Investigating the effect of cell substrate on cancer cell stiffness by optical tweezers

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17 kPa respectively). Breast cancer cells lines, namely HBL-100, MCF-7 and MDA-MB-231 with different level of aggressiveness are cultured on these substrates and their local elasticity is investigated by vertical indentation of the cell membrane. Our preliminary results show an unforeseen behavior of the MDA-MB-231 cells. When cultured on glass substrate as isolated cells, they are less stiff than the other two types of cells, in agreement with the general statement that more aggressive and metastatic cells are softer. However, when connected to other cells the stiffness of MDA-MB-231 cells becomes similar to the other two cell lines. Moreover, the stiffness of MDA-MB-231 cells cultured on soft PDMS substrates is significantly higher than the stiffness of the other cell types, demonstrating thus the strong influence of the environmental conditions on the mechanical properties of the cells.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

Interleukin-1-β and Tumor Necrosis Factor-α Increase Peripheral-Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.     

Variation of Pleurotus ostreatus (Jacq. Ex Fr.) P. Kumm. (1871) performance subjected to differentdoses and timings of nano-urea

Supplementation of the growing substrate by nitrogenous additives has been known to improve the production of oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. (1871)). However, the application of nano-additives has not been reported in such cultivation yet. The study investigated the effect of nano-urea added in two different doses (3 g and 5 g per kg substrate), once (at spawning or after first flush) or twice (at spawning and after first flush) to the growing substrate consisting of wheat straw and spent oyster substrate (1:1, w/w). Results showed that the application of nano-urea once has induced the highest number of mushroom flushes (four flushes) despite the dose applied. Contrarily to early findings, where high doses of nitrogen have caused inhibition of mushroom growth and production, nano-urea application has had better effects when applied twice. With 5 g/kg, it induced the shortest period between the first and the third flush (15 days). With 3 g/kg, it resulted in the highest biological and economic yields at the third flush (332.7 g/bag and 283.1 g/bag respectively), in total (973.4 g/bag and 854.0 g/bag respectively), the highest biological efficiency (109.6%), and pileus diameter/stipe length ratio (2.8). Experimental findings of the current study may be potentially applied at commercial scale.     

Differential human impact and vegetation history in two adjacent Pyrenean valleys in the Ariège basin,southern France,from 3000 B.P. to the present

Detailed palynological studies in two adjoining French Pyrenean valleys, complemented by the study of archives, demonstrate that under similar climatic conditions, the forest history of each valley from the Bronze Age to present time was essentially determined by socio-economical constraints, possibly modified by natural characteristics such as topography. The studies show why the expansion of Fagus (beech) at c. 4000 B.P. was asynchronous on the northern slope of the Pyrenees and emphasize the effects of the human impact on the recent lowering of the tree-line.A contribution to the 8th IPC, Aix-en-Provence, 1992     

Chick brain glutamine synthetase and Mn2+−Mg2+ interactions

Glutamine synthetase (GS) from the chick brain was purified to apparent homogeneity by ammonium sulfate fractionation followed by affinity chromatography, electrofocusing and Sephadex G-150 chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate analysis in polyacrylamide gel. By sedimentation equilibrium analysis and gel electrophoresis analysis, it was shown that the enzyme has a subunit molecular weight of 45,000 and a native molecular weight of 364,000, which is consistent with an octameric structure. Sedimentation analysis in the presence of Mg²⁺ revealed three different forms of macromolecules corresponding respectively to a monomer, a tetramer and an octamer. Among eight cations tested (Ca²⁺, Co²⁺, Fe²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Zn²⁺) only Co²⁺, Mg²⁺ and Mn²⁺ supported GS activity; the order of activatory ability was Mg²⁺>Co²⁺>Mn²⁺. The maximum activating effect of Mn²⁺ occurs only within a very narrow range of concentration: with an excess of cation causing strong inhibition of GS activity. For each cation, maximal GS activity occurs at a defined cation/ATP ratio. A regulatory system in which Mn²⁺, modulates the Mg²⁺ dependent GS activity, is proposed; such cation interactions may be of significance in the intracellular control of glutamine synthesis.     

The limited proteolysis of tumor necrosis factor-α

The limited proteolysis of human recombinant TNF- by trypsin yields two stable products resulting from cleavage after Arg6 and Arg44. In solution these two products remain associated together in a trimer with a Stokes' radius slightly greater than the radius of intact TNF- and, therefore, could not be separated from each other under nondenaturing conditions. This limited digest retains at least 20% of the activity of the original TNF- sample, and has a tertiary structure that is similar to that of the native protein by circular dichroism. On the other hand, incorrectly folded, inactive TNF- undergoes extensive digestion following similar treatment with trypsin. These results indicate that the active form of TNF- has a tight core structure which is maintained afterN-terminal cleavage and removal.