Plant programmed cell death : Can't live with it; Can't live without it

The decision of whether a cell should live or die is fundamental for the wellbeing of all organisms. Despite intense investigation into cell growth and proliferation, only recently has the essential and equally important idea that cells control/programme their own demise for proper maintenance of cellular homeostasis gained recognition. Furthermore, even though research into programmed cell death (PCD) has been an extremely active area of research there are significant gaps in our understanding of the process in plants. In this review, we discuss PCD during plant development and pathogenesis, and compare/contrast this with mammalian apoptosis. © 2008 Blackwell Publishing Ltd.

Metabolic and ecological study of environmental pentose utilizing bacteria (E-PUB)

Lignocellulosic materials, such as sugar cane bagasse, a waste product of the sugarcane processing industry, agricultural residues and herbaceous crops, may serve as an abundant and comparatively cheap feedstock for largescale industrial fermentation, resulting in the production of marketable end-products. However, the complex structure of lignocellulosic materials, the presence of various hexose and pentose sugars in the hemicellulose component, and the presence of various compounds that inhibit the organisms selected for the fermentation process, all constitute barriers that add to the production costs and make full scale industrial production economically less feasible. The work presented in this thesis was conducted in order to screen microorganisms for ability to utilize pentose sugars derived from the sugar mill industrial waste. A large number of individual bacterial strains were investigated from hemi-cellulose rich material collected at the Proserpine and Maryborough sugar mills, notably soil samples from the mill sites. The research conducted to isolation of six pentose-capable Gram-positive organisms from the actinomycetes group by using pentose as a sole carbon source in the cultivation process. The isolates were identified as Corynebacterium glutamicum, Actinomyces odontolyticus, Nocardia elegans, and Propionibacterium freudenreichii all of which were isolated from the hemicellulose-enriched soil. Pentose degrading microbes are very rare in the environment, so this was a significant discovery. Previous research indicated that microbes could degrade pentose after genetic modification but the microbes discovered in this research were able to naturally utilize pentose. Six isolates, identified as four different genera, were investigated for their ability to utilize single sugars as substrates (glucose, xylose, arabinose or ribose), and also dual sugars as substrates (a hexose plus a pentose). The results demonstrated that C. glutamicum, A. odontolyticus, N. elegans, and P. freudenreichii were pentose-capable (able to grow using xylose or other pentose sugar), and also showed diauxie growth characteristics during the dual-sugar (glucose, in combination with xylose, arabinose or ribose) carbon source tests. In addition, it was shown that the isolates displayed very small differences in growth rates when grown on dual sugars as compared to single sugars, whether pentose or hexose in nature. The anabolic characteristics of C. glutamicum, A. odontolyticus, N. elegans and P. freudenreichii were subsequently investigated by qualitative analysis of their end-products, using high performance liquid chromatography (HPLC). All of the organisms produced arginine and cysteine after utilization of the pentose substrates alone. In addition, P. freudenreichii produced alanine and glycine. The end-product profile arising from culture with dual carbon sources was also tested. Interestingly, this time the product was different. All of them produced the amino acid glycine, when grown on a combination substrate-mix of glucose with xylose, and also glucose with arabinose. Only N. elegans was able to break down ribose, either singly or in combination with glucose, and the end-product of metabolism of the glucose plus ribose substrate combination was glutamic acid. The ecological analysis of microbial abundance in sugar mill waste was performed using denaturing gradient gel electrophoresis (DGGE) and also the metagenomic microarray PhyloChip method. Eleven solid samples and seven liquid samples were investigated. A very complex bacterial ecosystem was demonstrated in the seven liquid samples after testing with the PhyloChip method. It was also shown that bagasse leachate was the most different, compared to all of the other samples, by virtue of its richness in variety of taxa and the complexity of its bacterial community. The bacterial community in solid samples from Proserpine, Mackay and Maryborough sugar mills showed huge diversity. The information found from 16S rDNA sequencing results was that the bacterial genera Brevibacillus, Rhodospirillaceae, Bacillus, Vibrio and Pseudomonas were present in greatest abundance. In addition, Corynebacterium was also found in the soil samples. The metagenomic studies of the sugar mill samples demonstrate two important outcomes: firstly that the bagasse leachate, as potentially the most pentose-rich sample tested, had the most complex and diverse bacterial community; and secondly that the pentose-capable isolates that were initially discovered at the beginning of this study, were not amongst the most abundant taxonomic groups discovered in the sugar mill samples, and in fact were, as suspected, very rare. As a bioprospecting exercise, therefore, the study has discovered organisms that are naturally present, but in very small numbers, in the appropriate natural environment. This has implications for the industrial application of E-PUB, in that a seeding process using a starter culture will be necessary for industrial purposes, rather than simply assuming that natural fermentation might occur.

Exploring plant genomes by RNA-induced gene silencing

The nucleotide sequences of several animal, plant and bacterial genomes are now known, but the functions of many of the proteins that they are predicted to encode remain unclear. RNA interference is a gene-silencing technology that is being used successfully to investigate gene function in several organisms - for example, Caenorhabditis elegans. We discuss here that RNA-induced gene silencing approaches are also likely to be effective for investigating plant gene function in a high-throughput, genome-wide manner.

Construct design for efficient, effective and high-throughput gene silencing in plants

Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.

Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Identification of small juvenile scombrids from northwest tropical Australia using mitochondrial DNA cytochrome b sequences

Small juveniles of the nine species of scombrids in Australian waters are morphologically similar to one another and, consequently, difficult to identify to species level. We show that the sequence of the mitochondrial DNA cytochrome b gene region is a powerful tool for identification of these young fish. Using this method, we identified 50 juvenile scombrids collected from Exmouth Bay, Western Australia. Six species of scombrids were apparent in this sample of fish: narrow-barred Spanish mackerel (Scomberomorus commerson), Indian mackerel (Rastrelliger kanagurta), frigate tuna (Auxis thazard), bullet tuna (Auxis rochei), leaping bonito (Cybiosarda elegans), and kawakawa (Euthynnus affinis). The presence of Indian mackerel, frigate tuna, leaping bonito, and kawakawa is the first indication that coastal waters may be an important spawning habitat for these species, although offshore spawning may also occur. The occurrence of small juvenile S. commerson was predicted from the known spawning patterns of that species, but other mackerel species (Scomberomorus munroi, Scomberomorus queenslandicus, Scomberomorus semifasiciatus) likely to be spawning during the sampling period were not detected among the 50 small juveniles analyzed here.

Zebrafish as a model of Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. Its etiology is unknown and no disease-modifying drugs are available. Thus, more information concerning its pathogenesis is needed. Among other genes, mutated PTEN-induced kinase 1 (PINK1) has been linked to early-onset and sporadic PD, but its mode of action is poorly understood. Most animal models of PD are based on the use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is metabolized to MPP+ by monoamine oxidase B (MAO B) and causes cell death of dopaminergic neurons in the substantia nigra in mammals. Zebrafish has been a widely used model organism in developmental biology, but is now emerging as a model for human diseases due to its ideal combination of properties. Zebrafish are inexpensive and easy to maintain, develop rapidly, breed in large quantities producing transparent embryos, and are readily manipulated by various methods, particularly genetic ones. In addition, zebrafish are vertebrate animals and results derived from zebrafish may be more applicable to mammals than results from invertebrate genetic models such as Drosophila melanogaster and Caenorhabditis elegans. However, the similarity cannot be taken for granted. The aim of this study was to establish and test a PD model using larval zebrafish. The developing monoaminergic neuronal systems of larval zebrafish were investigated. We identified and classified 17 catecholaminergic and 9 serotonergic neuron populations in the zebrafish brain. A 3-dimensional atlas was created to facilitate future research. Only one gene encoding MAO was found in the zebrafish genome. Zebrafish MAO showed MAO A-type substrate specificity, but non-A-non-B inhibitor specificity. Distribution of MAO in larval and adult zebrafish brains was both diffuse and distinctly cellular. Inhibition of MAO during larval development led to markedly elevated 5-hydroxytryptamine (serotonin, 5-HT) levels, which decreased the locomotion of the fish. MPTP exposure caused a transient loss of cells in specific aminergic cell populations and decreased locomotion. MPTP-induced changes could be rescued by the MAO B inhibitor deprenyl, suggesting a role for MAO in MPTP toxicity. MPP+ affected only one catecholaminergic cell population; thus, the action of MPP+ was more selective than that of MPTP. The zebrafish PINK1 gene was cloned in zebrafish, and morpholino oligonucleotides were used to suppress its expression in larval zebrafish. The functional domains and expression pattern of zebrafish PINK1 resembled those of other vertebrates, suggesting that zebrafish is a feasible model for studying PINK1. Translation inhibition resulted in cell loss of the same catecholaminergic cell populations as MPTP and MPP+. Inactivation of PINK1 sensitized larval zebrafish to subefficacious doses of MPTP, causing a decrease in locomotion and cell loss in one dopaminergic cell population. Zebrafish appears to be a feasible model for studying PD, since its aminergic systems, mode of action of MPTP, and functions of PINK1 resemble those of mammalians. However, the functions of zebrafish MAO differ from the two forms of MAO found in mammals. Future studies using zebrafish PD models should utilize the advantages specific to zebrafish, such as the ability to execute large-scale genetic or drug screens.

Sex Determination - A Hypothesis Based On Noncoding Dna

Certain recent models of sex determination in mammals, Drosophila melanogaster, Caenorhabditis elegans, and snakes are examined in the light of the hypothesis that the relevant genetic regulatory mechanisms are similar and interrelated. The proposed key element in each of these instances is a noncoding DNA sequence, which serves as a high-affinity binding site for a repressor-like molecule regulating the activity of a major "sex-determining" gene. On this basis it is argued that, in several eukaryotes, (i) certain DNA sequences that are sex-determining are noncoding, in the sense that they are not the structural genes of a sex-determining protein; (ii) in some species these noncoding sequences are present in one sex and absent in the other, while in others their copy number or accessibility to regulatory molecules is significantly unequal between the two sexes; and (iii) this inequality determines whether the embryo develops into a male or a female.

A core metabolic enzyme mediates resistance to phosphine gas

Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Small-scale capture, transport and tank adaptation of live, medium-sized Scombrids using "Tuna Tubes"

The transport of live fish is a crucial step to establish fish culture in captivity, and is especially challenging for species that have not been commonly cultured before, therefore transport and handling methods need to be optimized and tailored. This study describes the use of tuna tubes for small-scale transport of medium-sized pelagic fish from the Scombridae family. Tuna tubes are an array of vertical tubes that hold the fish, while fresh seawater is pumped up the tubes and through the fish mouth and gills, providing oxygen and removing wastes. In this study, 19 fish were captured using rod and line and 42% of the captured fish were transported alive in the custom-designed tuna tubes to an on-shore holding tank: five mackerel tuna (Euthynnus affinis) and three leaping bonito (Cybiosarda elegans). Out of these, just three (15.8% of total fish) acclimatized to the tank's condition. Based on these results, we discuss an improved design of the tuna tubes that has the potential to increase survival rates and enable a simple and low cost method of transporting of live pelagic fish.

Free and ATP-bound structures of Ap(4)A hydrolase from Aquifex aeolicus V5

Asymmetric diadenosine tetraphosphate (Ap(4)A) hydrolases degrade the metabolite Ap(4)A back into ATP and AMP. The three-dimensional crystal structure of Ap(4)A hydrolase (16 kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95 angstrom resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an alpha beta alpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap(4)A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

How do heterogametic females survive without gene dosage compensation?

When the male is the heterogametic sex (XX♀-XY♂ or XX♀-XO♂), as inDrosophila, orthopteran insects, mammals andCaenorhabditis elegans, X-linked genes are subject to dosage compensation: the single X in the male is functionally equivalent to the two Xs in the female. However, when the female is heterogametic (ZZ♂-ZW♀), as in birds, butterflies and moths, Z-linked genes are apparently not dosage-compensated. This difference between X-linked and Z-linked genes raises fundamental questions about the role of dosage compensation. It is argued that (i) genes which require dosage compensation are primarily those that control morphogenesis and the prospective body plan; (ii) the products of these genes are required in disomic doses especially during oogenesis and early embryonic development; (iii) heterogametic females synthesize and store during oogenesis itself morphogenetically essential gene products - including those encoded by Z-linked genes — in large quantities; (iv) the abundance of these gene products in the egg and their persistence relatively late into embryogenesis enables heterogametic females to overcome the monosomic state of the Z chromosome in ZW embryos. Female heterogamety is predominant in birds, reptiles and amphibians, all of which have megalecithal eggs containing several thousand times more maternal RNA and other maternal messages than eggs of mammals,Caenorhabditis elegans, orDrosophila. This increase in egg size, yolk content and, concomitantly, the size of the maternal legacy to the embryo, may have facilitated female heterogamety and the absence of dosage compensation.

LIN28B, LIN28A, KISS1, and KISS1R in idiopathic central precocious puberty

Abstract Background Pubertal timing is a strongly heritable trait, but no single puberty gene has been identified. Thus, the genetic background of idiopathic central precocious puberty (ICPP) is poorly understood. Overall, the genetic modulation of pubertal onset most likely arises from the additive effect of multiple genes, but also monogenic causes of ICPP probably exist, as cases of familial ICPP have been reported. Mutations in KISS1 and KISSR, coding for kisspeptin and its receptor, involved in GnRH secretion and puberty onset, have been suggested causative for monogenic ICPP. Variation in LIN28B was associated with timing of puberty in genome-wide association (GWA) studies. LIN28B is a human ortholog of the gene that controls, through microRNAs, developmental timing in C. elegans. In addition, Lin28a transgenic mice manifest the puberty phenotypes identified in the human GWAS. Thus, both LIN28B and LIN28A may have a role in pubertal development and are good candidate genes for monogenic ICPP. Methods Thirty girls with ICPP were included in the study. ICPP was defined by pubertal onset before 8 yrs of age, and a pubertal LH response to GnRH testing. The coding regions of LIN28B, LIN28A, KISS1, and KISS1R were sequenced. The missense change in LIN28B was also screened in 132 control subjects. Results No rare variants were detected in KISS1 or KISS1R in the 30 subjects with ICPP. In LIN28B, one missense change, His199Arg, was found in one subject with ICPP. However, this variant was also detected in one of the 132 controls. No variation in LIN28A was found. Conclusions We did not find any evidence that mutations in LIN28B or LIN28A would underlie ICPP. In addition, we confirmed that mutations in KISS1 and KISS1R are not a common cause for ICPP.

Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

Local demodulation of holograms using the Riesz transform with application to microscopy

We propose a Riesz transform approach to the demodulation of digital holograms. The Riesz transform is a higher-dimensional extension of the Hilbert transform and is steerable to a desired orientation. Accurate demodulation of the hologram requires a reliable methodology by which quadrature-phase functions (or simply, quadratures) can be constructed. The Riesz transform, by itself, does not yield quadratures. However, one can start with the Riesz transform and construct the so-called vortex operator by employing the notion of quasi-eigenfunctions, and this approach results in accurate quadratures. The key advantage of using the vortex operator is that it effectively handles nonplanar fringes (interference patterns) and has the ability to compensate for the local orientation. Therefore, this method results in aberration-free holographic imaging even in the case when the wavefronts are not planar. We calibrate the method by estimating the orientation from a reference hologram, measured with an empty field of view. Demodulation results on synthesized planar as well as nonplanar fringe patterns show that the accuracy of demodulation is high. We also perform validation on real experimental measurements of Caenorhabditis elegans acquired with a digital holographic microscope. (c) 2012 Optical Society of America