Automated fluorescent in situ hybridization (FISH) analysis of t(9;22)(q34;q11) in interphase nuclei.

BACKGROUND: For chronic myeloid leukemia, the FISH detection of t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes is an alternative method to bone marrow karyotyping for monitoring treatment. With automation, several drawbacks of manual analysis may be circumvented. In this article, the capabilities of a commercially available automated image acquisition and analysis system were determined by detecting t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes. METHODS: Three peripheral blood samples of normal adults, 21 samples of CML patients, and one sample of a t(9;22)(q34;q11) positive cell-line were used. RESULTS: Single nuclei with correctly detected signals amounted to 99.6% of nuclei analyzed after exclusion of overlapping nuclei and nuclei with incorrect signal detection. A cut-off value of 0.84 mum was defined to discriminate between translocation positive and negative nuclei based on the shortest distance between signals. Using this value, the false positive rate of the automated analysis for negative samples was 7.0%, whereas that of the manual analysis was 5.8%. Automated and manual results showed strong correlation (R(2) = 0.985), the mean difference of results was only 3.7%. CONCLUSIONS: A reliable and objective automated analysis of large numbers of cells is possible, avoiding interobserver variability and producing statistically more accurate results than manual evaluation.     

ENVIRONMENTAL FACTORS CORRELATED WITH CHRYSOPHYTE CYST ASSEMBLAGES IN LOW ARCTIC LAKES OF SOUTHWEST GREENLAND1

We analyzed the relationship between chrysophyte cyst assemblages in surface sediment samples and limnological and geographical variables for 70 lakes located along Søndre Strømfjord in southwest Greenland. Over 247 stomatocysts were identified and of these, 153 were sufficiently abundant for use in statistical analyses. Eight stomatocysts were considered to be new and are described formally. Canonical correspondence analysis indicated that conductivity was the dominant variable explaining cyst distribution, reflecting the large conductivity gradient in lake water chemistry in this area. High conductivity lakes had distinctive cyst assemblages with lower diversity than low alkalinity lakes, where assemblages were similar to alpine soft‐water lakes elsewhere. The high conductivity lakes, however, had similar cysts to other saline lakes elsewhere in the arctic. Additionally, pH, calcium, maximum depth, longitude, sulfate, total phosphorus, and altitude all explained significant amounts of variability of cyst assemblages. Longitude was the only geographical variable that explained cyst variability independently of other variables (i.e. had a unique effect), which suggests that the climatic gradient from the coast to the head of the fjord has a structuring effect on cyst assemblages. Conductivity (weighted‐averaging partial least squares, r²=0.917; root mean square error=0.142; r²_jack=0.861, root mean square error of prediction=0.191) and pH inference models (weighted averaging, r²=0.924; root mean square error=0.158; r²_jack=0.826, root mean square error of prediction=0.240) were developed. For the pH model, high conductivity lakes (>800 μ S₂₀·cm^?1) were removed. Both models are statistically robust and could be applied to lakes in west Greenland to reconstruct conductivity and/or pH. Such paleolimnological reconstructions provide the means of acquiring long‐term data for use in the evaluation of, for example, regional paleoclimatic models.     

Die Feinstruktur des ektopischen Schmelzorgans bei beschleunigter Eruption des Rattenschneidezahnes

Zusammenfassung Durch regelmäßig wiederholtes mechanisches Kürzen des rechten unteren Rattenschneidezahnes wurde eine beschleunigte Eruption hervorgerufen. Nach 15–17 Tagen trat eine Störung der Schmelzentwicklung in Form einer Produktion von kreideweißem statt normal pigmentiertem Schmelz und ein rotbräunliches Häutchen im Bereich des gingivalen Epithelverschlusses in Erscheinung. Dieses Häutchen wurde nach intravitaler Fixierung mit Glutaraldehyd abpräpariert und licht- und elektronenmikroskopisch untersucht.Das Häutchen besteht aus mehreren übereinander gelagerten Zellschichten: aus Ameloblasten, einem typischen Papillarorgan, einer Bindegewebsschicht und aus flachen Deckzellen. Die Ameloblasten im Bereich des Epithelverschlusses befinden sich im Stadium der frühen, die Ameloblasten des aus dem Epithelverschluß hervorwachsenden Häutchens dagegen im Stadium der späten Maturation. Die Ultrastruktur des gesamten Papillarorgans läßt auf eine intensive metabolische Funktion und auf Ionentransport schließen. Pigmentgranula, die bei normaler Eruption im Maturationsstadium sowohl im Papillarorgan als auch in den Ameloblasten vorkommen, wurden nur im Bereiche des Epithelverschlusses in vaskulären Elementen und in perivaskulären Bindegewebszellen gefunden. Die Oberfläche des Häutchens wird von einer schmalen Schicht flacher, organellenarmer Zellen gebildet.Das bei beschleunigter Eruption aus dem Epithelansatz hervorwachsende Häutchen ist ein ektopisches Schmelzorgan, dessen rotbräunliche Färbung von der reichen Gefäßversorgung herrührt.

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The fine structure of the ectopic enamel organ in accelerated eruption of the rat incisor

Summary An accelerated eruption of the right mandibular rat incisor was induced by its repeated, mechanical shortening. Fifteen to seventeen days after beginning of the experiment a disturbance of enamel development occured, manifested by production of white, opaque enamel instead of normally pigmented enamel and by appearance of a redbrownish membrane overlying the enamel. This membrane was intravitally fixed with glutaraldehyde and after dissection processed for histologic and electronmicroscopic investigation.The membrane consists of ameloblasts, a typical papillary organ, a connective tissue layer and of flat surface cells. The ameloblasts near the epithelial attachment are in early maturation state, the ameloblasts of the outgrowing membrane show signs of reduction, typical for late maturation. The papillary organ of the entire membrane showes ultrastructural equivalents of high metabolic activity as well as of fluid transports.Pigment granules, which in normal eruption occur in the maturation state in the papillary cells and in ameloblasts, are absent in these cells of the membrane. They were only seen in perivascular elements and in connective tissue cells near the epithelial attachment. On the oral surface the membrane is covered by a thin layer of flat, degenerated cells poor in organelles. These findings support the concept, that the membrane growing out of the epithelial attachment in accelerated eruption of the rat incisor, is an ectopic enamel organ. The red-brownish colour is not due to pigment accumulation but to its rich vascularization.

     

Seaweed-Derived Biostimulant (Kelpak<Superscript>®</Superscript>) Influences Endogenous Cytokinins and Bioactive Compounds in Hydroponically Grown <Emphasis Type="Italic">Eucomis autumnalis</Emphasis>

The effect of a seaweed-derived biostimulant (Kelpak^® at 1, 2.5 and 5 % dilution; v/v) on the growth, endogenous cytokinin (CK) and phytochemical content in Eucomis autumnalis (Mill.) Chitt. under hydroponic conditions was evaluated. After 4 months, the stimulatory effect of Kelpak^® treatments was more noticeable in the underground organs than in the aerial organs. Total endogenous CK was also higher in plants treated with Kelpak^® (c.a. 1000–1200 pmol g^?1 DW) compared to control plants (860 pmol g^?1 DW). Isoprenoid CKs (which mainly accumulated in the aerial organs) were more dominant than aromatic-type CKs across all the treatments. A total of 11 bioactive chemicals (8 phenolic acids and 3 flavonoids) and eucomic acid known for their diverse biological activities were quantified in the samples. The most abundant compound was p-coumaric acid (6.5 µg g^?1 DW) and it was approximately sevenfold higher in 2.5 % Kelpak^®-treated plants than in the control. It was also noteworthy that syringic acid only occurred in the underground organs of 5 % Kelpak^®-treated plants. Eucomic acid which is a major bioactive compound in E. autumnalis was significantly enhanced in Kelpak^® treatments, and the leaves accounted for more than 70 % of the overall content. Thus, Kelpak^® elicited a significant influence on the growth, endogenous CK and phytochemical content in E. autumnalis. These findings provide additional evidence of the enormous potential of Kelpak^® as a useful biostimulant with practical applications in various agricultural endeavours.     

Impacts of forestry planting on primary production in upland lakes from north‐west Ireland

Planted forests are increasing in many upland regions worldwide, but knowledge about their potential effects on algal communities of catchment lakes is relatively unknown. Here, the effects of afforestation were investigated using palaeolimnology at six upland lake sites in the north‐west of Ireland subject to different extents of forest plantation cover (4–64% of catchment area). ²¹⁰Pb‐dated sediment cores were analysed for carotenoid pigments from algae, stable isotopes of bulk carbon (δ¹³C) and nitrogen (δ¹⁵N), and C/N ratios. In lakes with >50% of their catchment area covered by plantations, there were two‐ to sixfold increases in pigments from cryptophytes (alloxanthin) and significant but lower increases (39–116%) in those from colonial cyanobacteria (canthaxanthin), but no response from biomarkers of total algal abundance (β‐carotene). In contrast, lakes in catchments with <20% afforestation exhibited no consistent response to forestry practices, although all lakes exhibited fluctuations in pigments and geochemical variables due to peat cutting and upland grazing prior to forest plantation. Taken together, patterns suggest that increases in cyanobacteria and cryptophyte abundance reflect a combination of mineral and nutrient enrichment associated with forest fertilization and organic matter influx which may have facilitated growth of mixotrophic taxa. This study demonstrates that planted forests can alter the abundance and community structure of algae in upland humic lakes of Ireland and Northern Ireland, despite long histories of prior catchment disturbance.     

Human liver paraoxonase (PON1): Subcellular distribution and characterization

The subcellular localization and different biochemical properties of a human hepatic microsomal enzyme that hydrolyses paraoxon (paraoxonase, PON1) were studied and compared to the paraoxon hydrolase activity found in human plasma as well as in rat liver and plasma. Having evaluated the influence of the postmortem interval by a parallel experiment performed in rats, we conclude that the paraoxonase activity was preferentially localized in the microsomal fraction. The enzyme reaction was optimized according to temperature, pH, buffer, ionic strength, substrate concentration, and enzyme protein concentration. The characterization of human liver paraoxonase included the study of optimum pH, pH stability, heat inactivation assays, and kinetic parameters (K_m and V_max). In addition, the enzyme activity showed an absolute requirement for exogenous calcium. The activity was lost after incubation with EDTA and partially restored by the addition of calcium; however, other metals assayed were not able to activate the human liver enzyme as did calcium. Our results support the possible identity between human plasma and liver paraoxonases. In spite of the technical difficulties of this study and the possible interference of the postmortem changes in the results, this article represents the first systematic approach to the characterization of human liver paraoxonase. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 61–69, 1998     

Integrating species distribution modelling into decision-making to inform conservation actions

Species distribution models (SDMs) have been widely tagged as valuable tools in a variety of conservation assessments to address pressing conservation problems. However, these solutions could be hampered by difficulties to overcome the knowledge-action boundary between conservation and modelling practice. These difficulties have been well typified in the ecological modelling sphere, but a specific conceptual framework on how to bridge this gap is still lacking. This work reports successful examples on how to use SDMs to identify the most favourable habitats for implementing conservation management actions. We use these examples to discuss about the three main topics that deserve special attention to help enhance information flow between practitioners and modellers: the decision context, the modelling framework and the spatial products. Finally, we suggest some practical solutions to improve applications of effective conservation action on the ground. We emphasize the importance of matching modelling goals and decision targets by a close collaboration of modellers with decision makers and species experts. Moreover, we highlight the key role of clear and useful spatial products to provide relevant and timely feedback to increase understanding and promote utilisation by conservation practitioners, and to inform and involve targeted audiences.     

KU0063794, a Dual mTORC1 and mTORC2 Inhibitor,Reduces Neural Tissue Damage and Locomotor Impairment After Spinal Cord Injury in Mice

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal–lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.