Assessment of some tools for the characterization of the human osteoarthritic cartilage proteome.

Since the proteome of osteoarthritic articular cartilage has been poorly investigated as yet, we adapted proteomic technologies to the study of the proteins secreted or released by fresh human osteoarthritic cartilage in culture. Fresh cartilage explants were obtained from three donors undergoing surgery for knee joint replacement. The explants were dissected out, minced, and incubated in serum-free culture medium. After 48 h, proteins in the medium were identified by two-dimensional or off-gel electrophoresis coupled to tandem mass spectrometry, or by using an antibody-based protein microarray designed to detect angiogenic factors, growth factors, chemokines, and cytokines. We identified a series of 43 proteins. Some of these proteins were already described as secretion products of chondrocytes, such as YKL-39 or osteoprotegerin, while several other were known proteins but have never been reported previously in cartilage, such as the serum amyloid P-component, the vitamin D binding protein, the pigment epithelium derived factor, the pulmonary and activation-regulated chemokine, lyl-1, thrombopoietin, fibrinogen, angiogenin, gelsolin, and osteoglycin/mimecan. While this study enabled the identification of novel proteins secreted or released by human osteoarthritic cartilage, the goal of the present work was essentially to describe the technical approach necessary for a systematic study of osteoarthritic cartilages from a large population of donors, in order to be able to select the good markers and/or targets for this poorly explored disease.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Crustacean Species Richness in Temporary Pools: Relationships with Habitat Traits

We examined species richness separately for cladocerans and ostracods in 52 temporary pools in a small geographical area, relating species richness with habitat traits using multiple regressions. Habitat traits considered included surface area, water depth, permanence and sediment depth. Permanence was an important predictor of species richness of both cladocerans and ostracods. Additionally, variation in ostracod species richness was significantly explained by water depth (negative relationship) and sediment depth (positive relationship). Surface area was not a statistically significant factor in any of our analyses. The importance of permanence supports the hypothesis that extinction due to pool drying is a major driving force behind the structuring of microcrustacean communities in temporary pools.     

Human cell lines for biopharmaceutical manufacturing: history,status, and future perspectives

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.     

Identification and characterization of mRNAs differentially expressed in thyroid cells stimulated by a mitogenic treatment.

The aim of our work is to identify new genes and proteins involved in the control of the proliferation of thyroid cells as putative protooncogenes and antioncogenes. Several strategies are discussed. A first study has allowed to identify three new genes. Further search will use the differential display and gene arrays methodology. The role of the identified proteins coded by the genes is studied in vitro by the search of partner proteins by the double hybrid method and in vivo by mice gene knockout technology.     

Adenosine 3':5'-monophosphate metabolism and turnover in dog thyroid slices.

The metabolism and turnover of adenosine 3':5'-monophosphate (cyclic AMP) have been studied in intact thyroid cells incubated in vitro. Thyroid slices have been stimulated by 1 mU thyrotropin/ml, then washed with buffer, or with buffer containing thyrotropin antibody, or trypsin so as to cut off the stimulation. The decline of cyclic AMP levels has been followed and the time required to decrease this level to half of the initial value estimated. Computer simulation taking into account the penetration of trypsin in the slices, the kinetics of thyrotropin inactivation and the relation between thyrotropin concentration and cyclic AMP concentration at the steady state has made it possible to estimate the true cellular half-life of cyclic AMP in the stimulated cell to 1 min 50 s. The method provides an experimental approach to the demonstration in intact cells of effective on cyclic AMP disappearance. The methodology of the calculation of half-life and turnover from such data is discussed.