Defining the risks of mesenchymal stromal cell therapy

We address the issue of the potential for malignant transformation of cultured mesenchymal stromal cells (MSC) commonly used in clinical cell-therapy protocols and describe the culture conditions under which tumorigenesis is likely to be an extremely uncommon event.     

Polypyrimidine track-binding protein binding downstream of caspase-2 alternative exon 9 represses its inclusion

We have been using the caspase-2 pre-mRNA as a model system to study the importance of alternative splicing in the regulation of programmed cell death. Inclusion or skipping of a cassette-type exon in the 3' portion of this pre-mRNA leads to the production of isoforms with antagonistic activity in apoptosis. We previously identified a negative regulatory element (In100) located in the intron downstream of alternative exon 9. The upstream portion of this element harbors a decoy 3' acceptor site that engages in nonproductive commitment complex interactions with the 5' splice site of exon 9. This in turn confers a competitive advantage to the exon-skipping splicing pattern. Further characterization of the In100 element reveals a second, functionally distinct, domain located downstream from the decoy 3' acceptor site. This downstream domain harbors several polypyrimidine track-binding protein (PTB)-binding sites. We show that PTB binding to these sites correlates with the negative effect on exon 9 inclusion. Finally, we show that both domains of the In100 element can function independently to repress exon 9 inclusion, although PTB binding in the vicinity of the decoy 3' splice site can modulate its activity. Our results thus reveal a complex composite element that regulates caspase-2 exon 9 alternative splicing through a novel mechanism.     

Expanded bed adsorption as a unique unit operation for the isolation of bacteriocins from fermentation media

Expanded bed adsorption using a strong cation exchanger allowed the direct isolation of amylovorin L471, a bacteriocin from Lactobacillus amylovorusDCE 471, from the fermentation medium. The pH of the loading and elution buffer were optimised in a packed bed with cell-free culture supernatant. Bound bacteriocin was eluted with 1.0 M NaCl. The highest recovery (30%) was obtained at the lowest pH (3.6). At higher pH values the recovery was lower, namely 12%, 15% and 7% at pH 4.5, 6.5 and 8.0, respectively. In expanded bed mode, direct isolation of the bacteriocin from the fermentation medium at pH 3.6 (loading and elution) initially resulted in a recovery of 12%. After optimisation of the pH (loading and elution at pH 3.6 and 6.5, respectively), the recovery for amylovorin L471 increased up to 30% and higher. Recovery of enterocin A from Enterococcus faeciumCTC 492 fermentation medium averaged 15% (loading and elution at pH 3.6 and 6.0, respectively). With pediocin, produced by Pediococcus acidilactici ATCC 8042, 26% recovery was obtained at a pH of 6.5 during loading and elution. Low recoveries can be ascribed to non-optimal operation conditions (pH of loading and elution buffer), inactivation of the bacteriocin on a cationic resin, and the formation of more insoluble and less active, strongly hydrophobic bacteriocin aggregates upon further purification.     

Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg²⁺) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na⁺ pump andCl channels. In this study,we determined whether theNa⁺-K⁺-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg²⁺ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg²⁺inhibitsNa⁺-K⁺-Clcotransport, with inhibitor constant(K_i) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg²⁺ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg²⁺binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

     

Reexamination of hemocytes in brine shrimp (Crustacea, branchiopoda)

In 1941, a single type of hemocyte was described in the blood of the brine shrimp Artemia salina using light microscopy. This condition is unusual because most crustaceans examined using morphological, cytochemical, and functional methods have at least two types of hemoctyes. Upon examining A. franciscana, we found a single type of disk-shaped hemocyte, with a centrally located nucleus and about 15 large (6 microm diameter) granules. The granules stain for the presence of acid phosphatase and react with L-DOPA suggesting, respectively, that they are involved in degrading ingested material and possess the phenoloxidase system. Hemocytes require calcium for adhesion, bind together to mend small wounds in the body wall, and are able to phagocytose bacteria. Blood cells of A. franciscana are morphologically and functionally similar to those of the primitive chelicerate, Limulus polyphemus, and both forms have apparently given rise to more advanced taxa with multiple types of hemocytes. The major difference between the two species is the presence of the phenoloxidase system in the Crustacea and its apparent absence in the chelicerates.     

La 12′-hydroxyisostrychnobiline: Nouvel alcaloïde du Strychnos variabilis

The structure of a new bisindole alkaloid, 12′-hydroxyisostrychnobiline, has been proposed from the analysis of its 300 MHz ¹H NMR spectrum and comparison of the spectroscopic data with those of various monomeric and dimeric alkaloids, previously isolated from the same Strychnos species     

Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes

Cytoskeleton damage, particularly microtubule (MT) alterations, may play an important role in the pathogenesis of ischemia-induced myocardial injury. However, this disorganization has been scarcely confirmed in the cellular context. We evaluated MT network disassembly in myoblast cell line H9c2 and in neonatal rat cardiomyocytes in an in vitro substrate-free hypoxia model of simulated ischemia (SI). After different duration of SI from 30 up to 180 min, the cells were fixed and the microtubule network was revealed by immunocytochemistry. The microtubule alterations were quantified using a house-developed image analysis program. Additionally, the tubulin fraction were extracted and quantified by Western blotting. The cell respiration, the release of cellular LDH and the cell viability were evaluated at the same periods. An early MT disassembly was observed after 60 min of SI. The decrease in MT fluorescence intensity at 60 and 90 min was correlated with a microtubule disassembly. Conversely, SI-induced significant LDH release (35%) and decrease in cell viability (34%) occurred after 120 min only. These results suggest that the simulated ischemia-induced changes in MT network should not be considered as an ultrastructural hallmark of the cell injury and could rather be an early ultrastructural correlate of the cellular reaction to the metabolic challenge.     

Balance control during an arm raising movement in bipedal stance: which biomechanical factor is controlled?

In order to obtain new insight into the control of balance during arm raising movements in bipedal stance, we performed a biomechanical analysis of kinematics and dynamical aspects of arm raising movements by combining experimental work, large-scale models of the body, and techniques simulating human behavior. A comparison between experimental and simulated joint kinematics showed that the minimum torque change model yielded realistic trajectories. We then performed an analysis based on computer simulations. Since keeping the center of pressure (CoP) and the projection of the center of mass (CoM) inside the support area is essential for equilibrium, we modeled an arm raising movement where displacement of one or the other variable is limited. For this optimization model, the effects of adding equilibrium constraints on movement trajectories were investigated. The results show that: (a) the choice of the regulated variable influences the strategy adopted by the system and (b) the system was not able to regulate the CoM for very fast movements without compromising its balance. Consequently, we suggest that the system is able to maintain balance while raising the arm by only controlling the CoP. This may be done mainly by using hip mechanisms and controlling net ankle torque.     

Protein secondary structure assignment through Voronoï tessellation

We present a new automatic algorithm, named VoTAP (Vo ronoï T essellation A ssignment P rocedure), which assigns secondary structures of a polypeptide chain using the list of α‐carbon coordinates. This program uses three‐dimensional Voronoï tessellation. This geometrical tool associates with each amino acid a Voronoï polyhedron, the faces of which unambiguously define contacts between residues. Thanks to the face area, for the contacts close together along the primary structure (low‐order contacts) a distinction is made between strong and normal ones. This new definition yields new contact matrices, which are analyzed and used to assign secondary structures. This assignment is performed in two stages. The first one uses contacts between residues close together along the primary structure and is based on data collected on a bank of 282 well‐refined nonredundant structures. In this bank, associations were made between the prints defined by these low‐order contacts and the assignments performed by different automatic methods. The second step focuses on the strand assignment and uses contacts between distant residues. Comparison with several other automatic assignment methods are presented, and the influence of resolution on the assignment is investigated. Proteins 2004. © 2004 Wiley‐Liss, Inc.     

Incorporation of oxyphytosterols in tissues of hamster

Oxyphytosterols (OPS) were fed to hamsters, at different concentrations, in order to observe their eventual incorporation into plasma, aorta, liver, kidneys and heart. The animals receiving the very high level (2500 ppm) presented 7beta-hydroxycampesterol, beta-epoxycampesterol, campestanetriol, 7-ketocampesterol, 7beta-hydroxysitosterol, beta-epoxysitosterol, sitostanetriol and 7-ketositosterol in all tissues. The same compounds were observed in the tissues of animals receiving 500 ppm of OPS in their diet, but with much lower levels. In hamsters fed 100 ppm of OPS, as well as in control animals, in most cases, the only observed OPS was sitostanetriol, which seems to be difficult to eliminate from the animal.