A dialysis medium refreshment cell culture set-up for an osteoblast-osteoclast coculture

Culture medium exchange leads to loss of valuable auto- and paracrine factors produced by the cells. However, frequent renewal of culture medium is necessary for nutrient supply and to prevent waste product accumulation. Thus it remains the gold standard in cell culture applications. The use of dialysis as a medium refreshment method could provide a solution as low molecular weight molecules such as nutrients and waste products could easily be exchanged, while high molecular weight components such as growth factors, used in cell interactions, could be maintained in the cell culture compartment. This study investigates a dialysis culture approach for an in vitro bone remodeling model. In this model, both the differentiation of human mesenchymal stromal cells (MSCs) into osteoblasts and monocytes (MCs) into osteoclasts is studied. A custom-made simple dialysis culture system with a commercially available cellulose dialysis insert was developed. The data reported here revealed increased osteoblastic and osteoclastic activity in the dialysis groups compared to the standard nondialysis groups, mainly shown by significantly higher alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activity, respectively. This simple culture system has the potential to create a more efficient microenvironment allowing for cell interactions via secreted factors in mono- and cocultures and could be applied for many other tissues.     

Genotypic variation in anther culture and effect of ovary coculture in durum wheat

The response of anthers to in vitro culture and the effect of coculture of ovaries on anther culturability have been studied in responsive and recalcitrant cultivars of durum wheat (Triticum turgidum ssp. durum) from Morocco and ICARDA. A large genotypic-dependence of anther culture has been shown in 18 cultivars. Their response in term of callus and embryo induction varied from 0 to 13%. Coculture of ovaries with anthers enhanced the response of the most responsive genotype (cv. Sarif) and removed the recalcitrance in Cocorit and Isly cultivars. However, there was no effect of anther-ovary coculture on green plant regeneration. The implication of the genome and the media conditioning by the ovaries on anther response is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Abstract Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C₆-C₁₀) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic degradation of dicarboxylic acids with even-numbered carbon atoms, propionate accumulated intermediately, and butyrate was the intermediate product of degradation of those with an odd number of carbon atoms. Degradation of the long-chain dicarboxylates depended strictly on the presence of hydrogenotrophic methanogens. The primary attack in these processes was β-oxidation rather than decarboxylation. A general scheme of anaerobic degradation of long-chain dicarboxylic acids has been deduced from these results.     

<Emphasis Type="Italic">Symbiobacterium</Emphasis> Lost Carbonic Anhydrase in the Course of Evolution

Recent genetic studies have elucidated that carbonic anhydrase (CA; EC 4.2.1.1), a ubiquitous enzyme catalyzing interconversion between CO₂ and bicarbonate, is essential for microbial growth under ambient air but not under high-CO₂ air. The irregular distribution of the phylogenetically distinct types of CA in the prokaryotic genome suggests its complex evolutionary history in prokaryotes. This paper deals with the genetic defect of CA in Symbiobacterium thermophilum, a syntrophic bacterium that effectively grows on CO₂ generated by other bacteria. Phylogenetic analysis based on 31 ribosomal protein sequences demonstrated the affiliation of Symbiobacterium with the class Clostridia with 100% bootstrap support. The phylogeny of β- and γ-type CA distributed among Clostridia supported the view that S. thermophilum and several related organisms lost this enzyme during the course of evolution. The loss of CA could be based on the availability of a high level of CO₂ in their living environments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.