Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements

The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.     

Comparison of the archaeal community in the fermentative compartment and faeces of the cow and the rabbit

The archaeal community in the fermentative compartment and faeces of the cow and the rabbit were compared by analysis capillary electrophoresis single-stranded conformation polymorphism (CE-SSCP) profiles of 16S rRNA genes. Ruminal and faecal contents were sampled in five cows for three weeks. Hard and soft faeces were collected in 14 rabbits for three consecutive weeks and cæcal contents were sampled in the third week. The archaeal community differed according to the host species (ANOSIM-R = 0.53 and 0.72 respectively for the comparison of the fermentative compartments and faeces; P < 0.001) and to the location within the digestive tract of both species (ANOSIM-R = 0.37, 0.52 respectively for the cow and the rabbit; P < 0.001). In both species, the archaeal community of the digestive tract was stable over weeks and varied very little between individual animals. The structure (NS) and the richness index (9.9 ± 2.7, 10.1 ± 3.1 respectively, NS) of the archaeal community were similar for the cæcal content and the soft faeces which permitted to use the latter as a representative indicator.     

A transesterification reaction is implicated in the covalent binding of benzo[b]acronycine anticancer agents with DNA and glutathion

The benzo[b]acronycine derivative S23906-1 has been recently identified as a promising antitumor agent, showing remarkable in vivo activities against a panel of solid tumors. The anticancer activity is attributed to the capacity of the drug to alkylate DNA, selectively at the exocyclic 2-amino group of guanine residues. Hydrolysis of the C-1 and C-2 acetate groups of S23906-1 provides the diol compound S28907-1 which is inactive whereas the intermediate C-2 monoacetate derivative S28687-1 is both highly reactive toward DNA and cytotoxic. The reactivity of this later compound S28687-1 toward two bionucleophiles, DNA and the tripeptide glutathion, has been investigated by mass spectrometry to identify the nature of the (type II) covalent adducts characterized by the loss of the acetate group at position 2. On the basis of NMR and molecular modeling analyses, the reaction mechanism is explained by a transesterification process where the acetate leaving group is transferred from position C-2 to C-1. Altogether, the study validates the reaction scheme of benzo[b]acronycine derivative with its target.     

Piezoelectric poly(vinylidene fluoride) microstructure and poling state in active tissue engineering

Tissue engineering strategies rely on suitable membranes and scaffolds, providing the necessary physicochemical stimuli to specific cells. This review summarizes the main results on piezoelectric polymers, in particular poly(vinylidene fluoride), for muscle and bone cell culture. Further, the relevance of polymer microstructure and surface charge on cell response is demonstrated. Together with the necessary biochemical cues, the proper design of piezoelectric polymers can open the way to novel and more reliable tissue engineering strategies for cells in which electromechanical stimuli are present in their environment.     

Enhancement of the Influenza A Hemagglutinin (HA)-Mediated Cell-Cell Fusion and Virus Entry by the Viral Neuraminidase (NA)

Background

The major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutin (HA) protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity.

Methodology/Principal Findings

The role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogeneous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA.

Conclusion/Significance

The NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion.     

The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair. Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood. From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components. Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies. In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides. Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.     

The possible role of hydroxylation in the detoxification of atrazine in mature vetiver (Chrysopogon zizanioides Nash) grown in hydroponics

The resistance mechanism of vetiver (Chrysopogon zizanioides) to atrazine was investigated to evaluate its potential for phytoremediation of environment contaminated with the herbicide. Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones, conjugation catalyzed by glutathione-S-transferases and dealkylation probably mediated by cytochromes P450. All three possibilities were explored in mature vetiver grown in hydroponics during this research project. Here we report on the chemical role of benzoxazinones in the transformation of atrazine. Fresh vetiver roots and leaves were cut to extract and study their content in benzoxazinones known to hydroxylate atrazine, such as 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA), 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and their mono- and di-glucosylated forms. Identification of benzoxazinones was performed by thin layer chromatography (TLC) and comparison of retention factors (Rf) and UV spectra with standards: although some products exhibited the same Rf as standards, UV spectra were different. Furthermore, in vitro hydroxylation of atrazine could not be detected in the presence of vetiver extracts. Finally, vetiver organs exposed to [14C]-atrazine did not produce any significant amount of hydroxylated products, such as hydroxyatrazine (HATR), hydroxy-deethylatrazine (HDEA), and hydroxy-deisopropylatrazine (HDIA). Altogether, these metabolic features suggest that hydroxylation was not a major metabolic pathway of atrazine in vetiver.     

Analysis of TwoStaphylococcus epidermidisPlasmids Coding for Resistance to Streptogramin A

The twoStaphylococcus epidermidisplasmids pIP1629 (7.5 kb) and pIP1630 (14.4 kb) contain thevgagene conferring resistance to streptogramin A. All the sequences of pIP1629, except two of the four 22-nt iterons preceding the replication gene, were found in pIP1630. The additional 6.9-kb fragment of pIP1630 is similar to the mobilizableS. epidermidisplasmid pSK639, carrying thedfrA-thyE-orf140operon and thought to replicate by an iteron controlled theta-type replication mechanism. The replication-mobilization elements of pIP1629 and pSK639 are very similar despite having been isolated in France and in Australia, respectively, showing that they are geographically widely dispersed inS. epidermidis.The genethyEencoding thymidylate synthetase carried by pSK639 is not present in pIP1630. pIP1630 probably arose by the recombination of two homologous plasmids carrying distinct resistance determinants.