A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias

Allele-specific epigenetic modifications are crucial for several important biological functions, including genomic imprinting and X-inactivation in mammals. Consequently, an ever increasing number of investigations requires accurate quantification of the relative abundance of parental alleles of a specific sequence in a DNA sample. Here, combining the use of polymorphic restriction sites with real-time polymerase chain reaction (PCR) amplification, we describe a simple and quantitative assay to measure allele ratios. The efficiency of the assay was assessed on genomic DNA for several polymorphic restriction sites located in the mouse Igf2/H19 imprinted locus. The assay was also successfully applied to quantify allele ratio in cDNA samples. In addition, we provide an experimental procedure for detection and correction of potential PCR amplification bias which significantly extends the range of application of the assay.     

Crystal structure of a NO-forming nitrite reductase mutant: an analog of a transition state in enzymatic reaction

I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes. The mutant has no enzyme activity. Its crystal structure determined at 1.65A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the N epsilon atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.     

Cybip,a starfish cyclin B-binding protein,is involved in meiotic M-phase exit

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219-231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.     

Mapping the microtubule binding regions of calponin

The smooth muscle basic calponin interacts with F-actin and inhibits the actomyosin ATPase in a calmodulin or phosphorylation modulated manner. It also binds in vitro to microtubules and its acidic isoform, present in nonmuscle cells, and co-localizes with microfilaments and microtubules in cultured neurons. To assess the physiological significance and the molecular basis of the calponin-microtubule interaction, we have first studied the solution binding of recombinant acidic calponin to microtubules using quantitative cosedimentation analyses. We have also characterized, for the first time, the ability of both calponin isoforms to induce the inhibition of the microtubule-stimulated ATPase activity of the cytoskeletal, kinesin-related nonclaret dysjunctional motor protein (ncd) and the abolition of this effect by calcium calmodulin. This property makes calponin a potent inhibitor of all filament-activated motor ATPases and, therefore, a potential regulatory factor of many motor-based biological events. By combining the enzymatic measurements of the ncd-microtubules system with various in vitro binding assays employing proteolytic, recombinant and synthetic fragments of basic calponin, we further unambiguously identified the interaction of microtubules at two distinct calponin sites. One is inhibitory and resides in the segment 145-182, which also binds F-actin and calmodulin. The other one is noninhibitory, specific for microtubules, and is located on the COOH-terminal repeat-containing region 183-292. Finally, quantitative fluorescence studies of the binding of basic calponin to the skeletal pyrenyl F-actin in the presence of microtubules did not reveal a noticeable competition between the two sets of filaments for calponin. This result implies that calponin undergoes a concomitant binding to both F-actin and microtubules by interaction at the former site with actin and at the second site with microtubules. Thus, in the living cells, calponin could potentially behave as a cross-linking protein between the two major cytoskeletal filaments.     

Injection of FGF6 accelerates regeneration of the soleus muscle in adult mice

FGF6, a member of the fibroblast growth factor (FGF) family, accumulated almost exclusively in the myogenic lineage, supporting the finding that FGF6 could specifically regulate myogenesis. Using FGF6 (-/-) mutant mice, important functions in muscle regeneration have been proposed for FGF6 but remain largely controversial. Here, we examined the effect of a single injection of recombinant FGF6 (rhFGF6) on the regeneration of mouse soleus subjected to cardiotoxin injection, specifically looking for molecular and morphological phenotypes. The injection of rhFGF6 has two effects. First, there is an up-regulation of cyclin D1 mRNA, accounting for the regulating role of a high FGF6 concentration on proliferation, and second, differentiation markers such as CdkIs and MHC I and Tn I increase and cellular differentiation is accelerated. We also show a down-regulation of endogenous FGF6, acceleration of FGFR1 receptor expression and deceleration of the FGFR4 receptor expression, possibly accounting for biphasic effects of exogenous FGF6 on muscle regeneration.     

The novel retinoid AHPN/CD437 induces a rapid but incomplete apoptotic response in human myeloma cells

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) appears to possess an apoptotic activity superior to classical retinoids in vitro as in vivo. Numerous studies have shown that CD437-induced apoptosis is independent of its nuclear receptor activity, suggesting that CD437 might have a unique mechanism of action. The purpose of this study was to compare CD437- and all-trans retinoic acid (atRA)-induced cell death. CD437 provoked a rapid apoptotic phenotype immediately followed by secondary necrosis in RPMI 8226, U266 and L363 human myeloma cell lines. Nuclear apoptotic features were observed upon both CD437 and atRA treatments. In contrast, membrane blebbing and the subsequent formation of apoptotic bodies, a classical apoptotic event, was only observed upon atRA treatment. In addition, CD437, contrary to atRA, was unable to induce tissue transglutaminase (tTG), an intracellular enzyme involved in the formation of cross-linked protein polymers contributing to apoptotic morphological changes. Taken together, these data suggest that CD437 induces rapid but incomplete apoptotic phenotype in human myeloma cells.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

The fate of the prion protein in the prion/plasminogen complex

The cellular prion protein (PrP(c)) forms complexes with plasminogen. Here, we show that the PrP(c) in this complex is cleaved to yield fragments of PrP(c). The cleavage is accelerated by plasmin but does not appear to be dependent on it.     

Prolonged culture in low glucose induces apoptosis of rat pancreatic beta-cells through induction of c-myc

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