Seasonal dynamics of mobile carbohydrate pools in phloem and xylem of two alpine timberline conifers.

Recent studies on non-structural carbohydrate (NSC) reserves in trees focused on xylem NSC reserves, while still little is known about changes in phloem carbohydrate pools, where NSC charging might be significantly different. To gain insight on NSC dynamics in xylem and phloem, we monitored NSC concentrations in stems and roots of Pinus cembra (L.) and Larix decidua (Mill.) growing at the alpine timberline throughout 2011. Species-specific differences affected tree phenology and carbon allocation during the course of the year. After a delayed start in spring, NSC concentrations in L. decidua were significantly higher in all sampled tissues from August until the end of growing season. In both species, NSC concentrations were five to seven times higher in phloem than that in xylem. However, significant correlations between xylem and phloem starch content found for both species indicate a close linkage between long-term carbon reserves in both tissues. In L. decidua also, free sugar concentrations in xylem and phloem were significantly correlated throughout the year, while a lack of correlation between xylem and phloem free sugar pools in P. cembra indicate a decline of phloem soluble carbohydrate pools during periods of high sink demand.

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia.

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

No evidence for depletion of carbohydrate pools in Scots pine (Pinus sylvestris L.) under drought stress.

The physiological mechanisms leading to Scots pine (Pinus sylvestris L.) decline in the dry inner alpine valleys are still unknown. Testing the carbon starvation hypothesis, we analysed the seasonal course of mobile carbohydrate pools (NSC) of Scots pine growing at a xeric and a dry-mesic site within an inner alpine dry valley (750 m a.s.l., Tyrol, Austria) during 2009, which was characterised by exceptional soil dryness. Although, soil moisture content dropped to ca. 10% at both sites during the growing season, NSC concentrations rose in all tissues (branch, stem, root) until the end of July, except in needles, where maxima were reached around bud break. NSC concentrations were not significantly different in the analysed tissues at the xeric and the dry-mesic site. At the dry-mesic site, NSC concentrations in the aboveground tree biomass were significantly higher during the period of radial growth. An accumulation of NSC in roots at the end of July indicates a change in carbon allocation after an early cessation in aboveground growth, possibly due to elevated belowground carbon demand. In conclusion, our results revealed that extensive soil dryness during the growing season did not lead to carbon depletion. However, even though carbon reserves were not exhausted, sequestration of carbohydrate pools during drought periods might lead to deficits in carbon supply that weaken tree vigour and drive tree mortality.

Spatial and seasonal variations in mobile carbohydrates in Pinus cembra in the timberline ecotone of the Central Austrian Alps.

To test whether the altitudinal limit of tree growth is determined by carbons shortage or by a limitation in growth we investigated non structural carbohydrates and their components starch and total soluble sugars in Pinus cembra trees along an elevational gradient in the timberline ecotone of the Central Austrian Alps. NSC contents in needles, branches, stems, and coarse roots were measured throughout an entire growing season. At the tissue level NSC contents were not significantly more abundant in treeline trees as compared to trees at lower elevations. Along our 425 m elevational transect from the closed forest to the treeline we failed to find a stable elevational trend in the total NSC pool of entire trees and observed within season increases in the tree's NSC pool that can be attributed to an altitudinal increase in leaf mass as needles contained the largest NSC fraction of the whole tree NSC pool. Furthermore, whole tree NSC contents were positively correlated with net photosynthetic capacity. Although our observed NSC characteristics do not support the hypothesis that tree life at their upper elevational limit is determined by an insufficient carbon balance we found no consistent confirmation for the sink limitation hypothesis.

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells.

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.

Islet isolation and GMP, ISO 9001:2000: what do we need--a 3-year experience.

Pancreatic islet cell isolation and transplantation has been performed for many years at several institutions. Although all institutions aim to produce high-quality islets, applied standards widely deviate from standards in the pharmaceutical industry. The legal situation within the European Union has changed requirements for setting up and running such a laboratory. The process is now clearly defined as a production of a pharmaceutical and therefore must be licensed by federal authorities. Analysis of workload for establishing an islet isolation program that fulfil GMP and ISO 9001 criteria including an estimation of costs and the impact of such a system on the isolation process. The definition of quality parameters and documentation is a central issue of all islet isolation laboratories. Therefore, GMP and ISO 9001:2000 do not add additional work per se. On the other hand, clear guidelines, a clear policy, working place descriptions, forms, checklists, and, particularly standard operating procedures, are instrumental for smooth functioning within the department. Collection of data such as errors, improvement measures, and preventive measures reduces subsequent costs. A clear definition of responsibilities minimizes organizational problems. Steering of inspection devices prevents bias errors and validating the processes clearly points out incorrect assumptions. Documentation helps to prove the correctness of the production at any time and is of use also for scientific evaluations. We strongly feel that GMP criteria are mandatory and together with an ISO 9001:2000 quality management system offers significant advantages for the process of islet isolation and a continuous improvement process.

"Real-time" assessment of human islet preparations with confocal live cell imaging.

Pancreatic islet cell transplantation is a promising approach to restoring normoglycemia in diabetic patients. The outcome of islet transplantation is uncertain for two reasons: The quality of isolated islets is still poorly defined and the functional potential of transplanted islets is difficult to predict. Therefore, one of the primary challenges in islet transplantation is to identify and understand the changes taking place in islets after isolation and culture. Description of such changes in living islet cells offers insights not achievable by use of fixed-cell techniques. Three fluorescent dyes, dichlorodihydrofluorescein diacetate, tetramethylrhodamine methyl ester perchlorate (TMRM), and fluorescent wheat germ agglutinin, were used to assess either overall oxidative stress, time-dependent mitochondrial membrane potentials, or localization of oligosaccharides. Confocal microscopy was performed with a microlens-enhanced Nipkow disk-based confocal system mounted on an inverse microscope. We were able to show differences in the amount of oligosaccharides on the cell surface between endocrine and exocrine cells in freshly isolated human islet preparations. The study of the mitochondrial membrane potential via TMRM proved to be useful to early identification of damaged or stressed cells. Thus a combination of fluorescent dyes as subcellular markers, with a powerful live confocal imaging system may be of great value to isolation and culture.

In the search of potential human islet stem cells: is tetranectin showing us the way?

Pancreatic islet cell transplantation is a promising approach to restore the required mass of functional beta cells in diabetic patients as a means to achieve long-term normoglycemia. This therapy is, however, not yet widely used, in part because of the shortage of human islet cells. Gaining detailed knowledge of the physiological basis governing the processes of differentiation of pancreatic stem or progenitor cells and the mechanisms and molecules necessary for a successful engraftment of the transplanted cells into the liver is instrumental for the ambitious goal of engineering new pancreatic islets to cure type I diabetes. We describe the in vivo and in vitro localisation of tetranectin (TN) in human and murine islet cells. Similar to human islets, murine islets stain positive for tetranectin. The amount and localization of TN is influenced by different culture conditions. The ability of TN to bind plasminogen indicates that it may have a role in regulating pericellular proteolysis and proteolytic activation of latent forms of metalloproteinases and growth factors. Tetranectin may thereby play an important role in the survival of islets in the liver after islet transplantation. TN-positive cells can be isolated and maintained in culture after human islet isolation, thereby providing the possibility to further clarify its role and function in vivo as well as in the course of islet transplantation.