Dextran-coated iron oxide nanoparticle-induced nanotoxicity in neuron cultures.

Recent technological advances have introduced diverse engineered nanoparticles (ENPs) into our air, water, medicine, cosmetics, clothing, and food. However, the health and environmental effects of these increasingly common ENPs are still not well understood. In particular, potential neurological effects are one of the most poorly understood areas of nanoparticle toxicology (nanotoxicology), in that low-to-moderate neurotoxicity can be subtle and difficult to measure. Culturing primary neuron explants on planar microelectrode arrays (MEAs) has emerged as one of the most promising in vitro techniques with which to study neuro-nanotoxicology, as MEAs enable the fluorescent tracking of nanoparticles together with neuronal electrical activity recording at the submillisecond time scale, enabling the resolution of individual action potentials. Here we examine the dose-dependent neurotoxicity of dextran-coated iron oxide nanoparticles (dIONPs), a common type of functionalized ENP used in biomedical applications, on cultured primary neurons harvested from postnatal day 0-1 mouse brains. A range of dIONP concentrations (5-40 µg/ml) were added to neuron cultures, and cells were plated either onto well plates for live cell, fluorescent reactive oxidative species (ROS) and viability observations, or onto planar microelectrode arrays (MEAs) for electrophysiological measurements. Below 10 µg/ml, there were no dose-dependent cellular ROS increases or effects in MEA bursting behavior at sub-lethal dosages. However, above 20 µg/ml, cell death was obvious and widespread. Our findings demonstrate a significant dIONP toxicity in cultured neurons at concentrations previously reported to be safe for stem cells and other non-neuronal cell types.

Isolation and characterization of a lipid transfer protein expressed in ripening fruit of Capsicum chinense.

A novel LTP (CcLTP) from a Capsicum chinense cv Habanero was isolated from a fruit-specific SSH library. While this gene shares similarity with other LTPs, it is considerably larger than any lipid transfer protein reported to date and has a neutral predicted pI. CcLTP is consistently expressed in seedlings from three Capsicum species. It is also present at very high levels in ripening and mature fruit in C. chinense, but not in fruit of any C. annuum or C. frutescens varieties examined. We have obtained 3.8 kb of sequence containing the CcLTP gene and isolated two forms of mRNA transcripts which result from an alternative splicing event. Both transcripts are full-length cDNAs with putative open reading frames of 492 bp and 519 bp, encoding proteins of 164 and 173 amino acids, respectively, which differ only by an insertion of 9 amino acids. Both splice variants are detected consistently via RT-PCR. A 19 bp deletion in the promoter region differentiates C. chinense CcLTP from that of C. annuum and C. frutescens. The protein and its expression are characterized in C. chinense fruit, and a possible role in pepper fruit ripening and maturation is discussed.

A GH3-like gene, CcGH3, isolated from Capsicum chinense L. fruit is regulated by auxin and ethylene.

Auxin, which has been implicated in multiple biochemical and physiological processes, elicits three classes of genes (Aux/IAAs, SAURs and GH3s) that have been characterized by their early or primary responses to the hormone. A new GH3-like gene was identified from a suppressive subtraction hybridization (SSH) library of pungent pepper (Capsicum chinense L.) cDNAs. This gene, CcGH3, possessed several auxin- and ethylene-inducible elements in the putative promoter region. Upon further investigation, CcGH3 was shown to be auxin-inducible in shoots, flower buds, sepals, petals and most notably ripening and mature pericarp and placenta. Paradoxically, this gene was expressed in fruit when auxin levels were decreasing, consistent with ethylene-inducibility. Further experiments demonstrated that CcGH3 was induced by endogenous ethylene, and that transcript accumulation was inhibited by 1-methylcyclopropene, an inhibitor of ethylene perception. When over-expressed in tomato, CcGH3 hastened ripening of ethylene-treated fruit. These results implicate CcGH3 as a factor in auxin and ethylene regulation of fruit ripening and suggest that it may be a point of intersection in the signaling by these two hormones.

The Pun1 gene for pungency in pepper encodes a putative acyltransferase.

Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.

ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.

Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.