Embryonic cell lineage of the marine nematode Pellioditis marina

We describe the complete embryonic cell lineage of the marine nematode Pellioditis marina (Rhabditidae) up to somatic muscle contraction, resulting in the formation of 638 cells, of which 67 undergo programmed cell death. In comparison with Caenorhabditis elegans, the overall lineage homology is 95.5%; fate homology, however, is only 76.4%. The majority of the differences in fate homology concern nervous, epidermal, and pharyngeal tissues. Gut and, remarkably, somatic muscle is highly conserved in number and position. Partial lineage data from the slower developing Halicephalobus sp. (Panagrolaimidae) reveal a lineage largely, but not exclusively, built up of monoclonal sublineage blocs with identical fates, unlike the polyclonal fate distribution in C. elegans and P. marina. The fate distribution pattern in a cell lineage could be a compromise between minimizing the number of specification events by monoclonal specification and minimizing the need for migrations by forming the cells close at their final position. The latter could contribute to a faster embryonic development. These results reveal that there is more than one way to build a nematode.     

Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas RhoA is activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors, it is unknown how LPA receptors may signal to Rac. Here we report that the prototypic LPA(1) receptor (previously named Edg2), when expressed in B103 neuroblastoma cells, mediates transient activation of RhoA and robust, prolonged activation of Rac leading to cell spreading, lamellipodia formation, and stimulation of cell migration. LPA-induced Rac activation is inhibited by pertussis toxin and requires phosphoinositide 3-kinase activity. Strikingly, LPA fails to activate Rac in cell types that lack the Rac-specific exchange factor Tiam1; however, enforced expression of Tiam1 restores LPA-induced Rac activation in those cells. Tiam1-deficient cells show enhanced RhoA activation, stress fiber formation, and cell rounding in response to LPA, consistent with Tiam1/Rac counteracting RhoA. We conclude that LPA(1) receptors couple to a G(i)-phosphoinositide 3-kinase-Tiam1 pathway to activate Rac, with consequent suppression of RhoA activity, and thereby stimulate cell spreading and motility.     

Correcting mass isotopomer distributions for naturally occurring isotopes

In one method of metabolic flux analysis, simulated mass spectrometry data is fitted to measured mass distributions of metabolites that are isolated from cultures with defined feeding of (13)C-labeled substrates. Doing so, simulated mass distributions must be corrected for the presence of naturally occurring isotopes. A method that was recently introduced for this purpose consists of consecutive correction steps for each isotope of each element in the considered compound. Here we show that all isotopes of each individual element must, however, be corrected in one single step. Furthermore, it is shown that the source of information with respect to isotopic compositions of the elements needs to be chosen with care.     

Effects of Pb-EDTA and EDTA on oxidative stress reactions and mineral uptake in Phaseolus vulgaris

Sequestration of Pb by synthetic chelates has been reported to increase bioavailability, uptake, and translocation of this metal in plants. In this work the potential phytotoxic effects of Pb-EDTA were investigated in Phaseolus vulgaris L. cv. Limburgse vroege plants grown on hydroponics. Addition of 50 µ M Pb-EDTA to the nutrient solution caused a significant induction of syringaldazine peroxidase (SPOD; EC 1.11.1.7) in roots and primary leaves and guaiacol peroxidase (GPOD; EC 1.11.1.7) in leaves. Addition of 100 µ M Pb-EDTA further exacerbated ascorbate peroxidase (APOD; EC 1.11.1.11), GPOD, dehydroascorbate reductase (DHAR; EC 1.8.5.1), glutathione reductase (GR; EC 1.6.4.2) and malic enzyme (ME; EC 1.1.1.40) in roots and APOD and ME in primary leaves. Addition of 200 µ M Pb-EDTA also induced DHAR in leaves. This induction of peroxidases (SPOD, GPOD, APOD), enzymes of the ascorbate-glutathione cycle (DHAR, GR in roots) and of an NADP⁺ reducing enzyme in roots and primary leaves indicates that oxidative stress has been initiated. At 200 µ M Pb-EDTA, chlorophyll a and b content in leaves was significantly reduced while visible effects on root morphology and shoot length were observed, while no significant morphological effects were found in the leaves, confirming the sensitive character of the measured enzymes as plant stress indicators. Elevation of the Pb-EDTA concentration in the growth medium significantly reduced the content of Ca, Fe, Mn and Zn taken up by plants, probably due to ion leakage as a result of observed toxicity. Addition of up to 200 µ M EDTA increased chelation of divalent cations in nutrient solution resulting in reduced plant uptake of Zn, Cu, Fe and Mn. This did not result in phytotoxicity.     

UDP-glucuronate decarboxylase, a key enzyme in proteoglycan synthesis: cloning, characterization, and localization

UDP-glucuronate decarboxylase (UGD) catalyzes the formation of UDP-xylose from UDP-glucuronate. UDP-xylose is then used to initiate glycosaminoglycan biosynthesis on the core protein of proteoglycans. In a yeast two-hybrid screen with the protein kinase Akt (protein kinase B), we detected interactions with a novel sequence, which we cloned and expressed. The expressed protein displayed UGD activity but did not display the activities of homologous nucleotide sugar epimerases or dehydratases. We did not detect phosphorylation of UGD by Akt nor did we detect any influence of Akt on UGD activity. Effects of UGD on Akt kinase activity were also absent. Northern blot and Western blot analyses revealed the presence of UGD in multiple tissues and brain regions. Subcellular studies and histochemistry localized UGD protein to the perinuclear Golgi where xylosylation of proteoglycan core proteins is known to occur.     

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.     

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.     

19F Nuclear Magnetic Resonance as a Tool To Investigate Microbial Degradation of Fluorophenols to Fluorocatechols and Fluoromuconates

A method was developed to study the biodegradation and oxidative biodehalogenation of fluorinated phenols by ¹⁹F nuclear magnetic resonance (NMR). Characterization of the ¹⁹F NMR spectra of metabolite profiles of a series of fluorophenols, converted by purified phenol hydroxylase, catechol 1,2-dioxygenase, and/or by the yeast-like fungus Exophiala jeanselmei, provided possibilities for identification of the ¹⁹F NMR chemical shift values of fluorinated catechol and muconate metabolites. As an example, the ¹⁹F NMR method thus defined was used to characterize the time-dependent metabolite profiles of various halophenols in either cell extracts or in incubations with whole cells of E. jeanselmei. The results obtained for these two systems are similar, except for the level of muconates observed. Altogether, the results of the present study describe a ¹⁹F NMR method which provides an efficient tool for elucidating the metabolic pathways for conversion of fluorine-containing phenols by microorganisms, with special emphasis on possibilities for biodehalogenation and detection of the type of fluorocatechols and fluoromuconates involved. In addition, the method provides possibilities for studying metabolic pathways in vivo in whole cells.     

Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.