Approximation techniques for neuromimetic calculus

Approximation Theory plays a central part in modern statistical methods, in particular in Neural Network modeling. These models are able to approximate a large amount of metric data structures in their entire range of definition or at least piecewise. We survey most of the known results for networks of neurone-like units. The connections to classical statistical ideas such as ordinary least squares (LS) are emphasized.     

IGF-II is up-regulated and myofibres are hypertrophied in regenerating soleus of mice lacking FGF6

Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage. However, the use of FGF6(-/-) mutant mice gave contradictory results and the role of FGF6 during myogenesis remains largely unclear. Using FGF6(-/-) mice, we first analysed the morphology of the regenerated soleus following cardiotoxin injection and showed hypertrophied myofibres in soleus of the mutant mice as compared to wild-type mice. Secondly, to examine the function of the IGF family in the hypertrophy process, we used semiquantitative and real-time RT-PCR assays and Western blots to monitor the expression of the insulin-like growth factors (IGF-I and IGF-II), their receptors [type I IGF receptor (IGF1R) and IGF-II receptor (IGF2R)], and of a binding protein IGFBP-5 in regenerating soleus muscles of FGF6(-/-) knockout mice vs. wild-type mice. In the mutant, both IGF-II and IGF2R, but not IGF-I and IGF1R, were strongly up-regulated, whereas IGFBP5 was down-regulated, strongly suggesting that, in the absence of FGF6, the mechanisms leading to myofibre hypertrophy were mediated specifically by an IGF-II/IGF2R signalling pathway distinct from the classic mechanism involving IGF-I and IGF1R previously described for skeletal muscle hypertrophy. The potential regulating role of IGFBP5 on IGF-II expression is also discussed. This report shows for the first time a specific role for FGF6 in the regulation of myofibre size during a process of in vivo myogenesis.     

Heterodimerization of endothelin-converting enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.     

Cytotoxic effector cells with antitumor activity can be amplified ex vivo from biopsies or blood of patients with renal cell carcinoma for cell therapy use

Adoptive immunotherapy with antitumor effector cells is an attractive therapeutic approach in metastatic renal cell carcinoma (RCC). The aim of the work was to enhance in vitro activation of lymphocytes with optimal cytotoxic activity against tumor cells. We evaluated a procedure based on the use of dendritic cells (DCs) loaded with irradiated tumor cells (DC-Tu) to stimulate lymphocytes. Experimental conditions were established with cells from healthy donors and melanoma cell lines. Procedures were then applied to cells from RCC patients. A total of 30 tumor biopsies, 14 proximal lymph nodes, and 17 peripheral blood samples from 30 patients were used. When lymphocytes were stimulated in vitro with DC-Tu, they responded to tumor cells with an increased cytolytic activity for all the assays with donor cells (n=18). For RCC patients, DC-Tu stimulation improved the final cytotoxic activity in only half of the assays (16/31). When significantly enhanced (>10%, n=8), responder cells resulted in a final 43% cytotoxicity against autologous RCC cells. Mechanism of lysis was at least in part class I mediated. Effector cells have no lytic activity against normal renal cells. Percentage of cells with regulatory T-cell phenotype was not found to be enhanced in the DC-Tu stimulated lymphocytes. Individual differences were observed in the characteristics of DCs generated from RCC patients in contrast to that observed in donors and could explain why lymphocyte stimulation was not improved by DC-Tu in half of the RCC assays. T-cell spreading was suitable for a therapeutic use (>10⁹ cells) irrespective of the procedure (with or without DC-Tu stimulation) or the tissular origin of lymphocytes from patients. Data show that precursors of selective antitumor effector cells are present in patients with RCC and can be amplified in vitro either with or without DC-Tu stimulation. One of these populations could be chosen for an adoptive transfer immunotherapy.This work was supported by grants from the Comité Grand Ouest de La Ligue Contre le Cancer and from the Faculté de Médecine de Rennes.     

Heritable transgene expression pattern imposed onto maize ubiquitin promoter by maize <Emphasis Type="Italic">adh-1</Emphasis> matrix attachment regions: tissue and developmental specificity in maize transgenic plants

Matrix attachment regions (MARs) have been used to enhance transgene expression and to reduce transgene expression instability in various organisms. In plants, contradictory data question the role of MAR sequences. To assess the use of MAR sequences in maize, we have used two well-characterized MARs from the maize adh-1 region. The MARs have been cloned either 5 to or at both sides of a reporter gene expression cassette to reconstitute a MAR-based domain. Histochemical staining revealed a new transgene expression pattern in roots of regenerated plants and their progeny. Furthermore, MARs systematically induced variegation. We show here that maize adh-1 MARs are able to modify transgene expression patterns as a heritable trait, giving a new and complementary outcome following use of MARs in genetic transformation.Abbreviations adh-1 Alcohol dehydrogenase 1 - GUS -Glucuronidase - HSC80 Heat shock cognate 80 gene - MAR Matrix attachment regions - Rsyn-7 Root specific synthetic promoter     

S-Ribosylhomocysteinase (LuxS) is a mononuclear iron protein

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce L-homocysteine and 4,5-dihydroxy-2,3-pentanedione (DHPD). This is a key step in the biosynthetic pathway of the type II autoinducer (AI-2) in both Gram-positive and Gram-negative bacteria. Previous studies demonstrated that LuxS contains a divalent metal cofactor, which has been proposed to be a Zn(2+) ion. To gain insight into the catalytic mechanism of this unusual reaction and the function of the metal cofactor, we developed an efficient expression and purification system to produce LuxS enriched in either Fe(2+), Co(2+), or Zn(2+). Comparison of the catalytic properties and stability of the metal-substituted LuxS with those of the native enzyme revealed that the native metal ion is Fe(2+). The electronic absorption spectrum of the Co(II)-substituted LuxS underwent dramatic catalysis-dependent changes, suggesting the direct involvement of the metal ion in catalysis. Site-directed mutagenesis studies showed that Glu-57 and Cys-84 are essential for catalysis, most likely acting as general acids/bases. Reaction in D(2)O resulted in the incorporation of deuterium at the C-1, C-2, and C-5 positions of the diketone product. These data suggest a catalytic mechanism in which the metal ion catalyzes an intramolecular redox reaction, shifting the carbonyl group from the C-1 position to the C-3 position of the ribose. Subsequent beta-elimination at the C-4 and C-5 positions releases homocysteine as a free thiol.     

Specific activation of the acetylcholine receptor subunit genes by MyoD family proteins

Whether the myogenic regulatory factors (MRFs) of the MyoD family can discriminate among the muscle gene targets for the proper and reproducible formation of skeletal muscle is a recurrent question. We have previously shown that, in Xenopus laevis, myogenin specifically transactivated muscle structural genes in vivo. In the present study, we used the Xenopus model to examine the role of XMyoD, XMyf5, and XMRF4 for the transactivation of the (nicotinic acetylcholine receptor) nAChR genes in vivo. During early Xenopus development, the expression patterns of nAChR subunit genes proved to be correlated with the expression patterns of the MRFs. We show that XMyf5 specifically induced the expression of the delta-subunit gene in cap animal assays and in endoderm cells of Xenopus embryos but was unable to activate the expression of the gamma-subunit gene. In embryos, overexpression of a dominant-negative XMyf5 variant led to the repression of delta-but not gamma-subunit gene expression. Conversely, XMyoD and XMRF4 activated gamma-subunit gene expression but were unable to activate delta-subunit gene expression. Finally, all MRFs induced expression of the alpha-subunit gene. These findings strengthen the concept that one MRF can specifically control a subset of muscle genes that cannot be activated by the other MRFs.     

A suppressive effect of the adenovirus 5 protein E1B 55K on apoptosis induced by IL-3 deprivation and gamma-irradiation

The murine IL-3-dependent myeloid cell line 32D undergoes a rapid death when deprived of interleukin-3 (IL-3), a process that is suppressed or delayed by the constitutive expression of Bcl-2 or the Bcl-2-related Bcl-xL survival protein. The adenovirus type 5 E1B region encodes an E1B 55K protein, that has been reported to bind and inactivate the p53 protein that plays an important role in the induction of apoptosis. In order to explore the potential effect of the E1B 55K protein on IL-3 deprival-induced cell death, we have established 32D cell lines overexpressing the adenovirus E1B 55K protein and compared its ability to modulate the cell death with that of the human Bcl-2 protein. We observed that E1B 55K, as Bcl-2, delays the cell death caused by either IL-3-deprivation or DNA damage induced by gamma-irradiation. Cell-cycle analysis after IL-3 deprivation indicated that surviving Bcl-2 transfectants accumulate predominantly in the G0/G1 phase of the cell cycle, while E1B 55K transfectants survive in both G0/G1 and the S and G2/M phases of the cell cycle. zVAD-fmk, a broad caspase inhibitor, prevented chromatin condensation and fragmentation, but not cell death, suggesting that IL-3 deprivation induces a cell death program in which the caspases are dispensable. In contrast, both E1B 55K and Bcl-2 allowed cell survival and prevented the typical features of programmed cell death, such as phosphatidyl-serine exposure, loss of mitochondrial membrane potential, and chromatin condensation and fragmentation. Our findings indicate that the adenovirus 5 E1B 55K protein has the capability to act as a survival factor, and suggest that E1B 55K exerts its effect upstream of the activation of effector caspases, by preventing the loss of mitochondrial membrane potential induced by IL-3 deprivation.