Insulin induces expression of adenosine kinase gene in rat lymphocytes by signaling through the mitogen-activated protein kinase pathway

The activity of adenosine kinase (AK) was significantly impaired in splenocytes isolated from diabetic rats. Administration of insulin to diabetic animals restored AK activity, protein, and mRNA levels in diabetic splenocytes. Experiments performed on cultured rat lymphocytes demonstrated that insulin did not change the stability of AK mRNA. Insulin induced AK gene expression in a dose- and time-dependent manner. Maximal increases in AK mRNA (3.9-fold) and activity level (3.7-fold) were observed at the fourth and fifth hours of cell incubation with 10 nM insulin, respectively. The insulin effect on AK expression was not influenced by dibutyryl cAMP (dcAMP). On the other hand dcAMP weakly increased (1.7-fold) basal expression of AK. Exposure of rat lymphocytes to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or rapamycin, an inhibitor of mTOR, did not affect the ability of insulin to stimulate expression of AK. Prior treatment of the cells with 10 microM PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK) completely blocked insulin-stimulated expression of AK gene. Insulin produced a significant transient increase in the tyrosine phosphorylation of ERK1/2, and PD98059 inhibited this phosphorylation. Furthermore exposure of cells to insulin has resulted in transient phosphorylation of Elk-1 on Ser-383 and sustained elevation of c-Jun and c-Fos protein. The maximal phosphorylation of Elk-1 was observed at 15 min, and was blocked by PD98059. We concluded that insulin stimulates AK gene expression through a series of events occurring sequentially. This includes activation of the MAPK cascade and subsequent phosphorylation of Elk-1 followed by increased expression of c-fos and c-jun genes.     

Enantioselective Dynamic Process Reduction of α- and β-Tetralone and Stereoinversion of Resulting Alcohols in a Selected Strain Culture

α-Tetralone and β-tetralone were subjected to biotransformation by 14 fungal strains. Enantiomeric purity of the products depended on the reaction time. 3-Day transformation of α-tetralone in Absidia cylindrospora culture gave S-(+)-1,2,3,4-tetrahydro-1-naftol of 92 % ee, whereas longer biotransformation time resulted in decrease of ee value. 3-Day transformation of β-tetralone by the same strain gave predominantly S-(-)-1,2,3,4-tetrahydro-2-naftol, whereas after 9 days of the reaction, the R-enantiomer with 85 % ee was isolated. Transformation of β-tetralone by Chaetomium sp. KCh 6651 gave pure (S)-(-)-1,2,3,4-tetrahydro-2-naftol in high yield at the concentration of 1 g/l. In this process, a non-selective carbonyl reduction was observed, followed by a selective oxidation of the R-alcohol.     

In Vivo Transfer of Intracellular Labels from Locally Implanted Bone Marrow Stromal Cells to Resident Tissue Macrophages

Intracellular labels such as dextran coated superparamagnetic iron oxide nanoparticles (SPION), bromodeoxyuridine (BrdU) or green fluorescent protein (GFP) are frequently used to study the fate of transplanted cells by in vivo magnetic resonance imaging or fluorescent microscopy. Bystander uptake of labeled cells by resident tissue macrophages (TM) can confound the interpretation of the presence of intracellular labels especially during direct implantation of cells, which can result in more than 70% cell death. In this study we determined the percentages of TM that took up SPION, BrdU or GFP from labeled bone marrow stromal cells (BMSCs) that were placed into areas of angiogenesis and inflammation in a mouse model known as Matrigel™ plaque perfusion assay. Cells recovered from digested plaques at various time points were analyzed by fluorescence microscopy and flow cytometry. The analysis of harvested plaques revealed 5% of BrdU⁺, 5–10% of GFP⁺ and 5–15% of dextran⁺ macrophages. The transfer of the label was not dependent on cell dose or viability. Collectively, this study suggests that care should be taken to validate donor origin of cells using an independent marker by histology and to assess transplanted cells for TM markers prior to drawing conclusions about the in vivo behavior of transplanted cells.     

Long-term pheromone-mediated mating disruption of the Mediterranean flour moth, Ephestia kuehniella, in a flourmill

Several studies have indicated that mating disruption (MD) successfully reduces population densities of stored product moths, for example, the Mediterranean flour moth, Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae). However, practical issues, such as finding control plots, replication, and lack of similarity between replicates, often incur problems in full-scale investigations and often draw focus from the added results. We here present results from long-term monitoring of E. kuehniella populations in two similar flourmills in Poland, one treated with pheromone for MD and one left untreated and used as control. Pheromone-mediated MD was applied during 2 years. Thirty pheromone dispensers (one per 100 m³ factory volume), each releasing about 2 mg ( Z,E )-9,12-tetradecadienyl acetate per day, were used. Pheromone-baited traps were used to monitor the moth populations in the MD mill and in a nearby untreated mill. The reduction in trap catch during the MD treatment was about 90% or more, compared with the untreated mill or pre-treatment periods of the MD mill. The reduction was larger during the 2nd year of MD than during the 1st year. These results contribute to the picture that MD is an effective method to control moth species infesting stored products.     

The activity of alpha1,6-fucosyltransferase during human megakaryocytic differentiation

alpha1,6-Fucosyltransferase (6FucT, E.C. 2.4.1.68) is one of the enzymes involved in the synthesis of N-linked glycans of the GpIIb/IIIa complex (CD41a) which is present on megakaryocytes (MKs) and platelets. In this study, we examined 6FucT activity in ex vivo cultures of immunoselected cord blood CD34(+) cells grown in a medium promoting megakaryocytopoiesis. Our results show that the activity of 6FucT increased ahead of, and thereafter concomitantly with, cells expressing the CD41a antigen. When the CD41a(+) subpopulation of cells was immunoselected (using anti-CD61 i.e. anti-GpIIIa antibodies), its 6FucT activity increased proportionally to the yield of CD61(+)(+)(+) cells. Taking into account the heavy load of 6FucT in platelets and megakaryocytes, we regard this enzyme as a candidate for the earliest marker of MK-commitment in cultured hematopoietic stem cells. Such a marker should allow an earlier detection and earlier transplantation of patients' own, ex vivo expanded, Mk progenitors.     

Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex.

The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S0.5 for 2-oxoglutarate, and in an increase of S0.5 for NAD. Changes in ionic strength over the range of 0.05-0.2 M have little, if any, effect on S0.5 for CoA. The Hill coefficient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively. At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4-7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca2+, increases in ionic strength lower the inhibitory effects of ATP. The Si0.5 for ATP in both presence and absence of Ca2+ was not affected by changes in ionic strength in the range of 0.1-0.2 M. In contrast, the Sa0.5 for ADP in the absence of Ca2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity.