Polynucleotide phosphorylase-based photometric assay for inorganic phosphate

Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5'-diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5'-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.     

Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.     

Preparative resolution of 1-aminoindan-1,5-dicarboxylic acid (AIDA) by chiral ligand-exchange chromatography

Chiral ligand-exchange chromatography has been shown to be effective in the resolution and semipreparative separation of 1-aminoindan-1,5-dicarboxylic acid (AIDA) enantiomers. In functional activity experiments, only (S)-AIDA was a potent and mGluR1 subtype selective antagonist.     

Ovarian follicle vascularization in fasted pig

The authors have investigated in the different classes of ovarian follicles the vascular area, the blood vessel distribution, the vascular endothelial growth factor (VEGF) mRNA expression and the VEGF secretion during equine chorionic gonadotropin (eCG) induced follicle growth in prepubertal gilts fed ad libitum or fasted. Immunohistochemistry staining of Von Willebrand factor showed that fasting caused a dramatic increase in the vascular area of medium-large tertiary follicles. The increase involved the two concentric vessel networks and the area between them that, becoming crossed by several anastomosis, modified the whole vessel architecture. Both in situ hybridization and in vitro culture experiments demonstrate that granulosa cells from medium-large follicles are engaged in a copious VEGF production upon eCG stimulation both in gilts fed ad libitum or fasted. More surprisingly, the production of VEGF becomes diffuse amongst theca cells of fasted animals thus recruiting a compartment that in condition of normal feeding regimen appears nearly quiescent. In conclusion, the data presented describe a local angiogenic process that develops in the follicle wall of growing antral follicle in case of acute severe food restriction. The mechanism, essentially confined to follicles that potentially approach ovulation, appears to assume the meaning of a local compensatory mechanism that may help maintaining adequate nutrient delivery to follicles that undergo ovulation.     

Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.     

Synthesis and pharmacological characterization of functionalized 2-pyridones structurally related to the cardiotonic agent milrinone

A new class of cardiotonic agents characterized by a 2-pyridone structure was synthesized. Appropriate sym-2-dimethylaminomethylene-1,3-diones reacted with methylcyanoacetate to afford the desired compounds. These derivatives were evaluated for their ability in inducing cardiotonic response on guinea pig isolated myocardial preparations. Compound 8b increased atrial contractility to an extent which is significantly higher than that of milrinone, the parent drug used as a reference compound. The pharmacological characterization and the docking studies performed on 8b highlighted its selective mechanism of action via type 3 PDE (PDE3) inhibition.     

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.     

The application of abstract topology to RAS-related signal transduction pathways

Ras is a protein related to cancer development. It is a convergence point for different signal transduction pathways that allow the cell to respond to external stimuli with different cell functions like growth, division, death, etc. In this paper, we analyze the signal pathways generated by different Ras effectors (Raf, RalGDS and PI3K), and the pathway relating Ras to the cell cycle control. We show that the interaction among different elements of these pathways induces a topologic structure in the set of elements. We discuss properties of this topology and give an algorithm to build it. The application of topological concepts makes easier the interaction analysis. Using a computational algorithm, we can create isolated, independently manageable sub-groups. Then we construct their hierarchical structure. The procedure allows us to visualize groups of elements related to the Ras effectors involved in cell growth, the elements involved in the cytoskeleton regulation, and the elements related to the cell cycle control. Thus the division in sub-groups does not only make easier the analysis, but it also provides a biologically meaningful subdivision.     

Human neoplastic submandibular intercalated duct cells express an acinar phenotype when cultured on a basement membrane matrix

Abstract. Culture of the human neoplastic submandibular gland intercalated duct cell line, HSG, on the basement membrane extract Matrigel induces dramatic morphologic changes and cytodifferentiation. Transmission electron microscopy demonstrated an acinar cell phenotype with polarized cells containing a well-developed Golgi apparatus, multiple microvilli-like projections from the apical surfaces into a lumenal-like area, and numerous granule-like organelles. Amylase, an acinar cell marker, was detected by both immunocytochemical and Northern blot analyses. A 50% reduction in [³H]thymidine incorporation by cells cultured on Matrigel, as compared to cells cultured on tissue culture plates, confirmed the differentiated phenotype of the cells. Multiple components of Matrigel appear to contribute to the morphologic differentiation of the HSG cells since antibodies to both laminin and collagen IV, as well as the lamininderived bioactive peptide containing SIKVAV, have potent inhibitory effects on HSG cell organization on Matrigel. Collectively, these data indicate that culture of HSG cells on Matrigel is a useful model to study salivary gland acinar development.