P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.     

The peptide nucleic acid targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma lacks immunogenicity: follow-up characterization of PNAEmu-NLS

The present study aims to evaluate the antigenicity of a PNA complementary to the Emu sequence (PNAEmu) with cancer therapeutic potential properties in Burkitt's lymphoma (BL). In BL cells, the c-myc oncogene is repositioned next to the Emu enhancer of the immunoglobulin (Ig) locus, due to chromosomal translocation, and up-regulated. PNAEmu linked to a nuclear localization signal peptide was shown specifically to block c-myc hyperexpression by inhibiting cell growth in vitro and in vivo. Recently, we reported that the administration of PNAEmu to mice, following inoculation with BL cells, hinders tumor growth without toxic effects. To investigate the potential use of PNAEmu in clinical applications further, we tested its antigenicity. Mice were inoculated with an emulsion of free PNA or PNA crosslinked to the immunogenic carrier keyhole limpet hemocyanin (KLH) with Freund's adjuvant. Antibodies to free PNA were undetected, whereas both IgG and IgM antibodies to PNA-KLH were detected in mouse serum 28 and 38 days after inoculation.     

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPS, indicated that only the AT₂ subtype was expressed.3. AT₂ receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT₂ receptors being expressed earlier than ACE. In contrast, high expression of AT₂ receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT₂ receptors to cell growth and cell–cell contact.     

Interactions of protein kinase CK2 subunits

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2 that has Asp 156 mutated to Ala (CK2A156) is able to bind the CK2 subunit and to compete effectively in this binding with wild-type subunits and . The interaction between CK2A156 and CK2 was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2A156 coprecipitated the subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2A156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2A156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2A156 affects the endogenous activity of CK2. Transfection experiments with CK2 and subunits and CK2A156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2 indicated that CK2 is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.     

An architectural hypothesis for direction selectivity in the visual cortex: the role of spatially asymmetric intracortical inhibition

Within a linear field approach, an architectural model for simple cell direction selectivity in the visual cortex is proposed. The origin of direction selectivity is related to recurrent intracortical interactions with a spatially asymmetric character along the axis of stimulus motion. No explicit asymmetric temporal mechanisms are introduced or adopted. The analytical investigation of network behavior, carried out under the assumption of a linear superposition of geniculate and intracortical contributions, shows that motion sensitivity of the resulting receptive fields emerges as a dynamic property of the cortical network without any feed-forward direction selectivity bias. A detailed analysis of the effects of the architectural characteristics of the cortical network on directionality and velocity-response curves was conducted by systematically varying the model's parameters. Received: 8 May 1998 / Accepted in revised form: 10 November 1998     

CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients

Purpose: Many years ago, diphtheria toxin (DT) showed antitumor activity in mice and in humans, but it was unclear whether this depended on the toxicity of the molecule only or on its strong inflammatory-immunological property as well. To deal with this open question, we planned to treat a group of cancer patients with cross-reacting material 197 (CRM197). CRM197 is a nontoxic mutant of DT that shares the immunological properties of the native molecule and its ability to bind to heparin-binding epidermal growth factor (HB-EGF), the specific cell-membrane receptor for DT that is often overexpressed in cancer. Methods: 25 outpatients with various advanced tumors who were refractory to standard therapies (23 subjects) or had refused, in whole or in part, conventional therapies (2 subjects) were treated with CRM197 injected subcutaneously in the abdominal wall, on alternate days, for 6 days. Three different dosages (1.7, 2.6, or 3.5 mg/day) were used according to the patients degree of immunological reactivity to DT/CRM197 (none, moderate, or high). Results: After the first administration of CRM197, a significant increase in the number of circulating neutrophils and in the serum level of TNF- was detected. Toxicities were minimal. Only patients with delayed-type hypersensitivity to DT/CRM197 had irritating skin reactions in the injection sites and a flu-like syndrome with fever. Pharmacokinetics showed a mean peak concentration (12.7 ng/ml) 12 h after the first injection and a mean half-life of 18.1 h. There were two complete and one partial responses (metastatic breast carcinoma, neuroblastoma, and metastatic breast carcinoma) lasting 4, 45+, and 15 months, respectively. Six cases of stable disease, lasting from 1 to 15 months, were also recorded. Conclusions: CRM197 injected subcutaneously elicited an inflammatory-immunological reaction, caused tolerable toxicities, was absorbed to a good extent into the circulatory system, and exerted some degree of biological antitumor activity. A possible role of neutrophils and TNF- in the mode of action of the molecule is hypothesized.     

6-aryl-2,4-dioxo-5-hexenoic acids, novel integrase inhibitors active against HIV-1 multiplication in cell-based assays

A series of 6-aryl-2,4-dioxo-5-hexenoic acids, were synthesized and tested against HIV-1 in cell-based assays and against recombinant HIV-1 integrase (rIN) in enzyme assays. Compound 8a showed potent antiretroviral activity (EC(50)=1.5 microM) and significant inhibition against rIN (strand transfer: IC(50)=7.9 microM; 3'-processing: IC(50)=7.0 microM). A preliminary molecular modeling study was carried out to compare the spatial conformation of 8a with those of L-731988 (4) and 5CITEP (7) in the IN core.     

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.     

Liquid chromatographic assay for riluzole in mouse plasma and central nervous system tissues

An isocratic, reversed-phase high-performance liquid chromatographic procedure (HPLC) was developed for determination of the neuroprotective agent riluzole in mice plasma, brain and spinal cord. The procedure is based on isolation of the compound and the internal standard from plasma and central nervous system tissues using a Bakerbond spe C8 cartridge, with satisfactory recovery and specificity. Separation was on a C18 column, coupled with an UV detector at 263 nm. The assay was linear over a wide range, with a lower limit of quantification of 100 ng ml(-1) or g(-1) using 0.1 ml of plasma and about 100mg of brain tissue. The precision and accuracy were within the acceptable limits for an HPLC assay. The method is currently used to support pharmacological studies of the activity of riluzole when given in combination with other potential neuroprotective agents in an animal model of familiar amyotrophic lateral sclerosis (SOD1-G93A transgenic mice).     

Mutagenesis of human DNA polymerase lambda: essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities

DNA polymerase (pol) λ is homologous to pol β and has intrinsic polymerase and terminal transferase activities. However, nothing is known about the amino acid residues involved in these activites. In order to precisely define the nucleotide-binding site of human pol λ, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol β, which are involved in nucleotide binding. Our analysis demonstrated that pol λ Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol λ, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.