Detection of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> in Alfalfa,Mung Bean,Cilantro, and Mamey Sapote (<Emphasis Type="Italic">Pouteria sapota</Emphasis>) Food Matrices Using DNA Microarray Chip Hybridization

Four different food matrices (alfalfa, cilantro, mamey sapote, and mung bean) were contaminated with three different dilutions 10⁶, 10⁴, and 10³ cfu/g of Yersinia enterocolitica. DNA was isolated from each food mix and used in chromosomal amplifications. The amplified DNA was used as templates in single PCR reactions of the four genes (virF, ail, yst, and blaA) followed by mixing the four reactions for one PCR primer extension reaction. The presence and the limit of detection of four genes in four food matrices were established by microarray hybridization. Data revealed the diversity of signal intensities. Neither the microarray chip hybridization nor the single PCR amplification could detect virF’s presence located on a plasmid. Ail was detected in 10³ cfu/g, whereas blaA and yst were detected from 10⁵ to 10⁶ cfu/g in all food matrices. Therefore, the ail gene could be the gene of choice in identifying Y. enterocolitica in alfalfa, cilantro, mamey, and mung bean. Other genes—blaA, yst, virF—exhibited wide variability in hybridization signals, highlighting the need of a better DNA purification step prior to DNA microarray hybridization.     

Cytokinin Profiles in the Conifer Tree <Emphasis Type="Italic">Abies nordmanniana</Emphasis>: Whole-Plant Relations in Year-Round Perspective

Conifer trees are routinely manipulated hormonally to increase flowering, branching, or adjust crown shape for production purposes. This survey of internal cytokinin levels provides a background for such treatments in Abies nordmanniana, a tree of great economic interest. Reference points in the crown and root system were sampled destructively in 4- and 6-year-old trees and analyzed for a range of cytokinins by LC-MS/MS. No seasonal patterns were detected in the root samples, and a major portion of cytokinin was in conjugated forms. Dramatic and consistent seasonal changes occurred in the crown, at levels 17–65 times higher than in the root. Predominant among crown cytokinins was ZR, except in the needles where IPR was also prominent. Within the crown, cytokinin profiles in different organs differed consistently. The leader bud showed a pronounced mid-June minimum, and a maximum later in summer. Subapical buds showed the same June minimum but peaked in mid autumn at a much lower level. Maxima in these buds were preceded by peaks in the subapical stem. Parallel patterns were observed in homologous tissues on branches.This pattern is consistent with two surges beginning in the uppermost stem tissues leading to subsequent accumulation or stimulated production within the buds. Strong differential hormonal profiles between adjacent buds with different fates agree with recent evidence of localized cytokinin production. The data suggest a reduced role of root-derived cytokinins in crown development. Practical cytokinin treatments for crown-shape regulation require close attention to dosage as well as precise timing and positioning.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Epigenetic regulation of neuronal dendrite and dendritic spine development

Dendrites and the dendritic spines of neurons play key roles in the connectivity of the brain and have been recognized as the locus of long-term synaptic plasticity, which is correlated with learning and memory. The development of dendrites and spines in the mammalian central nervous system is a complex process that requires specific molecular events over a period of time. It has been shown that specific molecules are needed not only at the spine’s point of contact, but also at a distance, providing signals that initiate a cascade of events leading to synapse formation. The specific molecules that act to signal neuronal differentiation, dendritic morphology, and synaptogenesis are tightly regulated by genetic and epigenetic programs. It has been shown that the dendritic spine structure and distribution are altered in many diseases, including many forms of mental retardation (MR), and can also be potentiated by neuronal activities and an enriched environment. Because dendritic spine pathologies are found in many types of MR, it has been proposed that an inability to form normal spines leads to the cognitive and motor deficits that are characteristic of MR. Epigenetic mechanisms, including DNA methylation, chromatin remodeling, and the noncoding RNA-mediated process, have profound regulatory roles in mammalian gene expression. The study of epigenetics focuses on cellular effects that result in a heritable pattern of gene expression without changes to genomic encoding. Despite extensive efforts to understand the molecular regulation of dendrite and spine development, epigenetic mechanisms have only recently been considered. In this review, we will focus on epigenetic mechanisms that regulate the development and maturation of dendrites and spines. We will discuss how epigenetic alterations could result in spine abnormalities that lead to MR, such as is seen in fragile X and Rett syndromes. We will also discuss both general methodology and recent technological advances in the study of neuronal dendrites and spines.     

Fickean and Non-Fickean Water Desorption During Vacuum Drying of <Emphasis Type=Italic>L. bulgaricus</Emphasis>

The recovery of Lactobacillus bulgaricus was studied in correlation to the kinetics of cell drying. When bacteria were dehydrated at 30 °C, either in the presence or the absence of sucrose, the drying kinetics corresponds to a Fickean diffusion in correspondence with a short lag time. In contrast, when the bacteria were dehydrated at 70 °C in the absence of sugar, the kinetics corresponds to an anomalous diffusion, and the lag time is four to five times higher than that at 30 °C. However, when drying at 70 °C was carried out in the presence of sucrose, drying kinetics turned into a Fickean process parallel to a substantial decrease in the lag time. The pattern of water desorption was correlated with the critical water activity. When the drying kinetics corresponds to a Fickean diffusion, the lag time started to increase at 0.7 water activity, but when the cells were dried at 70 °C, the damage started at 0.5 water activity. This observation indicates that the drying rate affects the pattern of water desorption, and it can change the value of critical water activity. These results put into relevance that the cell recovery is due to the drying history and that the recovery increase produced by sucrose can be related to the maintenance of kinetic barriers for water desorption.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Low diversity and high levels of population genetic structuring in introduced eastern mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) in the greater Melbourne area,Australia

Eastern mosquitofish (Gambusia holbrooki) were introduced into Australia in 1925 and released to control mosquitoes. Gambusia holbrooki rapidly became invasive in recipient environments and now threaten native fauna. In this study, we used five polymorphic microsatellite loci and sequence from two mitochondrial genes, cytochrome b and cytochrome oxidase I, to evaluate genetic variation, colonisation and movement patterns of introduced G. holbrooki in the greater Melbourne area, and to assist in identifying the feasibility of local eradication. Microsatellite variation was consistently low within populations and there was evidence of bottleneck events for several populations. Populations displayed significant structuring associated with river basins rather than geographic distance, suggesting that habitat connectivity is important for dispersal. However, a few populations within river basins were more closely related to populations in other river basins than within their own basin, most likely reflecting a role of human-assisted dispersal in population establishment. Mitochondrial sequencing revealed only a single haplotype and suggested all populations were founded by individuals from a common source. These genetic data help delineate boundaries for local management strategies.     

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.     

H2AX: functional roles and potential applications

Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed γ-H2AX, is easily identified with antibodies and serves as a sensitive indicator of DNA DSB formation. This review focuses on the potential clinical applications of γ-H2AX detection in cancer and in response to other cellular stresses. In addition, the role of H2AX in homeostasis and disease will be discussed. Recent work indicates that γ-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.