of 1996/1997 and 2003. No females from the 1996 (n=3)/1997 (n=3) group gave birth to a second pouch young in the same breeding season. However, two females in this group gave birth to young the following season. In contrast, all the 2003 group of females (n=6) produced a second offspring in the same breeding season after removal of pouch young (RPY). The reason for the different response to RPY between the two groups is unknown. These studies confirm that southern hairy-nosed wombats are polyoestrus in the wild and are capable of producing more than one offspring in a single breeding season. Females that failed to return to oestrus in the breeding season that pouch young were removed bred again in the following season. Rapid replacement of southern hairy-nosed wombat pouch young in the same breeding season as RPY suggests that this procedure, linked to either hand-rearing or interspecific cross-fostering, should be seriously considered as a priority conservation action to increase the population size of the critically endangered sister species, the northern hairy-nosed wombat.

  2. Improved cardenolide production in Calotropis gigantea hairy roots using mechanical wounding and elicitation.

    PubMed

    Sun, Jian; Xiao, Jie; Wang, Xiaodong; Yuan, Xiaofan; Zhao, Bing

    2012-03-01

    A hairy root culture system of Calotropis gigantea was established and effects of mechanical wounding (MW) and elicitors [methyl jasmonate (MJ), yeast extract (YE) and chitosan (CS)] on cardenolide production were investigated. All treatments stimulated the production of cardenolide in hairy root cultures of C. gigantea. CS was the most effective elicitor, followed by MJ. YE and MW also improved cardenolide yield in individual treatments. The highest cardenolide yield (1,050 ± 55 mg/l) was obtained after adding 50 mg CS/l for 20 days, which was 2.7-fold higher than the control.

  3. First isolation of natural cyanamide as a possible allelochemical from hairy vetch Vicia villosa.

    PubMed

    Kamo, Tsunashi; Hiradate, Syuntaro; Fujii, Yoshiharu

    2003-02-01

    Cyanamide was isolated from the leaves and stems of hairy vetch (Vicia villosa), guided by plant growth inhibitory activity against lettuce (Lectuca sativa) seedlings. A large proportion of the inhibitory activity in the crude extract was explained by the presence of cyanamide, suggesting it to be a possible allelochemical in this species. The amount in a 9-day-old seedling, which had been grown without nutrients, reached approx. 40 times that of a nongerminated seed, demonstrating cyanamide biosynthesis in the seedlings. This is the first report on the isolation of a possible allelochemical from hairy vetch and also of the finding of cyanamide as a natural product.

  4. Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots.

    PubMed

    Salma, Mohammad; Engelmann-Sylvestre, Isabelle; Collin, Myriam; Escoute, Jacques; Lartaud, Marc; Yi, Jung-Yoon; Kim, Haeng-Hoon; Verdeil, Jean-Luc; Engelmann, Florent

    2014-05-01

    In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45 %) was already observed after the sucrose pretreatment; it increased to 54-68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.

  5. Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots.

    PubMed

    Salma, Mohammad; Engelmann-Sylvestre, Isabelle; Collin, Myriam; Escoute, Jacques; Lartaud, Marc; Yi, Jung-Yoon; Kim, Haeng-Hoon; Verdeil, Jean-Luc; Engelmann, Florent

    2014-05-01

    In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45 %) was already observed after the sucrose pretreatment; it increased to 54-68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment. PMID:24150426

  6. Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase.

    PubMed

    Lozovaya, Vera V; Lygin, Anatoliy V; Zernova, Olga V; Ulanov, Alexander V; Li, Shuxian; Hartman, Glen L; Widholm, Jack M

    2007-02-01

    Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen. PMID:16924535

  7. The transcription factor Rap1p is required for tolerance to cell-wall perturbing agents and for cell-wall maintenance in Saccharomyces cerevisiae.

    PubMed

    Azad, Gajendra Kumar; Singh, Vikash; Baranwal, Shivani; Thakare, Mayur Jankiram; Tomar, Raghuvir S

    2015-01-01

    Yeast repressor activator protein (Rap1p) is involved in genomic stability and transcriptional regulation. We explored the function of Rap1p in yeast physiology using Rap1p truncation mutants. Our results revealed that the N-terminal truncation of Rap1p (Rap1ΔN) leads to hypersensitivity towards elevated temperature and cell-wall perturbing agents. Cell wall analysis showed an increase in the chitin and glucan content in Rap1ΔN cells as compared with wild type cells. Accordingly, mutant cells had a twofold thicker cell wall, as observed by electron microscopy. Furthermore, Rap1ΔN cells had increased levels of phosphorylated Slt2p, a MAP kinase of the cell wall integrity pathway. Mutant cells also had elevated levels of cell wall integrity response transcripts. Taken together, our findings suggest a connection between Rap1p and cell wall homeostasis.

  8. A highly fluorescent AIE-active theranostic agent with anti-tumor activity to specific cancer cells

    NASA Astrophysics Data System (ADS)

    Zhao, Yueyue; Kwok, Ryan T. K.; Lam, Jacky W. Y.; Tang, Ben Zhong

    2016-06-01

    A tetraphenylethene derivative with a structure resembling Tamoxifen is designed and synthesized as a theranostic agent for cell imaging and anti-breast cancer therapy. Its high brightness, excellent photostability and long-term cell tracing properties enable elucidation of its working mechanism and hence provide new insights into drug development.A tetraphenylethene derivative with a structure resembling Tamoxifen is designed and synthesized as a theranostic agent for cell imaging and anti-breast cancer therapy. Its high brightness, excellent photostability and long-term cell tracing properties enable elucidation of its working mechanism and hence provide new insights into drug development. Electronic supplementary information (ESI) available: Detailed synthesis and characterization of TPE-OH and TPE-TMX PL spectra of TPE-TMX fluorescent photographs of TPE-TMX taken under UV irradiation; various concentrations of TPE-TMX with different incubation times. See DOI: 10.1039/c5nr08782a

  9. Suppression of NRF2-ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells.

    PubMed

    Peng, Hui; Wang, Huihui; Xue, Peng; Hou, Yongyong; Dong, Jian; Zhou, Tong; Qu, Weidong; Peng, Shuangqing; Li, Jin; Carmichael, Paul L; Nelson, Bud; Clewell, Rebecca; Zhang, Qiang; Andersen, Melvin E; Pi, Jingbo

    2016-02-01

    Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2-ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As2O3), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2-ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As2O3-challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2-ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As2O3-induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As2O3-induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2-ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents.

  10. Profilin potentiates chemotherapeutic agents mediated cell death via suppression of NF-κB and upregulation of p53.

    PubMed

    Zaidi, Adeel H; Raviprakash, Nune; Mokhamatam, Raveendra B; Gupta, Pankaj; Manna, Sunil K

    2016-04-01

    The molecular mechanism by which Profilin acts as a tumor suppressor is still unclear. Several chemotherapeutic agents, used till date either have unfavorable side effects or acquired resistance in tumor cells. Our findings show that Profilin enhances cell death mediated by several chemotherapeutic-agents. The activation of NF-κB and its dependent genes, mediated by paclitaxel and vinblastine, was completely inhibited in Profilin overexpressing cells. This inhibition was due to the Profilin mediated attenuation of IκBα degradation, thereby preventing p65 nuclear translocation and low NF-κB DNA binding activity.Moreover, Profilin increases level of p53 in the presence of known inducers, such as doxorubicin, vinblastine, and benzofuran. This increased p53 level leads to enhanced cell death as indicated by activation of caspases 3, 8, 9, which results in cleavage of PARP.Furthermore, knocking down of p53 in Profilin overexpressing cells leads to decreased cell death. Ectopic expression of Profilin in HCT116 p53 knock out cells showed lesser cell death as compared to the HCT116 p53 wild type cells. For the first time, we provide evidences, which suggest that Profilin synergizes with chemotherapeutic drugs to induce tumor cell death by regulating NF-κB and p53. Thus, modulation of Profilin may be a useful strategy for effective combination therapy. PMID:26842845

  11. The Sarin-like Organophosphorus Agent bis (isopropyl methyl)phosphonate Induces Apoptotic Cell Death and COX-2 Expression in SK-N-SH Cells.

    PubMed

    Arima, Yosuke; Yoshimoto, Kanji; Namera, Akira; Makita, Ryosuke; Murata, Kazuhiro; Nagao, Masataka

    2016-03-01

    Organophosphorus compounds, such as sarin, are highly toxic nerve agents that inhibit acetylcholinesterase (AChE), but not cholinesterase, via multiple mechanisms. Recent studies have shown that organophosphorus compounds increase cyclooxygenase-2 (COX-2) expression and induce neurotoxicity. In this study, we examined the toxicity of the sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate (BIMP) and the effects of BIMP on COX-2 expression in SK-N-SH human neuroblastoma cells. Exposure to BIMP changed cell morphology and induced caspase-dependent apoptotic cell death accompanied by cleavage of caspase 3, caspase 9, and poly (ADP-ribose) polymerase (PARP). It also increased COX-2 expression, while pretreatment with a COX inhibitor, ibuprofen, decreased BIMP-dependent cell death and COX-2 expression in SK-N-SH cells. Thus, our findings suggest that BIMP induces apoptotic cell death and upregulates COX-2 expression. PMID:27348899

  12. Block copolymer as a nanostructuring agent for high-efficiency and annealing-free bulk heterojunction organic solar cells.

    PubMed

    Renaud, Cédric; Mougnier, Sébastien-Jun; Pavlopoulou, Eleni; Brochon, Cyril; Fleury, Guillaume; Deribew, Dargie; Portale, Giuseppe; Cloutet, Eric; Chambon, Sylvain; Vignau, Laurence; Hadziioannou, Georges

    2012-04-24

    The addition of a block copolymer to the polymer/fullerene blend is a novel approach to the fabrication of organic solar cells. The block copolymer (P3HT-b-P4VP) is used as nanostructuring agent in the active layer. A significant enhancement of the cell efficiency is observed, in correlation with morphology control, both before (as-cast) and after the annealing process.

  13. The potent microtubule-stabilizing agent (+)-discodermolide induces apoptosis in human breast carcinoma cells--preliminary comparisons to paclitaxel.

    PubMed

    Balachandran, R; ter Haar, E; Welsh, M J; Grant, S G; Day, B W

    1998-01-01

    (+)-Discodermolide, a sponge-derived natural product, stabilizes microtubules more potently than paclitaxel despite the lack of any obvious structural similarities between the drugs. It competitively inhibits the binding of paclitaxel to tubulin polymers, hypernucleates microtubule assembly more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant ovarian and colon carcinoma cells. Because paclitaxel shows clinical promise for breast cancer treatment, its effects in a series of human breast cancer cells were compared to those of (+)-discodermolide. Growth inhibition, cell and nuclear morphological, and electrophoretic and flow cytometric analyses were performed on (+)-discodermolide-treated MCF-7 and MDA-MB231 cells. (+)-Discodermolide potently inhibited the growth of both cell types (IC50 < 2.5 nM) at concentrations similar to those observed with paclitaxel. Complete inhibition of growth occurred with 10 nM or greater of each drug and was not reversed by removal. (+)-Discodermolide-treated cells exhibited condensed and highly fragmented nuclei. Flow cytometric comparison of cells treated with either drug at 10 nM, a concentration well below that achieved clinically with paclitaxel, showed both caused cell cycle perturbation and induction of a hypodiploid cell population. (+)-Discodermolide caused these effects more extensively and at earlier time points. The timing and type of high molecular weight DNA fragmentation induced by the two agents was consistent with induction of apoptosis. The results suggest that (+)-discodermolide has promise as a new chemotherapeutic agent against breast and other cancers.

  14. The cyclophilin-binding agent Sanglifehrin A is a dendritic cell chemokine and migration inhibitor.

    PubMed

    Immecke, Sabrina N; Baal, Nelli; Wilhelm, Jochen; Bechtel, Juliane; Knoche, Angela; Bein, Gregor; Hackstein, Holger

    2011-01-01

    Sanglifehrin A (SFA) is a cyclophilin-binding immunosuppressant but the immunobiology of action is poorly understood. We and others have reported that SFA inhibits IL-12 production and antigen uptake in dendritic cells (DC) and exhibits lower activity against lymphocytes. Here we show that SFA suppresses DC chemokine production and migration. Gene expression analysis and subsequent protein level confirmation revealed that SFA suppressed CCL5, CCL17, CCL19, CXCL9 and CXCL10 expression in human monocyte-derived DC (moDC). A systems biology analysis, Onto Express, confirmed that SFA interferes with chemokine-chemokine receptor gene expression with the highest impact. Direct comparison with the related agent cyclosporine A (CsA) and dexamethasone indicated that SFA uniquely suppresses moDC chemokine expression. Competitive experiments with a 100-fold molar excess of CsA and with N-Methyl-Val-4-cyclosporin, representing a nonimmunosuppressive derivative of CsA indicated chemokine suppression through a cyclophilin-A independent pathway. Functional assays confirmed reduced migration of CD4+ Tcells and moDCs to supernatant of SFA-exposed moDCs. Vice versa, SFA-exposed moDC exhibited reduced migration against CCL19. Moreover, SFA suppressed expression of the ectoenzyme CD38 that was reported to regulate DC migration and cytokine production. These results identify SFA as a DC chemokine and migration inhibitor and provide novel insight into the immunobiology of SFA. PMID:21483789

  15. The Cyclophilin-Binding Agent Sanglifehrin A Is a Dendritic Cell Chemokine and Migration Inhibitor

    PubMed Central

    Immecke, Sabrina N.; Baal, Nelli; Wilhelm, Jochen; Bechtel, Juliane; Knoche, Angela; Bein, Gregor; Hackstein, Holger

    2011-01-01

    Sanglifehrin A (SFA) is a cyclophilin-binding immunosuppressant but the immunobiology of action is poorly understood. We and others have reported that SFA inhibits IL-12 production and antigen uptake in dendritic cells (DC) and exhibits lower activity against lymphocytes. Here we show that SFA suppresses DC chemokine production and migration. Gene expression analysis and subsequent protein level confirmation revealed that SFA suppressed CCL5, CCL17, CCL19, CXCL9 and CXCL10 expression in human monocyte-derived DC (moDC). A systems biology analysis, Onto Express, confirmed that SFA interferes with chemokine-chemokine receptor gene expression with the highest impact. Direct comparison with the related agent cyclosporine A (CsA) and dexamethasone indicated that SFA uniquely suppresses moDC chemokine expression. Competitive experiments with a 100-fold molar excess of CsA and with N-Methyl-Val-4-cyclosporin, representing a nonimmunosuppressive derivative of CsA indicated chemokine suppression through a cyclophilin-A independent pathway. Functional assays confirmed reduced migration of CD4+ Tcells and moDCs to supernatant of SFA-exposed moDCs. Vice versa, SFA-exposed moDC exhibited reduced migration against CCL19. Moreover, SFA suppressed expression of the ectoenzyme CD38 that was reported to regulate DC migration and cytokine production. These results identify SFA as a DC chemokine and migration inhibitor and provide novel insight into the immunobiology of SFA. PMID:21483789

  16. Metabolic shift from withasteroid formation to phenylpropanoid accumulation in cryptogein-cotransformed hairy roots of Withania somnifera (L.) Dunal.

    PubMed

    Sil, Bipradut; Mukherjee, Chiranjit; Jha, Sumita; Mitra, Adinpunya

    2015-07-01

    Cotransformed hairy roots containing a gene that encodes a fungal elicitor protein, β-cryptogein, were established in Withania somnifera, a medicinal plant widely used in Indian systems of medicine. To find out whether β-cryptogein protein endogenously elicits the pathway of withasteroid biosynthesis, withaferin A and withanolide A contents along with transcript accumulation of farnesyl pyrophosphate (FPP) synthase, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), and sterol glycosyltransferase (SGT) were analyzed in both cryptogein-cotransformed and normal hairy roots of W. somnifera. It was observed that the withaferin A and withanolide A contents were drastically higher in normal hairy roots than cryptogein-cotransformed ones. Similar trends were also observed on the levels of transcript accumulation. Subsequently, the enzyme activity of phenylalanine ammonia lyase (PAL), one of the key enzymes of phenylpropanoid pathway, was measured in both cryptogein-cotransformed and normal hairy roots of W. somnifera along with the levels of PAL transcript accumulation. Upliftment of PAL activity was observed in cryptogein-cotransformed hairy roots as compared to the normal ones, and the PAL expression also reflected a similar trend, i.e., enhanced expression in the cryptogein-cotransformed lines. Upliftment of wall-bound ferulic acid accumulation was also observed in the cryptogein-cotransformed lines, as compared to normal hairy root lines. Thus, the outcome of the above studies suggests a metabolic shift from withanolide accumulation to phenylpropanoid biosynthesis in cryptogein-cotransformed hairy roots of W. somnifera.

  17. Production of the biopesticide azadirachtin by hairy root cultivation of Azadirachta indica in liquid-phase bioreactors.

    PubMed

    Srivastava, Smita; Srivastava, Ashok K

    2013-11-01

    Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2 g l(-1)dry weight and azadirachtin yield of 3.2 mg g(-1) leading to a volumetric productivity of azadirachtin as 1.14 mg l(-1) day(-1). The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97 % at a concentration of 2 % w/v and 83 % at a concentration of 0.05 % (w/v), respectively, in ethanol).

  18. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

    PubMed

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-01-01

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

  19. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

    PubMed Central

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-01-01

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

  20. The seminiferous epithelial cycle and microanatomy of the koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) testis.

    PubMed

    Oishi, Motoharu; Takahashi, Mei; Amasaki, Hajime; Janssen, Tina; Johnston, Stephen D

    2013-03-01

    The koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) are iconic Australian fauna that share a close phylogenetic relationship but there are currently no comparative studies of the seminiferous epithelial cell or testicular microanatomy of either species. Koala and wombat spermatozoa are unusual for marsupials as they possess a curved stream-lined head and lateral neck insertion that superficially is similar to murid spermatozoa; the koala also contains Sertoli cells with crystalloid inclusions that closely resemble the Charcot-Bottcher crystalloids described in human Sertoli cells. Eighteen sexually mature koalas and four sexually mature southern hairy-nosed (SHN) wombats were examined to establish base-line data on quantitative testicular histology. Dynamics of the seminiferous epithelial cycle in the both species consisted of eight stages of cellular association similar to that described in other marsupials. Both species possessed a high proportion of the pre-meiotic (stages VIII, I - III; koala - 62.2 ± 1.7% and SHN wombat - 66.6 ± 2.4%) when compared with post-meiotic stages of the seminiferous cycle. The mean diameters of the seminiferous tubules found in the koalas and the SHN wombats were 227.8 ± 6.1 and 243.5 ± 3.9 μm, respectively. There were differences in testicular histology between the species including the koala possessing (i) a greater proportion of Leydig cells, (ii) larger Sertoli cell nuclei, (iii) crystalloids in the Sertoli cell cytoplasm, (iv) a distinctive acrosomal granule during spermiogenesis and (v) a highly eosinophilic acrosome. An understanding of the seminiferous epithelial cycle and microanatomy of testis is fundamental for documenting normal spermatogenesis and testicular architecture; recent evidence of orchitis and epididymitis associated with natural chlamydial infection in the koala suggest that this species might be useful as an experimental model for understanding Chlamydia

  1. The seminiferous epithelial cycle and microanatomy of the koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) testis.

    PubMed

    Oishi, Motoharu; Takahashi, Mei; Amasaki, Hajime; Janssen, Tina; Johnston, Stephen D

    2013-03-01

    The koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) are iconic Australian fauna that share a close phylogenetic relationship but there are currently no comparative studies of the seminiferous epithelial cell or testicular microanatomy of either species. Koala and wombat spermatozoa are unusual for marsupials as they possess a curved stream-lined head and lateral neck insertion that superficially is similar to murid spermatozoa; the koala also contains Sertoli cells with crystalloid inclusions that closely resemble the Charcot-Bottcher crystalloids described in human Sertoli cells. Eighteen sexually mature koalas and four sexually mature southern hairy-nosed (SHN) wombats were examined to establish base-line data on quantitative testicular histology. Dynamics of the seminiferous epithelial cycle in the both species consisted of eight stages of cellular association similar to that described in other marsupials. Both species possessed a high proportion of the pre-meiotic (stages VIII, I - III; koala - 62.2 ± 1.7% and SHN wombat - 66.6 ± 2.4%) when compared with post-meiotic stages of the seminiferous cycle. The mean diameters of the seminiferous tubules found in the koalas and the SHN wombats were 227.8 ± 6.1 and 243.5 ± 3.9 μm, respectively. There were differences in testicular histology between the species including the koala possessing (i) a greater proportion of Leydig cells, (ii) larger Sertoli cell nuclei, (iii) crystalloids in the Sertoli cell cytoplasm, (iv) a distinctive acrosomal granule during spermiogenesis and (v) a highly eosinophilic acrosome. An understanding of the seminiferous epithelial cycle and microanatomy of testis is fundamental for documenting normal spermatogenesis and testicular architecture; recent evidence of orchitis and epididymitis associated with natural chlamydial infection in the koala suggest that this species might be useful as an experimental model for understanding Chlamydia

  2. The seminiferous epithelial cycle and microanatomy of the koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) testis

    PubMed Central

    Oishi, Motoharu; Takahashi, Mei; Amasaki, Hajime; Janssen, Tina; Johnston, Stephen D

    2013-01-01

    The koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) are iconic Australian fauna that share a close phylogenetic relationship but there are currently no comparative studies of the seminiferous epithelial cell or testicular microanatomy of either species. Koala and wombat spermatozoa are unusual for marsupials as they possess a curved stream-lined head and lateral neck insertion that superficially is similar to murid spermatozoa; the koala also contains Sertoli cells with crystalloid inclusions that closely resemble the Charcot–Bottcher crystalloids described in human Sertoli cells. Eighteen sexually mature koalas and four sexually mature southern hairy-nosed (SHN) wombats were examined to establish base-line data on quantitative testicular histology. Dynamics of the seminiferous epithelial cycle in the both species consisted of eight stages of cellular association similar to that described in other marsupials. Both species possessed a high proportion of the pre-meiotic (stages VIII, I – III; koala – 62.2 ± 1.7% and SHN wombat – 66.6 ± 2.4%) when compared with post-meiotic stages of the seminiferous cycle. The mean diameters of the seminiferous tubules found in the koalas and the SHN wombats were 227.8 ± 6.1 and 243.5 ± 3.9 μm, respectively. There were differences in testicular histology between the species including the koala possessing (i) a greater proportion of Leydig cells, (ii) larger Sertoli cell nuclei, (iii) crystalloids in the Sertoli cell cytoplasm, (iv) a distinctive acrosomal granule during spermiogenesis and (v) a highly eosinophilic acrosome. An understanding of the seminiferous epithelial cycle and microanatomy of testis is fundamental for documenting normal spermatogenesis and testicular architecture; recent evidence of orchitis and epididymitis associated with natural chlamydial infection in the koala suggest that this species might be useful as an experimental model for understanding Chlamydia

  3. Selecting bioactive phenolic compounds as potential agents to inhibit proliferation and VEGF expression in human ovarian cancer cells

    PubMed Central

    HE, ZHIPING; LI, BO; RANKIN, GARY O.; ROJANASAKUL, YON; CHEN, YI CHARLIE

    2015-01-01

    Ovarian cancer is a disease that continues to cause mortality in female individuals worldwide. Ovarian cancer is challenging to treat due to emerging resistance to chemotherapy, therefore, the identification of effective novel chemotherapeutic agents is important. Polyphenols have demonstrated potential in reducing the risk of developing numerous types of cancer, as well reducing the risk of cancer progression, due to their ability to reduce cell viability and vascular endothelial growth factor (VEGF) expression. In the present study, eight phenolic compounds were screened in two human ovarian cancer cell lines (OVCAR-3 and A2780/CP70) to determine their effect on proliferation suppression and VEGF protein secretion inhibition, in comparison to cisplatin, a conventional chemotherapeutic agent. The current study identified that 40 μM gallic acid (GA) exhibited the greatest inhibitory effect on OVCAR-3 cell viability, compared with all of the phenolic compounds investigated. Similarly to cisplatin, baicalein, GA, nobiletin, tangeretin and baicalin were all identified to exhibit significant VEGF inhibitory effects from ELISA results. Furthermore, western blot analysis indicated that GA effectively decreased the level of the VEGF-binding protein hypoxia-inducible factor-1α in the ovarian cancer cell line. Considering the results of the present study, GA appears to inhibit cell proliferation and, thus, is a potential agent for the treatment of ovarian cancer. PMID:25663929

  4. Reactive Oxygen Species (ROS) Inducible DNA Cross-Linking Agents and Their Effect on Cancer Cells and Normal Lymphocytes

    PubMed Central

    2015-01-01

    Reducing host toxicity is one of the main challenges of cancer chemotherapy. Many tumor cells contain high levels of ROS that make them distinctively different from normal cells. We report a series of ROS-activated aromatic nitrogen mustards that selectively kill chronic lymphocytic leukemia (CLL) over normal lymphocytes. These agents showed powerful DNA cross-linking abilities when coupled with H2O2, one of the most common ROS in cancer cells, whereas little DNA cross-linking was detected without H2O2. Consistent with chemistry observation, in vitro cytotoxicity assay demonstrated that these agents induced 40–80% apoptosis in primary leukemic lymphocytes isolated from CLL patients but less than 25% cell death to normal lymphocytes from healthy donors. The IC50 for the most potent compound (2) was ∼5 μM in CLL cells, while the IC50 was not achieved in normal lymphocytes. Collectively, these data provide utility and selectivity of these agents that will inspire further and effective applications. PMID:24801734

  5. The sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate induces ER stress in human astrocytoma cells.

    PubMed

    Arima, Yosuke; Shiraishi, Hiroaki; Saito, Atsushi; Yoshimoto, Kanji; Namera, Akira; Makita, Ryosuke; Murata, Kazuhiro; Imaizumi, Kazunori; Nagao, Masataka

    2016-01-01

    Organophosphorus (OP) compounds such as sarin are toxic agents that irreversibly inhibit the enzyme acetylcholinesterase. A recent study showed that OP compounds also have multiple toxicity mechanisms, and another suggested that endoplasmic reticulum (ER) dysfunction contributes to OP toxicity. However, the signaling pathway and mechanisms involved are poorly understood. We examined whether the sarin-like OP agent bis(isopropyl methyl)phosphonate (BIMP), which exhibits toxicity similar to that of sarin, induced ER stress in human astrocytoma CCF-STTG1 cells. Our results demonstrate that BIMP exposure reduced cell viability. Moreover, it induced changes in mitochondrial membrane potential and increased cleavage of caspase 3. Treatment with BIMP increased the mRNA levels of the ER stress marker genes binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP). Furthermore, BIMP increased the protein expressions and phosphorylation of BiP, CHOP, and protein kinase RNA-like ER kinase and the phosphorylation of eukaryotic translation initiation factor 2. Compared to BIMP treatment alone, pretreatment with the CHOP siRNA, siCHOP, decreased BIMP-dependent CHOP expression and improved CCF-STTG1 cell viability. Our findings suggest that BIMP induced mitochondrial dysfunction and apoptotic cell death event mediated by ER stress in CCF-STTG1 cells and that treatment targeted at managing ER stress has the potential to attenuate the toxicity of OP nerve agents. PMID:27665771

  6. Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents

    SciTech Connect

    Wan, X. Steven; Ware, Jeffrey H.; Zhou, Zhaozong; Donahue, Jeremiah J.; Guan, Jun; Kennedy, Ann R. . E-mail: akennedy@mail.med.upenn.edu

    2006-04-01

    Purpose: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. Methods and Materials: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, {alpha}-lipoic acid, L-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, {gamma}-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. Results: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. Conclusion: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects.

  7. A comparison of strategies for multiple-gene co-transformation via hairy root induction.

    PubMed

    Huang, Yu; Su, Ching-Yueh; Kuo, Han-Jung; Chen, Yi-Hung; Huang, Pung-Ling; Lee, Kung-Ta

    2013-10-01

    Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90 % of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1 %, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes. PMID:23812331

  8. Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of two-chemical elicitors, salicylic acid and methyl jasmonate, on the production of gossypol, 6-methoxy gossypol, and 6,6'-dimethoxy gossypol in Gossypium barbadense hairy roots was examined. Methyl jasmonate, but not salicylic acid, was found to increase the production of gossypol and ...

  9. Thermally Reversible Physically Cross-Linked Hybrid Network Hydrogels Formed by Thermosensitive Hairy Nanoparticles.

    PubMed

    Wright, Roger A E; Henn, Daniel M; Zhao, Bin

    2016-08-18

    This Article reports on thermally induced reversible formation of physically cross-linked, three-dimensional network hydrogels from aqueous dispersions of thermosensitive diblock copolymer brush-grafted silica nanoparticles (hairy NPs). The hairy NPs consisted of a silica core, a water-soluble polyelectrolyte inner block of poly(2-(methacryloyloxy)ethyltrimethylammonium iodide), and a thermosensitive poly(methoxydi(ethylene glycol) methacrylate) (PDEGMMA) outer block synthesized by sequential surface-initiated atom transfer radical polymerizations and postpolymerization quaternization of tertiary amine moieties. Moderately concentrated dispersions of these hairy nanoparticles in water underwent thermally induced reversible transitions between flowing liquids to self-supporting gels upon heating. The gelation was driven by the lower critical solution temperature (LCST) transition of the PDEGMMA outer block, which upon heating self-associated into hydrophobic domains acting as physical cross-linking points for the gel network. Rheological studies showed that the sol-gel transition temperature decreased with increasing hairy NP concentration, and the gelation was achieved at concentrations as low as 3 wt %. PMID:27455167

  10. Effectiveness of herbicides for control of hairy vetch (Vicia villosa) in winter wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We conducted a field experiment in 2009-10 at Pennsylvania and Maryland locations, and repeated it in 2010-11, to test the effectiveness of post-emergent herbicides applied at fall and spring timings on seeded hairy vetch in winter wheat. We tested 16 herbicide treatment combinations that included ...

  11. Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch genotypes with resident soil rhizobia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Presence of compatible rhizobia strains is essential for nodulation and BNF of hairy vetch (Vicia villosa, HV). We evaluated how past HV cultivation affects nodulation and nitrogen fixation across host genotypes. Five groups of HV genotypes were inoculated with soil dilutions from six paired fields,...

  12. Genetic diversity of resident soil rhizobia isolated from nodules of distinct hairy vetch genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hairy vetch (Vicia villosa Roth) is widely grown as a legume cover crop throughout the U.S.A., with biological nitrogen fixation (BNF) through symbiosis with Rhizobium leguminosarum biovar viciae (Rlv) being one of the most sought after benefits of its cultivation. This study determined if HV culti...

  13. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.

    PubMed

    Kai, Guoyin; Xu, Hui; Zhou, Congcong; Liao, Pan; Xiao, Jianbo; Luo, Xiuqin; You, Lijia; Zhang, Lin

    2011-05-01

    Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.

  14. Induction of functional CD154 (CD40 ligand) in neonatal T cells by cAMP-elevating agents

    PubMed Central

    Suárez, A; Mozo, L; Gayo, A; Simó, A; Gutiérrez, C

    2000-01-01

    A deficiency of neonatal T lymphocytes to express CD154 antigen in response to ionomycin and phorbol 12-myrsistate 13-acetate (PMA) stimulation or after CD3 cross-linking has been described. In the present report we describe that CD45RA+ newborn cells are able to synthesize and express CD154 at similar or even higher levels than adult cells in response to ionomycin and cAMP-elevating agents which trigger the protein kinase A (PKA) -mediated metabolic pathway. Peak CD154 protein concentrations in newborn cells were found between 4 and 8 hr after stimulation with ionomycin and dibutyryl cAMP. These agents, however, did not induce expression of the early activation antigen CD69. Surface levels of CD154 did not correlate with specific mRNA concentration, indicating that dibutyryl cAMP up-regulates CD154 by acting at a post-transcriptional stage. The CD154 antigen induced by PKA activation of newborn cells was functional, since upon binding to CD40 on B lymphocytes in the presence of interleukin-4 (IL-4), it promoted immunoglobulin heavy-class switching to IgE. We also found a different pattern of cytokine production between neonatal and adult CD4+ T cells. In response to ionomycin and dibutyryl cAMP, cord blood cells were more prone than adult lymphocytes to secrete the T helper type 2-derived immunosuppressive cytokines IL-4 and IL-10. Taking into account that the feto–maternal environment is rich in cAMP-elevating agents, the reduced risk of graft versus host disease associated with cord blood trasplantation, as compared with the risk with adult bone marrow cell transplants, may be due to the bias of neonatal cells to differentiate towards the T helper type 2 functional cell subset. PMID:10929069

  15. Transformation of Nasturtium officinale, Barbarea verna and Arabis caucasica for hairy roots and glucosinolate-myrosinase system production.

    PubMed

    Wielanek, Marzena; Królicka, Aleksandra; Bergier, Katarzyna; Gajewska, Ewa; Skłodowska, Maria

    2009-06-01

    Hairy roots of Nasturtium officinale, Barbarea verna and Arabis caucasica with active glucosinolate-myrosinase system were obtained after transformation with Agrobacterium rhizogenes. Hairy roots of N. officinale produced phenylalanine-derived gluconasturtiin and glucotropaeolin (max. 24 and 7 mg g(-1) DW). B. verna and A. caucasica hairy roots produced gluconasturtiin (max. 41 mg g(-1) DW) and methionine-derived glucoiberverin (max. 32 mg g(-1) DW), respectively. Treatment of the roots with amino acid precursors of glucosinolate or/and cysteine biosynthesis increased levels of glucosinolate production, combinations of phenylalanine with cysteine (for gluconasturtiin and glucotropaeolin) and methionine with o-acetylserine (for glucoiberverin) were the most effective.

  16. Severe respiratory distress at birth caused by a hairy polyp of the Eustachian tube: Transoral endoscopy-guided treatment.

    PubMed

    Cantarella, Giovanna; Gaffuri, Michele; Pugni, Lorenza; Pignataro, Lorenzo; Mosca, Fabio

    2015-08-01

    Hairy polyps are rare developmental lesions, which present as masses mainly consisting of fatty tissue covered by skin, seldom localized in the nasopharynx, causing respiratory obstruction. We describe the case of a female newborn affected by a hairy polyp arising from the left Eustachian tube, who presented severe respiratory distress soon after birth. The polyp was successfully removed transorally under videoendoscopic guidance. This case highlights the importance of including hairy polyp in the differential diagnosis of respiratory distress at birth because this type of tumor can be lethal and requires prompt treatment. A transoral endoscopy-guided approach can allow successful and minimally invasive excision even in a newborn.

  17. Echium acanthocarpum hairy root cultures, a suitable system for polyunsaturated fatty acid studies and production

    PubMed Central

    2011-01-01

    Background The therapeutic and health promoting role of highly unsaturated fatty acids (HUFAs) from fish, i.e. eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are well known. These same benefits may however be shared by some of their precursors, the polyunsaturated fatty acids (PUFAs), such as stearidonic acid (SDA, 18:4 n-3). In order to obtain alternative sources for the large-scale production of PUFAs, new searches are being conducted focusing on higher plants oils which can contain these n-3 and n-6 C18 precursors, i.e. SDA and GLA (18:3n-6, γ-linolenic acid). Results The establishment of the novel Echium acanthocarpum hairy root cultures represents a powerful tool in order to research the accumulation and metabolism of fatty acids (FAs) in a plant particularly rich in GLA and SDA. Furthermore, this study constitutes the first example of a Boraginaceae species hairy root induction and establishment for FA studies and production. The dominant PUFAs, 18:2n-6 (LA, linoleic acid) and 18:3n-6 (GLA), accounted for about 50% of total FAs obtained, while the n-3 PUFAs, 18:3n-3 (ALA, α-linolenic acid) and 18:4n-3 (SDA), represented approximately 5% of the total. Production of FAs did not parallel hairy root growth, and the optimal productivity was always associated with the highest biomass density during the culture period. Assuming a compromise between FA production and hairy root biomass, it was determined that sampling times 4 and 5 gave the most useful FA yields. Total lipid amounts were in general comparable between the different hairy root lines (29.75 and 60.95 mg/g DW), with the major lipid classes being triacylglycerols. The FAs were chiefly stored in the hairy roots with very minute amounts being released into the liquid nutrient medium. Conclusions The novel results presented here show the utility and high potential of E. acanthocarpum hairy roots. They are capable of biosynthesizing and accumulating a large range of

  18. Nucleotide sequence variation of GLABRA1 contributing to phenotypic variation of leaf hairiness in Brassicaceae vegetables.

    PubMed

    Li, Feng; Zou, Zhongwei; Yong, Hui-Yee; Kitashiba, Hiroyasu; Nishio, Takeshi

    2013-05-01

    GLABRA1 (GL1) belongs to the group of R2R3-MYB transcription factors and is known to be essential for trichome initiation in Arabidopsis. In our previous study, we identified a GL1 ortholog in Brassica rapa as a candidate for the gene controlling leaf hairiness by QTL analysis and suggested that a 5-bp deletion (B-allele) and a 2-bp deletion (D-allele) in the exon 3 of BrGL1 and a non-synonymous SNP (C-allele) in the second nucleotide of exon 3 possibly cause leaf hairlessness. In this study, we transformed a B. rapa line having the B-allele with the A-allele (wild type) or the C-allele of BrGL1 under the control of the CaMV 35S promoter. The transgenic plants with the A-allele showed dense coverage of seedling tissues including stems, young leaves and hypocotyls with trichomes, whereas the phenotypes of those with the C-allele were unchanged. In order to obtain more information about allelic variation of GL1 in different plant lineages and its correlation with leaf hairiness, two GL1 homologs, i.e., RsGL1a and RsGL1b, in Raphanus sativus were analyzed. Allelic variation of RsGL1a between a hairless line and a hairy line was completely associated with hairiness in their BC1F1 population. Comparison of the full-length of RsGL1a in the hairless and hairy lines showed great variation of nucleotides in the 3' end, which might be essential for its function and expression.

  19. Alkylating agent melphalan augments the efficacy of adoptive immunotherapy using tumor-specific CD4+ T cells

    PubMed Central

    Lu, Xiaoyun; Ding, Zhi-Chun; Cao, Yang; Liu, Chufeng; Habtetsion, Tsadik; Yu, Miao; Lemos, Henrique; Salman, Huda; Xu, Hongyan; Mellor, Andrew L.; Zhou, Gang

    2014-01-01

    In recent years the immune-potentiating effects of some widely used chemotherapeutic agents have been increasingly appreciated. This provides a rationale for combining conventional chemotherapy with immunotherapy strategies to achieve durable therapeutic benefits. Previous studies have implicated the immunomodulatory effects of melphalan, an alkylating agent commonly used to treat multiple myeloma, but the underlying mechanisms remain obscure. In the current study, we investigated the impact of melphalan on endogenous immune cells as well as adoptively transferred tumor-specific CD4+ T cells in tumor-bearing mice. We showed that melphalan treatment resulted in a rapid burst of inflammatory cytokines and chemokines during the cellular recovery phase after melphalan-induced myelo-leukodepletion. After melphalan treatment, tumor cells exhibited characteristics of immunogenic cell death, including membrane translocation of the endoplasmic reticulum resident calreticulin (CRT), and extracellular release of high-mobility group box 1 (HMGB1). In addition, there was enhanced tumor antigen uptake by dendritic cells in the tumor-draining lymph node. Consistent with these immunomodulatory effects, melphalan treatment of tumor-bearing mice led to the activation of the endogenous CD8+ T cells, and more importantly, effectively drove the clonal expansion and effector differentiation of adoptively transferred tumor-specific CD4+ T cells. Notably, the combination of melphalan and CD4+ T-cell adoptive cell therapy (ACT) was more efficacious than either treatment alone in prolonging the survival of mice with advanced B-cell lymphomas or colorectal tumors. These findings provide mechanistic insights into melphalan’s immunostimulatory effects, and demonstrate the therapeutic potential of combining melphalan with adoptive cell therapy utilizing antitumor CD4+ T cells. PMID:25560408

  20. Use of non-melanocytic HEK293 cells stably expressing human tyrosinase for the screening of anti-melanogenic agents.

    PubMed

    Kim, Mijin; An, Sang Mi; Koh, Jae-Sook; Jang, Dong-Il; Boo, Yong Chool

    2011-01-01

    Tyrosinase (TYR) from mushrooms has been inappropriately used in the screening assay for hypopigmenting agents even though its biochemical properties are different from those of human TYR. Cell-free extracts of human epidermal melanocyes (HEMs) could be another choice for the assay, but HEMs grow too slowly to get a sufficient amount of cell-free extracts. In the present study, human embryonic kidney (HEK) 293 cells were transfected with a human TYR construct to establish a cell line that grows rapidly and expresses human TYR constitutively. Cell-free extracts of the established cell line, HEK293-TYR, were tentatively used in the screening assays for 11 phenylpropanoids that have chemical structures similar to that of L-tyrosine, the substrate of TYR. Of the 11 compounds, the strongest inhibition of TYR activity was shown by p-coumaric acid (IC50, 3 μM), followed by 3-(4-hydroxyphenyl)propionic acid (50 μM) and 3-(4-hydroxyphenyl)lactic acid (70 μM). The results indicate that p-coumaric acid has an optimal chemical structure for the inhibition of TYR. The effects of these phenylpropanoids on melanin synthesis in HEMs correlated well with their effects on TYR activity in vitro. This study demonstrated that HEK293-TYR cells can be a good source of the human TYR enzymes needed in the screening assay of anti-melanogenic agents. PMID:22152495

  1. Use of non-melanocytic HEK293 cells stably expressing human tyrosinase for the screening of anti-melanogenic agents.

    PubMed

    Kim, Mijin; An, Sang Mi; Koh, Jae-Sook; Jang, Dong-Il; Boo, Yong Chool

    2011-01-01

    Tyrosinase (TYR) from mushrooms has been inappropriately used in the screening assay for hypopigmenting agents even though its biochemical properties are different from those of human TYR. Cell-free extracts of human epidermal melanocyes (HEMs) could be another choice for the assay, but HEMs grow too slowly to get a sufficient amount of cell-free extracts. In the present study, human embryonic kidney (HEK) 293 cells were transfected with a human TYR construct to establish a cell line that grows rapidly and expresses human TYR constitutively. Cell-free extracts of the established cell line, HEK293-TYR, were tentatively used in the screening assays for 11 phenylpropanoids that have chemical structures similar to that of L-tyrosine, the substrate of TYR. Of the 11 compounds, the strongest inhibition of TYR activity was shown by p-coumaric acid (IC50, 3 μM), followed by 3-(4-hydroxyphenyl)propionic acid (50 μM) and 3-(4-hydroxyphenyl)lactic acid (70 μM). The results indicate that p-coumaric acid has an optimal chemical structure for the inhibition of TYR. The effects of these phenylpropanoids on melanin synthesis in HEMs correlated well with their effects on TYR activity in vitro. This study demonstrated that HEK293-TYR cells can be a good source of the human TYR enzymes needed in the screening assay of anti-melanogenic agents.

  2. Superparamagnetic Iron Oxide Nanoparticles as MRI contrast agents for Non-invasive Stem Cell Labeling and Tracking

    PubMed Central

    Li, Li; Jiang, Wen; Luo, Kui; Song, Hongmei; Lan, Fang; Wu, Yao; Gu, Zhongwei

    2013-01-01

    Stem cells hold great promise for the treatment of multiple human diseases and disorders. Tracking and monitoring of stem cells in vivo after transplantation can supply important information for determining the efficacy of stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be the most effective and safest non-invasive technique for stem cell tracking in living bodies. Commercial superparamagnetic iron oxide nanoparticles (SPIONs) in the aid of transfection agents (TAs) have been applied to labeling stem cells. However, owing to the potential toxicity of TAs, more attentions have been paid to develop novel SPIONs with specific surface coating or functional moieties which facilitate effective cell internalization in the absence of TAs. This review aims to summarize the recent progress in the design and preparation of SPIONs as cellular MRI probes, to discuss their applications and current problems facing in stem cell labeling and tracking, and to offer perspectives and solutions for the future development of SPIONs in this field. PMID:23946825

  3. Statistical estimation of red blood cell osmotic damage during cryoprotective agent removal from cryopreserved blood.

    PubMed

    Gong, Liangquan; Ding, Weiping; Ma, Yuncong; Sun, Sijie; Zhao, Gang; Gao, Dayong

    2013-10-01

    Statistical estimation of the osmotic damage of red blood cells (RBCs) during the removal of cryoprotective agents (CPAs) from cryopreserved blood has been a very difficult issue. In this paper, the discrete mass transfer model developed in our previous work is modified to study the volume variation of individual RBCs and thereby to estimate the osmotic damage of all RBCs statistically during CPA removal by the dilution-concentration method we proposed recently. The model is validated with respect to the experimental results either with or without RBCs. Then, it is used to investigate the effects of blood volume, hematocrit, blood and diluent flow rates on the osmotic damage of RBCs, as well as the washing time of CPAs. Our results show that both the increase of blood flow rates and the decrease of diluent flow rates can bring about a reduction in osmotic damage of RBCs; however, only the former can cause a decrease in the washing time of CPAs. The blood volume could also affect the osmotic damage of RBCs. For a given flow condition, there could exist an optimal blood volume range for the dilution-concentration system. The effect of blood volume could be alleviated by an increase in the dilution region volume. In addition, the osmotic damage of RBCs decreases as the hematocrit decreases. Therefore, in practice, the increase of blood flow rates is the best solution to reduce both the osmotic damage of RBCs and the washing time of CPAs simultaneously. A lower hematocrit in the cryopreserved blood and/or longer tubing in the dilution region are also recommended to achieve better performance for the dilution-concentration method. PMID:24835261

  4. Use of trimetasphere metallofullerene MRI contrast agent for the non-invasive longitudinal tracking of stem cells in the lung.

    PubMed

    Murphy, Sean V; Hale, Austin; Reid, Tanya; Olson, John; Kidiyoor, Amritha; Tan, Josh; Zhou, Zhiguo; Jackson, John; Atala, Anthony

    2016-04-15

    Magnetic Resonance Imaging (MRI) is a commonly used, non-invasive imaging technique that provides visualization of soft tissues with high spatial resolution. In both a research and clinical setting, the major challenge has been identifying a non-invasive and safe method for longitudinal tracking of delivered cells in vivo. The labeling and tracking of contrast agent labeled cells using MRI has the potential to fulfill this need. Contrast agents are often used to enhance the image contrast between the tissue of interest and surrounding tissues with MRI. The most commonly used MRI contrast agents contain Gd(III) ions. However, Gd(III) ions are highly toxic in their ionic form, as they tend to accumulate in the liver, spleen, kidney and bones and block calcium channels. Endohedral metallofullerenes such as trimetallic nitride endohedral metallofullerenes (Trimetasphere®) are one unique class of fullerene molecules where a Gd3N cluster is encapsulated inside a C80 carbon cage referred to as Gd3N@C80. These endohedral metallofullerenes have several advantages over small chelated Gd(III) complexes such as increased stability of the Gd(III) ion, minimal toxic effects, high solubility in water and high proton relativity. In this study, we describe the evaluation of gadolinium-based Trimetasphere® positive contrast agent for the ​in vitro labeling and in vivo tracking of human amniotic fluid-derived stem cells within lung tissue. In addition, we conducted a 'proof-of-concept' experiment demonstrating that this methodology can be used to track the homing of stem cells to injured lung tissue and provide longitudinal analysis of cell localization over an extended time course.

  5. Use of trimetasphere metallofullerene MRI contrast agent for the non-invasive longitudinal tracking of stem cells in the lung.

    PubMed

    Murphy, Sean V; Hale, Austin; Reid, Tanya; Olson, John; Kidiyoor, Amritha; Tan, Josh; Zhou, Zhiguo; Jackson, John; Atala, Anthony

    2016-04-15

    Magnetic Resonance Imaging (MRI) is a commonly used, non-invasive imaging technique that provides visualization of soft tissues with high spatial resolution. In both a research and clinical setting, the major challenge has been identifying a non-invasive and safe method for longitudinal tracking of delivered cells in vivo. The labeling and tracking of contrast agent labeled cells using MRI has the potential to fulfill this need. Contrast agents are often used to enhance the image contrast between the tissue of interest and surrounding tissues with MRI. The most commonly used MRI contrast agents contain Gd(III) ions. However, Gd(III) ions are highly toxic in their ionic form, as they tend to accumulate in the liver, spleen, kidney and bones and block calcium channels. Endohedral metallofullerenes such as trimetallic nitride endohedral metallofullerenes (Trimetasphere®) are one unique class of fullerene molecules where a Gd3N cluster is encapsulated inside a C80 carbon cage referred to as Gd3N@C80. These endohedral metallofullerenes have several advantages over small chelated Gd(III) complexes such as increased stability of the Gd(III) ion, minimal toxic effects, high solubility in water and high proton relativity. In this study, we describe the evaluation of gadolinium-based Trimetasphere® positive contrast agent for the ​in vitro labeling and in vivo tracking of human amniotic fluid-derived stem cells within lung tissue. In addition, we conducted a 'proof-of-concept' experiment demonstrating that this methodology can be used to track the homing of stem cells to injured lung tissue and provide longitudinal analysis of cell localization over an extended time course. PMID:26546729

  6. Use of trimetasphere metallofullerene MRI contrast agent for the non-invasive longitudinal tracking of stem cells in the lung

    PubMed Central

    Murphy, Sean V.; Hale, Austin; Reid, Tanya; Olson, John; Kidiyoor, Amritha; Tan, Josh; Zhou, Zhiguo; Jackson, John; Atala, Anthony

    2016-01-01

    Magnetic Resonance Imaging (MRI) is a commonly used, non-invasive imaging technique that provides visualization of soft tissues with high spatial resolution. In both a research and clinical setting, the major challenge has been identifying a non-invasive and safe method for longitudinal tracking of delivered cells in vivo. The labeling and tracking of contrast agent labeled cells using MRI has the potential to fulfill this need. Contrast agents are often used to enhance the image contrast between the tissue of interest and surrounding tissues with MRI. The most commonly used MRI contrast agents contain Gd(III) ions. However, Gd(III) ions are highly toxic in their ionic form, as they tend to accumulate in the liver, spleen, kidney and bones and block calcium channels. Endohedral metallofullerenes such as trimetallic nitride endohedral metallofullerenes (Trimetasphere®) are one unique class of fullerene molecules where a Gd3N cluster is encapsulated inside a C80 carbon cage referred to as Gd3N@C80. These endohedral metallofullerenes have several advantages over small chelated Gd(III) complexes such as increased stability of the Gd(III) ion, minimal toxic effects, high solubility in water and high proton relativity. In this study, we describe the evaluation of gadolinium-based Trimetasphere® positive contrast agent for the in vitro labeling and in vivo tracking of human amniotic fluid-derived stem cells within lung tissue. In addition, we conducted a ‘proof-of-concept’ experiment demonstrating that this methodology can be used to track the homing of stem cells to injured lung tissue and provide longitudinal analysis of cell localization over an extended time course. PMID:26546729

  7. Cell proliferation in cancer prevention; effects of preventive agents on estrogen-related endometrial carcinogenesis model and on an in vitro model in human colorectal cells.

    PubMed

    Mori, H; Niwa, K; Zheng, Q; Yamada, Y; Sakata, K; Yoshimi, N

    2001-09-01

    Proto-oncogenes such as c-fos, c-jun and c-myc are known to relate to cell proliferation and differentiation. Some oriental herbal medicines like Glycyrrhizae radix or Juzen-taiho-to were found to suppress estradiol-17 beta (E2)-induced expression of c-fos/jun in uterine corpus and inhibited N-methyl-N-nitrosourea and E2-induced endometrial carcinogenesis in mice. It is suggested that the effects of such oriental drugs are exerted probably through suppression of estrogen-induced c-fos/jun expression and they are promising preventing agents for endometrial cancers. In the combined in vitro assay for cell proliferation (MTS assay) and apoptosis (DNA fragmentation) in human colorectal cancer cells (Colo 320), a number of naturally occurring chemopreventive agents such as curcumin, quercetin, auraptene, 1'-acetoxychavicol acetate (ACA) and indole-3-carbinol were shown to generate apoptosis as well as to inhibit cell proliferation. The results suggest a mode of action of these chemopreventive agents and also imply that such in vitro short term assay is useful for detection of new agents for cancer prevention.

  8. Differential responses of skin cancer-chemopreventive agents silibinin, quercetin, and epigallocatechin 3-gallate on mitogenic signaling and cell cycle regulators in human epidermoid carcinoma A431 cells.

    PubMed

    Bhatia, N; Agarwal, C; Agarwal, R

    2001-01-01

    Silibinin, quercetin, and epigallocatechin 3-gallate (EGCG) have been shown to be skin cancer-preventive agents, albeit by several different mechanisms. Here, we assessed whether these agents show their cancer-preventive potential by a differential effect on mitogenic signaling molecules and cell cycle regulators. Treatment of human epidermoid carcinoma A431 cells with these agents inhibited the activation of the epidermal growth factor receptor and the downstream adapter protein Shc, but only silibinin showed a marked inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase-1 and -2 activation. In terms of cell cycle regulators, silibinin treatment showed an induction of Cip1/p21 and Kip1/p27 together with a significant decrease in cyclin-dependent kinase (CDK)-4, CDK2, and cyclin D1. Quercetin treatment, however, resulted in a moderate increase in Cip1/p21 with no change in Kip1/p27 and a decrease in CDK4 and cyclin D1. EGCG treatment also led to an induction of Cip1/p21 but no change in Kip1/27, CDK2, and cyclin D1 and a decrease in CDK4 only at low doses. Treatment of cells with these agents resulted in a strong dose- and time-dependent cell growth inhibition. A high dose of silibinin and low and high doses of quercetin and EGCG also led to cell death by apoptosis, suggesting that a lack of their inhibitory effect on mitogen-activated protein kinase-extracellular signal-regulated kinase-1 and -2 activation possibly "turns on" an apoptotic cell death response associated with their cancer-preventive and anticarcinogenic effects. Together, these results suggest that silibinin, quercetin, and EGCG exert their cancer-preventive effects by differential responses on mitogenic signaling and cell cycle regulators.

  9. An agent-based model approach to multi-phase life-cycle for contact inhibited, anchorage dependent cells.

    PubMed

    Hoehn, Ross D; Schreder, Ashley M; Rez, Mohammed Fayez Al; Kais, Sabre

    2014-12-01

    Cellular agent-based models are a technique that can be easily adapted to describe nuances of a particular cell type. Within we have concentrated on the cellular particularities of the human Endothelial Cell, explicitly the effects both of anchorage dependency and of heightened scaffold binding on the total confluence time of a system. By expansion of a discrete, homogeneous, asynchronous cellular model to account for several states per cell (phases within a cell's life); we accommodate and track dependencies of confluence time and population dynamics on these factors. Increasing the total motility time, analogous to weakening the binding between lattice and cell, affects the system in unique ways from increasing the average cellular velocity; each degree of freedom allows for control over the time length the system achieves logistic growth and confluence. These additional factors may allow for greater control over behaviors of the system. Examinations of system's dependence on both seed state velocity and binding are also enclosed.

  10. Direct cellular effects of some mediators, hormones and growth factor-like agents on denervated (isolated) rat gastric mucosal cells.

    PubMed

    Bódis, B; Karádi, O; Nagy, L; Dohoczky, C; Kolega, M; Mózsik, G

    1997-01-01

    The brain-gut axis has an important role in the mechanism of gastric cytoprotection in vivo. The aim of this study was to evaluate the in vitro effect of protective agents without any central and peripheral innervation. A mixed population of rat gastric mucosal cells was isolated by the method of Nagy et al (Gastroenterology (1994) 77, 433-443). Cells were incubated for 60 min with cytoprotective drugs such as prostacyclin, histamine, pentagastrin and PL-10 substances (synthesized parts of BPC). At the end of this incubation cells were treated by 15% ethanol for 5 min. Cell viability was tested by trypan blue exclusion test and succinic dehydrogenase activity. The following results were obtained: 1) prostacyclin, histamine and pentagastrin had no direct cytoprotective effect on isolated cells; and 2) PL-10 substances significantly protected the cells against ethanol-induced cellular damage. This led to the following conclusions: 1) in the phenomenon of gastric cytoprotection only the growth factor-like agents have a direct cellular effect; and 2) the intact peripheral innervation is basically necessary for the development of mediators and hormone-induced gastric cytoprotection. PMID:9403792

  11. Esters of Bendamustine Are by Far More Potent Cytotoxic Agents than the Parent Compound against Human Sarcoma and Carcinoma Cells

    PubMed Central

    Huber, Stefan; Huettner, Johannes Philip; Hacker, Kristina; Bernhardt, Günther; König, Jörg; Buschauer, Armin

    2015-01-01

    The alkylating agent bendamustine is approved for the treatment of hematopoietic malignancies such as non-Hodgkin lymphoma, chronic lymphocytic leukemia and multiple myeloma. As preliminary data on recently disclosed bendamustine esters suggested increased cytotoxicity, we investigated representative derivatives in more detail. Especially basic esters, which are positively charged under physiological conditions, were in the crystal violet and the MTT assay up to approximately 100 times more effective than bendamustine, paralleled by a higher fraction of early apoptotic cancer cells and increased expression of p53. Analytical studies performed with bendamustine and representative esters revealed pronounced cellular accumulation of the derivatives compared to the parent compound. In particular, the pyrrolidinoethyl ester showed a high enrichment in tumor cells and inhibition of OCT1- and OCT3-mediated transport processes, suggesting organic cation transporters to be involved. However, this hypothesis was not supported by the differential expression of OCT1 (SLC22A1) and OCT3 (SLC22A3), comparing a panel of human cancer cells. Bendamustine esters proved to be considerably more potent cytotoxic agents than the parent compound against a broad panel of human cancer cell types, including hematologic and solid malignancies (e.g. malignant melanoma, colorectal carcinoma and lung cancer), which are resistant to bendamustine. Interestingly, spontaneously immortalized human keratinocytes, as a model of “normal” cells, were by far less sensitive than tumor cells against the most potent bendamustine esters. PMID:26196503

  12. In silico modelling of a cancer stem cell-targeting agent and its effects on tumour control during radiotherapy

    PubMed Central

    Marcu, Loredana G.; Marcu, David

    2016-01-01

    Head and neck cancers (HNC), like most solid tumours, contain a subpopulation of cancer stem cells (CSC) that are commonly responsible for treatment failure. Conventional therapies are unsuccessful in controlling CSCs, thus novel, targeting therapies are needed. A promising agent is ATRA (All-trans-retinoic acid) that was shown to induce CSC differentiation, cell cycle redistribution and CSCs radiosensitisation. To add to the limited data, this work simulated the effects of ATRA on a virtual HNC and evaluated tumour response to radiotherapy. A Monte Carlo technique was employed to grow a HNC consisting of all lineages of cancer cells. The biologically realistic input parameters led to a pre-treatment CSC population of 5.9%. The Linear Quadratic model was employed to simulate radiotherapy. ATRA-induced differentiation, cell arrest and apoptosis were modelled, based on literature data. While the effect of differentiation was marginal, the strongest influence on CSC subpopulation was displayed by ATRA’s cell arrest effect via an exponential behaviour of the dose-response curve. The apoptotic effect induced by ATRA shows linear correlation between the percentage of apoptotic cells and dose required to eradicate CSCs. In conclusion, ATRA is a potent CSC-targeting agent with viable impact on tumour control when combined with radiotherapy. PMID:27573059

  13. Robust specification of sensory neurons by dual functions of charlatan, a Drosophila NRSF/REST-like repressor of extramacrochaetae and hairy.

    PubMed

    Yamasaki, Yasutoyo; Lim, Young-Mi; Niwa, Nao; Hayashi, Shigeo; Tsuda, Leo

    2011-08-01

    Sensory bristle formation in Drosophila is a well-characterized system for studying sensory organ development at the molecular level. The master proneural genes of the achaete-scute (ac-sc) complex, which encode basic-helix-loop-helix (bHLH) transcription factors, are necessary and sufficient for sensory bristle formation. charlatan (chn) was originally identified as a transcriptional activator of ac-sc gene expression through interaction with its enhancer, an activity that promotes sensory bristle development. In contrast, Chn was also identified as a functional homologue of mammalian neuron-restrictive silencing factor or RE1 silencing transcription factor (NRSF/REST), an important transcriptional repressor during vertebrate neurogenesis and stem cell development that acts through epigenetic gene silencing. Here, we report that Chn acts as a repressor of extramacrochaetae (emc) and hairy, molecules that inhibit ac-sc expression. This double-negative mechanism, together with direct activation via the achaete enhancer, increases expression of achaete and ensures robust development of sensory neurons. A mutation in the C-terminal repressor motif of Chn, which causes Chn to lose its repression activity, converted Chn to an activator of emc and hairy, suggesting that Chn is a dual functional regulator of transcription. Because chn-like sequences are found among arthropods, regulation of neuronal development by Chn-like molecules may be widely conserved. PMID:21762412

  14. Transcription of a zebrafish gene of the hairy-Enhancer of split family delineates the midbrain anlage in the neural plate.

    PubMed

    Müller, M; von Weizsäcker, E; Campos-Ortega, J A

    1996-09-01

    her5 encodes a basic helix-loop-helix (bHLH) protein with all features characteristic of the Drosophila hairy-E(spl) family. her5 is expressed in a band of cells within the neural anlage from about 90% epiboly on to at least 36 h postfertilization (hpf). After completion of brain morphogenesis, her5-expressing cells are located in the caudal region of the midbrain, at the boundary with the rhombencephalon. Labelling of cells within the her5 expression domain in the neural plate by injection of fluorescein-dextran allows their labelled progeny to be localized in the 36-hpf-old embryo using an anti-fluorescein antibody. This shows that the her5 expression domain corresponds to the midbrain primordium, including both the tectum and the tegmentum, in the neural plate. A possible function for her5 in regionalization of the brain and/or control of the midbrain-hindbrain boundary is discussed.

  15. Phosphorylcholine-Coated Semiconducting Polymer Nanoparticles as Rapid and Efficient Labeling Agents for in vivo Cell Tracking

    PubMed Central

    Pu, Kanyi; Shuhendler, Adam J.; Valta, Maija P.; Cui, Lina; Saar, Matthias; Peehl, Donna M.

    2014-01-01

    Despite the pressing need to noninvasively monitor transplanted cells in vivo with fluorescence imaging, desirable fluorescent agents with rapid labeling capability, durable brightness, and ideal biocompatibility remain lacking. Herein we report phosphorylcholine-coated near-infrared (NIR) fluorescent semiconducting polymer nanoparticles (SPNs) as a new class of rapid, efficient and cytocompatible labeling nanoagents for in vivo cell tracking. The phosphorylcholine coating results in efficient and rapid endocytosis and allows the SPN to enter cells within 0.5 h in complete culture medium apparently independent of the cell type, while its NIR fluorescence leads to a tissue penetration depth of 0.5 cm. In comparison to quantum dots and Cy5.5, the SPN is tolerant to physiologically ubiquitous reactive oxygen species ROS, resulting in durable fluorescence both in vitro and in vivo. These desirable physical and physiological properties of the SPN permit cell tracking of human renal cell carcinoma (RCC) cells in living mice at a lower limit of detection of 10,000 cells with no obvious alteration of cell phenotype after 12 days. SPNs thus could provide unique opportunities for optimizing cellular therapy and deciphering pathological processes as a cell tracking label. PMID:24668903

  16. Resistance to DNA Damaging Agents Produced Invasive Phenotype of Rat Glioma Cells-Characterization of a New in Vivo Model.

    PubMed

    Stojković, Sonja; Podolski-Renić, Ana; Dinić, Jelena; Pavković, Željko; Ayuso, Jose M; Fernández, Luis J; Ochoa, Ignacio; Pérez-García, Victor M; Pešić, Vesna; Pešić, Milica

    2016-01-01

    Chemoresistance and invasion properties are severe limitations to efficient glioma therapy. Therefore, development of glioma in vivo models that more accurately resemble the situation observed in patients emerges. Previously, we established RC6 rat glioma cell line resistant to DNA damaging agents including antiglioma approved therapies such as 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ). Herein, we evaluated the invasiveness of RC6 cells in vitro and in a new orthotopic animal model. For comparison, we used C6 cells from which RC6 cells originated. Differences in cell growth properties were assessed by real-time cell analyzer. Cells' invasive potential in vitro was studied in fluorescently labeled gelatin and by formation of multicellular spheroids in hydrogel. For animal studies, fluorescently labeled cells were inoculated into adult male Wistar rat brains. Consecutive coronal and sagittal brain sections were analyzed 10 and 25 days post-inoculation, while rats' behavior was recorded during three days in the open field test starting from 25th day post-inoculation. We demonstrated that development of chemoresistance induced invasive phenotype of RC6 cells with significant behavioral impediments implying usefulness of orthotopic RC6 glioma allograft in preclinical studies for the examination of new approaches to counteract both chemoresistance and invasion of glioma cells. PMID:27355941

  17. Smart Plasmonic Glucose Nanosensors as Generic Theranostic Agents for Targeting-Free Cancer Cell Screening and Killing.

    PubMed

    Chen, Limei; Li, Haijuan; He, Haili; Wu, Haoxi; Jin, Yongdong

    2015-07-01

    Fast and accurate identification of cancer cells from healthy normal cells in a simple, generic way is very crucial for early cancer detection and treatment. Although functional nanoparticles, like fluorescent quantum dots and plasmonic Au nanoparticles (NPs), have been successfully applied for cancer cell imaging and photothermal therapy, they suffer from the main drawback of needing time-consuming targeting preparation for specific cancer cell detection and selective ablation. The lack of a generic and effective method therefore limits their potential high-throughput cancer cell preliminary screening and theranostic applications. We report herein a generic in vitro method for fast, targeting-free (avoiding time-consuming preparations of targeting moiety for specific cancer cells) visual screening and selective killing of cancer cells from normal cells, by using glucose-responsive/-sensitive glucose oxidase-modified Ag/Au nanoshells (Ag/Au-GOx NSs) as a smart plasmonic theranostic agent. The method is generic to some extent since it is based on the distinct localized surface plasmon resonance (LSPR) responses (and colors) of the smart nanoprobe with cancer cells (typically have a higher glucose uptake level) and normal cells.

  18. The differential short- and long-term effects of HIV-1 latency-reversing agents on T cell function

    PubMed Central

    Clutton, G.; Xu, Y.; Baldoni, P. L.; Mollan, K. R.; Kirchherr, J.; Newhard, W.; Cox, Kara; Kuruc, J. D.; Kashuba, A.; Barnard, R.; Archin, N.; Gay, C. L.; Hudgens, M. G.; Margolis, D. M.; Goonetilleke, N.

    2016-01-01

    Despite the extraordinary success of HIV-1 antiretroviral therapy in prolonging life, infected individuals face lifelong therapy because of a reservoir of latently-infected cells that harbor replication competent virus. Recently, compounds have been identified that can reverse HIV-1 latency in vivo. These latency- reversing agents (LRAs) could make latently-infected cells vulnerable to clearance by immune cells, including cytolytic CD8+ T cells. We investigated the effects of two leading LRA classes on CD8+ T cell phenotype and function: the histone deacetylase inhibitors (HDACis) and protein kinase C modulators (PKCms). We observed that relative to HDACis, the PKCms induced much stronger T cell activation coupled with non-specific cytokine production and T cell proliferation. When examining antigen-specific CD8+ T cell function, all the LRAs except the HDACi Vorinostat reduced, but did not abolish, one or more measurements of CD8+ T cell function. Importantly, the extent and timing of these effects differed between LRAs. Panobinostat had detrimental effects within 10 hours of drug treatment, whereas the effects of the other LRAs were observed between 48 hours and 5 days. These observations suggest that scheduling of LRA and CD8+ T cell immunotherapy regimens may be critical for optimal clearance of the HIV-1 reservoir. PMID:27480951

  19. Resistance to DNA Damaging Agents Produced Invasive Phenotype of Rat Glioma Cells-Characterization of a New in Vivo Model.

    PubMed

    Stojković, Sonja; Podolski-Renić, Ana; Dinić, Jelena; Pavković, Željko; Ayuso, Jose M; Fernández, Luis J; Ochoa, Ignacio; Pérez-García, Victor M; Pešić, Vesna; Pešić, Milica

    2016-06-27

    Chemoresistance and invasion properties are severe limitations to efficient glioma therapy. Therefore, development of glioma in vivo models that more accurately resemble the situation observed in patients emerges. Previously, we established RC6 rat glioma cell line resistant to DNA damaging agents including antiglioma approved therapies such as 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ). Herein, we evaluated the invasiveness of RC6 cells in vitro and in a new orthotopic animal model. For comparison, we used C6 cells from which RC6 cells originated. Differences in cell growth properties were assessed by real-time cell analyzer. Cells' invasive potential in vitro was studied in fluorescently labeled gelatin and by formation of multicellular spheroids in hydrogel. For animal studies, fluorescently labeled cells were inoculated into adult male Wistar rat brains. Consecutive coronal and sagittal brain sections were analyzed 10 and 25 days post-inoculation, while rats' behavior was recorded during three days in the open field test starting from 25th day post-inoculation. We demonstrated that development of chemoresistance induced invasive phenotype of RC6 cells with significant behavioral impediments implying usefulness of orthotopic RC6 glioma allograft in preclinical studies for the examination of new approaches to counteract both chemoresistance and invasion of glioma cells.

  20. Trientine, a copper-chelating agent, induced apoptosis in murine fibrosarcoma cells in vivo and in vitro.

    PubMed

    Hayashi, Masanobu; Nishiya, Hide; Chiba, Toshiaki; Endoh, Daiji; Kon, Yasuhiro; Okui, Toyo

    2007-02-01

    Anti-copper treatments have been investigated to determine whether they suppress angiogenesis and tumor development since Cu is widely accepted as being required for angiogenesis. We examined the effects of treatment with trientine, a copper-chelating agent, on tumor development in a murine xenograft model using fibrosarcoma-derived transplantable QRsp-11 cells and C57BL/6 mice and induction of apoptosis in tumor cells and endothelial cells in vivo and in vitro. The tumor volumes increased more slowly in trientine-treated mice than in untreated mice. Tumor volumes in the treated mice were significantly smaller than those in the untreated mice at 24 days postinoculation (d.p.i.) of tumor cells. A cluster of pyknotic tumor cells and morphological abnormalities in capillary endothelial cells were observed in the tumors of trientine-treated mice but not in the tumors of untreated mice. The proportions of apoptotic and necrotic cells in the tumors of treated mice were approximately 3.5-fold higher than those in the tumors of untreated mice at 14 d.p.i. When the cells were treated with trientine in vitro, mouse endothelial cells and bovine primary endothelial cells showed an approximately 10-fold higher sensitivity to trientine than QRsp-11 cells in terms of D37. However, the proportion of apoptotic cells in endothelial cells was significantly lower than that in QRsp-11 cells after treatment with trientine. These results show that apoptosis was induced in tumor cells by treatment with trientine in vivo and in vitro.

  1. Effects of phytohormones and jasmonic acid on glucosinolate content in hairy root cultures of Sinapis alba and Brassica rapa.

    PubMed

    Kastell, Anja; Smetanska, Iryna; Ulrichs, Christian; Cai, Zhenzhen; Mewis, Inga

    2013-01-01

    Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species-Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis-were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg L(-1)) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg L(-1)) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.

  2. Methyl jasmonate influence on silymarin production and plant stress responses in Silybum marianum hairy root cultures in a bioreactor.

    PubMed

    Rahimi, Shadi; Hasanloo, Tahereh; Najafi, Farzaneh; Khavari-Nejad, Ramezan Ali

    2012-01-01

    In this article our aim was to evaluate mass cultivation of S. marianum hairy roots in a bioreactor to produce silymarin. The effects of methyl jasmonate (MJ) elicitation on the accumulation of silymarin and the extent of the MJ-induced oxidative damage were investigated in bioreactor hairy root cultures of S. marianum. The growth rate of the bioreactor hairy root cultures was higher than that of those in a shake flask after 3 weeks. Silymarin accumulation was increased from 0.13 mg g⁻¹ DW in non-treated hairy roots to 0.22 mg g⁻¹ DW in hairy roots 72 h after 100 µM MJ treatment. Guaiacol peroxidase and ascorbate peroxidase were activated by MJ 72 h after treatment, being 3.2- and 1.3-fold higher, respectively, than that of the control. An increase in enzymatic activity suggests increased scavenging of reactive oxygen species, indicating the tolerance to MJ stress. These results suggest that MJ elicitation is beneficial for silymarin production using bioreactor hairy root cultures.

  3. cAMP-synthesis in a medullary thyroid carcinoma cell line: response to adrenergic agents and prostaglandines.

    PubMed

    Mertens, P R; Goretzki, P E; Keck, E

    1999-01-01

    Calcitonin secretion by C-cells is mediated through intracellular 3'5'-cyclic adenosine monophosphate (cAMP) and calcium signaling. Calcitonin release stimulation tests may take advantage of both signaling cascades in screening for medullary thyroid carcinomas (MTC). To elucidate the regulation of the adenylyl cyclase system we have determined cAMP levels of a calcitonin-expressing MTC cell line (RG) after exposure to adrenergic agents and prostaglandines. In early passages (20-30) cAMP concentrations were significantly elevated in RG cells after exposure to beta-adrenergic agents and prostaglandines E1 and E2. In advanced passages (60-80) the beta-adrenergic response was no longer detectable and adrenergic receptors were uncoupled from the adenylyl cyclase complex; while the effect of prostaglandines E1 and E2 remained unaffected. Preincubation with dexamethasone, in a process requiring protein new synthesis, re-established the adrenergic response in later passages, indicating that RG cells dedifferentiated in culture over time. Our in vitro findings suggest that MTC cell dedifferentiation may be accompanied by adrenergic receptor-uncoupling from the adenylate cyclase system and that this process may be reversed by dexamethasone incubation.

  4. The effect of varying type and volume of sedimenting agents on leukocyte harvesting and labelling in sickle cell patients.

    PubMed

    Webber, D; Nunan, T O; O'Doherty, M J

    1994-09-01

    Leukocyte labelling in patients with sickle cell anaemia has been reported as difficult if not impossible due to the slow erythrocyte sedimentation rate (ESR) in these patients. This study investigated standard sedimentation methods in patients with sickle cell disease (n = 16) and compared the results obtained with those following changes in the amount and type of sedimenting agent used. Labelling with either 111In-oxine or 99Tcm-exametazime was attempted in only five patients. Replacement of the commonly used 6% Hetastarch (Hespan) with Dextran or Haemaccel did not improve leukocyte harvesting, even when the proportions used of these agents were increased. In most cases where standard procedures for leukocyte collection did not lead to harvesting of viable samples, it was possible to obtain adequate leukocyte labelling in the majority of sickle cell patients using a minor modification of standard techniques. In this group of patients a ratio of 8 ml of Hespan to 16 ml of blood should be used for cell separation. If this fails then donor cells, anti-granulocyte antibody labelling or HIG should be considered.

  5. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

    PubMed

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M

    2016-01-01

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents. PMID:27147293

  6. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design

    NASA Astrophysics Data System (ADS)

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M.

    2016-05-01

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

  7. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design

    NASA Astrophysics Data System (ADS)

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M.

    2016-05-01

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared – non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

  8. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design

    PubMed Central

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M.

    2016-01-01

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared – non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents. PMID:27147293

  9. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

    PubMed

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M

    2016-05-05

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

  10. Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

    SciTech Connect

    McDonald, Gail T.; Sullivan, Richard; Pare, Genevieve C.; Graham, Charles H.

    2010-11-15

    Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G{sub 1} phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}; and knockdown of p27{sup kip1} with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.

  11. On-lattice agent-based simulation of populations of cells within the open-source Chaste framework.

    PubMed

    Figueredo, Grazziela P; Joshi, Tanvi V; Osborne, James M; Byrne, Helen M; Owen, Markus R

    2013-04-01

    Over the years, agent-based models have been developed that combine cell division and reinforced random walks of cells on a regular lattice, reaction-diffusion equations for nutrients and growth factors; and ordinary differential equations for the subcellular networks regulating the cell cycle. When linked to a vascular layer, this multiple scale model framework has been applied to tumour growth and therapy. Here, we report on the creation of an agent-based multi-scale environment amalgamating the characteristics of these models within a Virtual Physiological Human (VPH) Exemplar Project. This project enables reuse, integration, expansion and sharing of the model and relevant data. The agent-based and reaction-diffusion parts of the multi-scale model have been implemented and are available for download as part of the latest public release of Chaste (Cancer, Heart and Soft Tissue Environment; http://www.cs.ox.ac.uk/chaste/), part of the VPH Toolkit (http://toolkit.vph-noe.eu/). The environment functionalities are verified against the original models, in addition to extra validation of all aspects of the code. In this work, we present the details of the implementation of the agent-based environment, including the system description, the conceptual model, the development of the simulation model and the processes of verification and validation of the simulation results. We explore the potential use of the environment by presenting exemplar applications of the 'what if' scenarios that can easily be studied in the environment. These examples relate to tumour growth, cellular competition for resources and tumour responses to hypoxia (low oxygen levels). We conclude our work by summarizing the future steps for the expansion of the current system. PMID:24427527

  12. Secondary Metabolites from Plants Inhibiting ABC Transporters and Reversing Resistance of Cancer Cells and Microbes to Cytotoxic and Antimicrobial Agents

    PubMed Central

    Wink, Michael; Ashour, Mohamed L.; El-Readi, Mahmoud Zaki

    2012-01-01

    Fungal, bacterial, and cancer cells can develop resistance against antifungal, antibacterial, or anticancer agents. Mechanisms of resistance are complex and often multifactorial. Mechanisms include: (1) Activation of ATP-binding cassette (ABC) transporters, such as P-gp, which pump out lipophilic compounds that have entered a cell, (2) Activation of cytochrome p450 oxidases which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) Activation of glutathione transferase, which can conjugate xenobiotics. This review summarizes the evidence that secondary metabolites (SM) of plants, such as alkaloids, phenolics, and terpenoids can interfere with ABC transporters in cancer cells, parasites, bacteria, and fungi. Among the active natural products several lipophilic terpenoids [monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes] but also some alkaloids (isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids) function probably as competitive inhibitors of P-gp, multiple resistance-associated protein 1, and Breast cancer resistance protein in cancer cells, or efflux pumps in bacteria (NorA) and fungi. More polar phenolics (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) directly inhibit proteins forming several hydrogen and ionic bonds and thus disturbing the 3D structure of the transporters. The natural products may be interesting in medicine or agriculture as they can enhance the activity of active chemotherapeutics or pesticides or even reverse multidrug resistance, at least partially, of adapted and resistant cells. If these SM are applied in combination with a cytotoxic or antimicrobial agent, they may reverse resistance in a synergistic fashion. PMID:22536197

  13. Classification of agents using Syrian hamster embryo (SHE) cell transformation assay (CTA) with ATR-FTIR spectroscopy and multivariate analysis.

    PubMed

    Ahmadzai, Abdullah A; Trevisan, Júlio; Pang, Weiyi; Riding, Matthew J; Strong, Rebecca J; Llabjani, Valon; Pant, Kamala; Carmichael, Paul L; Scott, Andrew D; Martin, Francis L

    2015-09-01

    The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher's linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.

  14. Sarcoma Cell Line Screen of Oncology Drugs and Investigational Agents Identifies Patterns Associated with Gene and microRNA Expression.

    PubMed

    Teicher, Beverly A; Polley, Eric; Kunkel, Mark; Evans, David; Silvers, Thomas; Delosh, Rene; Laudeman, Julie; Ogle, Chad; Reinhart, Russell; Selby, Michael; Connelly, John; Harris, Erik; Monks, Anne; Morris, Joel

    2015-11-01

    The diversity in sarcoma phenotype and genotype make treatment of this family of diseases exceptionally challenging. Sixty-three human adult and pediatric sarcoma lines were screened with 100 FDA-approved oncology agents and 345 investigational agents. The investigational agents' library enabled comparison of several compounds targeting the same molecular entity allowing comparison of target specificity and heterogeneity of cell line response. Gene expression was derived from exon array data and microRNA expression was derived from direct digital detection assays. The compounds were screened against each cell line at nine concentrations in triplicate with an exposure time of 96 hours using Alamar blue as the endpoint. Results are presented for inhibitors of the following targets: aurora kinase, IGF-1R, MEK, BET bromodomain, and PARP1. Chemical structures, IC50 heat maps, concentration response curves, gene expression, and miR expression heat maps are presented for selected examples. In addition, two cases of exceptional responders are presented. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sarcoma.cancer.gov. These data provide a unique resource to the cancer research community. PMID:26351324

  15. Trapping and dynamic manipulation of polystyrene beads mimicking circulating tumor cells using targeted magnetic/photoacoustic contrast agents

    PubMed Central

    Wei, Chen-Wei; Xia, Jinjun; Hu, Xiaoge; Gao, Xiaohu; O’Donnell, Matthew

    2012-01-01

    Abstract. Results on magnetically trapping and manipulating micro-scale beads circulating in a flow field mimicking metastatic cancer cells in human peripheral vessels are presented. Composite contrast agents combining magneto-sensitive nanospheres and highly optical absorptive gold nanorods were conjugated to micro-scale polystyrene beads. To efficiently trap the targeted objects in a fast stream, a dual magnet system consisting of two flat magnets to magnetize (polarize) the contrast agent and an array of cone magnets producing a sharp gradient field to trap the magnetized contrast agent was designed and constructed. A water-ink solution with an optical absorption coefficient of 10  cm−1 was used to mimic the optical absorption of blood. Magnetomotive photoacoustic imaging helped visualize bead trapping, dynamic manipulation of trapped beads in a flow field, and the subtraction of stationary background signals insensitive to the magnetic field. The results show that trafficking micro-scale objects can be effectively trapped in a stream with a flow rate up to 12  ml/min and the background can be significantly (greater than 15 dB) suppressed. It makes the proposed method very promising for sensitive detection of rare circulating tumor cells within high flow vessels with a highly absorptive optical background. PMID:23223993

  16. Trapping and dynamic manipulation of polystyrene beads mimicking circulating tumor cells using targeted magnetic/photoacoustic contrast agents.

    PubMed

    Wei, Chen-Wei; Xia, Jinjun; Pelivanov, Ivan; Hu, Xiaoge; Gao, Xiaohu; O'Donnell, Matthew

    2012-10-01

    Results on magnetically trapping and manipulating micro-scale beads circulating in a flow field mimicking metastatic cancer cells in human peripheral vessels are presented. Composite contrast agents combining magneto-sensitive nanospheres and highly optical absorptive gold nanorods were conjugated to micro-scale polystyrene beads. To efficiently trap the targeted objects in a fast stream, a dual magnet system consisting of two flat magnets to magnetize (polarize) the contrast agent and an array of cone magnets producing a sharp gradient field to trap the magnetized contrast agent was designed and constructed. A water-ink solution with an optical absorption coefficient of 10  cm⁻¹ was used to mimic the optical absorption of blood. Magnetomotive photoacoustic imaging helped visualize bead trapping, dynamic manipulation of trapped beads in a flow field, and the subtraction of stationary background signals insensitive to the magnetic field. The results show that trafficking micro-scale objects can be effectively trapped in a stream with a flow rate up to 12  ml/min and the background can be significantly (greater than 15 dB) suppressed. It makes the proposed method very promising for sensitive detection of rare circulating tumor cells within high flow vessels with a highly absorptive optical background.

  17. Trapping and dynamic manipulation of polystyrene beads mimicking circulating tumor cells using targeted magnetic/photoacoustic contrast agents

    NASA Astrophysics Data System (ADS)

    Wei, Chen-Wei; Xia, Jinjun; Pelivanov, Ivan; Hu, Xiaoge; Gao, Xiaohu; O'Donnell, Matthew

    2012-10-01

    Results on magnetically trapping and manipulating micro-scale beads circulating in a flow field mimicking metastatic cancer cells in human peripheral vessels are presented. Composite contrast agents combining magneto-sensitive nanospheres and highly optical absorptive gold nanorods were conjugated to micro-scale polystyrene beads. To efficiently trap the targeted objects in a fast stream, a dual magnet system consisting of two flat magnets to magnetize (polarize) the contrast agent and an array of cone magnets producing a sharp gradient field to trap the magnetized contrast agent was designed and constructed. A water-ink solution with an optical absorption coefficient of 10 cm-1 was used to mimic the optical absorption of blood. Magnetomotive photoacoustic imaging helped visualize bead trapping, dynamic manipulation of trapped beads in a flow field, and the subtraction of stationary background signals insensitive to the magnetic field. The results show that trafficking micro-scale objects can be effectively trapped in a stream with a flow rate up to 12 ml/min and the background can be significantly (greater than 15 dB) suppressed. It makes the proposed method very promising for sensitive detection of rare circulating tumor cells within high flow vessels with a highly absorptive optical background.

  18. Surfactant-free Gd3+-ion-containing carbon nanotube MRI contrast agents for stem cell labeling

    NASA Astrophysics Data System (ADS)

    Gizzatov, Ayrat; Hernández-Rivera, Mayra; Keshishian, Vazrik; Mackeyev, Yuri; Law, Justin J.; Guven, Adem; Sethi, Richa; Qu, Feifei; Muthupillai, Raja; Cabreira-Hansen, Maria Da Graça; Willerson, James T.; Perin, Emerson C.; Ma, Qing; Bryant, Robert G.; Wilson, Lon J.

    2015-07-01

    There is an ever increasing interest in developing new stem cell therapies. However, imaging and tracking stem cells in vivo after transplantation remains a serious challenge. In this work, we report new, functionalized and high-performance Gd3+-ion-containing ultra-short carbon nanotube (US-tube) MRI contrast agent (CA) materials which are highly-water-dispersible (ca. 35 mg ml-1) without the need of a surfactant. The new materials have extremely high T1-weighted relaxivities of 90 (mM s)-1 per Gd3+ ion at 1.5 T at room temperature and have been used to safely label porcine bone-marrow-derived mesenchymal stem cells for MR imaging. The labeled cells display excellent image contrast in phantom imaging experiments, and TEM images of the labeled cells, in general, reveal small clusters of the CA material located within the cytoplasm with 109 Gd3+ ions per cell.There is an ever increasing interest in developing new stem cell therapies. However, imaging and tracking stem cells in vivo after transplantation remains a serious challenge. In this work, we report new, functionalized and high-performance Gd3+-ion-containing ultra-short carbon nanotube (US-tube) MRI contrast agent (CA) materials which are highly-water-dispersible (ca. 35 mg ml-1) without the need of a surfactant. The new materials have extremely high T1-weighted relaxivities of 90 (mM s)-1 per Gd3+ ion at 1.5 T at room temperature and have been used to safely label porcine bone-marrow-derived mesenchymal stem cells for MR imaging. The labeled cells display excellent image contrast in phantom imaging experiments, and TEM images of the labeled cells, in general, reveal small clusters of the CA material located within the cytoplasm with 109 Gd3+ ions per cell. Electronic supplementary information (ESI) available: NMRD profiles, the Fourier transforms of the EXAFS data, EXAFS curve fitting data, cell viability data. See DOI: 10.1039/c5nr02078f

  19. The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells.

    PubMed

    Yoshii, J; Yoshiji, H; Kuriyama, S; Ikenaka, Y; Noguchi, R; Okuda, H; Tsujinoue, H; Nakatani, T; Kishida, H; Nakae, D; Gomez, D E; De Lorenzo, M S; Tejera, A M; Fukui, H

    2001-12-15

    Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

  20. An outbreak of Vicia villosa (hairy vetch) poisoning in grazing Aberdeen Angus bulls in Argentina.

    PubMed

    Odriozola, E; Paloma, E; Lopez, T; Campero, C

    1991-06-01

    Vicia villosa (hairy vetch) is used as a forage source in some cattle-producing areas in Argentina. The plant had no previous reports of toxicity in this country. A herd of 33 Aberdeen Angus bulls grazed during 20 days in October on a pasture composed mainly of hairy vetch. Eight animals developed conjunctivitis, rinitis, dermatitis, loss of hair and fever. All of them died within 15 d after the development of signs with a marked loss of body condition. No more animals became sick 5 d after the removal of the herd from the pasture. Serum parameters tested (calcium, phosphorus, magnesium, GOT, alfa-GT and bilirubin) enlarged liver and spleen, generalized hemorrhage in the abomasum, dilated kidneys and multiple pale areas on the heart. Severe necrotizing granulomatous myocarditis, interstitial nephritis, and necrotizing cholangitis were the most striking microscopic changes. Close observation of animals feeding on pastures in which V villosa is dominant is the only prevention. PMID:1858312

  1. Hairy Root Cultures of Gymnema sylvestre R. Br. to Produce Gymnemic Acid.

    PubMed

    Rajashekar, J; Kumar, Vadlapudi; Veerashree, V; Poornima, D V; Sannabommaji, Torankumar; Gajula, Hari; Giridhara, B

    2016-01-01

    Gymnema sylvestre R. Br. (Asclepiadaceae) is an endangered species extensively used in the management of diabetes, obesity, and treatment of various diseases. Uncontrolled exploitation to meet the increasing demand and low seed viability hastens the disappearance of the plant from its natural habitat. Hairy root culture provides a suitable alternative for the enhanced production of active principles. The current protocol provides the optimized culture conditions for the establishment of hairy root cultures and elicitation studies and also confirmation of stable integration of A. rhizogenes plasmid T-DNA into host genetic material by PCR and RT-PCR. Furthermore, it also discusses the suitable methods for the extraction procedures, and qualitative and quantitative analysis of gymnemic acid by HPTLC and HPLC. PMID:27108334

  2. Root tip-dependent, active riboflavin secretion by Hyoscyamus albus hairy roots under iron deficiency.

    PubMed

    Higa, Ataru; Miyamoto, Erika; ur Rahman, Laiq; Kitamura, Yoshie

    2008-04-01

    Hyoscyamus albus hairy roots with/without an exogenous gene (11 clones) were established by inoculation of Agrobacterium rhizogenes. All clones cultured under iron-deficient condition secreted riboflavin from the root tips into the culture medium and the productivity depended on the number and size of root tips among the clones. A decline of pH was observed before riboflavin production and root development. By studying effects of proton-pump inhibitors, medium acidification with external organic acid, and riboflavin addition upon pH change and riboflavin productivity, we indicate that riboflavin efflux is not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs as a response to an internal requirement in H. albus hairy roots under iron deficiency. PMID:18367404

  3. Hairy black holes in AdS5 × S 5

    NASA Astrophysics Data System (ADS)

    Markeviciute, Julija; Santos, Jorge E.

    2016-06-01

    We use numerical methods to exhaustively study a novel family of hairy black hole solutions in AdS5. These solutions can be uplifted to solutions of type IIB supergravity with AdS5 × S 5 asymptotics and are thus expected to play an important role in our understanding of AdS/CFT. We find an intricate phase diagram, with the aforementioned family of hairy black hole solutions branching from the Reissner-Nordström black hole at the onset of the superradiance instability. We analyse black holes with spherical and planar horizon topology and explain how they connect in the phase diagram. Finally, we detail their global and local thermodynamic stability across several ensembles.

  4. Resveratrol induces catalytic bioscavenger paraoxonase 1 expression and protects against chemical warfare nerve agent toxicity in human cell lines.

    PubMed

    Curtin, Bryan F; Seetharam, Karthik I; Dhoieam, Pilin; Gordon, Richard K; Doctor, Bhupendra P; Nambiar, Madhusoodana P

    2008-04-01

    Current advances in enzyme bioscavenger prophylactic therapy against chemical warfare nerve agent (CWNA) exposure are moving towards the identification of catalytic bioscavengers that can degrade large doses of organophosphate (OP) nerve agents without self destruction. This is a preferred method compared to therapy with the purified stoichiometric bioscavenger, butyrylcholinesterase, which binds OPs 1:1 and would thus require larger doses for treatment. Paraoxonase-1 (PON-1) is one such catalytic bioscavenger that has been shown to hydrolyze OP insecticides and contribute to detoxification in animals and humans. Here we investigated the effects of a common red wine ingredient, Resveratrol (RSV), to induce the expression of PON-1 in the human hepatic cell line HC04 and evaluated the protection against CWNA simulants. Dose-response curves showed that a concentration of 20 microM RSV was optimal in inducing PON-1 expression in HC04 cells. RSV at 20 microM increased the extracellular PON-1 activity approximately 150% without significantly affecting the cells. Higher doses of RSV were cytotoxic to the cells. Resveratrol also induced PON-1 in the human lung cell line A549. RSV pre-treatment significantly (P = 0.05) protected the hepatic cells against exposure to 2x LD(50) of soman and sarin simulants. However, lung cells were protected against soman simulant exposure but not against sarin simulant exposure following RSV treatment. In conclusion, these studies indicate that dietary inducers, such as RSV, can up-regulate PON-1, a catalytic bioscavenger, which can then hydrolyze and protect against CWNA-induced toxicity, providing a prospective new method to protect against CWNA exposure.

  5. A new class of flavonol-based anti-prostate cancer agents: Design, synthesis, and evaluation in cell models.

    PubMed

    Li, Xiang; Chen, Guanglin; Zhang, Xiaojie; Zhang, Qiang; Zheng, Shilong; Wang, Guangdi; Chen, Qiao-Hong

    2016-09-01

    Flavonoids are a large class of polyphenolic compounds ubiquitously distributed in dietary plants with an array of biological activities. Flavonols are a major sub-class of flavonoids featuring a hydroxyl group at C-3. Certain natural flavonols, such as quercetin and fisetin, have been shown by in vitro cell-based and in vivo animal experiments to be potential anti-prostate cancer agents. However, the Achilles' heel of flavonols as drug candidates is their moderate potency and poor pharmacokinetic profiles. This study aims to explore the substitution effect of 3-OH in flavonols on the in vitro anti-proliferative potency against both androgen-sensitive and androgen-insensitive human prostate cancer cell lines. Our first lead flavonol (3',4'-dimethoxyflavonol), eight 3-O-alkyl-3',4'-dimethoxyflavonols, and six 3-O-aminoalkyl-3',4'-dimethoxyflavonols have been synthesized through aldol condensation and the Algar-Flynn-Oyamada (AFO) reaction. The WST-1 cell proliferation assay indicates (i) that all synthesized 3-O-alkyl-3',4'-dimethoxyflavonols and 3-O-aminoalkyl-3',4'-dimethoxyflavonols are more potent than the parent 3',4'-dimethoxyflavonol and the natural flavonol quercetin in suppressing prostate cancer cell proliferation; and (ii) that incorporation of a dibutylamino group to the 3-OH group through a three- to five-carbon linker leads to the optimal derivatives with up to 292-fold enhanced potency as compared with the parent flavonol. Flow cytometry analysis showed that the most potent derivative 22 can activate PC-3 cell cycle arrest at the G2/M phase and induce PC-3 cell apoptosis. No inhibitory ability of 22 up to 50μM concentration was observed against PWR-1E normal human epithelial prostate cells, suggesting its in vitro safety profile. The results indicate that chemical modulation at 3-OH is a vital strategy to optimize flavonols as anti-prostate cancer agents. PMID:27476422

  6. Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis).

    PubMed

    Nishiguchi, Tomoki; Cho, Kichul; Yasutomi, Masumi; Ueno, Mikinori; Yamaguchi, Kenichi; Basti, Leila; Yamasaki, Yasuhiro; Takeshita, Satoshi; Kim, Daekyung; Oda, Tatsuya

    2016-10-01

    A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both

  7. Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis).

    PubMed

    Nishiguchi, Tomoki; Cho, Kichul; Yasutomi, Masumi; Ueno, Mikinori; Yamaguchi, Kenichi; Basti, Leila; Yamasaki, Yasuhiro; Takeshita, Satoshi; Kim, Daekyung; Oda, Tatsuya

    2016-10-01

    A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both

  8. Paired-agent imaging for resection during surgery (PAIRS) of head and neck squamous cell carcinomas (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Samkoe, Kimberley S.; Tichauer, Kenneth M.; Chen, Eunice; Gunn, Jason R.; Hoopes, P. Jack; Wells, Wendy A.; Hasan, Tayyaba; Pogue, Brian W.

    2016-03-01

    Ninety percent of patients with head and neck squamous cell carcinomas (HNSCC) have overexpression of epidermal growth factor receptor (EGFR), which is correlated with poor prognosis. Complete surgical resection of HNSCC tumors has a large impact on patient survival, where detection of tumor at or close to surgical margins increases the risk of death at 5-years by 90%. In addition, large surgical margins can greatly increase the morbidity experienced by the patient due to functional and cosmetic damage of oral and facial structures. Single fluorescence targeting agents are often used for tumor detection in in vivo pre-clinical imaging; however, the arising signal is qualitative at best because it is a complex mixture of vascular perfusion, vascular leakage, inhibited lymphatic clearance, and receptor binding. In vivo ratiometric receptor concentration imaging (RCI) allows quantification of receptor expression (hence identification of cancerous tissue) by utilizing co-administered paired-agents consisting of a targeted agent and non-targeted perfusion agent to reference the plasma delivery and leakage. A panel of HNSCC tumors with varying levels of EGFR expression (SCC-15 >SCC-25 > SCC-09) have been imaged using ABY-029, a clinically relevant anti-EGFR affibody labeled with IRDye 800CW, and affibody control imaging agent labeled with IRDye 680RD. RCI maps of in vivo tissue have been created and are spatially correlated with EGFR and CD31 immunohistochemistry and basic H and E staining. The RCI threshold parameters for distinguishing tumor from normal tissues (skin and muscle) and the accuracy of margin detection in these tumors will be presented. RCI surgical resection will be further developed using a novel multi-channel, gated fluorescence-guided surgery (FGS) imaging system that is capable of performing RCI in normal room light.

  9. Application of membrane tubing aeration and perfluorocarbon To improve oxygen delivery to hairy root cultures

    PubMed

    Kanokwaree; Doran

    1998-05-01

    Growth and atropine production by Atropa belladonna hairy roots were studied in bioreactor cultures using porous polypropylene membrane tubing as a supplementary aeration device and with FC-43 perfluorocarbon emulsion added to the medium. Both these treatments were applied to improve oxygen transfer to the roots. Membrane tubing aeration allowed direct delivery of oxygen within the root clump, thus overcoming mass transfer resistances associated with poor intraclump penetration of liquid convective currents. Combined air sparging and membrane tubing aeration in a gas-driven bioreactor supported biomass levels 32-65% higher than sparging only of air or oxygen-enriched air at the same total gas flow rate. The optimal air flow rate ratio between the sparger and membrane tubing giving the maximum final biomass concentration was 0.2:0.4 L min-1. Intraclump dissolved oxygen tensions at high biomass densities were generally greater using air delivered by combined sparger-membrane tubing aeration than with sparging only of air or oxygen-enriched air. Specific atropine levels were not significantly affected by membrane tubing aeration. Indicators of anaerobic metabolism, such as lactic acid, ethanol, and ADH activity levels, were not significantly different in sparged and membrane-aerated systems; A. belladonna hairy roots also did not produce aerenchyma in response to oxygen limitations. Addition of perfluorocarbon emulsion to Murashige and Skoog medium in sparged stirred tank bioreactors did not improve growth, even when the emulsion was continuously recycled for re-aeration in an external vessel. Perfluorocarbons are associated with enhancement of gas-liquid oxygen transfer, so their ineffectiveness in this work most likely reflects the dominance of liquid-solid transfer resistances in hairy root cultures. The results of this investigation highlight the importance of developing new approaches for site-directed aeration of hairy root cultures, targeting oxygen delivery

  10. Application of Brassica napus hairy root cultures for phenol removal from aqueous solutions.

    PubMed

    Coniglio, María S; Busto, Victor D; González, Paola S; Medina, María I; Milrad, Silvia; Agostini, Elizabeth

    2008-07-01

    Phenolic compounds present in the drainage from several industries are harmful pollutants and represent a potential danger to human health. In this work we have studied the removal of phenol from water using Brassica napus hairy roots as a source of enzymes, such as peroxidases, which were able to oxidise phenol. These hairy roots were investigated for their tolerance to highly toxic concentrations of phenol and for the involvement of their peroxidase isoenzymes in the removal of phenol. Roots grew normally in medium containing phenol in concentrations not exceeding 100 mg l(-1), without the addition of H(2)O(2). However, roots were able to remove phenol concentrations up to 500 mg l(-1), in the presence of H(2)O(2), reaching high removal efficiency, within 1h of treatment and over a wide range of pH (4-9). Hairy roots could be re-used, at least, for three to four consecutive cycles. Peroxidase activity gradually decreased to approximately 20% of the control, at the fifth cycle. Basic and near neutral isoenzymes (BNP) decreased along time of recycling while acidic isoenzymes (AP) remained without changes. Although both group of isoenzymes would be involved in phenol removal, AP showed higher affinity and catalytic efficiency for phenol as substrate than BNP. In addition, AP retained more activity than BNP after phenol treatment. Thus, AP appears to be a promising isoenzyme for phenol removal and for application in continuous treatments. Furthermore, enzyme isolation might not be necessary and the entire hairy roots, might constitute less expensive enzymatic systems for decontamination processes. PMID:18499219

  11. Production of chlorogenic acid and its derivatives in hairy root cultures of Stevia rebaudiana.

    PubMed

    Fu, Xiao; Yin, Zhong-Ping; Chen, Ji-Guang; Shangguan, Xin-Chen; Wang, Xiaoqiang; Zhang, Qing-Feng; Peng, Da-Yong

    2015-01-14

    Chlorogenic acid and its derivatives (CADs) are valuable bioactive plant secondary metabolites with many health benefits. In the present study, Stevia rebaudiana hairy root cultures were established, and the culture conditions for the production of CADs were optimized. The hairy roots were induced by coculture of S. rebaudiana leaves and Agrobacterium rhizogenes (C58C1) after infection, which were further verified by PCR detection of rolB and rolC genes. HPLC-MS and HPLC analysis showed that chlorogenic acid (3-caffeoylquinic acid, 3-CQA), 3,5-dicaffeoylquinic acid (3,5-CQA), and 4,5-dicaffeoylquinic acid (4,5-CQA) were the major CADs in the hairy roots. Eight single roots with rapid growth rate were selected. Among them, T3 had the highest yield of CADs. B5 medium supplemented with 40 g/L sucrose was more suitable for the production of CADs than others. Under optimal culture conditions, the total content of these three compounds reached 105.58 mg/g and total yield was 234.40 mg/100 mL.

  12. Genetic Diversity and Symbiotic Phenotype of Hairy Vetch Rhizobia in Japan.

    PubMed

    Yuan, Kun; Miwa, Hiroki; Iizuka, Maki; Yokoyama, Tadashi; Fujii, Yoshiharu; Okazaki, Shin

    2016-06-25

    Hairy vetch (Vicia villosa Roth) is a leguminous crop widely used as green manure and a cover crop in Japan. It exhibits strong weed-suppressing activity, high resistance to insect pests, and the ability to fix nitrogen through symbiotic interactions with soil bacteria known as rhizobia. Few studies have investigated the rhizobia that form nodules on hairy vetch in Japan, and the biological resources available for selecting high nitrogen-fixing rhizobia are limited. In the present study, we isolated 110 hairy vetch rhizobia from 13 different areas in Japan. Based on their 16S rRNA gene sequences, 73% of the isolates were identified as Rhizobium leguminosarum. A comparative analysis of nodC and 16S rRNA gene phylogenies revealed that several isolates possessed congruent nodC sequences despite having divergent 16S rRNA gene sequences, suggesting that the horizontal transfer of nod genes occurred during the evolution of rhizobia. Inoculation tests showed that isolates closely related to R. leguminosarum had better plant growth-promoting effects than other strains, thereby providing a promising agricultural resource for inoculating crops. PMID:27151657

  13. Genetic Diversity and Symbiotic Phenotype of Hairy Vetch Rhizobia in Japan

    PubMed Central

    Yuan, Kun; Miwa, Hiroki; Iizuka, Maki; Yokoyama, Tadashi; Fujii, Yoshiharu; Okazaki, Shin

    2016-01-01

    Hairy vetch (Vicia villosa Roth) is a leguminous crop widely used as green manure and a cover crop in Japan. It exhibits strong weed-suppressing activity, high resistance to insect pests, and the ability to fix nitrogen through symbiotic interactions with soil bacteria known as rhizobia. Few studies have investigated the rhizobia that form nodules on hairy vetch in Japan, and the biological resources available for selecting high nitrogen-fixing rhizobia are limited. In the present study, we isolated 110 hairy vetch rhizobia from 13 different areas in Japan. Based on their 16S rRNA gene sequences, 73% of the isolates were identified as Rhizobium leguminosarum. A comparative analysis of nodC and 16S rRNA gene phylogenies revealed that several isolates possessed congruent nodC sequences despite having divergent 16S rRNA gene sequences, suggesting that the horizontal transfer of nod genes occurred during the evolution of rhizobia. Inoculation tests showed that isolates closely related to R. leguminosarum had better plant growth-promoting effects than other strains, thereby providing a promising agricultural resource for inoculating crops. PMID:27151657

  14. Enhanced Diterpene Tanshinone Accumulation and Bioactivity of Transgenic Salvia miltiorrhiza Hairy Roots by Pathway Engineering.

    PubMed

    Shi, Min; Luo, Xiuqin; Ju, Guanhua; Li, Leilei; Huang, Shengxiong; Zhang, Tong; Wang, Huizhong; Kai, Guoyin

    2016-03-30

    Tanshinones are health-promoting diterpenoids found in Salvia miltiorrhiza and have wide applications. Here, SmGGPPS (geranylgeranyl diphosphate synthase) and SmDXSII (1-deoxy-D-xylulose-5-phosphate synthase) were introduced into hairy roots of S. miltiorrhiza. Overexpression of SmGGPPS and SmDXSII in hairy roots produces higher levels of tanshinone than control and single-gene transformed lines; tanshinone production in the double-gene transformed line GDII10 reached 12.93 mg/g dry weight, which is the highest tanshinone content that has been achieved through genetic engineering. Furthermore, transgenic hairy root lines showed higher antioxidant and antitumor activities than control lines. In addition, contents of chlorophylls, carotenoids, indoleacetic acid, and gibberellins were significantly elevated in transgenic Arabidopsis thaliana plants. These results demonstrate a promising method to improve the production of diterpenoids including tanshinone as well as other natural plastid-derived isoprenoids in plants by genetic manipulation of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway.

  15. Keeping warm with fur in cold water: entrainment of air in hairy surfaces

    NASA Astrophysics Data System (ADS)

    Nasto, Alice; Regli, Marianne; Brun, Pierre-Thomas; Clanet, Christophe; Hosoi, Anette

    2015-11-01

    Instead of relying on a thick layer of body fat for insulation as many aquatic mammals do, fur seals and otters trap air in their dense fur for insulation in cold water. Using a combination of model experiments and theory, we rationalize this mechanism of air trapping underwater for thermoregulation. For the model experiments, hairy surfaces are fabricated using laser cut molds and casting samples with PDMS. Modeling the hairy texture as a network of capillary tubes, the imbibition speed of water into the hairs is obtained through a balance of hydrostatic pressure and viscous stress. In this scenario, the bending of the hairs and capillary forces are negligible. The maximum diving depth that can be achieved before the hairs are wetted to the roots is predicted from a comparison of the diving speed and imbibition speed. The amount of air that is entrained in hairy surfaces is greater than what is expected for classic Landau-Levich-Derjaguin plate plunging. A phase diagram with the parameters from experiments and biological data allows a comparison of the model system and animals.

  16. Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare.

    PubMed

    Habibi, Peyman; de Sa, Maria Fatima Grossi; da Silva, André Luís Lopes; Makhzoum, Abdullah; da Luz Costa, Jefferson; Borghetti, Ivo Albertto; Soccol, Carlos Ricardo

    2016-04-01

    Origanum vulgare L is commonly known as a wild marjoram and winter sweet which has been used in the traditional medicine due to its therapeutic effects as stimulant, anticancer, antioxidant, antibacterial, anti-inflammatory and many other diseases. A reliable gene transfer system via Agrobacterium rhizogenes and plant regeneration via hairy roots was established in O. vulgare for the first time. The frequency of induced hairy roots was different by modification of the co-cultivation medium elements after infection by Agrobacterium rhizogenes strains K599 and ATCC15834. High transformation frequency (91.3 %) was achieved by co-cultivation of explants with A. rhizogenes on modified (MS) medium. The frequency of calli induction with an 81.5 % was achieved from hairy roots on MS medium with 0.25 mg/L(-1) 2,4-D. For shoot induction, initiated calli was transferred into a medium containing various concentrations of BA (0.1, 0.25, 0.5, 0.75 and 1 mg/L(-1)). The frequency of shoot generation (85.18 %) was achieved in medium fortified with 0.25 mg/L(-1) of BA. Shoots were placed on MS medium with 0.25 mg/l IBA for root induction. Roots appeared and induction rate was achieved after 15 days. PMID:27436918

  17. Genetic Diversity and Symbiotic Phenotype of Hairy Vetch Rhizobia in Japan.

    PubMed

    Yuan, Kun; Miwa, Hiroki; Iizuka, Maki; Yokoyama, Tadashi; Fujii, Yoshiharu; Okazaki, Shin

    2016-06-25

    Hairy vetch (Vicia villosa Roth) is a leguminous crop widely used as green manure and a cover crop in Japan. It exhibits strong weed-suppressing activity, high resistance to insect pests, and the ability to fix nitrogen through symbiotic interactions with soil bacteria known as rhizobia. Few studies have investigated the rhizobia that form nodules on hairy vetch in Japan, and the biological resources available for selecting high nitrogen-fixing rhizobia are limited. In the present study, we isolated 110 hairy vetch rhizobia from 13 different areas in Japan. Based on their 16S rRNA gene sequences, 73% of the isolates were identified as Rhizobium leguminosarum. A comparative analysis of nodC and 16S rRNA gene phylogenies revealed that several isolates possessed congruent nodC sequences despite having divergent 16S rRNA gene sequences, suggesting that the horizontal transfer of nod genes occurred during the evolution of rhizobia. Inoculation tests showed that isolates closely related to R. leguminosarum had better plant growth-promoting effects than other strains, thereby providing a promising agricultural resource for inoculating crops.

  18. Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid.

    PubMed

    Grzegorczyk, Izabela; Królicka, Aleksandra; Wysokińska, Halina

    2006-01-01

    Shoots of Salvia officinalis, a medicinally important plant, were infected with Agrobacterium rhizogenes strains ATCC 15834 and A4 which led to the induction of hairy roots in 57% and 37% of the explants, respectively. Seven lines of hairy roots were established in WP liquid medium under light and dark conditions. The transformed nature of the root lines was confirmed by polymerase chain reaction using rolB and rolC specific primers. Transformed root cultures of Salvia officinalis showed variations in biomass and rosmarinic acid production depending on the bacterial strain used for transformation and the root line analyzed. Both parameters (growth and rosmarinic acid content) of ATCC 15834-induced lines were significantly higher than the A4-induced lines. The maximum accumulation of rosmarinic acid (about 45 mg g(-1) of dry weight) was achieved by hairy root line 1 (HR-1) at the end of the culture period (45-50 days). The level was significantly higher than that found in untransformed root culture (19 mg g(-10 of dry wt).

  19. Production of oleanolic acid glycosides by hairy root established cultures of Calendula officinalis L.

    PubMed

    Długosz, Marek; Wiktorowska, Ewa; Wiśniewska, Anita; Pączkowski, Cezary

    2013-01-01

    In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with β-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.

  20. Enhancement of ginsenoside biosynthesis and secretion by Tween 80 in Panax ginseng hairy roots.

    PubMed

    Liang, Yanlong; Wu, Jianjun; Li, Yao; Li, Jian; Ouyang, Yong; He, Zhi; Zhao, Shoujing

    2015-01-01

    We evaluated the effect of Tween 80 permeabilization on ginsenoside secretion in Panax ginseng hairy roots. Tween 80 (1.2%, w/v) had no significant effect on hairy root vitality. After a 25-day treatment with Tween 80, approximately 76% of the total ginsenosides was released into the surrounding medium. In the case of control, the ginsenosides secreted into the medium were negligible. Furthermore, when compared with control, the level of total ginsenosides was enhanced by approximately threefold under Tween treatment. Additionally, secretion of the typical ginsenoside monomers including Rb1 , Rg1 , and Re was analyzed, indicating that the most of them were released into the medium. Moreover, it was observed that dammarenediol synthase, a key enzyme involved in ginsenoside biosynthesis, was upregulated at both gene expression and enzyme activity levels. The expression of genes CYP716A47 and CYP716A53v2 encoding Cyt P450 enzymes catalyzing the formation of protopanaxadiol from dammarenediol and protopanaxatriol from protopanaxadiol, respectively, was slightly upregulated. These results clearly demonstrated that Tween 80 could act not only as an efficient permeabilizer to enhance ginsenoside secretion from the hairy roots, but also as an elicitor to promote the biosynthesis of ginsenoside.

  1. Growth and rutin production in hairy root cultures of buckwheat (Fagopyrum esculentum M.).

    PubMed

    Lee, Sook-Young; Cho, Soo-In; Park, Min-Hee; Kim, Yong-Kyung; Choi, Jae-Eul; Park, Sang-Un

    2007-01-01

    Buckwheat (Fagopyrum esculentum Moench.) is a potentially important source of rutin, a natural flavonoid with antihyperglycemic, antihypertensive, and antioxidative properties. To examine in vitro production of rutin, we established a hairy root culture of buckwheat by infecting leaf explants with Agrobacterium rhizogenes R1000, and tested the growth conditions and rutin production rates of these cultures. Ten hairy root clones were established; their growth and rutin production rates ranged from 233 to 312 (mg dry wt per 30 mL flask, and 0.8 to 1.2 (mg/g dry wt), respectively. Clone H8, which had high growth and rutin production rates (312 mg dry wt per 30 mL flask and 1.2 mg/g dry wt, respectively), was selected for further experiments. H8 showed maximal growth and rutin content at 30 days in culture in MS medium. Of four tested culture media, half-strength MS medium was found to induce the highest levels of growth (378 mg dry wt per 30 mL flask) and rutin production (1.4 mg/g dry wt) by clone H8. In contrast, supplementation with auxins (0.1-1 mg/l IAA, IBA and NAA) increased the growth rate, but had no significant effect on rutin production by H8. Collectively, these findings indicate that hairy root cultures of buckwheat culture could be a valuable alternative approach for rutin production.

  2. Isolation and characterization of alborixin from Streptomyces scabrisporus: A potent cytotoxic agent against human colon (HCT-116) cancer cells.

    PubMed

    Shah, Aabid Manzoor; Wani, Abubakar; Qazi, Parvaiz H; Rehman, Shakeel-U; Mushtaq, Saleem; Ali, Shiekh Abid; Hussain, Aehtesham; Shah, Aiyatullah; Qazi, Asif Khurshid; Makhdoomi, Ubaid Sharif; Hamid, Abid; Kumar, Ajay

    2016-08-25

    The ethyl acetate extract from the fermentation broth of an actinomycete strain, identified as Streptomyces scabrisporus isolated from soil of Kashmir Himalayas - India, exhibited significant cytotoxic activity against a panel of human cancer cell lines. The active fraction subjected to column chromatography led to the isolation of pharmacologically potent anticancer compound whose structure was established to be alborixin on the basis of spectral data analysis. The compound exhibited antiproliferative activity against panel of cell lines N2a, MCF-7, MiaPaca-2, PC-3, HCT-116, MDA-MB-231, HL-60 and A-549 cells with IC50 of 9.7, 15.4, 7.2, 8.1, 3.2, 9.7, 7.5 and 11.5 μM respectively. Alborixin displayed the maximum cytotoxic activity against HCT-116 human colon carcinoma cells and therefore further studies were carried on this cell line. Alborixin decreased the clonogenic potential of HCT-116 cells in a dose dependent manner. It induced apoptotic cell death in HCT116 cells that were confirmed by Flow cytometric analysis of Annexin V/PI staining and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by mitochondrial membrane potential loss, decreasing the expression profile of anti-apoptotic protein Bcl-2, whereas it augments cleavage of caspase-3 and PARP-1, activates caspase-8 and 9 with concomitant increase in expression of proapoptotic protein Bax in a dose dependent manner. These results indicate that alborixin obtained from Streptomyces scabrisporus IIIM55 induces apoptotic cell death in colon cancer cells HCT-116 and can be further evaluated for its potential as an anticancer agent. PMID:27378626

  3. Induced cancer stem-like cells as a model for biological screening and discovery of agents targeting phenotypic traits of cancer stem cell.

    PubMed

    Nishi, Mayuko; Akutsu, Hidenori; Kudoh, Ayumi; Kimura, Hirokazu; Yamamoto, Naoki; Umezawa, Akihiro; Lee, Sam W; Ryo, Akihide

    2014-09-30

    Cancer stem cells (CSCs) retain the capacity to propagate themselves through self-renewal and to produce heterogeneous lineages of cancer cells constituting the tumor. Novel drugs that target CSCs can potentially eliminate the tumor initiating cell population therefore resulting in complete cure of the cancer. We recently established a CSC-like model using induced pluripotent stem cell (iPSC) technology to reprogram and partially differentiate human mammary epithelial MCF-10A cells. Using the induced CSC-like (iCSCL) model, we developed a phenotypic drug assay system to identify agents that inhibit the stemness and self-renewal properties of CSCs. The selectivity of the agents was assessed using three distinct assays characterized by cell viability, cellular stemness and tumor sphere formation. Using this approach, we found that withaferin A (WA), an Ayurvedic medicine constituent, was a potent inhibitor of CSC stemness leading to cellular senescence primarily via the induction of p21Cip1 expression. Moreover, WA exhibited strong anti-tumorigenic activity against the iCSCL. These results indicate that our iCSCL model provides an innovative high throughput platform for a simple, easy, and cost-effective method to search for novel CSC-targeting drugs. Furthermore, our current study identified WA as a putative drug candidate for abrogating the stemness and tumor initiating ability of CSCs.

  4. Peloruside A is a microtubule-stabilizing agent with exceptional anti-migratory properties in human endothelial cells

    PubMed Central

    Ganguly, Anutosh; Cabral, Fernando; Yang, Hailing; Patel, Kamala D.

    2015-01-01

    Peloruside A is a novel antimitotic drug originally isolated from the marine sponge Mycale hentschieli. Previous studies showed that peloruside A stabilizes microtubules by binding to a site on tubulin distinct from paclitaxel, another microtubule stabilizing drug. Peloruside A blocks mitosis, but little is known about the effects on other cellular activities. Here we report that peloruside A is the most potent microtubule inhibitor yet tested for its ability to block endothelial cell migration. Quantitative analysis indicated that it inhibits microtubule dynamics and endothelial cell migration at 1/200th of the concentration needed to inhibit cell division (the cytotoxic concentration), indicating that it could potentially have a large margin of safety when used to specifically target angiogenesis. By comparison, paclitaxel, a well-known cancer therapeutic drug, suppresses cell migration at 1/13th of its cytotoxic concentration; and vinblastine suppresses cell migration at just slightly below its cytotoxic antimitotic concentration. Thus, different microtubule targeted drugs have varying relative potencies for inhibition of cell migration versus cell division. The results suggest that peloruside A may be an especially useful agent for anti-angiogenesis therapy and point to the likelihood that other antimitotic drugs might be found with an even larger potential margin of safety. PMID:26244166

  5. Modification of in vitro and in vivo BCG cell wall-induced immunosuppression by treatment with chemotherapeutic agents or indomethacin

    SciTech Connect

    DeSilva, M.A.; Wepsic, H.T.; Mizushima, Y.; Nikcevich, D.A.; Larson, C.H.

    1985-04-01

    The in vitro inhibition of spleen cell blastogenesis response and the in vivo enhancement of tumor growth are phenomena associated with BCG cell wall (BCGcw) immunization. What effect treatment with chemotherapeutic agents and the prostaglandin inhibitor indomethacin would have on the in vitro and in vivo responses to BCGcw immunization was evaluated. In vitro blastogenesis studies showed that chemotherapy pretreatment prior to immunization with BCGcw resulted in a restoration of the spleen cell blastogenesis response. In blastogenesis addback studies, where BCGcw-induced irradiated splenic suppressor cells were admixed with normal cells, less inhibition of blastogenesis occurred when spleen cells were obtained from rats that had received the combined treatment of chemotherapy and BCGcw immunization versus only BCGcw immunization. The cocultivation of spleen cells from BCGcw-immunized rats with indomethacin resulted in a 30-40% restoration of the blastogenesis response. In vivo studies showed that BCGcw-mediated enhancement of intramuscular tumor growth of the 3924a ACI rat tumor could be abrogated by either pretreatment with busulfan or mitomycin or by the feeding of indomethacin.

  6. Agent-Based Computational Modeling of Cell Culture: Understanding Dosimetry In Vitro as Part of In Vitro to In Vivo Extrapolation

    EPA Science Inventory

    Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assu...

  7. The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

    PubMed

    Sharifi, Sara; Sattari, Taher Nejad; Zebarjadi, Alireza; Majd, Ahmad; Ghasempour, Hamidreza

    2014-01-01

    We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10-14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g(-1) of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L(-1) naphthaleneacetic acid and 2 mg L(-1) 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots. PMID:24554840

  8. The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment.

    PubMed

    Annovazzi, Laura; Caldera, Valentina; Mellai, Marta; Riganti, Chiara; Battaglia, Luigi; Chirio, Daniela; Melcarne, Antonio; Schiffer, Davide

    2015-01-01

    Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas.

  9. Subcellular SIMS imaging of gadolinium isotopes in human glioblastoma cells treated with a gadolinium containing MRI agent

    NASA Astrophysics Data System (ADS)

    Smith, Duane R.; Lorey, Daniel R.; Chandra, Subhash

    2004-06-01

    Neutron capture therapy is an experimental binary radiotherapeutic modality for the treatment of brain tumors such as glioblastoma multiforme. Recently, neutron capture therapy with gadolinium-157 has gained attention, and techniques for studying the subcellular distribution of gadolinium-157 are needed. In this preliminary study, we have been able to image the subcellular distribution of gadolinium-157, as well as the other six naturally abundant isotopes of gadolinium, with SIMS ion microscopy. T98G human glioblastoma cells were treated for 24 h with 25 mg/ml of the metal ion complex diethylenetriaminepentaacetic acid Gd(III) dihydrogen salt hydrate (Gd-DTPA). Gd-DTPA is a contrast enhancing agent used for MRI of brain tumors, blood-brain barrier impairment, diseases of the central nervous system, etc. A highly heterogeneous subcellular distribution was observed for gadolinium-157. The nuclei in each cell were distinctly lower in gadolinium-157 than in the cytoplasm. Even within the cytoplasm the gadolinium-157 was heterogeneously distributed. The other six naturally abundant isotopes of gadolinium were imaged from the same cells and exhibited a subcellular distribution consistent with that observed for gadolinium-157. These observations indicate that SIMS ion microscopy may be a viable approach for subcellular studies of gadolinium containing neutron capture therapy drugs and may even play a major role in the development and validation of new gadolinium contrast enhancing agents for diagnostic MRI applications.

  10. Effect of passage number on cellular response DNA-damaging agents: cell survival and gene expression

    SciTech Connect

    Chang-Liu, Chin-Mei; Wolschak, G.E.

    1996-03-01

    The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or {gamma}-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to {sup 60}Co {gamma} rays or 254-m UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a ``crisis`` period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant- than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of {gamma}-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following {gamma}-ray exposure of the intermediate (passage 45) epithelial cells. Differential display, however, revealed changes in expression of several transcripts following exposure to ionizing and ultraviolet radiations. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

  11. Antitumor Activity of HM781-36B, alone or in Combination with Chemotherapeutic Agents, in Colorectal Cancer Cells

    PubMed Central

    Kang, Mi Hyun; Moon, Sung Ung; Sung, Ji Hea; Kim, Jin Won; Lee, Keun Wook; Lee, Hye Seung; Lee, Jong Seok; Kim, Jee Hyun

    2016-01-01

    Purpose HM781-36B is a novel and irreversible pan-human epidermal growth factor receptor (HER) inhibitor with TEC cytoplasmic kinase inhibition. The aim of this study is to evaluate the antitumor activity and mechanism of action for HM781-36B in CRC cell lines. Materials and Methods The CRC cell lines were exposed to HM781-36B and/or oxaliplatin (L-OHP), 5-fluorouracil (5-FU), SN-38. The cell viability was examined by Cell Titer-Glo luminescent cell viability assay kit. Change in the cell cycle and protein expression was determined by flow cytometry and immunoblot analysis, respectively. Synergism between 2 drugs was evaluated by the combination index. Results The addition of HM781-36B induced potent growth inhibition in both DiFi cells with EGFR overexpression and SNU-175 cells (IC50 = 0.003 and 0.005 μM, respectively). Furthermore, HM781-36B induced G1 arrest of the cell cycle and apoptosis, and reduced the levels of HER family and downstream signaling molecules, pERK and pAKT, as well as nonreceptor/cytoplasmic tyrosine kinase, BMX. The combination of HM781-36B with 5-FU, L-OHP, or SN-38 showed an additive or synergistic effect in most CRC cells. Conclusion These findings suggest the potential roles of HM781-36B as the treatment for EGFR-overexpressing colon cancer, singly or in combination with chemotherapeutic agents. The role of BMX expression as a marker of response to HM781-36B should be further explored. PMID:25761479

  12. Expression of mammalian O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents.

    PubMed

    Dolan, M E; Norbeck, L; Clyde, C; Hora, N K; Erickson, L C; Pegg, A E

    1989-09-01

    Chinese hamster ovary cells (CHO) were co-transfected with pSV2neo and sheared DNA from either a human cell line (HT29) expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) or from a cell line (BE) deficient in this activity. Cells expressing the selectable marker were obtained by exposure to G418 and colonies resistant to alkylation damage isolated by growth in the presence of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The number of colonies of cells expressing AGT activity arising after transfection with DNA from BE cells was similar to the number arising from cells exposed to HT29 DNA. Although the amount of AGT repair protein expressed in the transfectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-deficient cells by transfection of human DNA from both repair-deficient and proficient cells. A separate transfection of CHOMG cells [a mutant of CHO cells resistant to the drug, methylglyoxal bis(guanylhydrazone) (MGBG)] with HT29 DNA and pSV2neo followed by selection of G418 and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in three colonies with high AGT levels. These transfectants had different growth rates and expressed levels of the AGT protein between 230 and 300 fmol/mg protein. The transfectants were as resistant to the cytotoxic effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) as HT29 cells which were much more resistant than the parental CHOMG cells. Pretreatment of transfectant cells with 0.4 mM O6-methylguanine for 24 h reduced AGT activity to 14% basal levels, which upon removal of the base increased to approximately 74% basal level within 8 h. The sensitivity to the cytotoxic effects of both the chloroethylating and methylating agents was enhanced by treatment with O6-methylguanine. In the same manner, the number of BCNU-induced DNA interstrand cross-links increased in transfectant

  13. A hairy fall: syncope resulting from topical application of minoxidil.

    PubMed

    Dubrey, S W; VanGriethuysen, J; Edwards, C M B

    2015-01-01

    We describe the case of a young man who developed syncope after using a high strength formulation of topical minoxidil as a hair growth restorer. Other potential cardiovascular and endocrine causes were excluded, and his symptoms resolved on discontinuation of the product. While syncope is a recognised side effect of using this powerful systemic antihypertensive agent, few cases are documented in the literature, which we illustrate in our discussion. PMID:26347235

  14. Anticancer agent xanthohumol inhibits IL-2 induced signaling pathways involved in T cell proliferation.

    PubMed

    Liu, Yongbo; Gao, Xiaohua; Deeb, Dorrah; Arbab, Ali S; Dulchavsky, Scott A; Gautam, Subhash C

    2012-01-01

    Xanthohumol (XN), a prenylated chalcone present in hops exhibits anti-inflammatory, antioxidant and anticancer activity. In the present study we show that XN inhibits the proliferation of mouse lymphoma cells and IL-2 induced proliferation and cell cycle progression in mouse splenic T cells. The suppression of T cell proliferation by XN was due to the inhibition of IL-2 induced Janus kinase/signal transducers and activators of transcription (Jak/STAT) and extracellular signal-regulated kinase 1 and 2 (Erk1/2) signaling pathways. XN also inhibited proliferation-related cellular proteins such as c-Myc, c-Fos and NF-kappaB and cyclin D1. Thus, understanding of IL-2 induced cell signaling pathways in normal T cells, which are constitutively turned on in T cell lymphomas may facilitate development of XN for the treatment of hematologic cancers. PMID:22946339

  15. Anticancer agent xanthohumol inhibits IL-2 induced signaling pathways involved in T cell proliferation

    PubMed Central

    Liu, Yongbo; Gao, Xiaohua; Deeb, Dorrah; Arbab, Ali S.; Dulchavsky, Scott A.; Gautam, Subhash C.

    2013-01-01

    Xanthohumol (XN), a prenylated chalcone present in hops exhibits anti-inflammatory, antioxidant and anticancer activity. In the present study we show that XN inhibits the proliferation of mouse lymphoma cells and IL-2 induced proliferation and cell cycle progression in mouse splenic T cells. The suppression of T cell proliferation by XN was due to the inhibition of IL-2 induced Janus kinase/signal transducers and activators of transcription (Jak/STAT) and extracellular signal-regulated kinase 1 and 2 (Erk1/2) signaling pathways. XN also inhibited proliferation-related cellular proteins such as c-Myc, c-Fos and NF-κB and cyclin D1. Thus, understanding of IL-2 induced cell signaling pathways in normal T cells, which are constitutively turned on in T cell lymphomas may facilitate development of XN for the treatment of hematologic cancers. PMID:22946339

  16. Alternative agents versus prophylactic platelet transfusion for preventing bleeding in patients with haematological disorders after chemotherapy or stem cell transplantation

    PubMed Central

    Estcourt, Lise J; Gregg, Richard; Stanworth, Simon; Doree, Carolyn; Trivella, Marialena; Murphy, Michael F; Tinmouth, Alan

    2014-01-01

    This is the protocol for a review and there is no abstract. The objectives are as follows: To determine whether alternative agents (e.g. artificial platelet substitutes, platelet-poor plasma, fibrinogen, rFVIIa, thrombopoietin mimetics) are as effective and safe as the use of platelet transfusions for the prevention of bleeding (prophylactic platelet transfusion) in patients with haematological disorders who are undergoing myelosuppressive chemotherapy or stem cell transplantation. Antifibrinolytics (lysine analogues) will not be included in this review because they have been the focus of another Cochrane review (Wardrop 2013). PMID:25722650

  17. NAMPT inhibition synergizes with NQO1-targeting agents in inducing apoptotic cell death in non-small cell lung cancer cells.

    PubMed

    Liu, Hui-Ying; Li, Qing-Ran; Cheng, Xue-Fang; Wang, Guang-Ji; Hao, Hai-Ping

    2016-08-01

    Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for a number of enzymes and regulatory proteins involved in a variety of cellular processes, including deacetylation enzyme SIRT1 which modulates several tumor suppressors such as p53 and FOXO. Herein we report that NQO1 substrates Tanshione IIA (TSA) and β-lapachone (β-lap) induced a rapid depletion of NAD(+) pool but adaptively a significant upregulation of NAMPT. NAMPT inhibition by FK866 at a nontoxic dose significantly enhanced NQO1-targeting agent-induced apoptotic cell death. Compared with TSA or β-lap treatment alone, co-treatment with FK866 induced a more dramatic depletion of NAD(+), repression of SIRT1 activity, and thereby the increased accumulation of acetylated FOXO1 and the activation of apoptotic pathway. In conclusion, the results from the present study support that NAMPT inhibition can synergize with NQO1 activation to induce apoptotic cell death, thereby providing a new rationale for the development of combinative therapeutic drugs in combating non-small lung cancer. PMID:27608947

  18. Determination of seasonality in southern hairy-nosed wombats (Lasiorhinus latifrons) by analysis of fecal androgens.

    PubMed

    Hamilton, R A; Stanton, P G; O'Donnell, L; Steele, V R; Taggart, D A; Temple-Smith, P D

    2000-08-01

    Little is known about the reproductive biology of Australia's critically endangered northern hairy-nosed wombat (Lasiorhinus krefftii), largely due to its cryptic nature and the difficulty in accessing the small remaining population of about 70 animals. Using the noninvasive technique of fecal steroid analysis, we have examined the endocrinology of the more common yet closely related southern hairy-nosed wombat (Lasiorhinus latifrons). The aims of this study were to 1) develop and validate fecal androgen analysis in this species, 2) examine and compare seasonal differences in fecal and plasma androgens in male wombats, and 3) correlate seasonal differences in androgens with changes in male accessory glands (prostate and bulbourethral gland). Fecal androgens were extracted in ether; concentrated; separated by HPLC into testosterone (T), dihydrotestosterone (DHT), and 5 alpha-androstane-3 alpha,17 beta-diol (Adiol) fractions; and quantitated by RIA. The concentrations of androgens in fecal pellets from 14 wild southern hairy-nosed wombats as determined by RIA varied over the range 6.6-25.0 ng/g dry weight for T, 4.0-24.2 ng/g dry weight for DHT, and 0-34.8 ng/g dry weight for Adiol. For each androgen, a highly significant linear correlation was observed between plasma and fecal concentrations. When individuals were grouped into either breeding season (pellets collected between August-November) or nonbreeding season (collected between February-April), significant (P < 0.05) differences between seasons were observed for both plasma and fecal T, plasma DHT, and fecal Adiol. For all androgens, the mean fecal and plasma concentrations were higher during the breeding season than the nonbreeding season. A significant (P < 0.001) correlation was observed between fecal T and prostate weight, while DHT and Adiol correlations were nonsignificant. Significant correlations were observed, however, between all three fecal androgens and bulbourethral gland weight. These studies

  19. Determination of seasonality in southern hairy-nosed wombats (Lasiorhinus latifrons) by analysis of fecal androgens.

    PubMed

    Hamilton, R A; Stanton, P G; O'Donnell, L; Steele, V R; Taggart, D A; Temple-Smith, P D

    2000-08-01

    Little is known about the reproductive biology of Australia's critically endangered northern hairy-nosed wombat (Lasiorhinus krefftii), largely due to its cryptic nature and the difficulty in accessing the small remaining population of about 70 animals. Using the noninvasive technique of fecal steroid analysis, we have examined the endocrinology of the more common yet closely related southern hairy-nosed wombat (Lasiorhinus latifrons). The aims of this study were to 1) develop and validate fecal androgen analysis in this species, 2) examine and compare seasonal differences in fecal and plasma androgens in male wombats, and 3) correlate seasonal differences in androgens with changes in male accessory glands (prostate and bulbourethral gland). Fecal androgens were extracted in ether; concentrated; separated by HPLC into testosterone (T), dihydrotestosterone (DHT), and 5 alpha-androstane-3 alpha,17 beta-diol (Adiol) fractions; and quantitated by RIA. The concentrations of androgens in fecal pellets from 14 wild southern hairy-nosed wombats as determined by RIA varied over the range 6.6-25.0 ng/g dry weight for T, 4.0-24.2 ng/g dry weight for DHT, and 0-34.8 ng/g dry weight for Adiol. For each androgen, a highly significant linear correlation was observed between plasma and fecal concentrations. When individuals were grouped into either breeding season (pellets collected between August-November) or nonbreeding season (collected between February-April), significant (P < 0.05) differences between seasons were observed for both plasma and fecal T, plasma DHT, and fecal Adiol. For all androgens, the mean fecal and plasma concentrations were higher during the breeding season than the nonbreeding season. A significant (P < 0.001) correlation was observed between fecal T and prostate weight, while DHT and Adiol correlations were nonsignificant. Significant correlations were observed, however, between all three fecal androgens and bulbourethral gland weight. These studies

  20. Discovery of agents that eradicate leukemia stem cells using an in silico screen of public gene expression data

    PubMed Central

    Hassane, Duane C.; Guzman, Monica L.; Corbett, Cheryl; Li, Xiaojie; Abboud, Ramzi; Young, Fay; Liesveld, Jane L.; Carroll, Martin

    2008-01-01

    Increasing evidence indicates that malignant stem cells are important for the pathogenesis of acute myelogenous leukemia (AML) and represent a reservoir of cells that drive the development of AML and relapse. Therefore, new treatment regimens are necessary to prevent relapse and improve therapeutic outcomes. Previous studies have shown that the sesquiterpene lactone, parthenolide (PTL), ablates bulk, progenitor, and stem AML cells while causing no appreciable toxicity to normal hematopoietic cells. Thus, PTL must evoke cellular responses capable of mediating AML selective cell death. Given recent advances in chemical genomics such as gene expression-based high-throughput screening (GE-HTS) and the Connectivity Map, we hypothesized that the gene expression signature resulting from treatment of primary AML with PTL could be used to search for similar signatures in publicly available gene expression profiles deposited into the Gene Expression Omnibus (GEO). We therefore devised a broad in silico screen of the GEO database using the PTL gene expression signature as a template and discovered 2 new agents, celastrol and 4-hydroxy-2-nonenal, that effectively eradicate AML at the bulk, progenitor, and stem cell level. These findings suggest the use of multicenter collections of high-throughput data to facilitate discovery of leukemia drugs and drug targets. PMID:18305216

  1. Chromatin-modifying agents for epigenetic reprogramming and endogenous neural stem cell-mediated repair in stroke.

    PubMed

    Qureshi, Irfan A; Mehler, Mark F

    2011-03-01

    The recent explosion of interest in epigenetics and chromatin biology has made a significant impact on our understanding of the pathophysiology of cerebral ischemia and led to the identification of new treatment strategies for stroke, such as those that employ histone deacetylase inhibitors. These are key advances; however, the rapid pace of discovery in chromatin biology and innovation in the development of chromatin-modifying agents implies there are emerging classes of drugs that may also have potential benefits in stroke. Herein, we discuss how various chromatin regulatory factors and their recently identified inhibitors may serve as drug targets and therapeutic agents for stroke, respectively. These factors primarily include members of the repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor macromolecular complex, polycomb group (PcG) proteins, and associated chromatin remodeling factors, which have been linked to the pathophysiology of cerebral ischemia. Further, we suggest that, because of the key roles played by REST, PcG proteins and other chromatin remodeling factors in neural stem and progenitor cell (NSPC) biology, chromatin-modifying agents can be utilized not only to mitigate ischemic injury directly but also potentially to promote endogenous NSPC-mediated brain repair mechanisms. PMID:24014083

  2. HDAC Inhibitors Increase NRF2-Signaling in Tumour Cells and Blunt the Efficacy of Co-Adminstered Cytotoxic Agents

    PubMed Central

    McMahon, Michael; Campbell, Kathryn H.; MacLeod, A. Kenneth; McLaughlin, Lesley A.; Henderson, Colin J.; Wolf, C. Roland

    2014-01-01

    The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process. PMID:25427220

  3. HDAC inhibitors increase NRF2-signaling in tumour cells and blunt the efficacy of co-adminstered cytotoxic agents.

    PubMed

    McMahon, Michael; Campbell, Kathryn H; MacLeod, A Kenneth; McLaughlin, Lesley A; Henderson, Colin J; Wolf, C Roland

    2014-01-01

    The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process.

  4. Chromatin-Modifying Agents for Epigenetic Reprogramming and Endogenous Neural Stem Cell-Mediated Repair in Stroke

    PubMed Central

    Qureshi, Irfan A.; Mehler, Mark F.

    2013-01-01

    The recent explosion of interest in epigenetics and chromatin biology has made a significant impact on our understanding of the pathophysiology of cerebral ischemia and led to the identification of new treatment strategies for stroke, such as those that employ histone deacetylase inhibitors. These are key advances; however, the rapid pace of discovery in chromatin biology and innovation in the development of chromatin-modifying agents implies there are emerging classes of drugs that may also have potential benefits in stroke. Herein, we discuss how various chromatin regulatory factors and their recently identified inhibitors may serve as drug targets and therapeutic agents for stroke, respectively. These factors primarily include members of the repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor macromolecular complex, polycomb group (PcG) proteins, and associated chromatin remodeling factors, which have been linked to the pathophysiology of cerebral ischemia. Further, we suggest that, because of the key roles played by REST, PcG proteins and other chromatin remodeling factors in neural stem and progenitor cell (NSPC) biology, chromatin-modifying agents can be utilized not only to mitigate ischemic injury directly but also potentially to promote endogenous NSPC-mediated brain repair mechanisms. PMID:24014083

  5. Application of High-Resolution Magic-Angle Spinning NMR Spectroscopy to Define the Cell Uptake of MRI Contrast Agents

    NASA Astrophysics Data System (ADS)

    Calabi, Luisella; Alfieri, Goffredo; Biondi, Luca; De Miranda, Mario; Paleari, Lino; Ghelli, Stefano

    2002-06-01

    A new method, based on proton high-resolution magic-angle spinning ( 1H HR-MAS) NMR spectroscopy, has been employed to study the cell uptake of magnetic resonance imaging contrast agents (MRI-CAs). The method was tested on human red blood cells (HRBC) and white blood cells (HWBC) by using three gadolinium complexes, widely used in diagnostics, Gd-BOPTA, Gd-DTPA, and Gd-DOTA, and the analogous complexes obtained by replacing Gd(III) with Dy(III), Nd(III), and Tb(III) (i.e., complexes isostructural to the ones of gadolinium but acting as shift agents). The method is based on the evaluation of the magnetic effects, line broadening, or induced lanthanide shift (LIS) caused by these complexes on NMR signals of intra- and extracellular water. Since magnetic effects are directly linked to permeability, this method is direct. In all the tests, these magnetic effects were detected for the extracellular water signal only, providing a direct proof that these complexes are not able to cross the cell membrane. Line broadening effects (i.e., the use of gadolinium complexes) only allow qualitative evaluations. On the contrary, LIS effects can be measured with high precision and they can be related to the concentration of the paramagnetic species in the cellular compartments. This is possible because the HR-MAS technique provides the complete elimination of bulk magnetic susceptibility (BMS) shift and the differentiation of extra- and intracellular water signals. Thus with this method, the rapid quantification of the MRI-CA amount inside and outside the cells is actually feasible.

  6. Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster

    SciTech Connect

    Holdridge, C.; Dorsett, D. )

    1991-04-01

    The suppressor of hairy-wing [su(Hw)] locus of Drosophila melanogaster encodes a zinc finger protein that binds a repeated motif in the gypsy retroposon. Mutations of su(Hw) suppress the phenotypes associated with mutations caused by gypsy insertions. To examine the mechanisms by which su(Hw) alters gene expression, a fragment of gypsy containing multiple su(Hw) protein-binding sites was inserted into various locations in the well-characterized Drosophila hsp70 heat shock gene promoter. The authors found no evidence for activation of basal hsp70 transcription by su(Hw) protein in cultured Drosophila cells but observed that it can repress heat shock-induced transcription. Repression occurred only when su(Hw) protein-binding sites were positioned between binding sites for proteins required for heat shock transcription. They propose that su(Hw) protein interferes nonspecifically with protein-protein interactions required for heat shock transcription, perhaps sterically, or by altering the ability of DNA to bend or twist.

  7. The effects of UV-B stress on the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots.

    PubMed

    Binder, Bernard Y K; Peebles, Christie A M; Shanks, Jacqueline V; San, Ka-Yiu

    2009-01-01

    In nature, plants generate protective secondary metabolites in response to environmental stresses. Such metabolites include terpenoid indole alkaloids (TIAs), which absorb UV-B light and serve putatively to protect the plant from harmful radiation. Catharanthus roseus plants, multiple shoot cultures, and cell suspension cultures exposed to UV-B light show significant increases in the production of TIAs, including precursors to vinblastine and vincristine, which have proven effective in the treatment of leukemia and lymphoma. Here, the effect of UV-B light on C. roseus hairy roots was examined. Analysis of alkaloid concentrations up to 168 h after UV-B exposure shows significant increases in the concentrations of lochnericine and significant decreases in the concentration of hörhammericine over time (ANOVA, P < 0.05). Our results also indicate that increasing UV-B exposure time up to 20 min caused significant increases in lochnericine, serpentine, and ajmalicine and a decrease in hörhammericine (t-test, p < 0.05). PMID:19479674

  8. Increased adenosine triphosphate production by peripheral blood CD4+ cells in patients with hematologic malignancies treated with stem cell mobilization agents.

    PubMed

    Manga, Kiran; Serban, Geo; Schwartz, Joseph; Slotky, Ronit; Patel, Nita; Fan, Jianshe; Bai, Xiaolin; Chari, Ajai; Savage, David; Suciu-Foca, Nicole; Colovai, Adriana I

    2010-07-01

    Hematopoietic stem cell (HSC) transplantation is an important therapeutic option for patients with hematologic malignancies. To explore the immunomodulatory effects of HSC mobilization agents, we studied the function and phenotype of CD4(+) T cells from 16 adult patients with hematologic malignancies undergoing HSC mobilization treatment for autologous transplantation. Immune cell function was determined using the Immuknow (Cylex) assay by measuring the amount of adenosine triphosphate (ATP) produced by CD4(+) cells from whole blood. ATP activity measured in G-CSF-treated patients was significantly higher than that measured in healthy individuals or "nonmobilized" patients. In patients treated with G-CSF, CD4(+) T cells were predominantly CD25(low)FOXP3(low), consistent with an activated phenotype. However, T-cell depletion did not abrogate ATP production in blood samples from G-CSF-treated patients, indicating that CD4(+) myeloid cells contributed to the increased ATP levels observed in these patients. There was a significant correlation between ATP activity and patient survival, suggesting that efficient activation of CD4(+) cells during mobilization treatment predicts a low risk of disease relapse. Monitoring immune cell reactivity using the Immuknow assay may assist in the clinical management of patients with hematologic malignancies and optimization of HSC mobilization protocols.

  9. MET-Independent Lung Cancer Cells Evading EGFR Kinase Inhibitors are Therapeutically Susceptible to BH3 Mimetic Agents

    PubMed Central

    Fan, Weiwen; Tang, Zhe; Yin, Lihong; Morrison, Bei; Hafez-Khayyata, Said; Fu, Pingfu; Huang, Honglian; Bagai, Rakesh; Jiang, Shan; Kresak, Adam; Howell, Scott; Vasanji, Amit; Flask, Chris A.; Halmos, Balazs; Koon, Henry; Ma, Patrick C.

    2011-01-01

    Targeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after initial responses. To define early molecular changes within residual tumor cells that persist after treatment, we analyzed drug sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible EGFR inhibitors, alone or in combination with MET kinase inhibitors, to characterize the adaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependence on MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversed after withdrawal of targeted inhibitors. Findings were validated in a xenograft model, demonstrating BCL-2 induction and p-STAT3[Y705] activation within the residual tumor cells surviving the initial anti-tumor response to targeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cells using BH3 mimetic agents such as ABT-737, or by dual RNAi-mediated knockdown of BCL-2/BCL-XL, was sufficient to eradicate the early resistant lung tumor cells evading targeted inhibitors. Similarly, in a xenograft model the preemptive co-treatment of lung tumor cells with an EGFR inhibitor and a BH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response with prolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combined with targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung cancer treatment. PMID:21555370

  10. Aryl-Alkyl-Lysines: Agents That Kill Planktonic Cells, Persister Cells, Biofilms of MRSA and Protect Mice from Skin-Infection

    PubMed Central

    Ghosh, Chandradhish; Manjunath, Goutham B.; Konai, Mohini M.; Uppu, Divakara S. S. M.; Hoque, Jiaul; Paramanandham, Krishnamoorthy; Shome, Bibek R.; Haldar, Jayanta

    2015-01-01

    Development of synthetic strategies to combat Staphylococcal infections, especially those caused by methicillin resistant Staphyloccus aureus (MRSA), needs immediate attention. In this manuscript we report the ability of aryl-alkyl-lysines, simple membrane active small molecules, to treat infections caused by planktonic cells, persister cells and biofilms of MRSA. A representative compound, NCK-10, did not induce development of resistance in planktonic cells in multiple passages and retained activity in varying environments of pH and salinity. At low concentrations the compound was able to depolarize and permeabilize the membranes of S. aureus persister cells rapidly. Treatment with the compound not only eradicated pre-formed MRSA biofilms, but also brought down viable counts in bacterial biofilms. In a murine model of MRSA skin infection, the compound was more effective than fusidic acid in bringing down the bacterial burden. Overall, this class of molecules bears potential as antibacterial agents against skin-infections. PMID:26669634

  11. Establishment of hairy root cultures of Rhaponticum carthamoides (Willd.) Iljin for the production of biomass and caffeic acid derivatives.

    PubMed

    Skała, Ewa; Kicel, Agnieszka; Olszewska, Monika A; Kiss, Anna K; Wysokińska, Halina

    2015-01-01

    The aim of the study was to obtain transformed roots of Rhaponticum carthamoides and evaluate their phytochemical profile. Hairy roots were induced from leaf explants by the transformation of Agrobacterium rhizogenes strains A4 and ATCC 15834. The best response (43%) was achieved by infection with A4 strain. The effects of different liquid media (WPM, B5, SH) with full and half-strength concentrations of macro- and micronutrients on biomass accumulation of the best grown hairy root line (RC3) at two different lighting conditions (light or dark) were investigated. The highest biomass (93 g L(-1) of the fresh weight after 35 days) was obtained in WPM medium under periodic light. UPLC-PDA-ESI-MS(3) and HPLC-PDA analyses of 80% aqueous methanol extracts from the obtained hairy roots revealed the presence of eleven caffeoylquinic acids and their derivatives and five flavonoid glycosides. The production of caffeoylquinic acids and their derivatives was elevated in hairy roots grown in the light. Only light-grown hairy roots demonstrated the capability for the biosynthesis of such flavonoid glycosides as quercetagetin, quercetin, luteolin, and patuletin hexosides. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid and a tentatively identified tricaffeoylquinic acid derivative were detected as the major compounds present in the transformed roots.

  12. Establishment, Culture, and Scale-up of Brugmansia candida Hairy Roots for the Production of Tropane Alkaloids.

    PubMed

    Cardillo, Alejandra Beatriz; Rodriguez Talou, Julián; Giulietti, Ana María

    2016-01-01

    Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6β-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed. PMID:27108317

  13. Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor.

    PubMed

    Patra, Nivedita; Srivastava, Ashok K

    2014-11-01

    Artemisinin is an important drug commonly used in the treatment of malaria as a combination therapy. It is primarily produced by a plant Artemisia annua, however, its supply from plant is significantly lower than its huge demand and therefore alternative in vitro production routes are sought. Hairy root cultivation could be one such alternative production protocol. Agrobacterium rhizogenes was used to induce hairy roots of A. annua. Statistical optimization of media was thereafter attempted to maximize the biomass/artemisinin production. The growth and product formation kinetics and the significant role of O2 in hairy root propagation were established in optimized media. Mass cultivation of hairy roots was, thereafter, attempted in a modified 3-L Stirred Tank Bioreactor (Applikon Dependable Instruments, The Netherlands) using optimized culture conditions. The reactor was suitably modified to obtain profuse growth of hairy roots by segregating and protecting the growing roots from the agitator rotation in the reactor using a perforated Teflon disk. It was possible to produce 18 g biomass L(-1) (on dry weight basis) and 4.63 mg L(-1) of artemisinin in 28 days, which increased to 10.33 mg L(-1) by the addition of elicitor methyl jasmonate.

  14. Establishment of hairy root cultures of Rhaponticum carthamoides (Willd.) Iljin for the production of biomass and caffeic acid derivatives.

    PubMed

    Skała, Ewa; Kicel, Agnieszka; Olszewska, Monika A; Kiss, Anna K; Wysokińska, Halina

    2015-01-01

    The aim of the study was to obtain transformed roots of Rhaponticum carthamoides and evaluate their phytochemical profile. Hairy roots were induced from leaf explants by the transformation of Agrobacterium rhizogenes strains A4 and ATCC 15834. The best response (43%) was achieved by infection with A4 strain. The effects of different liquid media (WPM, B5, SH) with full and half-strength concentrations of macro- and micronutrients on biomass accumulation of the best grown hairy root line (RC3) at two different lighting conditions (light or dark) were investigated. The highest biomass (93 g L(-1) of the fresh weight after 35 days) was obtained in WPM medium under periodic light. UPLC-PDA-ESI-MS(3) and HPLC-PDA analyses of 80% aqueous methanol extracts from the obtained hairy roots revealed the presence of eleven caffeoylquinic acids and their derivatives and five flavonoid glycosides. The production of caffeoylquinic acids and their derivatives was elevated in hairy roots grown in the light. Only light-grown hairy roots demonstrated the capability for the biosynthesis of such flavonoid glycosides as quercetagetin, quercetin, luteolin, and patuletin hexosides. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid and a tentatively identified tricaffeoylquinic acid derivative were detected as the major compounds present in the transformed roots. PMID:25811023

  15. Establishment of Hairy Root Cultures of Rhaponticum carthamoides (Willd.) Iljin for the Production of Biomass and Caffeic Acid Derivatives

    PubMed Central

    Skała, Ewa; Kicel, Agnieszka; Olszewska, Monika A.; Kiss, Anna K.

    2015-01-01

    The aim of the study was to obtain transformed roots of Rhaponticum carthamoides and evaluate their phytochemical profile. Hairy roots were induced from leaf explants by the transformation of Agrobacterium rhizogenes strains A4 and ATCC 15834. The best response (43%) was achieved by infection with A4 strain. The effects of different liquid media (WPM, B5, SH) with full and half-strength concentrations of macro- and micronutrients on biomass accumulation of the best grown hairy root line (RC3) at two different lighting conditions (light or dark) were investigated. The highest biomass (93 g L−1 of the fresh weight after 35 days) was obtained in WPM medium under periodic light. UPLC-PDA-ESI-MS3 and HPLC-PDA analyses of 80% aqueous methanol extracts from the obtained hairy roots revealed the presence of eleven caffeoylquinic acids and their derivatives and five flavonoid glycosides. The production of caffeoylquinic acids and their derivatives was elevated in hairy roots grown in the light. Only light-grown hairy roots demonstrated the capability for the biosynthesis of such flavonoid glycosides as quercetagetin, quercetin, luteolin, and patuletin hexosides. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid and a tentatively identified tricaffeoylquinic acid derivative were detected as the major compounds present in the transformed roots. PMID:25811023

  16. Proteomic analysis of human glioblastoma cell lines differently resistant to a nitric oxide releasing agent.

    PubMed

    Leone, Roberta; Giussani, Paola; De Palma, Sara; Fania, Chiara; Capitanio, Daniele; Vasso, Michele; Brioschi, Loredana; Riboni, Laura; Viani, Paola; Gelfi, Cecilia

    2015-06-01

    Glioblastoma multiforme is the most aggressive astrocytoma characterized by the development of resistant cells to various cytotoxic stimuli. Nitric oxide (NO) is able to overcome tumor resistance in PTEN mutated rat C6 glioma cells due to its ability to inhibit cell growth by influencing the intracellular distribution of ceramide. The aim of this study is to monitor the effects of NO donor PAPANONOate on ceramide trafficking in human glioma cell lines, CCF-STTG1 (PTEN-mutated, p53-wt) and T98G (PTEN-harboring, p53-mutated), together with the assessment of their differential molecular signature by 2D-DIGE and MALDI mass spectrometry. In the CCF-STTG1 cell line, the results indicate that treatment with PAPANONOate decreased cell proliferation (<50%) and intracellular trafficking of ceramide, assessed by BODIPY-C5Cer, while these events were not observed in the T98G cell line. Proteomic results suggest that CCF-STTG1 cells are characterized by an increased expression of proteins involved in NO-associated ER stress (i.e. protein disulfide-isomerase A3, calreticulin, 78 kDa glucose-regulated protein), which could compromise ceramide delivery from ER to Golgi, leading to ceramide accumulation in ER and partial growth arrest. Conversely, T98G cell lines, resistant to NO exposure, are characterized by increased levels of cytosolic antioxidant proteins (i.e. glutathione-S-transferase P, peroxiredoxin 1), which might buffer intracellular NO. By providing differential ceramide distribution after NO exposure and differential protein expression of two high grade glioma cell lines, this study highlights specific proteins as possible markers for tumor aggressiveness. This study demonstrates that, in two different high grade glioma cell lines, NO exposure results in a different ceramide distribution and protein expression. Furthermore, this study highlights specific proteins as possible markers for tumor aggressiveness. PMID:25797839

  17. Norlittorine and norhyoscyamine identified as products of littorine and hyoscyamine metabolism by (13)C-labeling in Datura innoxia hairy roots.

    PubMed

    Al Balkhi, Mohamad Houssam; Schiltz, Séverine; Lesur, David; Lanoue, Arnaud; Wadouachi, Anne; Boitel-Conti, Michèle

    2012-02-01

    The presence of two compounds, norlittorine and norhyoscyamine, has been reported in leaves and roots of Datura innoxia; however their metabolic origin in the tropane alkaloid pathway has remained unknown. Precise knowledge of this pathway is a necessary pre-requisite to optimize the production of hyoscyamine and scopolamine in D. innoxia hairy root cultures. The exact structure of norlittorine and norhyoscyamine was confirmed by LC-MS/MS and NMR analyses. Isotopic labeling experiments, using [1-(13)C]-phenylalanine, [1'-(13)C]-littorine and [1'-(13)C]-hyoscyamine, combined with elicitor treatments, using methyl jasmonate, coronalon and 1-aminocyclopropane-1-carboxylic acid, were used to investigate the metabolic origin of the N-demethylated tropane alkaloids. The results suggest that norlittorine and norhyoscyamine are induced under stress conditions by conversion of littorine and hyoscyamine. We propose the N-demethylation of tropane alkaloids as a mechanism to detoxify cells in overproducing conditions.

  18. Alkylating agent methyl methanesulfonate (MMS) induces a wave of global protein hyperacetylation: Implications in cancer cell death

    SciTech Connect

    Lee, Min-Young; Kim, Myoung-Ae; Kim, Hyun-Ju; Bae, Yoe-Sik; Park, Joo-In; Kwak, Jong-Young; Chung, Jay H.; Yun, Jeanho . E-mail: yunj@dau.ac.kr

    2007-08-24

    Protein acetylation modification has been implicated in many cellular processes but the direct evidence for the involvement of protein acetylation in signal transduction is very limited. In the present study, we found that an alkylating agent methyl methanesulfonate (MMS) induces a robust and reversible hyperacetylation of both cytoplasmic and nuclear proteins during the early phase of the cellular response to MMS. Notably, the acetylation level upon MMS treatment was strongly correlated with the susceptibility of cancer cells, and the enhancement of MMS-induced acetylation by histone deacetylase (HDAC) inhibitors was shown to increase the cellular susceptibility. These results suggest protein acetylation is important for the cell death signal transduction pathway and indicate that the use of HDAC inhibitors for the treatment of cancer is relevant.

  19. NOTCH1 inhibition enhances the efficacy of conventional chemotherapeutic agents by targeting head neck cancer stem cell

    PubMed Central

    Zhao, Zhi-Li; Zhang, Lu; Huang, Cong-Fa; Ma, Si-Rui; Bu, Lin-Lin; Liu, Jian-Feng; Yu, Guang-Tao; Liu, Bing; Gutkind, J. Silvio; Kulkarni, Ashok B.; Zhang, Wen-Feng; Sun, Zhi-Jun

    2016-01-01

    Cancer stem cells (CSCs) are considered responsible for tumor initiation and chemoresistance. This study was aimed to investigate the possibility of targeting head neck squamous cell carcinoma (HNSCC) by NOTCH1 pathway inhibition and explore the synergistic effect of combining NOTCH inhibition with conventional chemotherapy. NOTCH1/HES1 elevation was found in human HNSCC, especially in tissue post chemotherapy and lymph node metastasis, which is correlated with CSCs markers. NOTCH1 inhibitor DAPT (GSI-IX) significantly reduces CSCs population and tumor self-renewal ability in vitro and in vivo. Flow cytometry analysis showed that NOTCH1 inhibition reduces CSCs frequency either alone or in combination with chemotherapeutic agents, namely, cisplatin, docetaxel, and 5-fluorouracil. The combined strategy of NOTCH1 blockade and chemotherapy synergistically attenuated chemotherapy-enriched CSC population, promising a potential therapeutic exploitation in future clinical trial. PMID:27108536

  20. Dioxonaphthoimidazoliums are Potent and Selective Rogue Stem Cell Clearing Agents with SOX2-Suppressing Properties.

    PubMed

    Ho, Si-Han Sherman; Ali, Azhar; Ng, Yi-Cheng; Lam, Kuen-Kuen Millie; Wang, Shu; Chan, Woon-Khiong; Chin, Tan-Min; Go, Mei-Lin

    2016-09-01

    Pluripotent stem cells are uniquely positioned for regenerative medicine, but their clinical potential can only be realized if their tumorigenic tendencies are decoupled from their pluripotent properties. Deploying small molecules to remove remnant undifferentiated pluripotent cells, which would otherwise transform into teratomas and teratomacarcinomas, offers several advantages over non-pharmacological methods. Dioxonapthoimidazolium YM155, a survivin suppressant, induced selective and potent cell death of undifferentiated stem cells. Herein, the structural requirements for stemotoxicity were investigated and found to be closely aligned with those essential for cytotoxicity in malignant cells. There was a critical reliance on the quinone and imidazolium moieties but a lesser dependence on ring substituents, which served mainly to fine-tune activity. Several potent analogues were identified which, like YM155, suppressed survivin and decreased SOX2 in stem cells. The decrease in SOX2 would cause an imbalance in pluripotent factors that could potentially prompt cells to differentiate and hence decrease the risk of aberrant teratoma formation. As phosphorylation of the NF-κB p50 subunit was also suppressed, the crosstalk between phospho-p50, SOX2, and survivin could implicate a causal role for NF-κB signaling in mediating the stem cell clearing properties of dioxonaphthoimidazoliums.

  1. Stem Cell-Derived Exosomes: A Potential Alternative Therapeutic Agent in Orthopaedics

    PubMed Central

    Burke, John; Kolhe, Ravindra; Hunter, Monte; Isales, Carlos; Hamrick, Mark; Fulzele, Sadanand

    2016-01-01

    Within the field of regenerative medicine, many have sought to use stem cells as a promising way to heal human tissue; however, in the past few years, exosomes (packaged vesicles released from cells) have shown more exciting promise. Specifically, stem cell-derived exosomes have demonstrated great ability to provide therapeutical benefits. Exosomal products can include miRNA, other genetic products, proteins, and various factors. They are released from cells in a paracrine fashion in order to combat local cellular stress. Because of this, there are vast benefits that medicine can obtain from stem cell-derived exosomes. If exosomes could be extracted from stem cells in an efficient manner and packaged with particular regenerative products, then diseases such as rheumatoid arthritis, osteoarthritis, bone fractures, and other maladies could be treated with cell-free regenerative medicine via exosomes. Many advances must be made to get to this point, and the following review highlights the current advances of stem cell-derived exosomes with particular attention to regenerative medicine in orthopaedics. PMID:26904130

  2. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction.

    PubMed

    Kam, Nadine Wong Shi; O'Connell, Michael; Wisdom, Jeffrey A; Dai, Hongjie

    2005-08-16

    Biological systems are known to be highly transparent to 700- to 1,100-nm near-infrared (NIR) light. It is shown here that the strong optical absorbance of single-walled carbon nanotubes (SWNTs) in this special spectral window, an intrinsic property of SWNTs, can be used for optical stimulation of nanotubes inside living cells to afford multifunctional nanotube biological transporters. For oligonucleotides transported inside living cells by nanotubes, the oligos can translocate into cell nucleus upon endosomal rupture triggered by NIR laser pulses. Continuous NIR radiation can cause cell death because of excessive local heating of SWNT in vitro. Selective cancer cell destruction can be achieved by functionalization of SWNT with a folate moiety, selective internalization of SWNTs inside cells labeled with folate receptor tumor markers, and NIR-triggered cell death, without harming receptor-free normal cells. Thus, the transporting capabilities of carbon nanotubes combined with suitable functionalization chemistry and their intrinsic optical properties can lead to new clas