Native membranes from human erythrocytes contain the following G proteins which are ADP-ribosylated by a number of bacterial toxins: Gi alpha and Go alpha (pertussis toxin), Gs alpha (cholera toxin), ...and three proteins of 27, 26 and 22 kDa (exoenzyme C3 from Clostridium botulinum). Three additional C3 substrates (18.5, 16.5 and 14.5 kDa) appeared in conditions of unrestrained proteolysis during hemolysis. SDS-PAGE separation of erythrocyte membrane proteins followed by electroblotting and incubation of nitrocellulose sheets with radiolabeled GTP revealed consistently four GTP-binding proteins with Mr values of 27, 26, 22 and 21 kDa. Although a 22 kDa protein was immunochemically identified as ras p21, the C3 substrate of 22 kDa is a different protein probably identifiable with a rho gene product. Accordingly, at least five distinct small molecular weight guanine nucleotide-binding proteins, whose functions are so far undetermined, are present in native human erythrocyte membranes.
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.
Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With 35SGTP gamma S this is 0.15 +/- 0.2 min-1. Treatment ...of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with Kd values of 10.2 +/- 2.3 nM 35SGTP gamma S, of 18.2 +/- 4.3 nM alpha-32PGTP and of 28.6 +/- 3.5 nM alpha-32PGDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65 +/- 7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.
3619 Background: T4 is widely recognized as one of the most important and independent prognostic factors in adjuvant setting for CC patients. However, the optimal management in terms of adjuvant ...treatment in stage II (pT4N0) as well as the impact of DNA mismatch repair deficiency (dMMR) in this subset of patients remain unclear. Here, we present a large, multicenter, real-world analysis of pT4N0 CC patients. Methods: A real-world database including pts treated at 9 Italian institutions from April 2010 to November 2023 was queried. Clinico-pathological characteristics of pts diagnosed with a stage II pT4N0 CC were analyzed. Pts were stratified according to duration of treatment, DNA mismatch repair (MMR) status, type of adjuvant chemotherapy (ACT). The primary endpoints were median relapse-free survival (RFS) and overall survival (OS). Results: A total of 380 pts was included, and outcomes data were available for 288 pts.Median age was 73 years and 49% of pts were male. 47 pts (16%) had dMMR CC, among which Lynch Syndrome was diagnosed in 19%. 10% of pts had BRAF mutant tumor, and 38% KRAS mutant tumor. After a median follow-up of 40.6 months (37.9-49.3), 3-yr RFS and 5-yr RFS were 70% and 55%, respectively, regardless treatment; 3-yr OS was 88% and 5-yr OS 73%. 154 pts (53%) received ACT: oxaliplatin (oxa)-based CT was given to 103 pts (67%), compared to 51 pts who received monotherapy (33%). Six-month ACT was given to 38 pts (74.5%) and 75 pts (72.8%) in the monotherapy and oxa-based group, respectively. Pts receiving 6-month ACT had a significant longer OS compared to those who did not receive ACT (HR 0.17; 0.08-0.34; p <.001). A trend towards improved OS was observed in pts receiving 6-month ACT versus pts receiving 3-month ACT (HR 0.46; 0.16-1.29; p 0.129). Similar results were observed in terms of RFS: 6-month ACT showed a longer RFS compared to 3-month ACT (HR 0.58; 0.32-1.05; p .068), and a significantly longer RFS compared to no-ACT (HR 0.50; 0.32-0.77; p .001). Oxa-based ACT was associated with a significant improvement in OS compared to monotherapy (HR 0.30; 0.11-0.85; p .016). Overall, pts with dMMR CC did not show a statistically significant difference in terms of OS and RFS (p .66 and .19 respectively) compared to proficient MMR pts. Conclusions: In this large real-world dataset, T4 was confirmed being a key poor prognostic factor, independently to MMR status. We observed that ACT provides large benefit in this population, and oxa-based ACT may be better than monotherapy. In addition, 6-month oxa-based ACT showed a trend towards a better outcome in comparison to 3-month ACT, even if not statistically significant. Given the retrospective nature of the study, these data should be validated in larger and prospective cohorts; however, this work may help oncologists in shared decision making in this specific subgroup of patients.
