Alteration of glucose consumption kinetics with progression of baculovirus infection in Spodoptera frugiperda cells

We have used the initial-rate approach to characterize changes in the glucose consumption kinetics of baculovirus-infected Spodoptera frugiperda clone 9 (Sf9) cells with the progression of the infection process. The specific glucose consumption rate (q _G) of cultured baculovirus-infected Sf9 cells was measured at 4, 8, 12, 16, and 24 h postinfection (h.p.i.) in media containing 4–35 mM glucose. Higher medium glucose concentrations resulted in higher final extracellular virus and recombinant β-galactosidase yields. q _G was related to the extracellular glucose concentration by means of a Michaelis-Menten relationship. The apparent Michaelis-Menten constant (K _m) for glucose consumption was found not to change significantly during the progression of the infection process, and remained between 6.2 and 7.2 mM. However, the maximal specific glucose consumption rate (q _Gmax) was found to rapidly increase after infection, peaking at 16 h.p.i. at a value four times that for uninfected Sf9 cells. The kinetic analysis of glucose consumption rates in baculovirus-infected Sf9 cells presented here will aid in the optimal design and operation of bioreactor systems for the large-scale production of recombinant products from the baculovirus/insect cell system.     

Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE₂ production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-κB DNA binding activity, IκBα degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.     

[4+2]-Cyclodimer of sabinone: formation,crystal structure,and NMR spectra

Oxidation of (+)-sabinol, (1S,3R,5S)-1-isopropyl-4-methylidenebicyclo[3.1.0]hexan-3-ol, by active MnO₂ afforded not the expected sabinone but only its [4+2]-cyclodimer. The molecular structure of the latter was established by X-ray diffraction analysis. The ¹H and ¹³C NMR spectra of this cyclodimer were interpreted using 2D NMR spectroscopy.     

(R)-4-Menthenone in the synthesis of optically pure sex pheromone of the peach leafminer moth (Lyonetia clerkella)

The synthesis of (14S)-methyloctadec-1-ene, sex pheromone of the peach leafminer moth (Lyonetia clerkella), is described to demonstrate a new potential of the synthetic use of (R)-4-menthenone.     

A new approach to arylaliphatic 1,5-, 1,6-, and 1,7-dicarbonyl compounds and their monoacetals based on direct anodic oxidation of 1-phenyl- and benzo[c]cycloalkenes

A new simple approach to -benzoylalkanals, 2-((-formylalkyl)benzaldehydes, and their monoacetals was developed based on direct anodic oxidation of I-phenylcycloalkenes and benzo[ccycloalkenes in methanol followed by acid hydrolysis of the electrolysis products. The target products are obtained in 53–72 % yields.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 2044–2046, August, 1996.     

Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3–5% and detection limits is 0.21 ng Cu g⁻¹.     

Thromboxane A2 modulates migration, proliferation, and differentiation of adipose tissue-derived mesenchymal stem cells

Prostanoid metabolites are key mediators in inflammatory responses, and accumulating evidence suggests that mesenchymal stem cells (MSCs) can be recruited to injured or inflamed tissues. In the present study, we investigated whether prostanoid metabolites can regulate migration, proliferation, and differentiation potentials of MSCs. We demonstrated herein that the stable thromboxane A₂ (TxA₂) mimetic U46619 strongly stimulated migration and proliferation of human adipose tissue-derived MSCs (hADSCs). Furthermore, U46619 treatment increased expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, in hADSCs, suggesting differentiation of hADSCs into smooth muscle-like cells. U46619 activated ERK and p38 MAPK, and pretreatment of the cells with the MEK inhibitor U0126 or the p38 MAPK inhibitor SB202190 abrogated the U46619-induced migration, proliferation, and α-SMA expression. These results suggest that TxA₂ plays a key role in the migration, proliferation, and differentiation of hADSCs into smooth muscle-like cells through signaling mechanisms involving ERK and p38 MAPK.     

Characterization of Citrus aurantifolia bioactive compounds and their inhibition of human pancreatic cancer cells through apoptosis

In the current study, we have isolated and purified five putative compounds from lime (Citrus aurantifolia Swingle) seeds using Soxhlet extraction and chromatographic separation. The purity and structures of the isolated compounds were identified by TLC, HPLC and mass spectra as limonin, limonexic acid (LNA), isolimonexic acid (ILNA), β-sitosterol glucoside (SG) and limonin glucoside (LG). These putative compounds exhibited significant inhibition of Panc-28 cells with IC₅₀ values in the range of 18–42 µM in MTT assay after incubation for 72 h, which was confirmed by viable cell count. Further, induction of apoptosis was confirmed through annexin-FITC staining of the cells and expression of apoptosis related proteins was also studied to understand the mechanism. Expression of apoptosis related protein showed apoptosis through induction of p53 by, p21 dependent manner and cytochrome-c mediated caspases driven intrinsic pathway. Expression of apoptosis favoring proteins in cells treated with isolated compounds were in the order of ILNA > LNA > SG > Limonin > LG based on the expression ratio of bax/bcl2. These results were further supported through fluorescent probed microscopic images. To the best of our knowledge, this is the first report on isolation and identification of LNA, SG and LG from lime and their mechanism of inhibition of human pancreatic cancer cells.     

The behavior of <Emphasis Type="Italic">p</Emphasis>-bis{3-[N-(3-chloro-buten-2-yl)pyrrolidinio(piperidinio or morpholinio)]propyn-1-yl}benzene dichlorides in an aqueous base medium

In aqueous base medium p-bis{3-[N-(3-chlorobuten-2-yl)pyrrolidinio (piperidinio or morpholinio)]-propyn-1-yl}benzene dichlorides undergo a two-way dehydrochlorination-cyclization reaction to form benzo[5,6-a:5′,6′-c]bis(2,2-tetramethylene- or -2,2-pentamethylene-4-methylisoindolinium) and benzo-[5,6-a:5′,6′-c]bisspiro(4-methylisoindoline-2,4′-morpholinium) dichlorides. Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 4, pp. 546–549, April, 2009.     

The mannich reaction in the synthesis of N,S-containing heterocycles 4. Aminomethylation of 6-amino-3,5-dicyano-1,4-dihydropyridine-2-thiolates: A convenient regioselective route to 3,5,7,11-tetraazatricyclo[7.3.1.02,7]tridec-2-ene derivatives

Treatment of N-methylmorpholinium 4-R-6-amino-3,5-dicyano-1,4-dihydropyridine-2-thiolates (R = 2-ClC₆H₄ and 2-MeOC₆H₄) with primary amines in the presence of an excess of formaldehyde gave 13-R-8-thioxo-3,5,7,11-tetraazatricyclo[7.3.1.0^2,7]tridec-2-ene-1,9-dicarbonitrile derivatives in high yields (66–95%). In a similar way, aminomethylation of 3-R-10-amino-7,11-dicyano-9-aza-3-azoniaspiro[5.5]undeca-7,10-diene-8-thiolates (R = Me and Et) afforded 1′-alkyl-8-thioxospiro[3,5,7,11-tetraazatricyclo[7.3.1.0^2,7]tridec-2-ene-13,4′-piperidine]-1,9-dicarbonitriles in 43–91% yields. Alternatively, these compounds were obtained by multicomponent cyclocondensation of N-alkylpiperidin-4-ones, cyanothioacetamide, primary amines, and aqueous formaldehyde. The starting 3-R-10-amino-7,11-dicyano-9-aza-3-azoniaspiro[5.5]undeca-7,10-diene-8-thiolates were prepared by a new method from N-alkylpiperidin-4-ones and cyanothioacetamide. The structure of 5,11-bis(4-ethoxyphenyl)-13-(2-methoxyphenyl)-8-thioxo-3,5,7,11-tetraazatricyclo[7.3.1.0^2,7]tridec-2-ene-1,9-dicarbonitrile was examined by X-ray diffraction analysis. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1014–1022, May, 2007.     

Magnetic properties of the spinel-type

The spinel sulphide CuCr₂S₄ is a metallic ferromagnet with a Curie temperature , while CuHf₂S₄ has no magnetic anomaly. Magnetic properties of the quaternary spinel-type Cu(Cr_1-xHf_x)₂S₄ system have been studied. With increasing x the ferromagnetic properties are weakened gradually from a predominant ferromagnetic, a spin-glass, finally to a simple paramagnetic behavior. For the composition of x0.50, a re-entrant spin-glass phase could emerge, even though the Curie temperature is ill-defined as a ferromagnetic phase boundary. Specimens with x≥0.90 remain paramagnetic down to 4.2 K. A spin crossover phenomenon is found around 160 K in the specimens of x=0.50–0.70. A step-like anomaly is manifestly detected in the magnetization, which corresponds with the change of the spin state. This crossover indicates that the spin state converts from high temperature S=2 into low temperature states. In the ordered states in , the magnetic moment originates from only Cr³⁺ ions.     

beta-Barrel transmembrane proteins: Geometric modelling, detection of transmembrane region, and structural properties

The location of the membrane lipid bilayer relative to a transmembrane protein structure is important in protein engineering. Since it is not present on the determined structures, it is essential to automatically define the membrane embedded protein region in order to test mutation effects or to design potential drugs. beta-Barrel transmembrane proteins, present in nature as outer membrane proteins (OMPs), comprise one of the two transmembrane protein fold classes. Lately, the number of their determined structures has increased and this enables the implementation and evaluation of structure-based annotation methods and their more comprehensive study. In this paper, we propose two new algorithms for (i) the geometric modelling of beta-barrels and (ii) the detection of the transmembrane region of a beta-barrel transmembrane protein. The geometric modelling algorithm combines a non-linear least square minimization method and a genetic algorithm in order to find the characteristics (axis, radius) of a shape with axial symmetry which best models a beta-barrel. The transmembrane region is detected by profiling the external residues of the beta-barrel along its axis in terms of hydrophobicity and existence of aromatic and charged residues. TbB-Tool implements these algorithms and is available in . A non-redundant set of 22 OMPs is used in order to evaluate the algorithms implemented and the results are very satisfying. In addition, we quantify the abundance of all amino acids and the average hydrophobicity for external and internal beta-stranded residues along the axis of beta-barrel, thus confirming and extending other researchers' results.     

Optical probes for detection and quantification of neutrophils’ oxidative burst. A review

Neutrophils, also known as polymorphonuclear leukocytes (PMN), are the most common type of white blood cells, comprising about 50-70% of all white blood cells. In the event of inflammatory processes, neutrophils display increased mobility, tissue influx ability, prolonged life span, and an increased phagocytic capacity, constituting the initial participants in the cellular defense of the organism. One of the most important defense systems of neutrophils corresponds to their ability to mediate a strong oxidative burst through the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). While oxidative burst is important for the elimination of invading microorganisms, the overproduction of ROS and RNS or the impairment of endogenous antioxidant defenses may result to detrimental effects to the host. The nature and the extent of ROS and RNS production by neutrophils in response to different stimuli is, consequently, a matter of extensive research, with scientific reports showing an enormous variability on the detection methodologies employed. This review attempts to provide a critical assessment of the most common approaches to identify and quantify reactive species formed during the neutrophils’ oxidative burst. The detection mechanisms and performance, as well as advantages and limitations of the different methodologies, are scrutinized, focusing on the use of fluorimetric, chemiluminometric and colorimetric probes.     

Thermodynamic and spectroscopic study of the interaction of Cu(II), Ni(II), Zn(II) and Ca(II) ions with 2-amino-N-(2-oxo-2-(2-(pyridin-2-yl)ethyl amino)ethyl)acetamide,a pseudo-mimic of human serum albumin

The protonation equilibria of 2-amino-N-(2-oxo-2-(2-(pyridin-2-yl)ethyl amino)ethyl)acetamide ([H₂(556)–N]) and the complexation of this ligand with Cu(II) Ca(II), Zn(II) and Ni(II) have been studied by glass electrode potentiometry and UV–visible spectrophotometry. From pH ∼2.00–11.00, five models for Cu(II) with the following complexes; MLH, ML, MLH₋₁, MLH₋₂ and MLH₋₃ were generated and observed to describe the experimental data equally well as far as the statistical criteria were concerned. The MLH₋₂ complex predominates at physiological pH in all five models, while the MLH₋₁ complex species exists only at low concentration in two models. The coordination in the MLH₋₂ complex suggested the involvement of one amino, two deprotonated peptides and one pyridyl nitrogen atoms. Molecular mechanics (MM) calculations confirmed the MLH₋₂ complex as the most stable species. Speciation calculations, using a blood plasma model, predicted that the Cu(II)–[H₂(556)–N] complex is able to mobilize Cu(II). Octanol/water partition of CuLH₋₂ showed that 30% of the complex went into the octanol phase, hence promoting percutaneous absorption of copper. The complex is a poor mimic of native copper–zinc superoxide dismutase.     

Common methods for the chiral determination of amphetamine and related compounds I. Gas, liquid and thin-layer chromatography

This article reviews the most common, useful methods for the chiral determination of amphetamine (AM) and AM-derived designer drugs in different of matrix, including blood, hair, urine, medicaments or standard solutions, taking into consideration articles published in the past 15 years. We consider chromatographic methods (e.g., gas, liquid, high-performance liquid, and thin layer). We describe several types of chiral derivatization reagent, mobile-phase additive and chiral stationary phase commonly used in the chromatographic methods. Tables summarize basic information about conditions (e.g., type of column and mobile phase), detection mode and reference data for each procedure.