Osteogenesis imperfecta: a heterogeneous morphologic phenotype in cultured dermal fibroblasts

Summary Osteogenesis imperfecta (OI) is a phenotype with clinical and biochemical heterogeneity. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n=6) and OI cell strains (n=10, representing the four major OI categories, Sillence classification) were compared by measuring the following: (i) days required in culture to reach confluence after plating at uniform density; (ii) cell density at confluence; (iii) width and length of cells (measured on phase contrast micrographs at 300xmagnification). Our results show that: (i) OI fibroblasts take longer (11–27 days, mean 20 days) than control cells (10–19 days, mean 16 days) to reach stationary phase; (ii) all OI phenotypes achieve a lower cell density (0.87x10⁶ cells/P60, range 0.3–1.6x10⁶) at stationary phase relative to control cells (2.2x10⁶ cells/P60, range 1.7–2.6x10⁶; F_4,77=56.1, p<0.01, indicating that OI cells are larger than normal). Cell shape (expressed as the width: length ratio) was also abnormal in OI cells. (F_4,730=37.6, p<0.01), types I and II OI cells have significantly increased ratios (p<0.01) relative to control, type III, and type IV cells. Intra-group phenotypic heterogeneity was also apparent in the OI categories and also within the control population. These findings confirm deviant morphologic phenotypes in OI dermal fibroblasts and further demonstrate interindividual heterogeneity in the expression of genes that determine size and shape of dermal fibroblasts in both OI and normal donors.Publication No. 84013 from the Montreal Children's Hospital Research Institute     

Getting to the heart of the matter: CCN2 plays a role in cardiomyocyte hypertrophy

Connective tissue growth factor (CTGF/CCN2) is overexpressed in diabetes. Diabetic rats possess myocardial and cardiomyocyte hypertrophy. In a recent report, Wang and colleagues (Am J Physiol Cell Physiol. 2009 Jul 22. [Epub ahead of print]) show that CCN2 directly mediates cardiomyocyte hypertrophy as well as that induced by high glucose and fatty acid. CCN2 acted via the TrkA receptor. These data are the subject of this commentary, and emphasize that CCN2 may be an excellent target for therapy in diabetes.     

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 μg/ml. Recoveries varied from 91 to 10% with coefficients of variation ranging from 0.387 to 7.95%.     

Influence of Particle Size and Concentration on Rheological Behaviour of Reconstituted Apple Purees

This work investigates the impact of structural parameters on the rheological behaviour of apple purees. Reconstructed apple purees from 0 g/100 g up to 2.32 g/100 g of insoluble solids content and varying in particle size were prepared. Three different particle size distributions were obtained by mechanical treatment only, to modify both size and morphology of the particles without modifying the intrinsic rigidity of the cell walls. Rheological measurements showed that the insoluble solids content have a first order effect on the rheological behaviour of the suspensions: three concentrations domains were observed in both dynamic and flow measurements. A model is proposed for each domain. The existence of a weak network between particles is clearly shown over a critical concentration of insoluble solids (cell walls) depending on particle size distribution (semi-diluted domain). In a concentrated domain, particles are on close packing conditions and their apparent volume begin to shrink. Particle size and shape also play an important role on the rheological behaviour of reconstructed apple puree. Due to their irregular shape, cell clusters clog the medium at lower concentration compared to individual cells.     

Possible regulation of pyruvate carboxylase fromThiobacillus novellus by hydroxypyruvate

Aspartate, glutamate, or dicarboxylic acids did not inhibit the activity of a highly purified but not homogeneous preparation of pyruvate carboxylase fromThiobacillus novellus. The only effective inhibitors were end-products of the reaction and to a lesser degree hydroxypyruvate. The latter has not been shown previously to regulate the enzyme's activity. Lineweaver-Burk plots revealed that it was uncompetitive with respect to acetyl CoA with a K_ii of 3.6 mM, and noncompetitive with respect to bicarbonate, magnesium ATP, and pyruvate with respective K_ii values of 7.1, 5.5, and 6.47 mM. The corresponding K_is values were 7.02, 5.4, and 4.25 mM. A mathematical model is presented that supports the findings.     

Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-<Superscript>19</Superscript>F-NMR

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using ¹⁹F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.