Targeting of proteins of the striatin family to dendritic spines: role of the coiled-coil domain

Striatin, SG2NA and zinedin, the three mammalian members of the striatin family are multimodular WD-repeat, calmodulin and calveolin-binding proteins. These scaffolding proteins, involved in both signaling and trafficking, are highly expressed in neurons. Using ultrastructural immunolabeling, we showed that, in Purkinje cells and hippocampal neurons, SG2NA is confined to the somatodendritic compartment with the highest density in dendritic spines. In cultured hippocampal neurons, SG2NA is also highly concentrated in dendritic spines. By expressing truncated forms of HA-tagged SG2NAbeta, we demonstrated that the coiled-coil domain plays an essential role in the targeting of SG2NA within spines. Furthermore, co-immunoprecipitation experiments indicate that this coiled-coil domain is also crucial for the homo- and hetero-oligomerization of these proteins. Thus, oligomerization of the striatin family proteins is probably an obligatory step for their routing to the dendritic spines, and hetero-oligomerization explains why all these proteins are often co-expressed in the neurons of the rat brain and spinal cord.     

Inhibition of lipid synthesis through activation of AMP kinase: an additional mechanism for the hypolipidemic effects of berberine

The alkaloid drug berberine (BBR) was recently described to decrease plasma cholesterol and triglycerides (TGs) in hypercholesterolemic patients by increasing expression of the hepatic low density lipoprotein receptor (LDLR). Using HepG2 human hepatoma cells, we found that BBR inhibits cholesterol and TG synthesis in a similar manner to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Significant increases in AMPK phosphorylation and AMPK activity were observed when the cells were incubated with BBR. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK, correlated with a subsequent increase in fatty acid oxidation. All of these effects were abolished by the mitogen-activated protein kinase kinase inhibitor PD98059. Treatment of hyperlipidemic hamsters with BBR decreased plasma LDL cholesterol and strongly reduced fat storage in the liver. These findings indicate that BBR, in addition to upregulating the LDLR, inhibits lipid synthesis in human hepatocytes through the activation of AMPK. These effects could account for the strong reduction of plasma TGs observed with this drug in clinical trials.     

Aurora-A kinase Ser349 phosphorylation is required during Xenopus laevis oocyte maturation

Xenopus laevis Aurora-A is phosphorylated in vivo onto three amino acids: Ser53, Thr295 and Ser349. The activation of the kinase depends on its autophosphorylation on Thr295 within the T-loop. The phosphorylation of Ser53 by still unknown kinase(s) prevents its degradation. The present work focused on the regulation of Aurora-A function via Ser349 phosphorylation. Mutagenesis of Ser349 to alanine (S349A) had few impact in vitro on the capability of the kinase to autophosphorylate as well as on its activity. These data in addition to in gel kinase assays and site-specific proteolytic digestion experiments prove that Ser349 is clearly neither a primary autophosphorylation site, nor an autophosphorylation site depending on the priming phosphorylation of Thr295. Using specific antibodies, we also show that the phosphorylation of Aurora-A Ser349 is a physiological event during Xenopus oocyte maturation triggered by progesterone. A peak of phosphorylation paralleled the decrease of Aurora activity observed between meiosis I and II. In response to progesterone, X. laevis stage VI oocytes microinjected with the Aurora-A S349A mutant proceeded normally to germinal vesicle breakdown (GVBD), but degenerated rapidly soon after. Since phosphorylation of Ser349 is responsible for a decrease in kinase activity, our results suggest that a down-regulation of Aurora-A activity involving Ser349 phosphorylation is required in the process of maturation.     

Proliferative effect of androst-4-ene-3,17-dione and its metabolites in the androgen-sensitive LNCaP cell line

As a therapeutic approach for the treatment of androgen-sensitive diseases, it would be tempting to lower the level of the potent androgens testosterone (T) and dihydrotestosterone (DHT) by using inhibitors of type 3 and type 5 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). However, the efficiency of such a strategy will be optimal only if androst-4-ene-3,17-dione (Delta4-dione), the precursor of T, does not possess per se agonist activity on the androgen receptor (AR). To determine if the proliferative effect previously observed on AR(+) cells for Delta4-dione originates from its direct (per se) action on AR or from its transformation into a metabolite, we started a series of experimentations using the human prostate cancer LNCaP cell line, which expresses a highly sensitive AR. By real-time RT-PCR analysis, we detected type 1 5alpha-reductase (5alpha-R), a small amount of type 5 17beta-HSD, but not type 2 5alpha-R nor type 3 17beta-HSD. We then studied the transformation of labeled Delta4-dione in LNCaP cells after 1-7 days and the most important metabolite detected was 5alpha-androstane-3,17-dione (A-dione), which is the product of 5alpha-R activity. We measured only low levels of androsterone (ADT) and epi-ADT. This result was next confirmed by using an inhibitor of 5alpha-R that completely inhibited the transformation of Delta4-dione into A-dione, and consequently into ADT and epi-ADT. The proliferative effect of Delta4-dione (carefully purified) on LNCaP (AR(+)) cells was next determined in presence or absence of the 5alpha-R inhibitor. Although the cells proliferate in the presence of Delta4-dione only, no cell proliferation was observed with a combination of Delta4-dione and 5alpha-R inhibitor, suggesting that Delta4-dione is not androgenic per se. We next determined that A-dione and epi-ADT stimulated cell growth with the same pattern and potency as Delta4-dione, whereas ADT had a 3.5-fold lower proliferative activity. In conclusion, Delta4-dione is not in itself an agonist steroid on LNCaP (AR(+)) cells, and its proliferative activity appears to be mediated by its transformation into A-dione and/or into epi-ADT.     

Antler mineral composition of Iberian red deer <Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis> is related to mineral profile of diet

Previous studies have suggested that antlers are costly bone structures whose mineral composition may change depending on physiological and other factors. This study examined whether nutrition variation associated with deer management influences antler mineral composition and structural characteristics of whole antler. Mineral distribution and bone structure were examined in antlers from two groups of adult Iberian red deer Cervus elaphus hispanicus Hilzheimer, 1909. They were kept under different feeding regimes at an experimental deer farm and a game estate in southeastern Spain. Protein and mineral contents differed between the diet of captive deer and that of deer in the wild. Significant differences were found for Na, Mg, K and protein. Antler composition seems to reflect the diet, as antlers of deer differed in protein, Na, Mg and K, but not in total mineral content, Ca, Fe or Zn. Thus, management conditions related to nutrition are reflected on antler composition.     

Programming an in vitro DNA oscillator using a molecular networking strategy

Living organisms perform and control complex behaviours by using webs of chemical reactions organized in precise networks. This powerful system concept, which is at the very core of biology, has recently become a new foundation for bioengineering. Remarkably, however, it is still extremely difficult to rationally create such network architectures in artificial, non‐living and well‐controlled settings. We introduce here a method for such a purpose, on the basis of standard DNA biochemistry. This approach is demonstrated by assembling de novo an efficient chemical oscillator: we encode the wiring of the corresponding network in the sequence of small DNA templates and obtain the predicted dynamics. Our results show that the rational cascading of standard elements opens the possibility to implement complex behaviours in vitro. Because of the simple and well‐controlled environment, the corresponding chemical network is easily amenable to quantitative mathematical analysis. These synthetic systems may thus accelerate our understanding of the underlying principles of biological dynamic modules.     

IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology

All osteogenic cells (osteoclasts, osteoblasts) contribute individually to bone remodeling. Their cellular interactions control their cellular activities and the bone remodeling intensity. These interactions can be established either through a cell-cell contact, involving molecules of the integrin family, or by the release of many polypeptidic factors and/or their soluble receptor chains. These factors can act directly on osteogenic cells and their precursors to control differentiation, formation and functions (matrix formation, mineralization, resorption...). Here, we present the involvement of three groups of cytokines which seem to be of particular importance in bone physiology: interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) (TNF-alpha)/IL-1, and the more recently known triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL). The interactions between these three groups are presented within the framework of bone resorption pathophysiology such as tumor associated osteolysis. The central role of the OPG/RANK/RANKL triad is pointed out.     

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O₂ ^•-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O₂ ^•- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O₂ ^•- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O₂ ^•- than those from controls. Nifedipine treatment considerably decreased O₂ ^•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O₂ ^•- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O₂ ^•- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.     

Magnitude and kinetics of rehydration influence the viability of dehydrated E. coli K-12

The influence of rehydration conditions on the recovery of Escherichia coli K-12 was studied. The results showed that the osmotic pressure gradient of rehydration shock realized before plating greatly affected cell viability. When rehydration occurred quickly from an hyperosmotic level of 133 MPa in glycerol solution before slow rehydration by plating on an agar surface to reach initial osmotic pressure (1.4 MPa), bacterial viability was strongly related to the intensity of the hypo-osmotic gradient used. Rehydration to 107 MPa resulted in a survival ratio of 41%, whereas strong rehydration to 1.4 MPa resulted in only 0.7% survival. These studies also demonstrated the influence of the rehydration kinetic on cell recovery. An optimal rehydration rate of 0.136 MPa x s(-1) increased cell recovery by a factor of 40 when compared with the faster and slower rates of 131.6 MPa x s(-1) and 0.006 MPa x s(-1), respectively.     

Lipid radical generation in polar (Laternula elliptica) and temperate (Mya arenaria) bivalves

Lipid peroxidation in Laternula elliptica was assessed by detecting lipid radicals by electronic paramagnetic resonance. The values were compared with data from the temperate mud clam Mya arenaria. Lipid radical content was higher in the Antarctic bivalve than in the temperate mud clam, even within the range of its habitat temperature. The rate of generation of lipid radicals was affected by the iron content in the samples. The iron content in individual samples of digestive glands in L. elliptica ranged from 3 to 6 nmol g⁻¹ fresh weight (fwt) and in M. arenaria from 0.6 to 2.7 nmol g⁻¹ fwt. Arrhenius plots, developed from the rates obtained in the presence of 25 μM iron, showed no significant differences between the activation energy calculated for digestive glands of L. elliptica and M. arenaria. The Fe³⁺ reduction rate in L. elliptica was higher than in M. arenaria (4.7±0.9 vs. 1.8±0.4 nmol mg⁻¹ protein min⁻¹, respectively). L. elliptica had a higher content of α-tocopherol and β-carotene than M. arenaria. Our data suggest that increased lipid radical content in the membranes of cold-adapted organisms could be related to iron content.