ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the 'universal' agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae,Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4omega6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3omega6, 20:3omega6 and AA, decreased, indicating an inhibition of the Delta12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 degrees C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 degrees C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.     

Membrane biogenesis in anoxygenic photosynthetic prokaryotes

Following the discovery of photosynthetic bacteria in the nineteenth century, technical developments of the 1950s led to their use in membrane biogenesis studies. These investigations had their origins in the isolation of subcellular particles designated as ‘chromatophores’ by Roger Stanier and colleagues, which were shown to be photosynthetically competent by Albert Frenkel, and to originate from the intracytoplasmic membrane (ICM) continuum observed in electron micrographs. These ultrastrucutral studies by the G. Drews group, Germaine Cohen-Bazire and others also suggested that the ICM originates by invagination of the cytoplasmic membrane, as later established in the biochemical and biophysical work of the R. Niederman and Drews groups. Through a combination of genetic approaches, first introduced in the early 1980s by Barry Marrs, and the atomic resolution structures determined for light-harvesting antennae and reaction centers, a detailed understanding is emerging of mechanisms regulating their levels in the membrane and the roles played by specific protein domains and additional factors in their assembly and supramolecular organization. Prospects for additional progress during the twenty-first century include further elucidation of molecular aspects of the assembly process and the application of newer spectroscopic probes to photosynthetic unit formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dephosphorylation of tau protein by calcineurin triturated into neural living cells

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Ser199/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.     

The mif gene is transcriptionally regulated by glucose in insulin-secreting cells

We have established that focal adhesion kinase (FAK)-transfected HL-60 (HL-60/FAK) cells were highly resistant to hydrogen peroxide and etoposide-induced apoptosis compared to vector-transfected cells. Mutagenesis study revealed that Y397 is required for anti-apoptotic activity in HL-60/FAK, since Y397F-mutated FAK (397FAK) lost anti-apoptotic function. Assuming that 397FAK functions as a dominant negative FAK, we introduced 397FAK cDNA into a human glioma cell line, T98G, using an adenoviral vector. We found that 397FAK induced marked apoptosis with significant FAK degradation. As PI3-kinase-Akt survival pathway was constitutively activated in T98G cells, we hypothesized that this pathway was shut off by 397FAK gene transfection. As expected, activation of PI3-kinase-Akt survival pathway was decreased by the 397FAK gene transfection. 397FAK activated mainly caspase-6 which induced degradation of transfected FAK as well as endogenous FAK. These results indicated that 397FAK induces apoptosis in T98G cells, by interrupting signals of FAK leading to the survival pathway in T98G glioma cells.     

Characterization of the OFD1/Ofd1 genes on the human and mouse sex chromosomes and exclusion of Ofd1 for the Xpl mouse mutant

Oral-facial-digital type 1 (OFD1) syndrome is an X-linked dominant condition characterized by malformations of the face, oral cavity, and digits. The responsible gene, OFD1, maps to human Xp22 and has an unknown function. We isolated and characterized the mouse Ofd1 gene and showed that it is subject to X-inactivation, in contrast to the human gene. Furthermore, we excluded a role for Ofd1 in the pathogenesis of the spontaneous mouse mutant Xpl, which had been proposed as a mouse model for this condition. Comparative sequence analysis demonstrated that OFD1 is conserved among vertebrates and absent in invertebrates. This analysis allowed the identification of evolutionarily conserved domains in the protein. Finally, we report the identification of 18 apparently nonfunctional OFD1 copies, organized in repeat units on the human Y chromosome. These degenerate OFD1-Y genes probably derived from the ancestral Y homologue of the X-linked gene. The high level of sequence identity among the different units suggests that duplication events have recently occurred during evolution.     

`Every dogma has its day'*: a personal look at carbon metabolism in photosynthetic bacteria

Dogmas are unscientific. What is perhaps the greatest biological dogma of all time, the `unity of biochemistry' is, in the main, still having its day. According to present knowledge, the exceptions to this dogma are mere details when seen in relation to the biosystem as a whole. Nevertheless the exceptions are scientifically interesting and the understanding of them has led to a better comprehension of photosynthesis and ecology. Until the discovery of ¹⁴C, photosynthetic CO₂ fixation was like a slightly opened black box. With ¹⁴C in hand scientists mapped out the path of carbon in green plant photosynthesis in the course of a few years. The impressive reductive pentose phosphate cycle was almost immediately assumed to be universal in autotrophs, including anoxygenic phototrophs, in spite of the odd observation to the contrary. A new dogma was born and held the field for about two decades. Events began to turn when green sulfur bacteria were found to contain ferredoxin-coupled ketoacid-oxidoreductases. This led to the formulation of a novel CO₂-fixing pathway, the reductive citric acid cycle, but its general acceptance required much work by many investigators. However, the ice had now been broken and after some years a third mechanism of CO₂ fixation was discovered, this time in Chloroflexus, and then a fourth in the same genus. One consequence of these discoveries is that it has become apparent that oxygen is an important factor that determines the kind of CO₂-fixing mechanism an organism uses. With the prospect of the characterization of hordes of novel bacteria forecast by molecular ecologists we can expect further distinctive CO₂ fixation mechanisms to turn up. This revised version was published online in August 2006 with corrections to the Cover Date.     

ATP-induced calcium oscillations and change of P2Y subtypes with culture conditions in HeLa cells

ATP, UTP, ADP and UDP induced intracellular Ca(2+) responses and oscillations in HeLa cells that sometimes lasted over 1 h. The response is due to the activation of P2Ys, G-protein coupled ATP receptors, because the oscillations persisted for several minutes even in Ca(2+)-free solution, and suramin and PPADS, antagonists of ATP receptors, partially inhibited the response. The potency of these nucleotides varied with the culture or cell conditions, i.e. UTP was generally most potent but in some cases UDP was more potent; responses to UDP were variable while those to ATP were constant. In addition, Ca(2+) responses to ATP and UDP were additive. These findings suggested the existence of two or more subtypes of P2Ys in HeLa cells. RT-PCR experiments revealed the existence of P2Y(2), P2Y(4) and P2Y(6). Recovery from starvation (culture in FBS-free medium overnight and re-addition of FBS) increased the responses to UTP and UDP but not to ATP, suggesting that the number or activity of P2Y(6) and/or P2Y(4) receptors may increase with cell proliferation in HeLa cells.