sFRP-1 binds via its netrin-related motif to the N-module of thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA-MB-231 breast carcinoma cell adhesion and migration

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K_d = 48 nM and the related sFRP-2 with a K_d = 95 nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited α3β1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.     

Cytotoxic effects of new trans-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines and their interaction with antitumoral drugs gemcitabine and paclitaxel on cellular lines of human breast cancer

Two tetrahydroquinoline compounds, called DM8 and DM12, from a new series of the cis-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines, were selected for cytotoxic effects studies on cellular lines of human breast cancer. The synergistic, additive and antagonistic effects in combination of these compounds with anticancer drugs, such as paclitaxel and gemcitabine, were studied. The isobolograms and their analysis demonstrated models of synergism, additivity and antagonism of these tetrahydroquinolines in the presence of paclitaxel and gemcitabine. Results showed that compounds DM8 and DM12 individually induced growth inhibition on breast cancer cell lines MCF-7 and SKBR3, and the addition of paclitaxel and gemcitabine intensified their cytotoxic activity on both cell lines at conc. below 1 μg/mL. During these studies the compound DM12 was identified as new, perspective and safe agent for adjuvant therapy.     

Organ printing: from bioprinter to organ biofabrication line

Organ printing, or the layer by layer additive robotic biofabrication of functional three-dimensional tissue and organ constructs using self-assembling tissue spheroid building blocks, is a rapidly emerging technology that promises to transform tissue engineering into a commercially successful biomedical industry. It is increasingly obvious that similar well-established industries implement automated robotic systems on the path to commercial translation and economic success. The use of robotic bioprinters alone however is not sufficient for the development of large industrial scale organ biofabrication. The design and development of a fully integrated organ biofabrication line is imperative for the commercial translation of organ printing technology. This paper presents recent progress and challenges in the development of the essential components of an organ biofabrication line.     

Restoring leptin signaling reduces hyperlipidemia and improves vascular stiffness induced by chronic intermittent hypoxia

Chronic intermittent hypoxia (IH) during sleep can result from obstructive sleep apnea (OSA), a disorder that is particularly prevalent in obesity. OSA is associated with high levels of circulating leptin, cardiovascular dysfunction, and dyslipidemia. Relationships between leptin and cardiovascular function in OSA and chronic IH are poorly understood. We exposed lean wild-type (WT) and obese leptin-deficient ob/ob mice to IH for 4 wk, with and without leptin infusion, and measured cardiovascular indices including aortic vascular stiffness, endothelial function, cardiac myocyte morphology, and contractile properties. At baseline, ob/ob mice had decreased vascular compliance and endothelial function vs. WT mice. We found that 4 wk of IH decreased vascular compliance and endothelial relaxation responses to acetylcholine in both WT and leptin-deficient ob/ob animals. Recombinant leptin infusion in both strains restored IH-induced vascular abnormalities toward normoxic WT levels. Cardiac myocyte morphology and function were unaltered by IH. Serum cholesterol and triglyceride levels were significantly decreased by leptin treatment in IH mice, as was hepatic stearoyl-Coenzyme A desaturase 1 expression. Taken together, these data suggest that restoring normal leptin signaling can reduce vascular stiffness, increase endothelial relaxation, and correct dyslipidemia associated with IH.     

Synthesis of α-mannosylated ergot alkaloids employing α-mannosidase

The ergot alkaloids elymoclavine, ergometrine and chanoclavine were α-mannosylated with α-mannosidase as catalyst. The kinetic reaction with p-nitrophenyl α-mannoside as glycosyl donor gave ca 28 % yield of chanoclavine α-mannoside, whereas the equilibrium reaction with mannose as the glycosyl donor gave ca 11 % yield. However, in the case of elymoclavine and ergometrine, higher yields of α-mannosides were obtained with the equilibrium approach (18 and 13 %).     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study

Thermus thermophilus catalase. Flash fluorescence studies indicate that the S₃ state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S₁ with an apparent halftime of ca. 0.4 s at about 18 °C, compared with a biphasic decay, with approximate halftimes of 28 s for S₃ to S₂ and 140 s for S₂ to S₁ in the absence of NO. Under similar conditions the S₂ state is reduced by NO to the S₁ state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S₁ state at −30°C, via the S₀ and S₋₁ states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S₃ state of the OEC, which is compatible with a metalloradical character of this state. Received: 16 June 1999 / Accepted: 28 February 2000     

Aerobic TNT Reduction via 2-Hydroxylamino-4,6-Dinitrotoluene by Pseudomonas aeruginosa Strain MX Isolated from Munitions-Contaminated Soil

Bioremediation of munitions-contaminated soil requires effective transformation and detoxification of high concentrations of 2,4,6-trinitrotoluene (TNT). Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, aerobically transformed TNT (100 mg/L) in culture medium within 15 h, causing transient accumulation of hydroxylaminodinitrotoluenes (HADNTs). The predominance of 2-hydroxylamino-4,6-dinitrotoluene (2HADNT), as well as 2-amino-4,6-dinitrotoluene (2ADNT) and 4,4' ,6,6' -tetranitro-2,2' -azoxytoluene (2,2'AZT), indicated preferential reduction of the TNT ortho nitro group. While only 12% of the TNT was transformed to 2ADNT, up to 65% was transformed to tetranitroazoxytoluenes (AZTs), which accumulated as a precipitate. The precipitate was formed by microscopic particles adhering to bacterial cells, which subsequently formed clusters containing lysed cells. Toxicity toward bacteria was primarily attributed to 2ADNT, because pure AZTs preincubated with sterile medium had little effect on the strain. While the culture medium containing TNT exhibited toxicity toward corn (Zea mays L.) and witchgrass (Panicum capillare L.), little phytotoxicity was observed after incubating with P. aeruginosa strain MX for 4 d. Strong binding of HADNTs to soil and low AZT bioavailability may further promote the detoxification of TNT in soil.     

Oxidative addition of 3,6-di-tert-butyl-o-benzoquinone and 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone to SnCl2

Addition of 3,6-di-tert-butyl-o-benzoquinone (3,6-DBBQ) to SnCl₂ in THF leads to the oxidation of Sn(II) to Sn(IV) with formation of catecholate complex (3,6-DBCat)SnCl₂ · 2THF (1), where 3,6-DBCat is 3,6-di-tert-butyl-catecholate dianion. The reaction of 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone (IBQ-Prⁱ) also proceeds on the oxidative-addition mechanism yielding bis-iminosemiquinonato species (ISQ-Prⁱ)₂SnCl₂(2), where ISQ-Prⁱ is anion-radical 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzosemiquinolate. The complexes have been characterized by IR, X-band EPR, ¹H NMR (for 1) spectroscopy and magnetochemistry (for 2). X-ray analysis data show the distorted octahedral environment of tin(IV) for both complexes. Complex 1 is diamagnetic (ground state S = 0), while 2 has triplet ground state (S = 1, biradical). Catecholate complex 1 is able to be a spin trap for different organic radicals.     

Prevalence of intron gain over intron loss in the evolution of paralogous gene families

The mechanisms and evolutionary dynamics of intron insertion and loss in eukaryotic genes remain poorly understood. Reconstruction of parsimonious scenarios of gene structure evolution in paralogous gene families in animals and plants revealed numerous gains and losses of introns. In all analyzed lineages, the number of acquired new introns was substantially greater than the number of lost ancestral introns. This trend held even for lineages in which vertical evolution of genes involved more intron losses than gains, suggesting that gene duplication boosts intron insertion. However, dating gene duplications and the associated intron gains and losses based on the molecular clock assumption showed that very few, if any, introns were gained during the last ~100 million years of animal and plant evolution, in agreement with previous conclusions reached through analysis of orthologous gene sets. These results are generally compatible with the emerging notion of intensive insertion and loss of introns during transitional epochs in contrast to the relative quiet of the intervening evolutionary spans.