Comparative study of in vitro expansion of bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BMD-MSCs) are of great interest for tissue engineering, but require expansion before they can be used for therapeutic applications. We compared three different culture techniques for their potential for large scale expansion of rat BMD-MSCs, i.e. monolayer cultures, stirred suspension cultures and pour-off cultures, and found that pour-off cultures supported the biggest expansion in BMD-MSCs as measured by the fibroblastic-colony forming unit assay (CFU-f). BMD-MSCs expanded in stirred suspension cultures stopped proliferating altogether and, although monolayer cultures allowed for expansion of BMD-MSCs, they favoured a differentiated phenotype over uncommitted MSCs. Only BMD-MSCs expanded in pour-off cultures were able to differentiate into both osteoblastic and adipocytic lineages and maintain CFU-f numbers. These data suggest that pour-off cultures are a viable method of BMD-MSC expansion.     

Stimulation of bone resorption by lipoxygenase metabolites of arachidonic acid

We have studied the effect of leukotrienes, (LT): B₄, C₄, D₄ and E₄ and the hydroxyeicosatetraenoic acids (HETEs) 5-HETE and 12-HETE on bone respiration . Resorption was measured by colorimetric assay of calcium released from neonatal mouse calvaria maintained in organ culture for 72h. All the LTs and HETEs stimulated bone resorption, with optimum responses at picomolar or nanomolar concentrations. The responses were biphasic, with a decreasing effect at higher concentrations. In contrast, prostaglandin E₂ (PGE₂) stimulated resorption only at 10nM and above. Indomethacin partially inhibited resorption by LTB4, LTC4 and LTD4, but did not affect resorption stimulated by LTE4, 5-HETE nd 12-HETE. These results indicate that liposygenase products of arachidonic acid are highly potent bone resorbing factors and may play an important role in the localised bone loss associated with inflammatory lesions.     

Molecular evolution of a repetitive region within the per gene of Drosophila

The clock gene period (per) controls a number of biological rhythms in Drosophila. In D. melanogaster, per has a repetitive region that encodes a number of alternating threonine-glycine residues. We sequenced and compared this region from several different Drosophila species belonging to various groups within the Drosophila and Sophophora subgenera. This part of per shows a great variability in both DNA sequence and length. Furthermore, analysis of the data suggests that changes in the length of this variable region might be associated with amino acid replacements in the more conserved flanking sequences.      

Male specific genes from dioecious white campion identified by fluorescent differential display

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.     

Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone in vitro

This study examines the capability of NIH3T3 fibroblasts to express osteoblastic markers following stimulation with a number of hormones and growth factors in vitro. Of the agents tested, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) dose-dependently induced alkaline phosphatase (ALP) activity in NIH3T3 cells, and this effect was enhanced by the addition of dexamethasone (Dex), which when administered alone caused no detectable ALP expression. The combined use of 1,25(OH)(2)D(3) and Dex also stimulated the synthesis of osteocalcin, and osteopontin. Furthermore, cells treated with the both hormones, in the presence of beta-glycerophosphate and l-ascorbic acid, formed mineralized plaques, indicating an osteoblast (OB) phenotype. By contrast, the differentiation induced by 1,25(OH)(2)D(3) or 1,25(OH)(2)D(3) plus Dex was significantly antagonized by transforming growth factor-beta1 and all trans-retinoic acid. These data indicate that NIH3T3 cells have the potential to adopt an OB-like phenotype and may prove to be a convenient model for studying the early events of osteogenic differentiation and the specific interactions of 1,25(OH)(2)D(3) with glucocorticoids in controlling this process in vitro.     

The identification of candidate genes for a reverse genetic analysis of development and function in the Arabidopsis gynoecium

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.