Phenotypic plasticity and targeting of Siglec-F(high) CD11c(low) eosinophils to the airway in a murine model of asthma.

Eosinophil recruitment in asthma is a multistep process, involving both trans-endothelial migration to the lung interstitium and trans-epithelial migration into the airways. While the trans-endothelial step is well studied, trans-epithelial recruitment is less understood. To contrast eosinophil recruitment between these two compartments, we employed a murine kinetics model of asthma. Eosinophils were phenotyped by multicolor flow cytometry in digested lung tissue and bronchoalveolar lavage (BAL) simultaneously, 6 h after each ovalbumin (OVA) challenge. There was an early expansion of tissue eosinophils after OVA challenge followed by eosinophil buildup in both compartments and a shift in phenotype over the course of the asthma model. Gradual transition from a Siglec-F(med) CD11c(-) to a Siglec-F(high) CD11c(low) phenotype in lung tissue was associated with eosinophil recruitment to the airways, as all BAL eosinophils were of the latter phenotype. Secondary microarray analysis of tissue-activated eosinophils demonstrated upregulation of specific integrin and chemokine receptor signature suggesting interaction with the mucosa. Using adhesion assays, we demonstrated that integrin CD11c mediated adhesion of eosinophils to fibrinogen, a significant component of epithelial barrier repair and remodeling. To the best of our knowledge, this is the only report to date dissecting compartmentalization of eosinophil recruitment as it unfolds during allergic inflammation. By capturing the kinetics of eosinophil phenotypic change in both tissue and BAL using flow cytometry and sorting, we were able to demonstrate a previously undocumented association between phenotypic shift of tissue-recruited eosinophils and their trans-epithelial movement, which implicates the existence of a specific mechanism targeting these cells to mucosal airways.

Tissue-resident alveolar macrophages reduce O3-induced inflammation via MerTK mediated efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3) - one of the six criteria air pollutants - is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung and their number increases following O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. Here, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage tracing experiments showed that 12, 24, and 72 h after exposure to O3 (2 ppm) for 3h all AMØs were tissue-resident origin. Similarly, in humans exposed to FA and O3 (200 ppb) for 135 minutes, we did not observe ~21h post-exposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØ demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK - a key receptor involved in efferocytosis - also resulted in impaired clearance of apoptotic neutrophils followed O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

Fourier transform ion cyclotron resonance mass spectrometer with coaxial multi-electrode cell ('O-trap'): first experimental demonstration.

The conceptual design of the O-trap Fourier transform ion cyclotron resonance (FT-ICR) cell addresses the speed of analysis issue in FT-ICR mass spectrometry. The concept of the O-trap includes separating the functions of ion excitation and detection between two different FT-ICR cell compartments. The detection compartment of the O-trap implements additional internal coaxial electrodes around which ions with excited cyclotron motion revolve. The expected benefits are higher resolving power and the lesser effect of the space charge. In this work we present the first experimental demonstration of the O-trap cell and its features, including the high ion transfer efficiency between two distinct compartments of an ICR cell after excitation of the coherent cyclotron motion. We demonstrate that utilization of the multiple-electrode detection in the O-trap provides mass resolving power enhancement (achieved over a certain time) equal to the order of the frequency multiplication. In an O-trap installed in a 5 T desk-top cryogen-free superconducting magnet, the resolving power of R = 80,000 was achieved for bradykinin [M + 2H](2+) (m/z 531; equivalent to 100,000 when recalculated for m/z 400) in 0.2 s analysis time (transient length), and R = 300,000 at m/z 531 for a 1 s transient. In both cases, detection on the third multiple of the cyclotron frequency was implemented. In terms of the acquisition speed at fixed resolving power, such performance is equivalent to conventional FT-ICR detection using a 15 T magnet.

[Synthesis of secasterol and 24-episecasterol and their toxicity in MCF-7 cells].

A convergent synthesis of biosynthetic precursors of brassinosteroids - secasterol and 24-episecasterol with Δ²-bond in cycle A is described. The key stages in the construction of the side chain of these compounds were Julia olefination of steroid 22-aldehyde followed by asymmetric Sharpless dihydroxylation of the intermediate Δ²²-olefin. Toxicity of synthesized compounds against breast carcinoma MCF-7 cells was studied.

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion.

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (T(reg)) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A-Z). In transgenic and WT mice, regardless of T(reg) depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After T(reg) depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4(+) T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA-d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.

[(22R,23R)-3beta-Hydroxy-22,23-epoxy-5alpha-ergost-8(14)-en-15-one: improved synthesis and toxicity to MCF-7 cells].

The chemical synthesis of (22R,23R)-3beta-hydroxy-22,23-epoxy-5alpha-ergost-8(14)-en-15-one from (22E)-3beta-acetoxy-5alpha-ergosta-7,14,22-triene was improved. The stages of obtaining and isomerizing (22E)-3beta-acetoxy-14alpha,15alpha-epoxy-5alpha-ergosta-7,22-diene were optimized. The introduction of the (22R,23R)-epoxide cycle was carried out by a basic treatment of intermediate (22S,23R)-3beta,23-diacetoxy-22-iodo-5alpha-ergost-8(14)-en-15-one. In cells of human mammary gland carcinoma MCF-7 (22R,23R)-3beta-hydroxy-22,23-epoxy-5alpha-ergost-8(14)-en-15-one showed a high toxicity (TC(50) = 0.4 +/- 0.1 microM for 48-h incubation in serum-free medium).

[Cytotoxic derivatives of (22R,23R)-22,23-dihydroxystigmastane].

(22R,23R)-22,23-dihydroxystigmast-4-en-3-one, (22R,23R)-22,23-dihydroxystigmast-4-en-3,6-dione, (22R,23R)-3beta,5alpha,6beta,22,23-pentahydroxystigmastane, (22R,23R)-5alpha,6alpha-oxido-3beta,22,23-trihydroxystigmastane, (22R,23R)-5beta,6beta-oxido-3beta,22,23-trihydroxystigmastane, and (22R,23R)-3beta,6beta,22,23-tetrahydroxystigmast-4-ene were synthesized. Their cytotoxicities were comparatively studied using the MCF-7 line of carcinoma cells of human mammary gland and cells of human hepatoma of the Hep G2 line.

[A simple synthesis of 3beta-acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene].

3beta-Acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene were synthesized from (22R,23R)-sitost-5-ene-3beta,22,23-triol in 66% overall yields.

[Delta5-7-Ketosterols with modified side chain: the synthesis and the effects on viability and cholesterol biosynthesis in Hep G2 cells].

(22E)-3beta-Hydroxysitosta-5,22-dien-7-one, (22R, 23R)-3beta,22,23-trihydroxysitost-5-en-7-one, and (22R, 23R)-3beta-hydroxy-22,23-isopropylidenedioxysitost-5-en-7-one were synthesized. The cytotoxicity and effects on cholesterol biosynthesis of the resulting 7-ketosterols, 7-ketocholesterol, and (22S,23S)-3beta-hydroxy-22,23-oxidositost-5-en-7-one were studied in hepatoblastoma Hep G2 cells.

[Interaction of novel oxazoline derivatives of 17(20)e-pregna-5,17(20)-diene with cytochrome P450 17A1].

In order to find novel inhibitors of 17a-hydroxylase-17,20-lyase (cytochrome P450 17A1, CYP17A1), a key enzyme of biosynthesis of androgens, molecular docking of six new oxazoline-containing derivatives 17(20)E-pregna-5,17(20)-diene has been carried out to the active site of the crystal structure of CYP17A1 (pdb 3ruk). Results of this study indicate that: 1) complex formation of docked compounds with CYP17A1 causes their isomerization in energetically less favorable 17(20)Z-isomer; 2) the localization of the steroid moiety of all compounds in the active site is basically the same; 3) the structure of the oxazoline moiety significantly influences its position relative to heme as well as the energy of complex formation; 4) coordination of the nitrogen atom of the oxazoline moiety and the heme iron is only possible in the 17(20)Z-conformation with anti oriented double bonds 17(20), and C=N; 5) the presence of two substituents at C4' of the oxazoline moiety significantly impairs ligand binding; 6) oxazoline--and benzoxazole-containing derivatives 17(20)E-pregna-5,17(20)-diene can effectively inhibit the catalytic activity CYP17A1 and may be of interest as a basis for the development of new drugs for the treatment of androgen-dependent cancer.

Binding of partially reassembled high-density lipoprotein to isolated human small intestine epithelial cells. Effect of lipid composition.

The binding of human high-density lipoprotein (HDL3), apolipoprotein A-I (apoA-I) and recombinants of apoA-I with cholesterol and/or dimyristoylphosphatidylcholine (DMPC) to the HDL receptor on isolated human small intestine epithelial cells was studied. ApoA-I competed for 125I-labelled HDL3 binding sites less effectively than HDL3, and a lower amount of 125I-labelled apoA-I than 125I-HDL3 was bound to cells. The apoA-I/DMPC recombinant competed for 125I-HDL3 binding sites nearly as well as HDL3, and 125I-apoA-I/DMPC recombinant bound to cells with at least the same efficiency as 125I-HDL3. The apoA-I/DMPC/cholesterol recombinant failed to compete for 125I-HDL3 binding sites, and the 125I-apoA-I/DMPC/cholesterol complex binding to cells was several-fold lower than that of other particles. All particles bound to cells with similar dissociation constants. Tetranitromethane-modified HDL3 failed to bind to high-affinity specific binding sites and compete with 125I-HDL3 for binding. The results obtained make it possible to assume that, while apoA-I may be a determinant of the HDL receptor, the lipid composition of the lipoprotein may affect its interaction with the receptor.

[New delta8(14)-ketosterols with an oxygenated side chain].

New analogues of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3beta-hydroxy-24-methyl-22,23-oxido-5alpha-cholest-8(14)-en-15-ones and (22RS,23xi,24S)-24-methyl-5alpha-cholesta-3beta,22,23-triol-15-one] were synthesized from (22E,24S)-3beta-acetoxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC50 0.9 +/- 0.2 and 0.7 +/- 0.2 microM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC50 4.0 +/- 0.5 microM), (22E,24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one (IC50 3.1 +/- 0.4 microM), and the 3beta,22,23-triol synthesized (IC50 6.0 +/- 1.0 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

[22,23-epoxides of sitosterol and related 7-oxygenated delta5-sterols].

(22S,23S)-22,23-Epoxysitosterol, (22R,23R)-22,23-epoxysitosterol, (22S,23S)-22,23-epoxy-7-ketositosterol, (22R,23R)-22,23-epoxy-7-ketositosterol, (22S,23S)-22,23-epoxy-7alpha-hydroxysitosterol, (22R,23R)-22,23-epoxy-7alpha-hydroxysitosterol, (22S,23S)-22,23-epoxy-7beta-hydroxysitosterol, and (22R,23R)-22,23-epoxy-7beta-hydroxysitosterol were synthesized. Their 1H and 13C NMR and the mass spectra of their trimethylsilyl derivatives were studied.

Does a HDL injection reduce the development of serum hyperlipidemia and progression of fatty streaks in cholesterol fed rabbits?

The influences of homologous (rabbit) or heterologous (human) high density lipoprotein (HDL) on the development of serum hyperlipidemia and progression of fatty streaks were studied in cholesterol fed rabbits. Three groups of New Zealand rabbits were fed a 0.5% cholesterol rich diet for 8 weeks. Additionally into these animals the following solutions were injected intravenously two times per week: group 1 (control): saline; group 2: human HDL dissolved in saline; group 3: rabbit HDL dissolved in saline. The animals of group 2 had lower serum cholesterol levels during the dietary period than rabbits of group 1 (p < 0.05) but the surface of intima covered with fatty streaks was the same as in group 1. On the other hand, the serum cholesterol level in rabbits of group 3 was the same as in group 1 during the whole experimental period, but the surface of aorta covered with fatty streaks was significantly lower (p < 0.05) in group 3 than in group 1. The results of this study support the hypothesis of an antiatherogenic action of HDL, which seems to be independent of the influence of HDL on the serum lipids but depends on the source of HDL.

[Interaction of spin-labeled analogues of vitamin B 6 with the active site of apotransaminase]. / Vzaimodeistvie spin-mechenykh analogov vitamina B6s aktivnym tsentrom apotransaminazy

Spin-labeled analogues of vitamin B6: 2, 2, 6, 6-tetramethyl-N-oxylpiperydinyl-4-(5' phosphopyridoxyl)-amine (1) and 2, 2, 6, 6-tetramethyl-N-oxyl-piperydinyl-4-(pyridoxal-5')-phosphate (II) are synthesized. There analogues were shown to interact in the equimolar ratio with the active site of cytosol aspartate transaminase. It was proved by CD-titration of apotransaminase with I and II and by competition between the coenzyme and synthesized analogues. The free valency of spin-labeled coenzymes immediately disappears after interaction with the apoenzyme due to iminoxyl group reduction. The binding of I and II with the apoenzyme is accompanied by oxidation of one of the inner cysteine residues. The reactivation of the modified apoenzyme with PLP is not less than 65% of original transaminase activity. The analysis of space-filling atomic models of synthesized compounds allows to conclude that the distance between the centre of pyridine ring of the coenzyme and the modified thiol group is not more than 8 A.

[Macromolecule rotative correlation time measurement by ESR for covalently bound spin label]. / Izmerenie vremeni vrashchatelnoi korreliatsii makromolekul metodom EPR v sluchae kovalentno sviazannoi spin-metki.

The dependence from temperature and viscosity of the shifts of the internal and external wide extremums in the ESR spectra of spin labelled bovine serum albumin has been studied. 2,2,6,6-tetramethylpiperidine-NI-oxyl-4-iodacetamide was used as a spin label. The obtained dependences was shown to be a consequence of the label participation in two types of rotations: an anisotropic fast rotation with tau less than 10(-9) sec relatively to a macromolecule, and the isotropic one with tau greater than 10(-8) sec due to rotation of the macromolecule itself. These conclusions were done on the basis of a model for complex rotation of the spin label. Comparison of theoretical and experimental data makes it possible to determined the correlation time for the protein moiety, to evaluate quantitatively the polarity of surroundings of the iminoxyl and to introduce a numerical parameter for the degree of mobility of the spin label relatively to protein molecule.

[Nonglycosidic analogues of nucleotides. IX. Self-association of 9-(omega'-hydroxyalkyl) adenine omega'-phosphates in aqueous solutions]. / Neglikozidnye analogi nukleotidov. 9. Samoassotsiatsiia 9-(Omera-oksialkil) adenin-omera-fosfatov v vodnyx rastvorakh.

The dependence of the NMR spectra of adenosine 5'-phosphate and phosphates of 9-(2'-hydroxyethyl)-, 9-(3'-hydroxypropyl)- and 9-(4'-hydroxybutyl)adenines on temperature and concentration has been investigated in aqueous solutions.

[Interaction of apolipoprotein A1 with phospholipids and structure of mixed micelles]. / Vzaimodeistviie apolipoproteina A1 s fosfolipidami i struktura smeshannykh mitsell.

The structural features of apolipoprotein A1 (apoA1) that provide for the formation of its stable micellar complexes with phosphatidylcholine are discussed. The results of studies on the secondary structure and functional properties of separate sites of the apoA1 polypeptide chain are analyzed. The preparation procedures for discoidal micellar complexes of apoA1 with phosphatidylcholine are discussed, and the characteristics of the complexes are described. The surface activity of apoA1 incorporated into the micellar complexes is characterized, and the recombination mechanisms of lipid-protein micelles in model systems are discussed. The data discussed indicate that all currently known structural-functional properties of apoA1 and its involvement in metabolic and physiological processes are completely determined by the surface activity of the amphiphilic fragments of its conformationally labile polypeptide chain.

[Features of the structure of apolipoprotein A1 as an HDL and complexes with dimyristoylphosphatidylcholine. A method for low temperature ESR-spectroscopy]. / Osobennosti struktury apolipoproteina A1 v sostave LVP i kompleksov s dimiristoilfosfatidilkholinom. Issledovanie metodom nizkotemperaturnoi EPR-spektroskopii.

Apolipoprotein A1 (apoA1) was labelled with the excess of 2,2,6,6-tetramethylpiperidinyl-N-oxyl-4-(2,4-dichloro-1,3,5- triazinyl-6)-amine at pH 9.8. The products, containing 7.2 (+/- 1) paramagnetic labels per 1 molecule of apoA1, showed spin-spin and dipole-dipole exchange interactions. The ESR spectra of the spin labelled A1 (at 77K) had d1/d values 0.76 (at pH 7.4), 0.59 (in 3 M NaCl), 0.55 (in 3 M guanidinium hydrochloride), 0.44 (in 40% 2-chloroethanol). Micellar complexes of spin labelled apoA1/DMPC (1 : 20 mol/mol and 1 : 190 mol/mol) and HDL containing spin labelled apoA1 were prepared. Comparison of ESR spectra at 77 K showed that apoA1 structure varies in the complexes with different stoichiometry and in spin labelled HDL. These data show the importance of hydrophobic protein-protein interactions for the structure of HDL and synthetic complexes of apoA1 with phosphatidyl choline.

[Stereospecific reduction of keto group in 3beta-triphenylmethoxy-5alpha-cholest-8(14)-en-15-one]. / Stereospetsifichnoe vosstanovlenie ketogruppy v 3beta-trifenilmetoksi-5alpha-kholest-8(14)-en-15-one.

The reduction of 3 beta-triphenylmethoxy-5 alpha-cholest-8(14)-en-15-one with lithium aluminum hydride resulted in a quantitative yield of 3 beta-triphenylmethoxy-5 alpha-cholest-8(14)-en-15 beta-ol.