A generic assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography for lead finding in natural products

A generic continuous-flow assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography (LC) has been developed. Operating the LC-biochemical assay in combination with mass spectrometry allows the fast detection and identification of inhibitors of these enzymes in complex mixtures. The assay is based on the detection of phosphate, released by the on-line continuous-flow enzymatic reaction, using a fluorescent probe. The probe consists of fluorophore-labeled phosphate-binding protein, which shows a strong fluorescence enhancement upon binding to inorganic phosphate. To detect very small changes of the phosphate concentration in a postcolumn enzymatic reaction medium, the enzymatic removal of phosphate impurities from solvents, reagents, and samples was optimized for application in continuous flow. The potential of the phosphate probe is demonstrated by monitoring the enzymatic activity, i.e., the phosphate release, from alkaline phosphatase. The selectivity of the phosphate readout, necessary to distinguish between phosphate containing substrate or product and free inorganic phosphate released after enzymatic conversion, is shown. The applicability of LC coupled to the enzymatic assay using the phosphate readout was demonstrated by detection of tetramisole in a plant extract as inhibitor of alkaline phosphatase. Parallel mass spectrometry allowed the simultaneous confirmation of the identity of the inhibitor.     

Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity

We showed recently that antisense plants with decreased activity of the plastidic ATP/ADP-transporter protein exhibit drastically reduced levels of starch and a decreased amylose/amylopectin ratio, whereas sense plants with increased activity of the transporter possessed more starch than wild-type plants and an increased amylose/amylopectin ratio. In this paper we investigate the effect of altered plastidic ATP/ADP-transporter protein expression on primary metabolism and granule morphology in more detail. Tuber tissues from antisense and sense plants exhibited substantially increased respiratory activity compared with the wild type. Tubers from antisense plants contained markedly increased levels of free sugars, UDP-Glc, and hexose phosphates, whereas phosphoenolpyruvate, isocitrate, ATP, ADP, AMP, UTP, UDP, and inorganic pyrophosphate levels were slightly decreased. In contrast, tubers from sense plants revealed a slight increase in adenine and uridine nucleotides and in the levels of inorganic pyrophosphate, whereas no significant changes in the levels of soluble sugars and metabolites were observed. Antisense tubers contained 50% reduced levels of ADP-Glc, whereas sense tubers contained up to 2-fold increased levels of this sole precursor for starch biosynthesis. Microscopic examination of starch grain morphology revealed that the size of starch grains from antisense tubers was substantially smaller (50%) compared with the wild type. The large starch grains from sense tubers appeared of a more angular morphology, which differed to the more ellipsoid shape of wild type grains. The results suggest a close interaction between plastidial adenylate transport and starch biosynthesis, indicating that ADP-Glc pyrophosphorylase is ATP-limited in vivo and that changes in ADP-Glc concentration determine starch yield, as well as granule morphology. Possible factors linking starch synthesis and respiration are discussed.     

The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids

Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8 microg/mg lipophorin compared to 3.1 microg/mg in the +Y+I mutant, 1.2 microg/mg in the YI mutant and 0.5 microg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62-77%), followed by beta-carotenes (22-38%). Although lutein and beta-carotene content of mutants' lipophorin differed significantly, the ratio of lutein to beta-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.     

Cultivation of Purified Primary Purkinje Cells from Rat Cerebella

Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.     

CD36 antisense expression in 3T3-F442A preadipocytes

An adipocyte membrane glycoprotein, FAT, homologous to CD36, has been implicated in the binding/transport of long-chain fatty acids. FAT/CD36 was identified by reaction with reactive long chain fatty acids derivatives under conditions where they inhibited FA uptake. Expression of CD36 in fibroblasts lacking the protein led to induction of a saturable high affinity, phloretinsensitive component of oleate uptake. In this report, we have examined the effects of FAT/CD36 antisense expression in 3T3-F442A preadipocyte cells, on FA uptake and cell differentiation. Cells were transfected with pSG5-TAF vector obtained by insertion of antisense coding sequence of FAT/CD36 into the BamH 1 site of pSG5. Four clones were selected based on expression of antisense CD36 mRNA. Levels of CD36 protein were determined by flow cytometry and correlated with rates of oleate uptake. Three clones, TAF13, TAF25, and TAF38 exhibited low CD36 expression and one clone TAF 18 had expression comparable to that of F442A control cells. FA uptake rates in clones TAF13, TAF25 and TAF3 8 were lower than those observed in TAF18. At confluence, adipocyte differentiation could be promoted by addition of insulin and triiodothyronine only in TAF18 cells but not in TAF13, TAF25 or TAF38. Addition of fatty acids to clones TAF13, TAF25 and TAF38 lead to an induction of CD36 expression, an enhancement of FA uptake and better cell differentiation. The data support a role of CD36 in the membrane uptake of long chain FA. CD36 expression and FA uptake appear to be closely linked to preadipocyte differentiation.     

Effect of salt on the binding of the linker histone H1 to DNA and nucleosomes

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.     

Activation of insulin-like growth factor type-1 receptor is required for H2O2-induced PKB phosphorylation in vascular smooth muscle cells

Evidence accumulated in recent years has revealed a potential role for reactive oxygen species (ROS) in the pathophysiology of cardiovascular diseases. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully established. Previous work from our laboratory has indicated that exogenous hydrogen peroxide (H2O2) activates several signaling protein kinases, such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) and protein kinase B (PKB) in A10 vascular smooth muscle cells (VSMC). However, the upstream elements responsible for this activation remain unclear. Although a role for epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK) in H2O2-induced ERK1/2 signaling has been suggested, the contribution of this PTK or other receptor or nonreceptor PTKs to PKB activation is not well defined in VSMC. In this study, we used pharmacological inhibitors to investigate the role of receptor and Src-family-PTKs in H2O2-induced PKB phosphorylation. AG1478, a specific inhibitor of EGFR, failed to attenuate the H2O2-induced increase in PKB Ser473 phosphorylation, whereas AG1024, an inhibitor of insulin-like growth factor type1 receptor (IGF-1R)-PTK, almost completely blocked this response. H2O2 treatment also enhanced tyrosine phosphorylation of the IGF-1Rbeta subunit, which was significantly inhibited by AG1024 pretreatment of cells. Furthermore, pharmacological inhibition of Src by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole(3,4-d) pyrimidine) decreased PKB phosphorylation. Moreover, H2O2-induced PKB phosphorylation was associated with increased tyrosine phosphorylation of c-Src and Pyk2 in an AG1024- and PP2-inhibitable manner. In conclusion, these data provide evidence of the contribution of IGF-1R-PTK in initiating H2O2-evoked PKB phosphorylation in A10 VSMC, with an intermediary role for c-Src and Pyk2 in this process.