Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A1-binding sequence.

BACKGROUND: Induction of the death pathway resulting from the specific interaction of the PP2A1 phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides. METHODS: Using biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences. RESULTS: We identified overlapping sequences, located within the N-terminal domain E4orf423-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A1 holoenzyme. We characterized DPT-E4orf44 and TAT-E4orf44, two bi-partite cell penetrating peptides containing the 12 PP2A1 binding residues of the canine type 2 E4orf427-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf44, in contrast to inactive TAT-E4orf44, adopted a well defined α-helical structure and co-precipitated PP2A1 from HeLa cell extracts. DPT-E4orf44 also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf44 or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf44 also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts. CONCLUSIONS: We characterized the transducing properties of a new α-helical DPT-E4orf44 peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein. GENERAL SIGNIFICANCE: Our results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.

[Observatory of the elderly over 80 years supported by the mobile emergency and resuscitation service]. / Observatoire du sujet âgé de plus de 80 ans pris en charge en urgence par le service mobile d'urgence et de réanimation.

Emergency medical services (EMS) received an increasing number of calls for patients aged 80 and older. The goal of the present study was to evaluate outcome and functional dependence of patients aged 80 and older who EMS managed in the prehospital theater. This prospective study was conducted over 1 year (September 2007-August 2008), all consecutive patients aged 80 and older managed by a medical team during the study period were included. Characteristics of patients, including previous health status and functional dependence, were recorded on-the scene by the attending physician. Three-month mortality was recorded, as well as ADL score. Data are expressed as mean values±standard deviations, medians and interquartile ranges (IQRs), and percentages and compared using univariate and multivariate analysis. P<0.05 was considered the threshold for significance. Five hundred twenty-three patients were included. Mean age was 86 ± 5. Median ADL index was 2 (IQR 0-9), and 63% of patients were living at home. At 3 months, the survival rate was 66% (n=273) and the proportion of patients living at home was 64% (P=0.9), the median ADL index of survivors was 2 (IQR 0-8) vs 1 (IQR 0-6) initially for this subpopulation, P=0.01. Our study confirms utility and efficacy of full access of elderly persons to advanced life support especially for self-patients and not restricted based on aging per se. The development and daily use of tools for rapid assessment of autonomy should enable practitioners to innovate and thus, adapt their management.

Carcinoembryonic gene member 2 mRNA expression as a marker to detect circulating enterocytes in the blood of colorectal cancer patients.

BACKGROUND: The aim of this study was to report our experience with a new molecular tool to detect circulating enterocytes in the blood of patients with colorectal cancer. METHODS: The study included 193 individuals: 78 patients with colorectal cancer and 115 controls composed of patients with benign colorectal diseases (n = 16), patients with noncolorectal cancer (n = 31), healthy individuals (n = 62), and healthy bone marrow transplantation donors (n = 6). A nested reverse transcriptase-polymerase chain reaction with specific primers for the carcinoembryonic gene member 2 (CGM2) was used to detect circulating enterocytes in the peripheral blood of 78 patients with colorectal cancer. The blood (n = 109) or the bone marrow (n = 6) of the 115 controls was studied to test the absence of CGM2 illegitimate transcription in nucleated blood cells and nucleated blood cell progenitors. The assay sensitivity was effective in detecting 1 CGM2-positive cell per 10(6) nucleated blood cells. RESULTS: Fifty-nine percent (46/78) of patients with colorectal cancer were found positive whereas all negative controls remained negative. Positivity rates were 38% (3/8) in Dukes' A classification, 43% (9/21) in Dukes' B, 77% (23/30) in Dukes' C, and 58% (11/19) in Dukes' D. CONCLUSIONS: The clinical significance of enterocyte detection in the blood of colorectal cancer patients by means of this CGM2 messenger RNA assay needs further evaluation.

Oxygen evolution loss and structural transitions in photosystem II induced by low intensity UV-B radiation of 280 nm wavelength.

UV-B radiation of 280 nm wavelength (UV280) and low intensity (2.0 W/m2) gives rise to an important oxygen evolution (OE) loss in photosystem II (PSII) particles isolated from the thylakoid membrane of plant chloroplasts on the one hand, and to structural changes, or transitions, in the proteins of the PSII complex on the other hand. The latter UV280 effect was studied in this work by Fourier transform infrared (FT-IR) spectroscopy. First, irradiation of the PSII particles with UV280 for about 40 min causes an almost complete loss of OE activity. The remaining OE after 15, 20, 30 and 40 min is respectively 52, 44, 27 and 12% of the OE activity in control PSH particles kept in darkness. Secondly, difference FT-IR spectra of PSII particles irradiated for 30 min, i.e., [PSII irradiated with UV280]-minus-[PSII non-irradiated], show that the UV280 light is at the origin of significant IR absorbance changes in several spectral regions: (i) amide I (1696-1620 cm(-1)) and amide II (1580-1520 cm(-1)), (ii) tyrosine side chain (1620-1580 cm(-1) and 1520-1500 cm(-1), i.e., the v8a, v8b and v19a vibrational modes, respectively), and (iii) chlorophylls (1750-1696 cm(-1)). Thirdly, comparison of the UV-B effect reported here with structural changes induced by heat-stress in PSII proteins [M. Joshi, M. Fragata, Z. Naturforsch. 54c (1999) 35-43] clearly indicates that the stability of the functional centers in the PSII complex is dependent on a dynamic equilibrium between a-helix conformers and extended chain (beta-strand) structures. In this framework, transient 'alpha-helix-to-beta-strand transitions' are susceptible of giving rise in vivo to recurrent changes in the activity of the PSII complex, and as such act as a control mechanism of the photosynthetic function in the thylakoid membrane.

System A neutral amino acid transporter regulation by interleukin-1beta in human osteoarthritic synovial cells: evidence for involvement of prostaglandin E(2) as a second messenger.

We studied the long-terms effects of interleukin-1beta (IL-1beta; 3 to 6 h) on alpha-(methylamino) isobutyric acid (MeAIB), a nonmetabolizable amino acid transported by system A. We found that IL-1beta induced a large decrease in MeAIB uptake by human osteoarthritic synovial cells and a concomitant increase in prostaglandin E(2) (PGE(2)) synthesis. Therefore, we investigated whether PGE(2) acts as a mediator for the long-term action of IL-1beta. We found that exogenous PGE(2) inhibited MeAIB uptake, and that AH6809, a PGE(2) receptor antagonist, inhibited IL-1beta-mediated MeAIB uptake. To identify the enzymes involved in the IL-1beta-mediated synthesis of PGE(2) that inhibits MeAIB uptake, we studied the expression of secreted (s) and cytosolic (c) phospholipase A(2) (PLA(2)). Because both were expressed, we selected a broad spectrum of inhibitors to determine which of the two PLA(2)s was involved. We used AACOCF3, a cPLA(2) inhibitor, and dithiothreitol (DTT) and bromophenacyl bromide (BPB), which are sPLA(2) inhibitors. Our results suggest that the PLA(2) involved in the IL-1beta-mediated synthesis of PGE(2) was sPLA(2). We also showed the expression of cyclooxygenase (COX)-2 and its partial involvement using a potent selective COX-2 inhibitor, L-745337. These findings provide insight into the mechanisms underlying the IL-1beta-mediated regulation of transport system A. The Il-1beta-induced inhibition of MeAIB uptake in human osteoarthritic synovial cells thus seems to be essentially mediated by PGE(2) production via the activation of sPLA(2) and the partial activation of COX-2.

[Evaluation and interest in new molecular markers in colon cancer]. / Evaluation et intérêt de nouveaux marqueurs moléculaires en cancérologie colique.

The presence of colonic tumor cells in the circulation may predict colorectal carcinoma recurrence and metastases. We have developed a highly sensitive nested RT-PCR assay, with primers derived from the cytokeratin 20 (CK20) and the carcinoembryonic gene CGM2, to detect occult microdisseminated enterocytes in blood of colorectal cancer patients. Among 82 healthy controls analyzed, 40.2% (33/82) have a positive expression of CK20 mRNA which is not statistically different from the 45.5% (15/33) of positive results found in colon cancer patients. This sensitive method may detect non-tissue specific constitutive low level (illegitimate) expression of CK20 mRNA in peripheral nucleated blood cells (PNBC) of a significant number of healthy control as well as in a number of normal bone marrow. The low specificity of this assay therefore hampers its value to detect blood colon cancer dissemination. In 47 patients with colorectal carcinoma, CGM2 primers detected circulating enterocytes in 25 of them (53%). In disseminated Dukes' stage C disease patients, 17 out of 29 (59%) were found positive whereas in localized adenocarcinoma (Dukes's stage A and B), CGM2 primers detected enterocytes in 44% suggesting that an hematogenous spillage of colonic cells may be a relatively early event in colon cancer. None of the patients suffering from benign colonic pathologies or from diverticulitis were found positive for this assay. The analysis of 56 healthy individuals without known colorectal cancer, of 20 non-colorectal cancer patients and of 6 normal bone marrows provide evidence that this assay is highly specific and may predict an hematogenous spread of colonic cells in patients with organ-confined disease. Nevertheless, the clinical significance of enterocyte detection and the potential applications of this molecular tool merit longer term follow-up.

Sedimentation field-flow fractionation of cellular species.

More than 15 years ago a pioneering report on the separation of cellular material using sedimentation field-flow fractionation (SdFFF) was published. Surprisingly, only a few reports on SdFFF applications to cell separations are available as yet. The major limitations which seemed to slow the development of SdFFF applications in biology appeared to be related to the development of specific instrumentation. Therefore, guidelines are given for setting up a biocompatible SdFFF apparatus. SdFFF elution of cells with different characteristics was performed to demonstrate the efficiency, completeness, and rapidity of cell separations by this method. Retention data describing the effect of lifting forces on nucleated cells are compared to those for red blood cells (RBC). The effects of flow rates, field intensity, and injection protocol were studied using normal human RBC eluted in isotonic medium as a probe for retention conditions. When possible, data were also compared to previously published reports. Guidelines for elution and separation optimization are given. Using mixtures of nucleated and living red blood cells, viability studies showed a surprisingly high recovery of each type of material.

IL-1 modifies system A transport activity in human rheumatoid synovial cells.

Given the importance of interleukin-1 in both rheumatic diseases and the modulation of cell metabolic activities, we studied the action of this cytokine on the neutral amino acid transport A system on rheumatoid synovial cells. In these cells IL-1 (1 ng/ml) induced amino transport stimulation from 5 min to 5 h. This effect was obtained only after a starvation period. No concentration-related effect was found for IL-1-stimulated MeAIB uptake, and the IL-1-mediated MeAIB uptake stimulation is independent of protein synthesis. Neosynthesis or post-translational maturation of protein transport is a prerequisite for obtaining this effect. In conclusion, rheumatoid synovial cells exhibit a higher sensitivity for IL-1 than osteoarthritic ones, probably related to their intense metabolic activity.

Action of anti-inflammatory drugs on interleukin-1 beta-mediated glucose uptake by synoviocytes.

Synovial cell cultures prepared from samples taken from osteoarthritic and rheumatoid patients were treated with different anti-inflammatory agents (cortisol, indomethacin, ibuprofen and piroxicam) to determine their 'anti-interleukin-1 beta' action, using inhibition of interleukin-1 beta-mediated glucose uptake stimulation as a biological test. Confluent cells were treated for 24 h with different concentrations of these drugs (10(-5), 10(-6) and 10(-7) mol/l) to study their effect on the inflammation process. 6 h before glucose uptake studies, interleukin-1 beta (1 ng/ml) was added. Whereas non-steroid anti-inflammatory agents were inefficient, cortisol inhibited the action of interleukin-1 beta on glucose uptake. In osteoarthritic cells, cortisol, 10(-5) mol/l, reduced interleukin-1 beta-mediated glucose uptake by 27% after a 24-h incubation. In rheumatoid cells, stimulated 2-deoxy-D-glucose uptake was reduced by 40.6%. Results were similar when interleukin-1 beta and cortisol were added simultaneously, 6 h before glucose uptake was measured. This rapid effect of cortisol was protein synthesis-dependent (inhibited by cycloheximide). Cortisol decreased glucose uptake by synoviocytes by acting on basal and interleukin-1 beta-mediated glucose uptake. This effect was more pronounced in rheumatoid synovial cells. The inhibition of interleukin-1 beta-mediated glucose uptake could be proposed as a new model for studying the anti-interleukin-1 beta effects of anti-rheumatic drugs.

Dependence of adaptative regulation for IL-1 beta action on system A activity in human synovial cells.

Human synovial cells are a suitable model for estimating the physiopathological effects of IL-1 beta (IL-1) in joint. Given the importance of this cytokine in the modulation of cell metabolic activities, we set out to study the action of IL-1 on the neutral amino acid transport A system, using the methyl (aminoisobutyric) acid (MeAIB), the most highly specific and nonmetabolizable substrate for the A system. Stimulation of system A activity by adaptative regulation is a prerequisite to obtain an increase of MeAIB uptake in IL-1-treated cells, since cells which had been grown in a normal medium did not express stimulation of system A activity when IL-1 was added. The IL-1-mediated MeAIB uptake is independent of protein synthesis, since cycloheximide (CHX) did not inhibit MeAIB uptake, and characterized by a decrease in the Michaelis constant K(m) (0.147 vs. 0.270 mmol/l, IL-1 vs. control) and a slight increase in maximal velocity (Vmax) (4.59 vs. 3.89 nmol/mg prot/10 min, IL-1 vs. control). These observations indicate that IL-1 induces modifications in both system A transporter affinity and number. Moreover, we indicate that system A should be responsive in vivo to IL-1 in the same way since derepression and IL-1 action occurred in the presence of human synovial fluid.

Haematogenous dissemination of prostatic epithelial cells during radical prostatectomy.

Radical prostatectomy is one treatment for organ-confined prostatic adenocarcinoma. Dissemination of malignant prostatic cells after radical prostatectomy could be partly due to prostate manipulation during dissection. We confirmed by assay of prostate-specific membrane antigen by reverse-transcription nested PCR that prostate manipulation seeded prostatic epithelial cells in the general circulation in 12 of 14 consecutive patients operated on for organ-confined prostate adenocarcinoma. Our results suggest that surgeons should approach radical prostatectomy with care to avoid seeding from the prostate gland. Antiandrogen therapy might reduce the haematogenous spread of prostatic cells during radical prostatectomy.