CYP90A1/CPD, a Brassinosteroid Biosynthetic Cytochrome P450 of Arabidopsis, Catalyzes C-3 Oxidation

Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.     

The effects of some Curdlan derivatives on Dectin-1 expression and cytokine production in human peripheral blood mononuclear cells

The cells of immune system such as monocytes and macrophages are in first line defence against dangerous signals. In the present paper the recognition of Dectin 1 receptors and the modulation of Interleukin-10 (IL-10) and Tumor Necrosis Factor-alpha (TNF-alpha) cytokine production by Curdlan and Curdlan derivatives in peripheral blood mononuclear cells (PBMCs) were studied. The effect of Curdlan or Curdlan derivatives on the expression of Dectin 1 receptors in PBMCs was revealed by flow-cytometry and the levels of IL-10 and TNFalpha were measured by ELISA kit in supernatants of PBMCs cultured in presence or absence of Curdlan, Curdlan derivatives and LPS. Our results suggested that Curdlan and Curdlan derivatives were able to increase the expression of Dectin-1 receptors on monocyte cells. The combined treatment of Curdlan/Curdlan derivatives and Pam3Cys produced an increase of CD14+ cells possessing Dectin-1 receptors. We demonstrated that Curdlan (at 20 microg unique dose) up-regulated TNF-alpha production and down-regulated IL-10 production in PBMCs. Conversely, Palm CM/SP-Curdlan (20 microg unique dose) was able to down-regulate TNF-alpha production and to up-regulate IL-10 production in PBMCs. For instance, Palm CM/SP-Curdlan determined a 5 times decrease of TNF-alpha production than Curdlan. Regarding the effect of Palm CM/SP-Curdlan on IL-10 production in PBMCs, we noticed that the level of IL-10 was about 4 times greater than Curdlan activity. We observed that a combined treatment of Curdlan/Curdlan derivatives and LPS induced about 5 times decrease in TNF-alpha production in PBMCs. IL-10 production induced by Palm CM/SP-Curdlan and LPS was about 6 times greater than the combined effect of Curdlan and LPS. The treatment of PBMCs with SP-Curdlan alone affected neither TNF-alpha production nor IL-10 production. Our results are in accordance with other studies demonstrating that Dectin-1 and TLR2/TLR6 signaling combine to enhance the responses triggered by each receptor and the signaling pathway induced by Dectin-1 could mediate the production of pro-inflammatory cytokines.     

Curdlan microspheres. Synthesis,characterization and interaction with proteins (enzymes,vaccines)

Microparticles of curdlan, synthesized through crosslinking with epichlorohydrin in organic suspension media, were chemically modified with the aim of introducing strongly and/or weakly acidic anionic and palmitoyl hydrophobic groups. Microparticles of both curdlan and curdlan derivatives were physico-chemically characterized. Study of the interaction with enzymes, such as lysozyme, and vaccines, such as tetanus anatoxin, showed a co-operative protein retention effect, induced by electrostatic and hydrophobic forces. The results of the in vitro release studies on support–protein complexes recommend them as potential controlled release systems.     

Substrate Compliance versus Ligand Density in Cell on Gel Responses

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on “rigid” glass or “stiff” gels than on “soft” gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.     

Design and synthesis of potent, orally active, inhibitors of carboxypeptidase U (TAFIa)

A series of 3-mercapto-propionic acid derivatives that function as reversible inhibitors of carboxypeptidase U have been prepared. We present a successful design strategy using cyclic, low basicity guanidine mimetics resulting in potent, selective and bioavailable inhibitors of carboxypeptidase U (TAFIa).     

Phase separation liquid-liquid extraction for the quantification of 8-iso-Prostaglandin F2 Alpha in human plasma by LC-MS/MS

BackgroundReactive oxygen species (ROS) are produced in the body during normal metabolism by means of enzymes and non-enzymatic chemical reduction of molecular oxygen. In case of the prevalence of ROS formation over their elimination, highly reactive free radicals can be accumulated and can cause multiple damages to the biomolecules and cells. Determination of isoprostanes in biological matrices is most often used to register free radical damage and requires selective, sensitive and specific techniques.MethodsThis study presents the development and validation of the LC-MS/MS method for the determination of 8-iso-Prostaglandin F2α in human plasma utilising a modified liquid-liquid extraction procedure with phase separation.ResultsModified sample preparation procedure assured higher extraction yield, clear separation of organic layer from the plasma water phase and protein precipitates, and better-purified product for instrumental analysis. Linearity was validated in the range 0.1-5.0 µg/L with R2 > 0.996; normalised matrix varied between 86.0% and 108.3%, accuracy ranged from 90.4 % to 113.9% and precision both within runs and between runs was less than 7%. With a run time of 10 min, a throughput of over 50 samples per working day could be performed.ConclusionsThe method meets all the current industrial validation criteria and allows the accurate and precise determination of 8-iso-PGF2α in human plasma at diagnostically significant concentration range.     

Über die chemische und photodynamische Wirkung von Neutralrot auf die Rotationsströmung beiHordeum vulgare L.

Zusammenfassung Die chemische und photodynamische Neutralrotwirkung auf die Rotationsströmung wurde in Abhängigkeit von den Absorptionsmaxima des Farbstoffes untersucht. Als Material dienten die Wurzelhaare der Gerste (Hordeum vulgare L.). Die Wurzelhaare wurden mit monochromatischem Licht verschiedener Wellenlängen (455, 499, 524, 553 und 654 nm) belichtet, dessen Wirkung auf die Kontrolle (nicht angefärbte Wurzelhaare) und die angefärbten Wurzelhaare getrennt verfolgt wurde.Die Kontrollwerte ergaben, daß das monochromatische Licht je nach dessen Wellenlänge eine verschiedene Wirkung auf die Rotationsströmung ausübte. Das blaue (455 nm) und das dunkelgrüne Licht (524 nm) induzierten die betonteste Stimulationswirkung. Das Rotlicht (654 nm) führte zu einer weit geringeren Stimulation der Rotation.Die Neutralrotwirkung war vom Absorptionsspektrum des Farbstoffes abhängig, jedoch mußte bei der Besprechung der Ergebnisse auch das Verhalten der nicht angefärbten Wurzelhaare unter monochromatischer Belichtung beachtet werden. Festzustellen ist, daß die betonteste Rotationshemmung im Dimeren-Bereich des Absorptionsspektrums des Farbstoffes (500 nm) stattfand. Sie ist der durch das Neutralrot induzierten photodynamischen Wirkung zu verdanken. In den Wellenlängen entsprechend der Maximalabsorption der Farbmoleküle (450 nm) und der monomeren Farbkationen (530 nm) war die photodynamische Wirkung schwächer als im Absorptionsbereich der dimeren Farbkationen. Im hellgrünen (V-Bande, 540–550 nm) und besonders im roten Licht (654 nm) fand vorzugsweise ein chemischer Neutralroteinfluß statt.Die erhaltenen Ergebnisse berechtigen zu der Annahme, daß das Neutralrot durch Adsorption labil an die kontraktilen Proteine des Cytoplasmas gebunden vorliegt. Die festgestellte betonte Hemmung im blaugrünen Licht (500 nm) läßt vermuten, daß der in dimerer Form an Proteine adsorbierte Anteil des Neutralrot eine besonders wichtige Rolle beim Zustandekommen des photodynamischen Effektes spielt.

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The chemical and photodynamic action of neutral red on rotational streaming in barley (Hordeum vulgare L.) root hairs

Summary The chemical and photodynamic action of neutral red on rotational streaming was studied in relation with absorption maxima of the dye. The barley (Hordeum vulgare L.) root hairs were employed. They were iluminated with monochromatic light of different wavelengths (455, 499, 524, 553, and 654 nm) and their effect were separately recorded both for control (root hairs without dye) and stained root hairs.The data from the control show, that monochromatic light has a different action on rotational streaming depending of its wavelength. The blue light (455 nm) and the dark green light (524 nm) has brought about a higher stimulation of rotational streaming. The red light (654 nm) has stimulated the rotational streaming in a little extent.The effect of neutral red was in relation with the absorption spectrum of the dye, but the discussion of the results must take into account also the behaviour of the control in the monochromatic light. The data show that the strongest inhibition of rotational streaming took place in the dimere region (500 nm) of the dye absorption spectrum. This inhibition was due to photodynamic action of neutral red. In the wavelengths corresponding to maximum absorption of light by the molecule (450 nm) and monomere (530 nm) of neutral red, the photodynamic action was weaker than in the dimere region of the spectrum. In light-green (540–550 nm) and especially in red (654 nm), only the chemical effect of neutral red was shown.The obtained data show that the neutral red is probably labile adsorbed on the cytoplasmic contractile proteins. The strongest inhibition in blue green light (500 nm) also points out that the proportion of neutral red adsorbed as a dimere on the proteine molecule plays an especial role in the promotion of photodynamic effect.

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Die Unkosten dieser Arbeit wurden von der Akademie der S.R. Rumänien getragen. Für die wertvollen Hinweise und Anleitungen sprechen wir unserem Lehrer, Prof. Dr.Emil pop, den herzlichsten Dank aus.     

The Effect of ATP (Disodium Salt) upon Rotational Streaming

Through the continuous treatment with various solutions of ATP disodium salt the rotational streaming of the cytoplasma in barley root hairs has been stimulated about 1.2–1.7 times. With the concentrations employed the stimulation of the streaming did not depend on the external ATP supply, but on the initial rate of streaming. It is assumed that the main source of energy supporting the protoplasmic streaming is ATP. Therefore, the results obtained may be interpreted on the basis of variations in ATP content and its degradation products. The differences between initial rates of streaming are supposed to be due to variations of the endogenous ATP level. The ATP taken up probably stimulates the rotational streaming both through the supply of delivered energy and by lowering the cytoplasm viscosity. On the contrary, products of ATP hydrolysis increase the cytoplasm viscosity and induce a lowering or even cessation of the streaming.