FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

The secE gene of Helicobacter pylori

Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori.     

A germline-specific gap junction protein required for survival of differentiating early germ cells

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.     

Genome-wide analysis of mRNAs targeted to yeast mitochondria

It is agreed that nuclear-encoded mitochondrial proteins are post-translationally targeted to mitochondria, even if, in some cases, a co-translational phase can assist the import of precursor proteins. We used yeast DNA microarrays to analyse the mRNA populations associated with free and mitochondrion-bound polysomes. As expected, many mRNAs, known to encode mitochondrial proteins, are localized to free cytoplasmic polysomes, but many are localized to mitochondrion-bound polysomes. Furthermore, the 3′-UTR of six randomly chosen mitochondrion-bound mRNAs contains sufficient information to target, in vivo, non-translatable RNA to the vicinity of mitochondria. Interestingly, genes producing mRNAs that are targeted to mitochondria are mainly of ancient bacterial origin, whereas those producing mRNAs that are translated in the cytoplasm are mainly of eukaryotic origin. These observations, which support the recent hypotheses concerning the dual origin of the mitochondrial proteome, provide new insights into the biogenesis of mitochondria.     

DNA immunisation. New histochemical and morphometric data

Splenic germinal center reactions were measured during primary response to a plasmidic DNA intramuscular injection. Cardiotoxin-pretreated Balb/c mice were immunized with DNA plasmids encodmg or not the SAG1 protein, a membrane antigen of Toxoplasma gondii. Specific anti-SAG1 antibodies were detected on days 16 and 36 after injection of coding plasmids. The results of ELISAs showed that the SAG1-specific antibodies are of the IgG2a class. Morphometric analyses were done on serial immunostained cryosections of spleen and draining or non-draining lymph nodes. This new approach made it possible to evaluate the chronological changes induced by DNA immunisation in the germinal centres (in number and in size). Significant increases in the number of germinal centres were measured in the spleen and only in draining lymph nodes after plasmid injection, the measured changes of the germinal centers appeared to result from the adjuvant stimulatory effect of the plasmidic DNA since both the coding and the noncoding plasmid DNA induced them. No measurable changes were recorded in the T-dependent zone of lymph organs.     

2-alkyl-3-Alkylamino-2H-Benzo- and pyridothiadiazine 1,1-dioxides: from K+ATP channel openers to Ca++ channel blockers?

A series of 2-alkyl-3-alkylamino-2H-benzo- and 2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides, structurally related to BPDZ 44 and BPDZ 73, two potent pancreatic B-cells K+ATP channel openers, were synthesized and tested on rat pancreatic islets (endocrine tissue) as well as on rat aorta rings (vascular smooth muscle tissue). Alkylation of the 2-position led to double bond tautomerization and formation of compounds with a 2H-conformation. In contrast to the previously described pyridothiadiazine dioxides, such as BPDZ 44, and 7-chlorobenzothiadiazine dioxides, such as BPDZ 73, the 2-alkyl-substituted analogs were found to be poorly active on the insulin releasing process although most drugs exhibited a vasorelaxant activity. As a result, the new 2-alkyl-substituted pyridinic compounds expressed a selectivity profile (vascular smooth muscle tissue vs pancreatic tissue) opposite to that of their non-alkyl-substituted counterparts, i.e. BPDZ 44. Additional investigations revealed that, in contrast to their non 2-alkyl-substituted analogs, the most interesting 2-methyl-substituted derivatives did not express the pharmacological profile of classical K+ATP channel openers. The pharmacological results rather suggest that alkylation of the 2-position of the thiadiazine ring led to drugs that could act as Ca2+ channel blockers rather than as potassium channel openers.     

Peptide fragments released from Phe-caseinomacropeptide in vivo in the rat

The aim of this study was to investigate the pharmacokinetics of bovine Phe-caseinomacropeptide (Phe-CMP) in the rat after oral administration. This polypeptide was monophosphorylated and mainly nonglycosylated: Phe-CMP-1P. During gastrointestinal digestion and absorption, Phe-CMP-1P was degraded. Intact Phe-CMP-1P and CMP-1P were rapidly released from the stomach. In contrast, partial hydrolysis by pancreatic enzymes was observed. In vitro hydrolysis by brush-border membrane vesicles also indicated that the peptide was degraded. In the blood, "CMP-immunoreactive material" appeared rapidly, reaching a maximum level of 5.5 microg/ml at 60 min.     

From autopoiesis to neurophenomenology: Francisco Varela's exploration of the biophysics of being

This paper reviews in detail Francisco Varela's work on subjectivity and consciousness in the biological sciences. His original approach to this "hard problem" presents a subjectivity that is radically intertwined with its biological and physical roots. It must be understood within the framework of his theory of a concrete, embodied dynamics, grounded in his general theory of autonomous systems. Through concepts and paradigms such as biological autonomy, embodiment and neurophenomenology, the article explores the multiple levels of circular causality assumed by Varela to play a fundamental role in the emergence of human experience. The concept of biological autonomy provides the necessary and sufficient conditions for characterizing biological life and identity as an emergent and circular self-producing process. Embodiment provides a systemic and dynamical framework for understanding how a cognitive self--a mind--can arise in an organism in the midst of its operational cycles of internal regulation and ongoing sensorimotor coupling. Global subjective properties can emerge at different levels from the interactions of components and can reciprocally constrain local processes through an ongoing, recursive morphodynamics. Neurophenomenology is a supplementary step in the study of consciousness. Through a rigorous method, it advocates the careful examination of experience with first-person methodologies. It attempts to create heuristic mutual constraints between biophysical data and data produced by accounts of subjective experience. The aim is to explicitly ground the active and disciplined insight the subject has about his/her experience in a biophysical emergent process. Finally, we discuss Varela's essential contribution to our understanding of the generation of consciousness in the framework of what we call his "biophysics of being."