Scale-up of a myoblast culture process

The effects of different types of cell carriers, strategies for cell transfer on carriers, and of several fusion inhibitors on the growth kinetics of primary human myoblasts culture were studied in order to develop a bioprocess suitable for the treatment of Duchenne muscular dystrophy based on the transplantation of unfused cells. Our results indicate that myoblast production is larger on Cytodex 1 and 3 than on polypropylene or polyester fabrics and on a commercial porous macrocarrier. Myoblast growth conditions with Cytodex 1 were further investigated to establish the bioprocess operating conditions. It was found that microcarrier density of 3 g DW l(-1), inoculum density of 2x10(5) cells ml(-1), and continuous agitation speed of 30-rpm result in final myoblast production comparable to static cultures. However, for all the culture conditions used, myoblasts growth kinetics exhibited a lag phase that lasted a minimum of 1 week prior to growth, the end of the lag phase correlating with the appearance of microcarrier aggregates. Based on this observation, we propose that aggregation promotes cell growth by offering a network of very large inter-particular pores that protect cells from mechanical stress. We took advantage of the presence of these aggregates for the scale-up of the culture process. Indeed, using myoblast-loaded microcarrier-aggregates instead of myoblast suspension to inoculate a fresh suspension of microcarriers significantly reduced the duration of the lag phase and allowed the scale-up of the bioprocess at the 500-ml scale. In order to ensure the production of unfused myoblasts, the efficiency of five different fusion inhibitors was investigated. Only calpeptin (9.1 microg ml(-1)) significantly inhibited the fusion of the myoblasts, while TGFbeta (50 ng ml(-1)) and LPA (10 microg ml(-1)) increased myoblasts growth but did not affect fusion, sphingosine (30 microg ml(-1)) induced a 50% death and NMMA (25 microg ml(-1)) had no effect on either growth or fusion. Finally, transplantation trials on severe combined immunodeficient mice showed that microcarrier-cultured human myoblasts grown using the optimized bioprocess resulted in grafts as successful as myoblasts grown in static cultures. The bioprocess, therefore, prove to be suitable for the large-scale production of myoblasts required for muscular dystrophy treatment.     

Catharanthus roseus genes regulated differentially by mollicute infections

A differential display of mRNAs was used to isolate periwinkle cDNAs differentially expressed following infection with one of three mollicutes: Spiroplasma citri, Candidatus Phytoplasma aurantifolia, and stolbur phytoplasma. Twenty-four differentially expressed cDNAs were characterized by Northern blots and sequence analysis. Eight of them had homologies with genes in databanks coding for proteins involved in photosynthesis, sugar transport, response to stress, or pathways of phytosterol synthesis. The regulation of these genes in periwinkle plants infected by additional phloem-restricted bacteria showed that they were not specific to a given mollicute, but correlations with particular symptoms could be established. Expression of transketolase was down regulated following infection with a pathogenic strain of S. citri. No down regulation was observed for the nonphytopathogenic mutant GMT553, which is deficient for fructose utilization.     

Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.     

Systemic arteriovenous fistula leads to pulmonary artery remodeling and abnormal vasoreactivity in the fetal lamb

Several cases of systemic arteriovenous fistula diagnosed in the human fetus have been associated with the postnatal development of persistent pulmonary hypertension. The aim of this study was to determine the effects of a prenatally created systemic arteriovenous fistula on the structure and reactivity of the pulmonary circulation in the fetal lamb. A fistula between the jugular vein and carotid artery was created in fetal lambs at 119-124 days of gestation. At delivery (134-139 days), left pulmonary artery (LPA) pressure was increased in the fistula group (n = 12) compared with controls (n = 11, P < 0.01). The pulmonary vascular resistance was significantly higher in the fistula group (P < 0.05), whereas mean LPA blood flow was not statistically different between the two groups. Morphometric analysis of the pulmonary vascular bed revealed an increase in the number of peripheral muscular arteries, together with an increase in pulmonary arterial medial thickness in the fistula group. There was no difference in the relative number or size of intraacinar arteries. In vitro organ bath studies on pulmonary arterial rings showed impaired endothelium-dependent relaxation in the fistula group compared with controls. However, endothelial nitric oxide synthase protein expression was similar in both groups, whereas endothelium-independent relaxation to sodium nitroprusside was greater in the fistula group compared with controls. A systemic arteriovenous fistula leads to both structural and functional alteration of the pulmonary vasculature, which might lead to the development of persistent pulmonary hypertension after birth.     

The combinatorics of tandem duplication trees

We developed a recurrence relation that counts the number of tandem duplication trees (either rooted or unrooted) that are consistent with a set of n tandemly repeated sequences generated under the standard unequal recombination (or crossover) model of tandem duplications. The number of rooted duplication trees is exactly twice the number of unrooted trees, which means that on average only two positions for a root on a duplication tree are possible. Using the recurrence, we tabulated these numbers for small values of n. We also developed an asymptotic formula that for large n provides estimates for these numbers. These numbers give a priori probabilities for phylogenies of the repeated sequences to be duplication trees. This work extends earlier studies where exhaustive counts of the numbers for small n were obtained. One application showed the significance of finding that most maximum-parsimony trees constructed from repeat sequences from human immunoglobins and T-cell receptors were tandem duplication trees. Those findings provided strong support to the proposed mechanisms of tandem gene duplication. The recurrence relation also suggests efficient algorithms to recognize duplication trees and to generate random duplication trees for simulation. We present a linear-time recognition algorithm.     

Common characteristics of the human and rhesus macaque CD1e molecules: conservation of biochemical and biological properties during primate evolution

In humans, a family of five genes encodes the CD1 molecules. Four of these proteins, CD1a, b, c, and d, are expressed on the plasma membrane and traffic between the cell surface and endocytic compartments, where they are loaded with antigenic glycolipids. The existence of human CD1e was demonstrated recently. This molecule surprisingly remains inside the cell, accumulating mainly in the Golgi compartments of immature dendritic cells and in the late endosomes of mature dendritic cells. In the latter compartments, CD1e is cleaved and becomes soluble. To determine whether these properties were specific to human CD1e, we investigated the presence and characteristics of CD1e in the rhesus macaque, an evolutionarily distant species of the primate lineage. Our results show that the cellular and biochemical properties of the human and simian CD1e molecules are similar, suggesting that the particular intracellular distribution of CD1e is important for its physiological and/or immunological function.     

Regulation of the anion channel of the chloroplast envelope from spinach

Several anions such as Cl^–, NO^– ₂, SO^2– ₄, and PO^3– ₄ are known to modulate the photosynthetic activity. Moreover, the chloroplast metabolism requires the exchange of both inorganic and organic (e.g., triose phosphate, dicarboxylic acid, ATP) anions between the cytoplasmand the stroma. A chloride channel form the chloroplast envelope was reconstituted in planar lipid bilayers. We show that the channel is active in conditions prevailing in the plant. The open probability increases with the ionic strength of the experimental solutions and is maximal at 0 mV. This suggests that the channel could play a role in the osmotic regulation of the chloroplast. Amino group reagents affect the channel activity in a way that demonstrated that lysine residues are important for channel gating but not for ATP binding. Together, our results provide new information on the functioning of this channel in the chloroplast envelope membranes. They indicate that the open probability of the channel is low (P _o 0.2) in vivo and that this channel can account for the chloride flux through the chloroplast envelope.