Isolation of a highly active H+-ATPase from beef heart mitochondria

The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H⁺-ATPase with high levels of P_i-ATP exchange activity (400–600 nmol × min^–1 × mg^–1). This activity is further enhanced (1400–1600 nmol × min^–1 × mg^–1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H⁺ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. P_i-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).     

Determinants of plasma uric acid

Summary Obesity, alcohol consumption, and hematocrit provide an index of plasma uric acid, which in path analysis has a cultural heritability of 0.11 in children and 0.23 in parents, a small maternal effect, and a genetic heritability of 0.25 in both generations. Preliminary evidence for a major locus is destroyed by the omission of one exceptional child. There is no evidence against the polygenic hypothesis for hyperuricemia in the Japanese-American population studied.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Epidermal growth factor stimulates tyrosine phosphorylation of human glucocorticoid receptor in cultured cells

Human breast epithelial HBL100 cells, which bind both epidermal growth factor (EGF) and glucocorticoids, were labelled to steady state specific activity with 32Pi and the glucocorticoid receptor was immunoprecipitated from cell lysates with polyclonal antiserum GR884. Immunoprecipitated receptor was resolved by NaDodSO4-polyacrylamide gel electrophoresis and identified by autoradiography. Immunoprecipitated receptor also was characterized by western blot analysis and affinity labelling with [3H]dexamethasone-21-mesylate. Phosphoamino acid analysis of 32P-glucocorticoid receptor revealed 89% phosphoserine and 11% phosphotyrosine. Treatment of steady state 32Pi-labelled cells with EGF stimulated total and alkali-stable phosphorylation in the 97 kDa receptor band by about 35%. Prior incubation with dexamethasone inhibited EGF stimulated, alkali-stable phosphorylation of the 97 kDa glucocorticoid receptor band.     

Androgen receptor predominance in human ovarian carcinoma

Cytosols of 94 untreated common epithelial ovarian cancer tissues were analysed for the presence of estrogen-, progesterone- and androgen receptors. Androgen receptors clearly predominated (90%) over and above estrogen- (55%) and progesterone receptors (52%). Further characterisation particularly of the androgen receptor revealed steroid-receptor complex with the sedimentation coefficient similar to ovalbumin (3,6 S). Only androgens, natural and synthetic, were able to alter the sedimentation profile. Estrogen appeared to slightly lower the peak, while progesterone and cortisol did not alter the profile at all. No difference in receptor concentrations between tumor tissues from pre-, peri- and postmenopausal women was found. The serum hormone levels (estradiol-17 beta, testosterone, FSH, LH) measured preoperatively in 20 postmenopausal patients did not correlate with the receptor status. Majority of the ovarian carcinomas studied contained androgen receptors. We therefore suggest consideration of the addition of anti-androgens to the therapeutic strategies applicable to ovarian cancer.     

A mathematical model for insulin kinetics and its application to protein-deficient (malnutrition-related) diabetes mellitus (PDDM)

A nonlinear mathematical model which incorporates both beta-cell kinetics and a glucose-insulin feedback system is proposed for describing the time variations of plasma glucose and insulin levels. Numerical simulations show that this model is consistent with experimental observations on normal groups. An analysis of the changes in the solutions with variations in the parameters showed that a decrease in a single parameter gave results consistent with experimental findings in protein-deficient (malnutrition-related) diabetes mellitus (PDDM). The model predicts that it is the function and not the number of beta cells which is reduced in PDDM.     

Fluorescence imaging in vivo: recent advances

In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.