Photochemical changes of fluorescent probes in membranes and their effect on the observed fluorescence anisotropy values

The fluorescence intensity of diphenylhexatriene (DPH) and of trimethylammonium-diphenylhexatriene (TMA-DPH) is measured when these probes are embedded in vesicles of dipalmitoyl- and dioleoylphosphatidylcholine (DPPC and DOPC), in mixtures of these vesicles as well as in vesicles of the mixed phospholipids, in trout intestinal brush border membranes and in mitoplasts of rat liver cells. The intensity in DOPC vesicles is found to be significantly higher than in DPPC vesicles. When these systems are irradiated with strong ultraviolet light radiation, a decrease in the fluorescence intensity is observed; this effect is much stronger in DOPC than in DPPC vesicles. The fluorescence anisotropy values in the mixture of vesicles as well as in the membranes show an initial increase with irradiation which is followed by a significant decrease. A transfer of DPH molecules between DPPC and DOPC vesicles is observed. For TMA-DPH this transfer takes place only from DPPC to DOPC vesicles, but not vice-versa. These results are related to intensity and anisotropy measurements of these probes in cell cultures.     

Release of different fractions of epidermal growth factor from human platelets in vitro: preferential release of 140 kDa fraction.

Platelet-rich plasma in acidic-citrate-dextrose anticoagulant was kept for 5 days in an oxygen-permeable bag at 22 degrees C in an incubator/rotator. Platelet count remained stable throughout the experiment. On days 0, 3 and 5, aliquots were removed; platelets were isolated by centrifugation at 22 degrees C, 1500 g for 20 min, reconstituted to the original volume with PBS buffer, and the contents of alpha-granules were released by repeated freezing and thawing. Epidermal growth factor (EGF) and beta-thromboglobulin (beta-TG) in the platelet-poor plasma and platelet lysates were determined by radioimmunoassays. Results indicated that in platelet-free plasma, both total EGF and beta-TG increased 3-5-fold after 5 days; this amount represented 10-20% of the factors stored in the platelets. Correspondingly, the EGF and beta-TG contents of the platelet lysates exhibited accompanying decreases. HPLC fractionation showed that the main EGF fraction which progressively decreased in the lysates and increased in plasma had a molecular mass of 140 kDa. The contents of the 67 kDa and 6 kDa fractions did not change substantially. We conclude that under these conditions, the 140 kDa fraction was released preferentially. In view of these and previous experiments, it seems likely that different organs contribute to plasma EGF fractions.     

Discovering the electronic circuit diagram of life: structural relationships among transition metal binding sites in oxidoreductases

Oxidoreductases play a central role in catalysing enzymatic electron-transfer reactions across the tree of life. To first order, the equilibrium thermodynamic properties of these proteins are governed by protein folds associated with specific transition metals and ligands at the active site. A global analysis of holoenzyme structures and functions suggests that there are fewer than approximately 500 fundamental oxidoreductases, which can be further clustered into 35 unique groups. These catalysts evolved in prokaryotes early in the Earth''s history and are largely responsible for the emergence of non-equilibrium biogeochemical cycles on the planet''s surface. Although the evolutionary history of the amino acid sequences in the oxidoreductases is very difficult to reconstruct due to gene duplication and horizontal gene transfer, the evolution of the folds in the catalytic sites can potentially be used to infer the history of these enzymes. Using a novel, yet simple analysis of the secondary structures associated with the ligands in oxidoreductases, we developed a structural phylogeny of these enzymes. The results of this ‘composome’ analysis suggest an early split from a basal set of a small group of proteins dominated by loop structures into two families of oxidoreductases, one dominated by α-helices and the second by β-sheets. The structural evolutionary patterns in both clades trace redox gradients and increased hydrogen bond energy in the active sites. The overall pattern suggests that the evolution of the oxidoreductases led to decreased entropy in the transition metal folds over approximately 2.5 billion years, allowing the enzymes to use increasingly oxidized substrates with high specificity.     

Modeling Magnetomyograms of Uterine Contractions during Pregnancy Using a Multiscale Forward Electromagnetic Approach

Understanding the mechanisms of uterine contractions during pregnancy is especially important in predicting the onset of labor and thus in forecasting preterm deliveries. Preterm birth can cause serious health problems in newborns, as well as large financial burdens to society. Various techniques such as electromyography (EMG) and magnetomyography (MMG) have been developed to quantify uterine contractions. However, no widely accepted method to predict labor based on electromagnetic measurement is available. Therefore, developing a biophysical model of EMG and MMG could help better understand uterine contractions, interpret real measurements, and detect labor. In this work, we propose a multiscale realistic model of uterine contractions during pregnancy. At the cellular level, building on bifurcation theory, we apply generalized FitzHugh-Nagumo (FHN) equations that produces both plateau-type and bursting-type action potentials. At the tissue level, we introduce a random fiber orientation model applicable to an arbitrary uterine shape. We also develop an analytical expression for the propagation speed of transmembrane potential. At the organ level, a realistic volume conductor geometry model is provided based on magnetic resonance images of a pregnant woman. To simulate the measurements from the SQUID Array for Reproductive Assessment (SARA) device, we propose a sensor array model. Our model is able to reproduce the characteristics of action potentials. Additionally, we investigate the sensitivity of MMG to model configuration aspects such as volume geometry, fiber orientation, and pacemaker location. Our numerical results show that fiber orientation and pacemaker location are the key aspects that greatly affect the MMG as measured by the SARA device. We conclude that sphere is appropriate as an approximation of the volume geometry. The initial step towards validating the model against real MMG measurement is also presented. Our results show that the model is flexible to mimic the limited-propagation magnetic signature during the emergence and decay of a uterine contraction.     

Epidermal growth factor in serum, urine, submandibular glands and kidneys of diabetic mice

Levels of epidermal growth factor (EGF) in serum were significantly decreased in streptozotocin (STZ)-diabetic mice (446 +/- 168 pg/ml after 1 week and 423 +/- 52 after 4 weeks vs 766 +/- 162 pg/ml in controls, P.002 and less than .001. respectively) and in genetically diabetic ob/ob mice (455 +/- 285 vs 962 +/- 453 pg/ml in nondiabetic ob/+ controls, P.043). The urinary excretion of EGF was significantly increased in STZ mice (104 +/- 53 vs 51 +/- 23 ng/h, P.013) but unchanged in ob/ob mice (33 +/- 9 vs 45 +/- 16 ng/h, P.134). However, when expressed per mg creatinine it was decreased in both cases: in STZ mice to 680 +/- 250 ng/mg at 1 week and 684 +/- 211 at 4 weeks vs 1250 +/- 303 ng/mg in controls (P less than .01); and in the ob/ob mice to 552 +/- 117 vs 1237 +/- 300 ng/mg in ob/+ controls (P less than .01). EGF content of the submandibular glands of STZ mice remained unchanged at 1 week (13.1 +/- 2.9 vs 11.0 +/- 1.8 micrograms/mg protein, P.170) but dropped by 4 weeks (4.7 +/- 1.2 micrograms/mg, P less than .001); in the ob/ob mice it was less than 20% that of controls (2.1 +/- 0.8 vs 12.2 +/- 3.6 micrograms/mg protein). In kidneys, the EGF content was not altered in either ob/ob (524 +/- 50 vs 571 +/- 33 pg/mg protein) or STZ mice (652 +/- 183 vs 665 +/- 80 pg/mg). The preproEGF mRNA level in STZ-treated mice was reduced after 4 weeks in submandibular glands but not in kidneys. The results show that diabetes affects EGF production, utilization and/or excretion in mice and that kidneys are spared from suppression of EGF synthesis that is pronounced in the submandibular glands.     

Media for cultivation of animal cells: an overview

The increasing interest in products from animal cells has caused an extensive research effort towards development of media for cell cultivation.The basic components in the media used for cultivation of animal cells vary depending upon the characters of the cells and the cultivation method. Basic components consist of an energy source, nitrogen source, vitamins, fats and fatty soluble components, inorganic salts, nucleic acid precursors, antibiotics, oxygen, pH buffering systems, hormones, growth factors and serum. Extensive efforts are directed towards developing serum-free or chemically defined media. Among the serum substitutes is a long list of hormones and growth factors.     

An immobilized hybridoma culture perfusion system for production of monoclonal antibodies

A fixed bed perfusion system for hybridoma cell immobilization is presented. The system consists of a culturing vessel (300 ml total volume) in which polyurethane (PU) sponges in the form of small cubes of about 5 mm sides are packed. Cells are immobilized by physical entrapment in the foam matrix. By entrapment of the cells in the pores of the matrix high cell concentration can be maintained in a mechanically protected environment. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber (700 ml total volume).Medium flow rate, feeding rate, dissolved oxygen, pH, nutrient uptake and waste product formation can be easily monitored and controlled. Steady state conditions are established with medium dilution rates of 1.0–1.5 reactor volume per day. The steady state is characterized by a constant cell density, constant culture volume and constant glucose and lactate levels. Cell-free supernatant is collected continuously in a cold room adjacent to the 37°C culture room. Monoclonal antibodies (MAb) are produced at a concentration of 150–200 g/ml for several weeks. An important feature of the system is the capacity to maintain a population of cells after the growth phase in a non-proliferating state for extended time periods expressing high titers of MAb.Abbreviations DO Dissolved Oxygen - FBS Fetal Bovine Serum - FBR Fixed Bed Reactor - MAb Monoclonal Antibody - PU polyurethane     

Selective regulation of arterial branching morphogenesis by synectin

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.