Proteolytic Remodeling of the Synaptic Cell Adhesion Molecules (CAMs) by Metzincins in Synaptic Plasticity

Cell adhesion molecules participate in the formation, maturation, function and plasticity of synaptic connections. The growing body of evidence indicates that in the regulation of the synaptic plasticity, in which these molecules play pivotal role, also the proteolytic processes are involved. This review focuses on extracellular proteolysis of the cell adhesion molecules by specific subgroup of the matrix metalloproteinases, a disintegrin and metalloproteases and a disintegrin and metalloproteinase with thrombospondin motifs, jointly referred to as metzincins, in driving coordinated synaptic structural and functional modifications underlying synaptic plasticity in the adult brain.     

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Cell cycle analysis of human peripheral blood T lymphocytes in long-term culture

We have studied the cell cycle of resting T lymphocytes from long-term (LT) cultures following stimulation with phytohemagglutinin (PHA) and recombinant Interleukin 2 (IL-2). We examined the kinetics of entry into S phase by autoradiography, the accumulation of cellular RNA by microfluorometric techniques, and ultrastructural morphology by electron microscopy. In addition, we examined the expression at the mRNA level of six cell cycle-dependent growth-regulated genes (c-fos, c-myc, KC-1, JE-3, vimentin, and histone H3). We show that T lymphocytes of LT cultures respond differently to mitogenic stimulation than the T lymphocytes of freshly isolated peripheral blood mononuclear cell cultures. At the ultrastructural, biochemical, and molecular levels, resting T lymphocytes of LT cultures can be distinguished from physiological (G0) lymphocytes of peripheral blood.     

Fibrinogen-sepharose interaction with prothrombin, prethrombin 1, prethrombin 2 and thrombin

Binding of prothrombin, prethrombin 1, prethrombin 2 and thrombin to fibrinogen-Sepharose was studied. Thrombin and prethrombin 2 bound to fibrinogen-Sepharose, while prethrombin 1 and prothrombin did not. Bound thrombin and prethrombin 2 were recovered from the column by eluting with 0.1 M NaCl/0.05 M Tris-HCl buffer (pH 7.4). The affinity of thrombin and prethrombin 2 to fibrinogen-Sepharose depended on ionic strength and reached a maximum at 50 mm concentration. Prethrombin 2 interacts with fibrinogen as well as thrombin; and prothrombin fragment 1.2 is not important in the formation of this complex. Thus, prethrombin 2, which is a precursor of thrombin without measurable enzymatic activity and which lacks the single cleavage at Arg-322-Ile-323 present in thrombin, has the same or very similar structural conformation as thrombin and has the same macromolecular substrate recognition site. These results confirm the earlier results that active center is not necessary in fibrinogen-thrombin interaction.     

Postirradiation recovery of haemopoiesis in Steel mutant mice

The recovery of haemopoiesis in Steel mutant mice following 1 Gy sublethal irradiation is described. Steel homozygotes (S1/S1) did not display the abortive phase of erythropoietic recovery while the secondary phase of erythropoietic recovery was more pronounced in S1/S1 than in control (+/+) animals. On the contrary, the neutrophilopoietic recovery in S1/S1 mice was defective only during the secondary phase of recovery. Steel heterozygotes (S1/+) manifested similar, albeit less pronounced, defects. In the course of studies of recovery of eosioniphils it was observed that neither wild-type nor mutant animals expressed the abortive rise. Moreover, the kinetics of recovery of eosinophils was essentially different from both erythropoietic and neutrophilopoietic recovery, and the preirradiation level was reached in both normal and mutant animals on day 60 postirradiation as opposed to 24 and 35 days for erythropoiesis and neutrophils respectively.     

Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.     

The use of graph theory in analysis of cells and cell nuclei in particular

Application of graph theory to morphological analysis of cells described by n parameters is presented. The analysis takes advantage of the possibility of describing structures in a multi-dimensional space. The method may be useful in determining similarity or differences between studied structures.     

Premiers stades de l'oogenèse de Rhabdophaga saliciperda (Cecidomyiidae,Diptera)

The females of Rhabdophaga saliciperda have in their somatic cells 8 chromosomes and the males 6. The type of sex determination is therefore: X₁X₁X₂X₂—♀; X₁X₂—♂. The cells of the germinal line have 46 chromosomes, but a variation of their number was observed. In the oogonia and spermatogonia the number of heterochromatic chromosomes may exceed the number of E chromosomes, i.e. 8. In the beginning of the growth stage of the oocytes an incorporation of somatic cells was observed. The nuclei of these somatic cells persist in the cytoplasm of the oocytes until the maturation divisions. The possibility of their participation in the reconstruction of the nucleus of the mature egg is envisaged. The metaphase of the I segmentation division has a complex character. During prophase of the first meiotic division the E chromosomes form 4 bunches of 6–8 chromosomes each. Some univalents may also be present. The 8 S chromosomes form 4 regular bivalents. The 4 groups of E chromosomes persist until metaphase I. During metaphase I a phenomenon of expulsion of the majority of E chromosomes from the metaphase spindle was observed. The 4 bivalents remain in the equatorial plain of the spindle with some E Chromosomes. After this expulsion 2 groups of chromosomes are formed. In connection with them 2 spindles develop. An irregular distribution of E chromosomes follows without their division. The bivalents are probably separated in regular manner. These 2 spindles correspond to the I maturation division. The II maturation division was not observed because of lack of respective stages.     

Microbial transformation of azacarbazoles X: Regioselective hydroxylation of 5,11-dimethyl-5H-indolo [2,3-b]quinoline, a novel DNA topoisomerase II inhibitor, by Rhizopus arrhizus

Summary Microbial transformation of cytotoxic 5,11-dimethyl-5H-indolo[2,3-b]quinoline (a compound displaying antitumor activity and affecting the activity of calf thymus DNA topoisomerase II) was performed by the Rhizopus arrhizus strain and yielded a 9-hydroxy derivative. The metabolite obtained displayed a stronger cytotoxity against KB cells than the parent compound (ID₅₀=0.001 mol/mL), and stimulated also the formation of calf thymus topoisomerase II mediated pSP65 DNA cleavage in vitro at the concentration of 3 M. Being analogous to 9-hydroxyellipticine (which is an antitumor alkaloid), this novel indolo[2,3-b] quinoline derivative can be regarded as a novel potential antitumor agent.     

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC ⁺ plasmid. Interestingly, while the frequency of UV-induced GC → AT transitions is greatly reduced, the frequency of AT → TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC ⁺; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.