Gene Silencing in Arabidopsis Spreads from the Root to the Shoot, through a Gating Barrier, by Template-Dependent, Nonvascular, Cell-to-Cell Movement

Upward long-distance mobile silencing has been shown to be phloem mediated in several different solanaceous species. We show that the Arabidopsis (Arabidopsis thaliana) seedling grafting system and a counterpart inducible system generate upwardly spreading long-distance silencing that travels not in the phloem but by template-dependent reiterated short-distance cell-to-cell spread through the cells of the central stele. Examining the movement of the silencing front revealed a largely unrecognized zone of tissue, below the apical meristem, that is resistant to the silencing signal and that may provide a gating or protective barrier against small RNA signals. Using a range of auxin and actin transport inhibitors revealed that, in this zone, alteration of vesicular transport together with cytoskeleton dynamics prevented or retarded the spread of the silencing signal. This suggests that small RNAs are transported from cell to cell via plasmodesmata rather than diffusing from their source in the phloem.

MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Adaptations to a subterranean environment and longevity revealed by the analysis of mole rat genomes

Subterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis) and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber). Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity.

Binding of polyphenols to plant cell wall analogues - Part 1: Anthocyanins

Anthocyanins are located within the vacuole of plant cells, and are released following cell rupture during eating or processing at which time they first come into contact with the plant cell wall. The extent of anthocyanin-cell wall interaction was investigated by monitoring the rate of anthocyanin depletion in the presence of pure cellulose or cellulose-pectin composites as cell wall models. It was found that anthocyanins interact with both cellulose and pectin over a two-stage process with initially (mins-hours) 13 similar to 18% of anthocyanins binding to cellulose or cellulose/pectincomposites. With prolonged exposure (days-weeks), a gradual increase in anthocyanin binding occurs, possibly due to anthocyanins stacking on top of a base layer. Binding of acylated and non-acylated anthocyanins followed a similar pattern with slightly more (5-10%) binding of the acylated forms. Composites with the highest pectin content had the greatest anthocyanin binding suggesting the existence of both ionic interactions (with pectin) and hydrophobic interactions (with cellulose) of anthocyanin with plant cell walls.

Effects of cell-cycle arrest agents on cleavage and development of loach (Misgurnus anguillicaudatus) embryos

The aim of this study was to determine the lowest concentration of nocodazole and colchicine to arrest blastomere division during the cleavage stage of loach embryos and to assess the reversibility and toxicity of the treatments in the treated embryos. Eight-cell loach embryos were incubated for 4, 8, 12, or 16 h in 1/10x Holtfreter supplemented with either nocodazole, an inhibitor of tubulin polymerization, or colchicine, an inhibitor of tubulin assembly. Complete arrest of cell cycle was observed, at a colchicine concentration of 0.996 mM and at a nocodazole concentration of 0.275 muM, respectively (the lowest effective concentration). No major morphological alteration in chromatin was observed. Reversibility and toxicity of both agents were dose and exposure period dependent. For both agents, prolonging cleavage arrest for more than 4 h (at the effective concentrations) is detrimental to development of embryos. Nocodazole treatment was less cytotoxic, whereas the concentrations of colchicine which induce cleavage arrest were detrimental to development beyond the blastula stage. Toxic effects beyond the blastula stage could be minimized for both agents by reducing the period of treatment and concentration.

Analysis of three marine fish cell lines by RAPD assay

We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG. SPH, and RSBK and as a possible tool to detect cross-contamination. Sixth commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical Patterns produced by 35-48% of the primers tested. the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851. indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic hand pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines.

Infrared thermography: A non-invasive window into thermal physiology

Infrared thermography is a non-invasive technique that measures mid to long-wave infrared radiation emanating from all objects and converts this to temperature. As an imaging technique, the value of modern infrared thermography is its ability to produce a digitized image or high speed video rendering a thermal map of the scene in false colour. Since temperature is an important environmental parameter influencing animal physiology and metabolic heat production an energetically expensive process, measuring temperature and energy exchange in animals is critical to understanding physiology, especially under field conditions. As a non-contact approach, infrared thermography provides a non-invasive complement to physiological data gathering. One caveat, however, is that only surface temperatures are measured, which guides much research to those thermal events occurring at the skin and insulating regions of the body. As an imaging technique, infrared thermal imaging is also subject to certain uncertainties that require physical modeling, which is typically done via built-in software approaches. Infrared thermal imaging has enabled different insights into the comparative physiology of phenomena ranging from thermogenesis, peripheral blood flow adjustments, evaporative cooling, and to respiratory physiology. In this review, I provide background and guidelines for the use of thermal imaging, primarily aimed at field physiologists and biologists interested in thermal biology. I also discuss some of the better known approaches and discoveries revealed from using thermal imaging with the objective of encouraging more quantitative assessment.

Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 (Ink4c) , a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

Establishment and characterization of a primary canine duodenal epithelial cell culture

Many mechanisms involved in the pathogenesis of chronic enteropathies or host-pathogen interactions in canine intestine have not been elucidated so far. Next to the clinical and in vivo research tools, an in vitro model of canine intestinal cell culture would be very helpful for studies at the cellular level. Therefore, the purpose of this study was to establish and characterize a primary canine duodenal epithelial cell culture. Neonatal duodenum was disrupted with trypsin-ethylenediaminetetraacetic acid (EDTA) and the mucosa scraped off and digested with collagenase and dispase. After centrifugation on a 2% sorbitol gradient, the cells were incubated at 37 degrees C in OptiMEM supplemented with Primocin, epidermal growth factor, insulin, hydrocortisone, and 10% fetal calf serum (FCS). After 24 h, the FCS concentration was reduced to 2.5%, and the temperature decreased to 33 degrees C. With this method, the cultures were growing to confluent monolayers within 5-6 d and remained viable for an average of 2 wk. Their epithelial nature was confirmed by electron microscopy and immunofluorescence staining using antibodies directed against specific cytokeratins, desmosomes, and tight junctions. The intestinal cells proliferated, as evidenced by immunolabeling with a Ki-67 antibody, and cryptal cell subpopulations could be identified. Furthermore, alkaline phosphatase and sucrase activity were detected.

Tilapia sex determination: Where temperature and genetics meet

This review deals with the complex sex determining system of Nile tilapia, Oreochromis niloticus, governed by the interactions between a genetic determination and the influence of temperature, shown in both domestic and wild populations. Naturally sex reversed individuals are strongly suggested in two wild populations. This can be due to the masculinising temperatures which some fry encounter during their sex differentiation period when they colonise shallow waters, and/or to the influence of minor genetic factors. Differences regarding a) thermal responsiveness of sex ratios between and within Nile tilapia populations, b) maternal and paternal effects on temperature dependent sex ratios and c) nearly identical results in offspring of repeated matings, demonstrate that thermosensitivity is under genetic control. Selection experiments to increase the thermosensitivity revealed high responses in the high and low sensitive lines. The high-line showed ~ 90% males after 2 generations of selection whereas the weakly sensitive line had 54% males. This is the first evidence that a surplus of males in temperature treated groups can be selected as a quantitative trait. Expression profiles of several genes (Cyp19a, Foxl2, Amh, Sox9a,b) from the gonad and brain were analysed to define temperature action on the sex determining/differentiating cascade in tilapia. The coexistence of GSD and TSD is discussed.

The role of insulin-like growth factor II and its receptor in mouse preimplantation development

Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.

Skeletal muscle and nuclear hormone receptors: Implications for cardiovascular and metabolic disease

Skeletal muscle is a major mass peripheral tissue that accounts for similar to 40% of the total body mass and a major player in energy balance. It accounts for > 30% of energy expenditure, is the primary tissue of insulin stimulated glucose uptake, disposal, and storage. Furthermore, it influences metabolism via modulation of circulating and stored lipid (and cholesterol) flux. Lipid catabolism supplies up to 70% of the energy requirements for resting muscle. However, initial aerobic exercise utilizes stored muscle glycogen but as exercise continues, glucose and stored muscle triglycerides become important energy substrates. Endurance exercise increasingly depends on fatty acid oxidation (and lipid mobilization from other tissues). This underscores the importance of lipid and glucose utilization as an energy source in muscle. Consequently skeletal muscle has a significant role in insulin sensitivity, the blood lipid profile, and obesity. Moreover, caloric excess, obesity and physical inactivity lead to skeletal muscle insulin resistance, a risk factor for the development of type II diabetes. In this context skeletal muscle is an important therapeutic target in the battle against cardiovascular disease, the worlds most serious public health threat. Major risk factors for cardiovascular disease include dyslipidemia, hypertension, obesity, sedentary lifestyle, and diabetes. These risk factors are directly influenced by diet, metabolism and physical activity. Metabolism is largely regulated by nuclear hormone receptors which function as hormone regulated transcription factors that bind DNA and mediate the pathophysiological regulation of gene expression. Metabolism and activity, which directly influence cardiovascular disease risk factors, are primarily driven by skeletal muscle. Recently, many nuclear receptors expressed in skeletal muscle have been shown to improve glucose tolerance, insulin resistance, and dyslipidernia. Skeletal muscle and nuclear receptors are rapidly emerging as critical targets in the battle against cardiovascular disease risk factors. Understanding the function of nuclear receptors in skeletal muscle has enormous pharmacological utility for the treatment of cardiovascular disease. This review focuses on the molecular regulation of metabolism by nuclear receptors in skeletal muscle in the context of dyslipidemia and cardiovascular disease. (c) 2005 Published by Elsevier Ltd.