Protein kinases in rat testes: evidence for different fractions of the enzyme.

Protein kinase activity of rat testis homogenate was separated into five fractions by means of pH 4.8 acidification and DEAE-cellulose chromatography. The five fractions showed a peculiar pattern of activity and cAMP dependency with the substrates used: casein, protamine, histone mixture, arginine-rich histone, lysine-rich histone, and phosvitin. The casein-sepharose substrate affinity column separated two fractions from the pH 4.8 precipitate. Peak number one phosphorylates histone preferently and is cAMP-dependent, while peak number tow has a strong affinity toward casein as substrate and is non cAMP-dependent.     

Alpha-Catulin Contributes to Drug-Resistance of Melanoma by Activating NF-κB and AP-1

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.     

Effect of changes in sympathetic vasomotor tonus on vasodilatatory effect of catecholamines]

The study of resistances modifications in the femoral vascular bed during bilateral carotide occlusion supports the hypothesis of an inhibitory action of isoprenaline on norepinephrine release by the sympathetic post-ganglionnic fibres. This action explain the relations between isoprenaline vasodilator potency and sympathetic activity level.     

Rapid effect of testosterone on rat Sertoli cell membrane potential. Relationship with K+ATP channels.

For this study, we investigated the changes in the electrophysiological parameters of Sertoli cells in seminiferous tubules from 17 - 19 day-old rats induced by testosterone. Using conventional intracellular microelectrode techniques, we analysed the membrane potential and its input resistance. The entire tubules were fixed in a superfusion chamber continuously perfused with Krebs-Ringer bicarbonate buffer (pH 7.4, 32 degrees C). Visual control of cell impalement was achieved using an inverted microscope. The parameters analysed were passed through an amplifier and recorded using a proprietary software system. The topical application of testosterone (0.1 to 10 microM) led to an immediate (within 30 seconds) and significant dose-dependent depolarization of the membrane potential of the cell at all concentrations used. Concomitantly, the input resistance of the cell membrane underwent a significant increment at 30 seconds. These changes returned to resting values after washout. Topical administration of 17beta-estradiol or progesterone (10 microM) did not change the membrane potential. The addition of the K +ATP channel agonist diazoxide to the perfusion buffer nullified the depolarization effect of testosterone at 30 seconds. This result suggests that the immediate action of testosterone is associated with the closing of K +ATP channels, thereby depolarizing the membrane.     

Predicting molecular properties of α-synuclein using force fields for intrinsically disordered proteins

Independent force field validation is an essential practice to keep track of developments and for performing meaningful Molecular Dynamics simulations. In this work, atomistic force fields for intrinsically disordered proteins (IDP) are tested by simulating the archetypical IDP α-synuclein in solution for 2.5 μs. Four combinations of protein and water force fields were tested: ff19SB/OPC , ff19SB/TIP4P-D , ff03CMAP/TIP4P-D , and a99SB-disp/TIP4P-disp , with four independent repeat simulations for each combination. We compare our simulations to the results of a 73 μs simulation using the a99SB-disp/TIP4P-disp combination, provided by D. E. Shaw Research. From the trajectories, we predict a range of experimental observations of α-synuclein and compare them to literature data. This includes protein radius of gyration and hydration, intramolecular distances, NMR chemical shifts, and ³J-couplings. Both ff19SB/TIP4P-D and a99SB-disp/TIP4P-disp produce extended conformational ensembles of α-synuclein that agree well with experimental radius of gyration and intramolecular distances while a99SB-disp/TIP4P-disp reproduces a balanced α-synuclein secondary structure content. It was found that ff19SB/OPC and ff03CMAP/TIP4P-D produce overly compact conformational ensembles and show discrepancies in the secondary structure content compared to the experimental data.     

Impacts of storms on Recent planktic foraminiferal test production and CaCO3 flux in the North Atlantic at 47 °N, 20 °W (JGOFS)

Planktic foraminiferal assemblages are well known to vary in accordance with seasonal fluctuations in ocean properties, periodic reproduction cycles, and variations between water masses. Here we report that storms also can significantly influence foraminiferal assemblages. During the RV Meteor cruise 21 to the Northeast Atlantic Ocean ( area), from March to May 1992, planktic foraminifera were sampled using a multiple opening-closing net. While sampling, two storms with wind forces up to 12 Beaufort caused intensified surface layer mixing with shifts in the depth of the upper ocean mixed-layer from 20–40 m to 170–240 m. Subsequently, planktic foraminiferal growth rates increased, resulting in an elevated quantity of small (100–150 μm) tests (Phase 1). When the wind strength increased a second time, the mixed-layer deepened to a depth below the former position of the pycnocline, and again the abundance of small tests increased (Phase 2). During Phase 2, the weight of calcite in specimens of the productive zone reached its maximum. In the export zone, an associated increase in empty tests occurred with a lag time depending on the test sinking velocity. In the upper export zone, down to 700 m water depth, CaCO₃ flux increased from 9.3 to 49.8 mg CaCO₃ m⁻² d⁻¹ after the first storm and from 8.9 to 19.9 mg CaCO₃ m⁻²d⁻¹ after the second storm. In the 700 to 2500 m depth interval, the flux increased from 5.1 mg CaCO₃ m⁻² d⁻¹ to about 9.2 mg CaCO, m⁻² d⁻¹. Thus, the standing stock of living foraminifera and export of empty tests from the productive zone increased after the storms, leading to pulses of CaCO₃ exported from the surface to deep water.     

A simplified method for growth of human microvascular endothelial cells results in decreased senescence and continued responsiveness to cytokines and growth factors

Summary Human dermal microvascular endothelial cells are used to analyze the functions of microvascular endothelium in vitro. However, the low yield and short lifespan of these cells in culture has limited the types of analysis that could be performed. Human microvascular endothelial cells are typically grown in media containing supplements such as dibutyryl cyclic AMP, hydrocortisone, bovine brain extract, and antifungal agents, each of which increase the complexity of experimental design and interpretation of results. In the present study, endothelial cells were transferred after Ulex europeus-I selection into a simplified medium consisting of Endothelial Basal Medium 131, 10% fetal bovine serum, and 2 ng/ml basic fibroblast growth factor and analyzed over 3 mo. The human microvascular endothelial cells expressed the endothelial markers von Willebrand factor, CD31, P-selectin, and E-selectin. In addition, the cells showed increased proliferation in the presence of basic fibroblast growth factor (0.5 ng/ml) or vascular endothelial cell growth factor (10 ng/ml). Tumor necrosis factor-α-induced expression of E-selectin was similar in cells at Passages 3, 6, and 12, indicating that the cells maintained responsiveness to cytokines over several weeks. Furthermore, the endothelial cells attained a typical cobblestone morphology with increased cellular density and also formed capillarylike tubes on Matrigel. In summary, the human dermal microvascular endothelial cells display the expected endothelial characteristics when grown for several passages and, therefore, provide a valuable in vitro model for human microvascular endothelium.     

The vertebrate pineal hormone melatonin is produced by the brown alga Pterygophora californica and mimics dark effects on growth rate in the light

Melatonin, a methoxylated indoleamine, plays a role as a mediator of darkness in animals as well as in the unicellular alga Gonyaulax polyedra Stein and was recently detected in higher plants. We report on the first finding of melatonin in a multicellular alga, the brown alga Pterygophora californica Rupr. Melatonin was identified in juvenile sporophytes of P. californica by two independent methods, reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection, and radioimmunoassay. Another indolic metabolite, 5-methoxytryptophol, was also indentified by HPLC. The rapid decline of growth rate upon the onset of darkness in P. californica is mimicked by melatonin in the light, with increasing efficiency from 5 × 10^–5M to 5 × 10^–4M, while no effect was obtained at 10^–5M.Abbreviations ANOVA analysis of variance - DMSO dimethyl sulfoxide - LD light-dark cycle K.L. and A.W. thank Petra Kadel for help with algal cultivation and evaluation of the experiments.     

Accumulation of Demolybdo Nitrate Reductase during Induction and Its Separation from Active Nitrate Reductase in Chlorella

During induction of nitrate reductase in Chlorella vulgaris,synthesis of the precursor, demolybdo cytochrome c reductase,exceeds the synthesis of active enzyme. Evidence is also presentedwhich shows that the purification procedure of Funkhouser etal. [(1980) Plant Physiol. 65: 939] separates demolybdo cytochromec reductase from active nitrate reductase. ¹Supported in part by a grant to B. V. from the Deutsche Forschungsgemeinschaftand a contribution of the Texas Agricultural Experiment Station. (Received July 27, 1983; Accepted September 13, 1983)