Iron excess in rice: from phenotypic changes to functional genomics of WRKY transcription factors.

Iron (Fe) is an essential microelement for all living organisms playing important roles in several metabolic reactions. Rice (Oryza sativa L.) is commonly cultivated in paddy fields, where Fe goes through a reduction reaction from Fe3+ to Fe2+. Since Fe2+ is more soluble, it can reach toxic levels inside plant cells, constituting an important target for studies. Here we aimed to verify morphological changes of different rice genotypes focusing on deciphering the underlying molecular network induced upon Fe excess treatments with special emphasis on the role of four WRKY transcription factors. The transcriptional response peak of these WRKY transcription factors in rice seedlings occurs at 4 days of exposition to iron excess. OsWRKY55-like, OsWRKY46, OsWRKY64, and OsWRKY113 are up-regulated in BR IRGA 409, an iron-sensitive genotype, while in cultivars Nipponbare (moderately resistant) and EPAGRI 108 (resistant) the expression profiles of these transcription factors show similar behaviors. Here is also shown that some cis-regulatory elements known to be involved in other different stress responses can be linked to conditions of iron excess. Overall, here we support the role of WRKY transcription factors in iron stress tolerance with other important steps toward finding why some rice genotypes are more tolerant than others.

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements.

Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

PERSONALITY TRAITS AND STRESS LEVELS AMONG SENIOR DENTAL STUDENTS: EVIDENCE FROM MALAYSIA AND SINGAPORE.

This study aimed to evaluate the association between dental students' personality traits and stress levels in relation to dental education programs among senior dental students in University Malaya (UM) in Malaysia and National University of Singapore (NUS). A cross-sectional survey using a self-administered questionnaire was conducted on UM and NUS senior dental students. The questionnaire comprised items on demographic background, the Big Five Inventory Personality Traits (BFIPT) test and a modified Dental Environment Stress (DES) scale. Rasch analysis was used to convert raw data to interval scores. Analyses were done by t-test, Pearson correlation, and Hierarchical regression statistics. The response rate was 100% (UM=132, NUS=76). Personality trait Agreeableness (mean=0.30) was significantly more prevalent among UM than NUS students (mean=0.15, p=0.016). In NUS, Neuroticism (mean=0.36) was significantly more prevalent than in UM (mean=0.14, p=0.002). The DES mean score was higher among NUS (mean=0.23) than UM students (mean=0.07). In UM, Neuroticism was significantly correlated with stress levels (r=0.338, p<0.001). In NUS, these were Neuroticism (r=0.278, p=0.015), Agreeableness (r=0.250, p=0.029) and Conscientiousness (r=-0.242, p=0.035) personality traits. The correlation was strongest for personality trait Neuroticism in both schools. Hierarchical regression analysis showed that gender and Neuroticism were significant predictors for students' stress levels (p<0.05) with the latter exerting a bigger effect size (R2=0.18) than gender (R2=004). This study showed that gender and Neuroticism personality trait were significant predictors for stress levels among selected groups of dental students in Southeast Asia. Information on students' personality may be useful in new students' intake, stress management counseling and future program reviews.

Thermal behaviour modelling of tapered optical fibres for scanning near-field microscopy.

A simple model allowing the calculation of the thermal field inside a metal-coated fibre tip is presented. The approach has been based on previous temperature measurements which operated in steady state and periodic rate. The modelhas been inspired from the general theory of heat transfer inside fins, after having divided the taper into a set of layers. The advantage of the method is the possibility to consider any taper shapes. Moreover, any kind of coating thickness and external heat transfer distributions can be considered. As a mean of comparison with some previous works, results obtained for simple configurations are presented. Then, a study of the main governing parameters provides the basic thermal behaviour analysis of optical tips and a comparison with experience is given in order to confirm the validity of our approach.

Seasonal mood patterns in eating disorders.

There has been an increasing body of research literature suggesting a seasonal pattern of mood fluctuations and eating behavior in bulimic patients. Fornari et al. [5] reported worsening of bulimic symptoms during winter. There is a logical connection between Seasonal Affective Disorder (SAD) and bulimia nervosa as both show increased appetite and carbohydrate craving and probably share a common neurobiologic abnormality such as serotonergic dysfunction. The aim of this study was to determine the prevalence of SAD in a sample of 259 consecutively evaluated outpatients admitted to an eating disorders clinic (254 women and 5 men). Eating disorder diagnosis was established on the basis of DSM-III-R criteria, and a modified version of the Seasonal Pattern Assessment Questionnaire was used to determine seasonality among patients. The sample was comprised of the following: 53.7% bulimics, 27.4% anorexics, 15.1% were classified as having an eating disorder not otherwise specified, and 3.9% had a diagnosis other than an eating disorder. The results indicated that 27.0% of the eating disorder patients met criteria for SAD. Of this group, 86 (71.4%) were bulimic, 35 (18.6%) were anorexic, and 20 (10.0%) were nonspecified. Details and additional findings are discussed.

Mkh1, a MEK kinase required for cell wall integrity and proper response to osmotic and temperature stress in Schizosaccharomyces pombe.

We have identified a Schizosaccharomyces pombe gene, mkh1, that encodes a MEK kinase (MEKK) homolog. The coding region of mkh1 is contained within a single exon encoding a 1,116-amino-acid protein. The putative catalytic domain of Mkh1 is 54% identical to the catalytic domain of S. cerevisiae Bck1, the most closely related protein. Deletion of mkh1 did not significantly affect cell growth or division under standard conditions. However, mkh1delta cell growth was inhibited by high KCl or NaCl concentrations. mkh1delta cells required a longer time to reenter the cell cycle after prolonged stationary-phase arrest. Also, mkh1delta cells exhibited a round cell shape, while overexpression of Mkh1 resulted in an elongated cell shape. mkh1delta cells exhibited a more dramatic phenotype when grown in nutrient-limiting conditions at high temperature or in hyperosmotic medium. In such conditions, completion of cytokinesis was inhibited, resulting in the growth of pseudohyphal filaments with multiple septa and nuclei. Also, mkh1delta cells were hypersensitive to beta-glucanase treatment. Together these results suggest that Mkh1 regulates cell morphology, cell wall integrity, salt resistance, cell cycle reentry from stationary-phase arrest, and filamentous growth in response to stress. These phenotypes are essentially identical to those exhibited by cells lacking Pmk1/Spm1, a recently identified mitogen-activated protein kinase. Our evidence suggests that Pmk1/Spm1 acts downstream from Mkh1 in a common pathway. Our results also suggest that Mkh1 and Pck2 act independently to maintain cell wall integrity, cell morphology, and salt resistance but act in opposition to regulate filamentous growth.

A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway.

Supracrestal circular collagen fiber network around osseointegrated nonsubmerged titanium implants.

Eight non-submerged titanium implant screws were placed in the first upper molar edentulous sites of monkeys and subsequently kept loaded with single crown prosthesis 1 month following implantation. The animals were killed after a further 14 months and specimens including implant and adjacent teeth were processed for light and electron microscopy. Histological pictures of all samples showed the neck and most of the screw body to be surrounded by new bone. The soft tissue surrounding the implant post included pocket epithelium and supra-crestal connective tissue displaying collagen fiber bundles comparable to gingival ligaments. These peri-implant collagen fiber bundles arose from the neighboring alveolar crest, root cementum of adjacent teeth or, superficially, from the epithelium and followed a circular array around the implant neck.

Different G1 cyclins control the timing of cell cycle commitment in mother and daughter cells of the budding yeast S. cerevisiae.

Growth of S. cerevisiae cells by budding gives rise to asymmetric progeny cells: a larger "mother" cell and a smaller "daughter" cell. The mother cell transits a brief G1 phase before forming a new bud and beginning DNA replication. The daughter cell stays in G1 for a longer period, growing in size before initiating a new cell cycle. We show that the timing of cell cycle initiation in mother and daughter cells is governed by different G1 cyclins. In daughter cells, transcription of CLN1 and CLN2 is induced in a size-dependent manner, and these cyclins are necessary for the normal timing of cell cycle initiation. CLN3 is not required in daughter cells, but is crucial for mother cells, in which the G1 phase is much longer in the absence of this cyclin.

Direct induction of G1-specific transcripts following reactivation of the Cdc28 kinase in the absence of de novo protein synthesis.

In Saccharomyces cerevisiae, the genes encoding the HO endonuclease, G1-specific cyclins CLN1 and CLN2, as well as most proteins involved in DNA synthesis, are periodically transcribed with maximal levels reached in late G1. For HO and the DNA replication genes, cell cycle stage-specific expression has been shown to be dependent on the Cdc28 kinase and passage through START. Here, we show that cells released from cdc28ts arrest in the presence of cycloheximide show wild-type levels of induction for HO, CLN1, and CDC9 (DNA ligase). Induction is gradual with a significant lag not seen in untreated cells where transcript levels fluctuate coordinately with the cell cycle. This lag may be due, at least in part, to association of the Cdc28 peptide with G1 cyclins to form an active kinase complex because overexpression of CLN2 prior to release in cycloheximide increases the rate of induction for CDC9 and HO. Consistent with this, release from pheromone arrest (where CLN1 and CLN2 are not expressed) in cycloheximide shows no induction at all. Transcriptional activation of CDC9 is likely to be mediated through a conserved promoter element also present in genes for other DNA synthesis enzymes similarly cell cycle regulated. The element contains an intact MluI restriction enzyme recognition site (consensus approximately 5'-A/TPuACGCGTNA/T-3'). Insertion of a 20-bp fragment from the CDC9 promoter (containing a MluI element) upstream of LacZ confers both periodic expression and transcriptional induction in cycloheximide following release from cdc28ts arrest. High levels of induction depended on both the MluI element and CDC28. These results suggest that the activity of trans-acting factors that operate through the MluI element may be governed by phosphorylation by the Cdc28 kinase.

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Differential replication of circular DNA molecules co-injected into early Xenopus laevis embryos.

Replication of co-injected supercoiled DNA molecules in fertilized Xenopus eggs was monitored through the blastula stage of development. The extent of replication, as measured by 32P-dTMP incorporation into form I DNA, was directly proportional to the number of molecules, rather than the size, of the plasmid injected. Although only a small fraction of molecules of either template was replicated, incorporation was predominantly into full length daughter molecules. Over at least a 20-fold concentration range of microinjected DNA, injection of equal masses of DNA resulted in greater incorporation into the smaller form I DNA present in molar excess. The extent of incorporation into supercoiled DNA for a particular plasmid was apparently independent of the concentration of a second, co-injected plasmid. The relative extents of replication of co-injected supercoiled templates could be altered simply by changing the molar ratios of the templates.

Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis.

The persistence and replication of defined circular and linear plasmid DNA molecules microinjected into fertilized eggs of Xenopus laevis were analyzed. For all plasmids tested, a small fraction of microinjected circular molecules was replicated; however, the overall copy numbers of either free form I or form II molecules usually did not increase through blastulation. In contrast, extensive amplification of input DNA sequences was seen whenever the microinjected DNA was assembled into high molecular weight concatemers. Moreover, the appearance and subsequent replication of injected sequences in high molecular weight DNA were enhanced when linear (form III), rather than circular, molecules were microinjected. The injected form III DNA was rapidly converted into long linear concatemers. All possible orientations of monomeric molecules within the concatemers were observed although, on occasion, head-to-tail orientations were favored. Long linear concatemers were replicated very efficiently, irrespective of the sequence of the input DNA. Form I and form II DNA molecules were also formed in the embryo from microinjected form III DNA. A small fraction of these circular forms was replicated, although overall copy numbers did not increase significantly. Form III molecules that remained monomeric were not observed to be replicated at all within our limits of detection. In some batches of embryos, form I and form II DNA molecules were replicated to the extent that overall copy number increased. Even in these cases, however, the amplification of long linear concatemers of the input DNA sequences was more efficient.

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis.

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.