Tips Cells Research Articles

Ca2+ involved in GABA signal transduction for phenolics accumulation in germinated hulless barley under NaCl stress

In this study, in order to investigate the role of Ca2+ in GABA signal transduction involved in phenolics accumulation in barley seedlings under NaCl stress, the seedlings were treated with exogenous GABA and its synthesis inhibitor, 3-mercaplopropionic acid (3-MP), as well as Ca2+ channel blockers La3+, Ca2+ chelator EGTA, and Ca2+ release channel inhibitor 2-aminoethoxydiphenyl borate (2-APB). The results showed that GABA significantly enhanced phenolics, calcium and calmodulin content. It also induced Ca2+ influx in barley root tips cells, and altered the distribution of Ca2+, making calcium precipitates more uniform and intensive. While, 3-MP treatment led to opposite changes, which suggested that GABA was essential for calcium content increase. In addition, accumulation of phenolics was inhibited by LaCl3, EGTA and 2-APB treatments, and this inhibition could be alleviated partly by exogenous GABA. Taken together, Ca2+ was involved in GABA signal transduction for phenolics accumulation in barley seedlings under NaCl stress.

Conventional farming reduces the activity of earthworms: Assessment of genotoxicity test of soil and vermicast

The activity of earthworms in an agricultural field and botanical garden was substantiated. Samples of vermicast produced by earthworms along with the soil were collected from an agricultural field (conventional farm) and a botanical garden and their genotoxic assessment were observed on the root tips cells of Allium cepa. The mitotic index (MI) was increased in both vermicast samples compared to soil. However, the mean (±SE) MI was greater in the vermicast (13.93 ± 0.67) collected from the botanical garden compared to the agricultural vermicast (11.40 ± 0.51). Chromosomal aberration levels were greater in the soil of the agricultural field (14.88%) compared to soil from the botanical garden (9.11%), whereas a reduction in chromosomal aberrations was observed in the vermicast of both sites. The number of earthworms was less on the conventional farm (18 earthworms/1800 cm2 area) compared to the botanical garden (72 earthworms/1800 cm2 area). Thus, this study showed that the activity of earthworms was less in an agricultural field where there may have been more application of chemical fertilizers and pesticides compared to a site using more organic manure with reduced genotoxicity.

Cadmium’s Effect on the Organization of Microtubular Cytoskeleton in Root Tips Cells of Salix matsudana Koidz

Cadmium’s Effect on the Organization of Microtubular Cytoskeleton in Root Tips Cells of Salix matsudana Koidz

Genotoxic effects of water extract of Nicotiana glauca (Garham,1828) on Allium cepa mitosis

The present study planned to find out the genotoxic effect of N. glauca water extract on rate cell division and chromosomes behavior in vivo in Allium cepa tips cells. The roots of this plant were treated with five different concentrations 100, 10, 1, 0.1, and 0.01 mg/ml of extract of N. glauca for 3, 6, 12 and 24 hours. Cytological analysis revealed a significant inhibition of mitotic index which increased by increasing concentration. All of the concentrations induced chromosomal abnormalities such as: micronuclei, chromosome stickiness, bridge, laggards, chromatide and chromosomes break.

Rates and spectra of chromosome aberrations in winter wheat cells after dimethylsulfate action

In the article we report about our investigation of cytogenetic parameters in chromosomal complex of the new Ukrainian winter wheat varieties and some relations amid value of cytological indexes and different concentrations of DMS (dimethylsulfate). We used the anaphases method for the investigation of chromosomal rearrangements and determination the mutagen action on plants and identification the nature of mutagen. We studied combined the sensitivity of genotype by the cytological analysis of mutagen treated population of wheat and revealed the reaction of different varieties depending upon the way of its origin. This was done for determining its connections and differences on cytogenetics level together with specificity of mutagen action on cell level. Dry seeds of seven varieties and one line of winter wheat were subjected to DMS in 0.0125, 0.025, and 0.05 % concentrations, which are the most useful for winter wheat in terms of genetic investigation programs. We investigated the rates and spectra of chromosomal aberrations in winter wheat (primary roots of tips cells) during the mitosis. We identified the following types of chromosomal rearrangements: chromatid and chromosome bridges, single and double fragments, micronuclei, and lagging chromosomes. Investigation of DMS action confirmed reliability of fragments-bridges ratio (prevalence of fragments under the bridges for chemical mutagens and vice versa for physical mutagens) for the mutagen nature identification.

Reliability of plant root comet assay in comparison with human leukocyte comet assay for assessment environmental genotoxic agents

Comet assay is an efficient test to detect genotoxic compounds based on observation of DNA damage. The aim of this work was to compare the results obtained from the comet assay in two different type of cells extracted from the root tips from Lactuca sativa L. and human blood. For this, Spent Pot Liner (SPL), and its components (aluminum and fluoride) were applied as toxic agents. SPL is a solid waste generated in industry from the aluminum mining and processing with known toxicity. Three concentrations of all tested solutions were applied and the damages observed were compared to negative and positive controls. It was observed an increase in the frequency of DNA damage for human leukocytes and plant cells, in all treatments. On human leukocytes, SPL induced the highest percentage of damage, with an average of 87.68%. For root tips cells of L. sativa the highest percentage of damage was detected for aluminum (93.89%). Considering the arbitrary units (AU), the average of nuclei with high levels of DNA fragmentation was significant for both cells type evaluated. The tested cells demonstrated equal effectiveness for detection of the genotoxicity induced by the SPL and its chemical components, aluminum and fluoride. Further, using a unique method, the comet assay, we proved that cells from root tips of Lactuca sativa represent a reliable model to detect DNA damage induced by genotoxic pollutants is in agreement of those observed in human leukocytes as model. So far, plant cells may be suggested as important system to assess the toxicological risk of environmental agents.

CNS angiogenesis and barriergenesis occur simultaneously

The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously.

Changes in radical scavenging activity of normal, endoreduplicated and depolyploid root tip cells of Allium cepa.

The plant cell responds to abiotic stress conditions by adjusting its cellular metabolism and various defensive mechanisms. Cellular metabolism involves changes in the cell cycle, in which the cell undergoes repeated rounds of endocycles leading to polyploidization. Defense mechanisms such as role of antioxidants are a key to understand plant adaptation. The present work describes endoreduplication and radical scavenging activity as two different defense mechanisms adapted by plants for their survival under stress condition. The work describes linkage of these two processes with each other under abiotic stress. Endoreduplicated root tip cells of Allium cepa were depolyploidized by exogenous phytohormones. Further, free radical scavenging activity from normal, endoreduplicated and depolyploidized root tips cells was observed to understand the role of phytohormones. Elevated free radical scavenging potential was observed in endoreduplicated cells compared to normal and depolyploidized cells. Based on these results, it was concluded that endoreduplication and antioxidant pathways are linked with each other through phytohormonal activities. The concentration of auxin and cytokinin regulates the activity of ascorbate oxidase enzyme, which in turn maintains the concentration of AsA within the cell. AsA level directs the prolyl-hydroxylation process of cell division proteins in quiescent center cells either toward endoreduplication process or cell division process.

Regeneration of tumor antigen-specific CTLs utilizing iPS technology.

Tumor immunotherapy, especially tumor antigen specific T cell therapy, is currently attracting attention. However, a critical issue still awaits resolution; it is difficult to efficiently expand tumor antigen-specific T cells. To solve this problem, we are now utilizing iPS cell technology. When iPS cells are established from tumor antigen specific T cells, T cells regenerated from these iPS cells are expected to express the same TCRs as the original T cells. In line with this concept, we succeeded in regenerating tumor antigen specific cytotoxic T cells. The regenerated T cells exhibited TCR specific killing activity comparable to that of the original cells, and were able to kill leukemia cells in an antigen-specific manner. We are currently endeavoring to apply this method clinically. In the future, we intend to establish an allogeneic transfusion system, in which various tumor antigen specific T-iPS cells from a wide range of HLA haplotype homozygous donors will be lined up as a "T-iPS cell bank", with the aim of making off-the-shelf tumor immunotherapy a reality.

Genotoxicity reduction in bagasse waste of sugar industry by earthworm technology.

The aim of the present study was to assess the genotoxicity reduction in post vermicompost feed mixtures of bagasse (B) waste using earthworm Eisenia fetida. The genotoxicity of bagasse waste was determined by using Allium cepa root chromosomal aberration assay. Bagasse was amended with cattle dung in different proportions [0:100 (B0) 25:75 (B25), 50:50 (B50), 75:25 (B75) and 100:0 (B100)] on dry weight basis. Genotoxic effects of initial and post vermicompost bagasse extracts were analysed on the root tips cells of Allium cepa. Root length and mitotic index (MI) was found to be increased in post vermicompost extracts when compared to initial bagasse waste. The maximum percent increase of root length was observed in the B50 bagasse extract (96.60 %) and the maximum MI was observed in B100 mixture (14.20 ± 0.60) 6 h treatment which was similar to the control. Genotoxicity analysis of post vermicompost extracts of bagasse revealed a 21–44 % decline in the aberration frequencies and the maximum reduction was found in B75 extract (44.50 %). The increase in root length and mitotic index, as well as decrease in chromosomal aberrations indicates that E. fetida has the ability to reduce the genotoxicity of the bagasse waste.

In Vitro Generation of Antigen-Specific T Cells from Induced Pluripotent Stem Cells of Antigen-Specific T Cell Origin.

Induced pluripotent stem (iPS) cells derived from T lymphocyte (T-iPS cells) preserve the T cell receptor (TCR) α and β gene rearrangements identical to the original T cell clone. Re-differentiated CD8 single positive αβ T cells from the T-iPS cells exhibited antigen-specific cytotoxicity, improved proliferative response, and elongation of telomere indicating rejuvenation of antigen specific T cell immunity in vitro. To regenerate antigen specific cytotoxic T lymphocytes (CTL), first, we have optimized a method for reprogramming-resistant CD8 T cell clones into T-iPS cells by using sendaiviral vectors. Second, we have optimized stepwise differentiation methods for inducing hematopoietic progenitor cells, T cell progenitors, and functionally matured CD8 single positive CTL. These protocols provide useful in vitro tools and models both for research of antigen-specific T cell immunotherapy and for research of normal and pathological thymopoiesis.

Genotoxicity of sulcotrione pesticide and photoproducts on Allium cepa root meristem

Contamination by toxic agents in the environment has become matters of concern to agricultural countries. Sulcotrione, a triketone herbicide used to control dicotyledonous weeds in maize culture is rapidly photolyzed on plant foliage and generate two main photoproducts the xanthene-1,9-dione-3,4-dihydro-6-methylsulfonyl and 2-chloro-4-mesylbenzoic acid (CMBA). The aim of this study was to analyze the potential toxicity of the herbicide and the irradiated herbicide cocktail. Cytotoxicity and genotoxicity of non irradiated and irradiated sulcotrione were investigated in Allium cepa test. The sulcotrione irradiation was monitored under sunlight simulated conditions to reach 50% of phototransformation. Concentrations of sulcotrione in the range 5×10−9–5×10−5M were tested. Cytological analysis of root tips cells showed that both non irradiated and irradiated sulcotrione caused a dose-dependent decrease of mitotic index with higher cytotoxicity for the irradiated herbicide which can lead to 24.2% reduction of mitotic index compared to water control. Concomitantly, chromosomal aberrations were observed in A.cepa root meristems. Both non irradiated sulcotrione and irradiated sulcotrione induced a dose-dependent increase of chromosomal abnormalities frequencies to a maximal value of 33.7%. A saturating effect in anomaly frequencies was observed in meristems treated with high concentrations of non irradiated sulcotrione only. These data suggest that photolyzed sulcotrione cocktail have a greater cytotoxicity and genotoxicity than parent molecule and question about the impact of photochemical process on environment.

Generation of Melanoma Antigen Specific T Cells From Human Ips Cells Using a Feeder Free System.

Abstract 2292iPS cells can be induced from various types of somatic cells by reprogramming them using Yamanaka factors (Oct4, Sox2, Klf4, and Myc). They are reported to be very similar to ES cells in many respects, such as gene expression pattern and multipotency. They are expected to be used as a cell source for production of various types of cells to be used in regeneration medicine. In this study, we planned to use iPS cells as a progenitor source for immune cell therapy. For this purpose, iPS cells derived from a lymphocyte that shows a certain antigen-specificity are preferable, because antigen specificity is inherited to iPS cells made from the lymphocyte. For example, if iPS cell are produced from cytotoxic T cells specific to a tumor antigen, T cells generated from these T-iPS can be used in cell therapy for patients bearing cancer.We have succeeded in establishing iPS cells from human mature T cells (T-iPS cells), namely from whole CD3+ cells or CD4−CD8+ cells of cord blood as well of adult peripheral blood. These T-iPS cells were confirmed to bear productively rearranged TCRβ chain gene. These T-iPS cells were differentiated to CD4+CD8+ double positive T cells and eventually into CD8+ single positive T cells expressing αβTCR in an in vitro co-culture system using OP9-DL1 stromal cells. Such T cell induction occurred more efficiently from T-iPS cells than from human ES cells or from iPS cells derived from other cell types. Sequence analysis of their TCRβ chain suggests that these T cells preserved their original antigen specificity. To confirm that T-iPS cells can generate antigen specific T cells we established T-iPS cells from mature cytotoxic T cells specific to MART-1 (Melanoma antigen recognized by T cells 1) epitope. These MART-1 T-iPS cells were differentiated in vitro until CD4+CD8+ double positive T cells. By stimulation with anti-CD3 antibody, DP cells generated in vitro from MART1-T-iPS cells turned into a large number of CD8+ T cells specific to MART-1, which produced a substantial amount of IFNg upon TCR stimulation. The present study thus provides a novel method for cloning and expanding CD8+ T cells specific to a given antigen, which can be potentially applied for cell therapy against cancer. Disclosures:No relevant conflicts of interest to declare.

Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes

Glandular secreting trichomes on the surface of tomato plants and many of its relatives in the Solanaceae produce a mixture of O-acyl sugars that contribute to insect resistance. The majority of acyl sucroses produced by the cultivated tomato (Solanum lycopersicum) contain three or four short chain aliphatic acyl esters, and tetra-acyl sucroses have an acetyl group as one of the acyl chains. We previously reported overlapping S. lycopersicum × Solanum pennellii introgression lines (ILs) that fail to accumulate high levels of acetylated tetra-acyl sucroses. A survey of the annotated genes in this region of cultivated tomato chromosome 1 revealed three candidate acyltransferases that were tested for function using virus-induced gene silencing. A member of the BAHD family of acyltransferases (Solyc01g105580, SlAT2) was shown to encode an acetyl-CoA-dependent acyltransferase enzyme capable of acyl sucrose acetylation in vitro. RNAi suppression of SlAT2 in transgenic S. lycopersicum cv. M82 resulted in reduced acyl sugar acetylation, whereas expression of the functional S. lycopersicum allele of SlAT2 in the triacyl sucrose producing IL1-3 restored the ability of the IL to synthesize acetylated tetra-acyl sugars. Transgenic plants with the SlAT2 promoter driving GFP expression showed fluorescence in tips cells of long, slender trichomes that is consistent with acyl sugar acetylation occurring in these cells.

Exposure of Vicia faba to sulcotrione pesticide induced genotoxicity

Potential genotoxicity of sulcotrione 2-(2-chloro-4-(methylsulfonyl)benzoyl)-1,3-cyclohexanedione, a selective triketonic herbicide was evaluated on Vicia faba seedlings in hydroponic culture conditions. Sulcotrione (10−5, 10−4 and 2×10−4M) treatments for 45h, caused a dose dependent increase in micronuclei frequencies in root meristematic cells. Cytological analysis of root tips cells showed aneugenic effects of the sulcotrione on the plant root meristems. Sulcotrione induced chromosomal alterations at the lowest concentration used (10−5M) when incubated for 42h, indicating the potent mutagenic effect of this element. This is the first report for the genotoxicity of such a sulcotrione herbicide.

Abstract B31: Potential for patient tailoring for prodrug of gemcitabine (LY2334737) by assessment carboxylesterase II expression in solid tumor biopsies

Abstract Carboxylesterase II (CES2), a serine ester hydrolase, is the major carboxylesterase responsible for the conversion of the prodrug of gemcitabine into its active form, gemcitabine (LY2334737), an anticancer chemotherapeutic. In humans, the ratio of plasma levels of prodrug to active form is 10:1. Therefore, increased availability of LY2334737 can result in cleavage of the prodrug into its active form at the tumor site and has potential for greater tumor cytotoxicity. Relatively high levels of expression of CES2 within non-neoplastic human tissue occur in the liver, kidney and gastrointestinal tract as visualized by immunohistochemistry (IHC). CES2 is also reported to be expressed in human cancers such as non-small cell lung carcinoma and colon adenocarcinomas. In this study, we analyzed CES2- transfected in vitro cell lines by real-time PCR, chromogenic IHC, and automated quantitative analysis (AQUA) of immunofluorescent in situ protein expression and showed that high levels of CES2 correlate with higher cytotoxicity. qRT-PCR analysis revealed that the mRNA levels of CES2 measured in formalin-fixed paraffin-embedded tumors correlate with protein levels. CES2 transcriptional profiling was performed to identify additional tumor types with high levels of expression of CES2. CES2 overexpression was detectable by IHC and/or AQUA analysis in human colon carcinoma, mesothelioma, non-small cell lung cancer, and tumors of the breast and ovary. CES2 IHC labeling in colonic adenocarcinomas was diffuse. However, labeling in non-neoplastic mucosa showed low levels in proliferating cells at the crypt base and increasingly higher expression toward the terminally differentiated cells at the tips—cells that would not progress through the cell cycle and should not be affected by antiproliferative agents. In conclusion, we have developed robust IHC and AQUA-based assays for differential expression of CES protein levels in a variety of archival human tumor types and have shown high correlation with CES2 transcript levels by qRT-PCR. These methodologies can identify tumors with high levels of CES2, a biomarker that may further be investigated in biomarker-driven clinical trials to identify patients who will more likely respond to LY2334737. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B31.

Effect of the aqueous extract of Banana Fruits Peal Musa paradisiaca on Mitosis in Plant and Mammalian cells

The aqueous extract of banana fruits peal was tested for its effect on mitosis . The root tips of Allium cepa were used as plant test system and the bone marrow cells of the albino mice Mus musculus were used as mammalians test system in vivo .Root tips of Allium cepa were treated for four hours with five concentrations of the extract (5 , 10 , 20 , 40 ,60 mg / ml.).The Metaphase was arrested in all the treatments , the highest percentage ( 100 % ) was recorded in the first concentration , the last concentration caused stickiness and clumping of the chromosomes. The treatments did not cause significant difference in the mitotic index. The peals extract (5 mg /ml) was compared with the extracts of fruits bulb, leaves and roots of banana plant, it was found that the extract of fruits peal is the best considering the highest percentages of arrested Metaphase in the root tips cells. The albino mice Mus musculus were injected intraperitonial with the peals extract ( 0.01 , 0.02, 0.04, 0.06, 0.08 mg / gm body weight), the percentages of arrested Metaphase in the bone marrow of these animals were comparable to the recorded percentages when the animals were injected with colchicine ( 0.01 mg / gm b.w.) .This study revealed the antimitotic activity of the aqueous extract of banana fruits peal on both the plant and mammalian cells in vivo. Studies will be conducted to investigate the effect of the extract and its components on the proliferation of cancer cells in vitro and in vivo.

Effect of the aqueous extract of Banana Fruits Peal Musa paradisiaca on Mitosis in Plant and Mammalian cells

The aqueous extract of banana fruits peal was tested for its effect on mitosis . The root tips of Allium cepa were used as plant test system and the bone marrow cells of the albino mice Mus musculus were used as mammalians test system in vivo .Root tips of Allium cepa were treated for four hours with five concentrations of the extract (5 , 10 , 20 , 40 ,60 mg / ml.).The Metaphase was arrested in all the treatments , the highest percentage ( 100 % ) was recorded in the first concentration , the last concentration caused stickiness and clumping of the chromosomes. The treatments did not cause significant difference in the mitotic index. The peals extract (5 mg /ml) was compared with the extracts of fruits bulb, leaves and roots of banana plant, it was found that the extract of fruits peal is the best considering the highest percentages of arrested Metaphase in the root tips cells. The albino mice Mus musculus were injected intraperitonial with the peals extract ( 0.01 , 0.02, 0.04, 0.06, 0.08 mg / gm body weight), the percentages of arrested Metaphase in the bone marrow of these animals were comparable to the recorded percentages when the animals were injected with colchicine ( 0.01 mg / gm b.w.) .This study revealed the antimitotic activity of the aqueous extract of banana fruits peal on both the plant and mammalian cells in vivo. Studies will be conducted to investigate the effect of the extract and its components on the proliferation of cancer cells in vitro and in vivo.

Distribution of Al<SUP>3+</SUP> in Subcellular Structure of Root Tips Cells and Aluminum Tolerance in Soybean

Distribution of Al<SUP>3+</SUP> in Subcellular Structure of Root Tips Cells and Aluminum Tolerance in Soybean

Microarray analysis of Arabidopsis genome response to aluminum stress

To better understand the mechanisms involved in aluminum toxicity and tolerance in plants, microarray technology was used to evaluate changes in gene expression in Arabidopsis thaliana under Al stress. With the use of Affymetrix Arabidopsis ATH1 Genechip, a comparison of RNA expression profiles was made between control and Al-treated Arabidopsis seedlings. A total of 256 genes were identified as Al-responsive. Ninety-four genes were shown to be up-regulated and 162 were down-regulated; comprising 1.1 % of the 24 000 Arabidopsis genes. Real-time RT-PCR was used to confirm the microarray data. The analysis showed that a large number of transcription factors and several putative signaling components were up-regulated by aluminum. Chloroplast structural and photosynthetic genes were, in general, down-regulated. A number of previously identified Al-responsive genes, e.g. GST, Auxin-regulated, Peroxidase, and Chitinase, were up-regulated by Al-stress, whereas Wali 3 and Wali 4 were down-regulated. We also identified several up-regulated genes involved in vacuolar signaling, sorting and docking. Three genes were also up-regulated by Al-stress, Ras GTP-binding protein, ABC-cassette binding, and the AtELP1 receptor genes, have previously been documented as responsive to drought and/or oxidative stress and may play important roles the detoxification of Al ions by transportation and storage into root vacuoles. Ultrastructural changes in the roots tips cells of Arabidopsis were evaluated using transmission electron microscopy and energy-dispersive X-ray analysis with scanning electron microscopy and results showed Al accumulation in the root tips of Arabidopsis.