A heat-shock inducible system for flexible gene expression in cereals.

BACKGROUND: Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana, they have not yet been implemented in grasses. RESULTS: Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). CONCLUSIONS: This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

NIN Acts as a Network Hub Controlling a Growth Module Required for Rhizobial Infection.

The symbiotic infection of root cells by nitrogen-fixing rhizobia during nodulation requires the transcription factor Nodule Inception (NIN). Our root hair transcriptomic study extends NIN's regulon to include Rhizobium Polar Growth and genes involved in cell wall modification, gibberellin biosynthesis, and a comprehensive group of nutrient (N, P, and S) uptake and assimilation genes, suggesting that NIN's recruitment to nodulation was based on its role as a growth module, a role shared with other NIN-Like Proteins. The expression of jasmonic acid genes in nin suggests the involvement of NIN in the resolution of growth versus defense outcomes. We find that the regulation of the growth module component Nodulation Pectate Lyase by NIN, and its function in rhizobial infection, are conserved in hologalegina legumes, highlighting its recruitment as a major event in the evolution of nodulation. We find that Nodulation Pectate Lyase is secreted to the infection chamber and the lumen of the infection thread. Gene network analysis using the transcription factor mutants for ERF Required for Nodulation1 and Nuclear Factor-Y Subunit A1 confirms hierarchical control of NIN over Nuclear Factor-Y Subunit A1 and shows that ERF Required for Nodulation1 acts independently to control infection. We conclude that while NIN shares functions with other NIN-Like Proteins, the conscription of key infection genes to NIN's control has made it a central regulatory hub for rhizobial infection.

Speed breeding is a powerful tool to accelerate crop research and breeding.

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand 1 . This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.

Biphasic organo-bioceramic fibrous composite as a biomimetic extracellular matrix for bone tissue regeneration.

In bone tissue engineering, the organo-ceramic composite, electrospun polycaprolactone/hydroxyapatite (PCL/HA) scaffold has the potential to support cell proliferation, migration, differentiation, and homeostasis. Here, we report the effect of PCL/HA scaffold in tissue regeneration using human mesenchymal stem cells (hMSCs). We characterized the scaffold by fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), scanning electron microscope (SEM) and assessed its biocompatibility. PCL/HA composite is superior as a scaffold compared to PCL alone. Furthermore, increasing HA content (5-10%) was more efficacious in supporting cell-scaffold attachment, expression of ECM molecules and proliferation. These results suggest that PCL/HA is useful as a scaffold for tissue regeneration.

Trace elements and heavy metals in the Grand Bay National Estuarine Reserve in the northern Gulf of Mexico.

The objectives of this study are to investigate distribution of trace elements and heavy metals in the salt marsh and wetland soil and biogeochemical processes in the Grand Bay National Estuarine Research Reserve of the northern Gulf of Mexico. The results show that Hg, Cd and to some extent, As and Pb have been significantly accumulated in soils. The strongest correlations were found between concentrations of Ni and total organic matter contents. The correlations decreased in the order: Ni>Cr>Sr>Co>Zn, Cd>Cu>Cs. Strong correlations were also observed between total P and concentrations of Ni, Co, Cr, Sr, Zn, Cu, and Cd. This may be related to the P spilling accident in 2005 in the Bangs Lake site. Lead isotopic ratios in soils matched well those of North American coals, indicating the contribution of Pb through atmospheric fallout from coal power plants.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

The root hair "infectome" of Medicago truncatula uncovers changes in cell cycle genes and reveals a requirement for Auxin signaling in rhizobial infection.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

Enhancement of arsenic trioxide-mediated changes in human induced pluripotent stem cells (IPS).

Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell, showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however, its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation, induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells, we hypothesize that it will induce alterations in morphology, inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0-9 µg/mL). Cell morphology, viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in a dose dependent manner. There were significant increases in tail length and moment as well as destruction of intact DNA as concentration increased. Exposure to ATO resulted in a reproducible dose dependent sequence of events marked by changes in morphology, decrease of cell viability, and induction of genotoxicity in IPS.

Synthetic biology approaches to engineering the nitrogen symbiosis in cereals.

Nitrogen is abundant in the earth's atmosphere but, unlike carbon, cannot be directly assimilated by plants. The limitation this places on plant productivity has been circumvented in contemporary agriculture through the production and application of chemical fertilizers. The chemical reduction of nitrogen for this purpose consumes large amounts of energy and the reactive nitrogen released into the environment as a result of fertilizer application leads to greenhouse gas emissions, as well as widespread eutrophication of aquatic ecosystems. The environmental impacts are intensified by injudicious use of fertilizers in many parts of the world. Simultaneously, limitations in the production and supply of chemical fertilizers in other regions are leading to low agricultural productivity and malnutrition. Nitrogen can be directly fixed from the atmosphere by some bacteria and Archaea, which possess the enzyme nitrogenase. Some plant species, most notably legumes, have evolved close symbiotic associations with nitrogen-fixing bacteria. Engineering cereal crops with the capability to fix their own nitrogen could one day address the problems created by the over- and under-use of nitrogen fertilizers in agriculture. This could be achieved either by expression of a functional nitrogenase enzyme in the cells of the cereal crop or through transferring the capability to form a symbiotic association with nitrogen-fixing bacteria. While potentially transformative, these biotechnological approaches are challenging; however, with recent advances in synthetic biology they are viable long-term goals. This review discusses the possibility of these biotechnological solutions to the nitrogen problem, focusing on engineering the nitrogen symbiosis in cereals.

Vitamin D3 potentiates the antitumorigenic effects of arsenic trioxide in human leukemia (HL-60) cells.

BACKGROUND: Arsenic trioxide (ATO) is a novel form of therapy that has been found to aid acute promyelocytic leukemia (APL) patients. Our laboratory has demonstrated that ATO-induced cytotoxicity in human leukemia (HL-60) cells is mediated by oxidative stress. Pro-oxidants have been known to play a role in free radical-mediated oxidative stress. Vitamin D3, (Vit D3) an active metabolite of vitamin D has been reported to inhibit the growth of number neoplasms such as prostate, breast, colorectal, leukemia, and skin cancers. The goal of the present research was to use (HL-60) cells as an in vitro test model to evaluate whether low doses of Vit D3 potentiate the toxicity of ATO and whether this toxic action is mediated via apoptotic mechanisms. METHOD: HL-60 cells were treated either with a pharmacologic dose of ATO alone and with several low doses of Vit D3. Cell survival was determined by MTT assay. Cell apoptosis was measured both by flow cytometry assessment, and DNA laddering assay. RESULTS: MTT assay indicated that Vit D3 co-treatment potentiates ATO toxicity in HL-60 cells in a dose dependent manner. A statistically significant and dose-dependent increase (p <0.05) was recorded in annexin V positive cells (apoptotic cells) with increasing doses of Vit D3 in ATO-treated cells. This finding was confirmed by the result of DNA laddering assay showing clear evidence of nucleosomal DNA fragmentation in vitamin and ATO co-treated cells. CONCLUSION: The present study indicates that Vit D3 potentiates the antitumor effects of ATO. This potentiation is mediated at least in part, through induction of phosphatidylserine externalization and nucleosomal DNA fragmentation. These findings highlight the potential impact of Vit D3 in promoting the pharmacological effect of ATO, suggesting a possible future role of Vit D3/ATO combination therapy in patients with acute promyelocytic leukemia (APL).

Rapid screening of heavy metals and trace elements in environmental samples using portable X-ray fluorescence spectrometer, A comparative study.

With industrialization, great amounts of trace elements and heavy metals have been excavated and released on the surface of the earth and dissipated into the environments. Rapid screening technology for detecting major and trace elements as well as heavy metals in variety of environmental samples is most desired. The objectives of this study were to determine the detection limits, accuracy, repeatability and efficiency of a X-ray fluorescence spectrometer (Niton XRF analyzer) in comparison with the traditional analytical methods, inductively coupled plasma optical emission spectrometer (ICP-OES) and inductively coupled plasma optical emission spectrometer (ICP-MS) in screening of major and trace elements of environmental samples including estuary soils and sediments, contaminated soils, and biological samples. XRF is a fast and non-destructive method in measuring the total concentration of multi--elements simultaneously. Contrary to ICP-OES and ICP-MS, XRF analyzer is characterized by the limited preparation required for solid samples, non-destructive analysis, increased total speed and high throughout, the decreased production of hazardous waste and the low running costs as well as multi-elemental determination and portability in the fields. The current comparative study demonstrates that XRF is a good rapid non-destructive method for contaminated soils, sediments and biological samples containing higher concentrations of major and trace elements. Unfortunately, XRF does not have sensitive detection limits of most major and trace elements as ICP-OES or ICP-MS but it may serve as a rapid screening tool for locating hot spots of uncontaminated field soils and sediments.

LegumeGRN: a gene regulatory network prediction server for functional and comparative studies.

Building accurate gene regulatory networks (GRNs) from high-throughput gene expression data is a long-standing challenge. However, with the emergence of new algorithms combined with the increase of transcriptomic data availability, it is now reachable. To help biologists to investigate gene regulatory relationships, we developed a web-based computational service to build, analyze and visualize GRNs that govern various biological processes. The web server is preloaded with all available Affymetrix GeneChip-based transcriptomic and annotation data from the three model legume species, i.e., Medicago truncatula, Lotus japonicus and Glycine max. Users can also upload their own transcriptomic and transcription factor datasets from any other species/organisms to analyze their in-house experiments. Users are able to select which experiments, genes and algorithms they will consider to perform their GRN analysis. To achieve this flexibility and improve prediction performance, we have implemented multiple mainstream GRN prediction algorithms including co-expression, Graphical Gaussian Models (GGMs), Context Likelihood of Relatedness (CLR), and parallelized versions of TIGRESS and GENIE3. Besides these existing algorithms, we also proposed a parallel Bayesian network learning algorithm, which can infer causal relationships (i.e., directionality of interaction) and scale up to several thousands of genes. Moreover, this web server also provides tools to allow integrative and comparative analysis between predicted GRNs obtained from different algorithms or experiments, as well as comparisons between legume species. The web site is available at http://legumegrn.noble.org.

Cytotoxicity patterns of arsenic trioxide exposure on HaCaT keratinocytes.

BACKGROUND: Arsenic is a ubiquitous environmental toxicant, and abnormalities of the skin are the most common outcomes of long-term, low-dose, chronic arsenic exposure. If the balance between keratinocyte proliferation, differentiation, and death is perturbed, pathologic changes of the epidermis may result, including psoriasis, atopic dermatitis, and certain forms of ichthyosis. Therefore, research investigations using in vitro human epidermal cells could help elucidate cellular and molecular processes in keratinocytes affected by arsenic. Data from such investigations could also provide the basis for developing cosmetic intervention for skin diseases caused by arsenic. METHODS: The viability of HaCaT keratinocyte cultures with or without prior exposure to low-dose arsenic trioxide was compared for varying concentrations of arsenic trioxide over a time course of 14 days because in untreated control cultures, approximately 2 weeks is required to complete cell differentiation. Long-term cultures were established by culturing HaCaT cells on collagen IV, and cells were subsequently exposed to 0 parts per million (ppm), 1 ppm, 5 ppm, 7.5 ppm, 10 ppm, and 15 ppm of arsenic trioxide. The percentages of viable cells as well as DNA damage after exposure were determined on Day 2, Day 5, Day 8, and Day 14. RESULTS: Using both statistical and visual analytics approaches for data analysis, we have observed a biphasic response at a 5 ppm dose with cell viability peaking on Day 8 in both chronic and acute exposures. Further, a low dose of 1 ppm arsenic trioxide enhanced HaCaT keratinocyte proliferation, whereas doses above 7.5 ppm inhibited growth. CONCLUSION: The time course profiling of arsenic trioxide cytotoxicity using long-term HaCaT keratinocyte cultures presents an approach to modeling the human epidermal cellular responses to varying doses of arsenic trioxide treatment or exposure. A low dose of arsenic trioxide appears to aid cell growth but concomitantly disrupts the DNA transcription process.

The effects of arsenic trioxide on DNA synthesis and genotoxicity in human colon cancer cells.

Colon cancer is the third leading cause of cancer-related deaths worldwide. Recent studies in our laboratory have demonstrated that arsenic trioxide is cytotoxic in human colon cancer (HT-29), lung (A549) and breast (MCF-7) carcinoma cells. The purpose of the present study is to investigate the effects of arsenic trioxide on DNA synthesis and the possible genotoxic effects on human colon cancer cells. HT-29 cells were cultured according to standard protocol, followed by exposure to various doses (0, 2, 4, 6, 8, 10, and 12 microg/mL) of arsenic trioxide for 24 h. The proliferative response (DNA synthesis) to arsenic trioxide was assessed by [(3)H]thymidine incorporation. The genotoxic effects of arsenic-induced DNA damage in a human colon cancer cell line was evaluated by the alkaline single cell gel electrophoresis. Results indicated that arsenic trioxide affected DNA synthesis in HT-29 cells in a biphasic manner; showing a slight but not significant increase in cell proliferation at lower levels of exposure (2, 4 and 6 microg/mL) followed by a significant inhibition of cell proliferation at higher doses (i.e., 8 and 10 microg/mL). The study also confirmed that arsenic trioxide exposure caused genotoxicity as revealed by the significant increase in DNA damage, comet tail-lengths, and tail moment when compared to non-exposed cells. Results of the [(3)H]thymidine incorporation assay and comet assay revealed that exposure to arsenic trioxide affected DNA synthesis and exhibited genotoxic effects in human colon cancer cells.

Deletion-based reverse genetics in Medicago truncatula.

The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. T-DNA and transposon-based mutagenesis has been widely employed but is limited to species in which transformation and tissue culture are efficient. In other species, TILLING (for Targeting Induced Local Lesions IN Genomes), based on chemical mutagenesis, has provided an efficient method for the identification of single base pair mutations, only 5% of which will be null mutations. Furthermore, the efficiency of inducing point mutations, like insertion-based mutations, is dependent on target size. Here, we describe an alternative reverse genetic strategy based on physically induced genomic deletions that, independent of target size, exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron mutagenesis and a sensitive polymerase chain reaction-based detection. A population of 156,000 Medicago truncatula plants has been structured as 13 towers each representing 12,000 M2 plants. The De-TILLING strategy allows a single tower to be screened using just four polymerase chain reaction reactions. Dual screening and three-dimensional pooling allows efficient location of mutants from within the towers. With this method, we have demonstrated the detection of mutants from this population at a rate of 29% using five targets per gene. This De-TILLING reverse genetic strategy is independent of tissue culture and efficient plant transformation and therefore applicable to any plant species. De-TILLING mutants offer advantages for crop improvement as they possess relatively few background mutations and no exogenous DNA.

EFD Is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula.

Mechanisms regulating legume root nodule development are still poorly understood, and very few regulatory genes have been cloned and characterized. Here, we describe EFD (for ethylene response factor required for nodule differentiation), a gene that is upregulated during nodulation in Medicago truncatula. The EFD transcription factor belongs to the ethylene response factor (ERF) group V, which contains ERN1, 2, and 3, three ERFs involved in Nod factor signaling. The role of EFD in the regulation of nodulation was examined through the characterization of a null deletion mutant (efd-1), RNA interference, and overexpression studies. These studies revealed that EFD is a negative regulator of root nodulation and infection by Rhizobium and that EFD is required for the formation of functional nitrogen-fixing nodules. EFD appears to be involved in the plant and bacteroid differentiation processes taking place beneath the nodule meristem. We also showed that EFD activated Mt RR4, a cytokinin primary response gene that encodes a type-A response regulator. We propose that EFD induction of Mt RR4 leads to the inhibition of cytokinin signaling, with two consequences: the suppression of new nodule initiation and the activation of differentiation as cells leave the nodule meristem. Our work thus reveals a key regulator linking early and late stages of nodulation and suggests that the regulation of the cytokinin pathway is important both for nodule initiation and development.

Differential effect of ascorbic acid and n-acetyl-L-cysteine on arsenic trioxide-mediated oxidative stress in human leukemia (HL-60) cells.

Arsenic trioxide (ATO) has been recommended for the treatment of refractory cases of acute promyelocytic leukemia (APL). Recent studies in our laboratory indicated that oxidative stress plays a key role in ATO-induced cytotoxicity in human leukemia (HL-60) cells. In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells coexposed to either ascorbic acid (AA) and ATO or to n-acetyl-L-cysteine (NAC) and ATO. The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. In contrary, the addition of NAC to ATO-treated HL-60 cells resulted in a dose-dependent decrease of MDA production. From these results, we conclude that the addition of the AA to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS), whereas the addition of NAC under the same experimental condition significantly (p < .05) decreases the level of ROS formation. On the basis of these direct in vitro findings, our studies provide evidence that AA may extend the therapeutic spectrum of ATO. The coadministration of NAC with ATO shows a potential specificity for tumor cells, indicating that it may not enhance the clinical outcome associated with ATO monotherapy in vivo.

ASCORBIC ACID - MODULATION OF ARSENIC TRIOXIDE TOXICITY: IMPLICATION FOR THE CLINICAL TREATMENT OF ACUTE PROMYELOCYTIC LEUKEMIA.

BACKGROUND: Acute Promyelocytic Leukemia (APL) is a subtype of acute leukemia which can affect people of any age. It strikes about 1,500 patients in the United States each year. Recent in vitro and in vivo studies have shown that arsenic trioxide (ATO) can induce clinical remission in de-novo and APL patients that have relapsed from conventional treatment. Ascorbic acid (AA) is an anti-oxidant and free radical scavenger effective against peroxyl- and hydroxyl-radicals, superoxide, singlet oxygen and peroxynitrite. Although research has shown that AA can prevent cancer by deactivating free radicals before they can damage DNA and initiate tumor growth, there are also published reports indicating that it may act as a pro-oxidant that helps the body's own free radical defense mechanism destroy tumors in their early stages. AIM: The aim of this research was to study the modulatory effect of AA on ATO-induced oxidative stress in leukemia cells. METHODS: In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells co-exposed to ascorbic acid (AA) and ATO. RESULTS: The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. From these results, we concluded that the addition of the ascorbic acid to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS). CONCLUSIONS: Based on these direct in vitro findings, our study provides evidence that AA may extend the therapeutic spectrum of ATO, and improve the clinical outcome associated with ATO monotherapy in vivo.

Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.

Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.