Effect of a modified thymine on the structure and stability of [d(TGGGT)]4 quadruplex

Telomeric guanine-rich sequence can adopt quadruplex structures that are important for their biological role in chromosomal stabilisation. G quartets are characterised by the cyclic hydrogen bonding of four guanine bases in a coplanar arrangement and their stability is ion-dependent. In this work we compare the stability of [d(TGGGT)]₄ and [d(T*GGGT)]₄ quadruplexes. The last one contains a modified thymine, where the hydroxyl group substitutes one hydrogen atom of the methyl group of the thymine in the [d(TGGGT)]₄ sequence. We used a combination of spectroscopic, calorimetric and computational techniques to characterise the G-quadruplex formation. NMR and CD spectra of [d(T*GGGT)]₄ were characteristic of parallel-stranded, tetramolecular quadruplex. CD and DSC melting experiments reveal that [d(T*GGGT)]₄ is less stable that unmodified quadruplex. Molecular models suggest possible explanation for the observed behaviour.     

Chemopreventive and Antioxidant Activity of the Chamazulene‐Rich Essential Oil Obtained from Artemisia arborescens L. Growing on the Isle of La Maddalena,Sardinia, Italy

The essential oils of Artemisia arborescens growing in Sardinia (Italy), collected during three plant growth stages, i.e., from the vegetative stage to post‐blooming time, were characterized. Moreover, the in vitro antiproliferative and antioxidant activities of the oil isolated from aerial parts collected in February were evaluated. The essential oils belonged to the β‐thujone/chamazulene chemotype, notably with the highest amount of chamazulene (ca. 52%) ever detected up to now in the genus Artemisia and, in general, in essential oils. Quantitative variations in the oil composition were observed as the plant passes from the vegetative to the blooming stage. The oil was tested for its potential tumor cell growth‐inhibitory effect on T98G, MDA‐MB 435S, A375, and HCT116 human cell lines, using the MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) assay. The highest activity was observed on A375 and HCT116 cell lines, with IC₅₀ values of 14 μg/ml. Moreover, the in vitro antioxidant and free radical‐scavenging assays revealed the oil to be an effective scavenger of the ABTS radical cation, with an activity comparable to that of Trolox^®. These results support the use of A. arborescens oil for the treatment of inflamed skin conditions. Finally, the composition of the polar fraction of the A. arborescens aerial parts was also examined, and the main component detected was 5‐O‐caffeoylquinic acid, which was identified for the first time in this plant.     

Peroxisomes as novel players in cell calcium homeostasis

Ca2+ concentration in peroxisomal matrix ([Ca2+](perox)) has been monitored dynamically in mammalian cells expressing variants of Ca2+-sensitive aequorin specifically targeted to peroxisomes. Upon stimulation with agonists that induce Ca2+ release from intracellular stores, peroxisomes transiently take up Ca2+ reaching peak values in the lumen as high as 50-100 microm, depending on cell types. Also in resting cells, peroxisomes sustain a Ca2+ gradient, [Ca2+](perox) being approximately 20-fold higher than [Ca2+] in the cytosol ([Ca2+](cyt)). The properties of Ca2+ traffic across the peroxisomal membrane are different from those reported for other subcellular organelles. The sensitivity of peroxisomal Ca2+ uptake to agents dissipating H+ and Na+ gradients unravels the existence of a complex bioenergetic framework including V-ATPase, Ca2+/H+, and Ca2+/Na+ activities whose components are yet to be identified at a molecular level. The different [Ca2+](perox) of resting and stimulated cells suggest that Ca2+ could play an important role in the regulation of peroxisomal metabolism.     

Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions

The effects of gold(I) complexes (auranofin, triethylphosphine gold and aurothiomalate), gold(III) complexes ([Au(2,2'-diethylendiamine)Cl]Cl(2), [(Au(2-(1,1-dimethylbenzyl)-pyridine) (CH(3)COO)(2)], [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine)(OH)](PF(6)), [Au(bipy(dmb)-H)(2,6-xylidine)](PF(6))), metal ions (zinc and cadmium acetate) and metal complexes (cisplatin, zinc pyrithione and tributyltin) on mitochondrial thioredoxin reductase and mitochondrial functions have been examined. Both gold(I) and gold(III) complexes are extremely efficient inhibitors of thioredoxin reductase showing IC(50) ranging from 0.020 to 1.42 microM while metal ions and complexes not containing gold are less effective, exhibiting IC(50) going from 11.8 to 76.0 microM. At variance with thioredoxin reductase, auranofin is completely ineffective in inhibiting glutathione peroxidase and glutathione reductase, while gold(III) compounds show some effect on glutathione peroxidase. The mitochondrial respiratory chain is scarcely affected by gold compounds while the other metal complexes and metal ions, in particular zinc ion and zinc pyrithione, show a more marked inhibitory effect that is reflected on a rapid induction of membrane potential decrease that precedes swelling. Therefore, differently from gold compounds, the various metal ions and metal complexes exert their effect on different targets indicating a lower specificity. It is concluded that gold compounds are highly specific inhibitors of mitochondrial thioredoxin reductase and this action influences other functions such as membrane permeability properties. Metal ions and metal complexes markedly inhibit the activity of thioredoxin reductase although to an extent lower than that of gold compounds. They also inhibit mitochondrial respiration, decrease membrane potential and, finally, induce swelling.     

The role of miRNA-221 and miRNA-126 in patients with benign metastasizing leiomyoma of the lung: an overview with new interesting scenarios

Molecular Biology Reports - Benign metastasizing leiomyoma (BML) is a rare disease characterized by extrauterine benign leiomyomatosis in patients with a previous or concomitant history of uterine...     

Neuronal nets and nerve cell interactions in vitro in insect systems

Summary The development of a synthetic medium that supports growth and differentiation of insect embryonic tissues afforded the possibility of studying the interactions between nerve and other cell types in long term cultures. The mechanical dissociation of embryonic nerve tissues results in survival of nerve cells but not of glial cells. The dissociated glial-free neurons produce a dense fibrillar network in the presence, but not in the absence, of foregut explants or other tissues from same donors. Nerve fiber bundles outgrowing from dissociated neurons enter foregut segments and establish synaptic connections with muscle cells. Foregut explants undergo differentiation and become contractile in long term cultures when innervated by dissociated nerve cells. The progressive deterioration of similar foregut tissues cultured alone contrasts with the excellent condition of innervated explants and suggests that this is due to trophic factors released by nerve fibers. The same in vitro systems provided the opportunity of studying the interaction between nerve fibers produced by the autonomic ingluvial ganglion, which adheres to the surface of the alimentary tract, and muscle cells. Multiple esophagus explants from cockroach embryos become interconnected by fibers emerging from ingluvial ganglia, when the explants are combined in vitro at short distance from each other. Muscle cells migrating from the esophagi line up on axons branching out in the medium, or form contractile ribbons which, in turn, establish connections with nerve fibers. The thigmotropism of muscle cells and strong affinity for nerve fibers reveal a new aspect of muscle cells-to-fibers interaction, amenable to further analysis in vitro. This work was supported in part by United States Public Health Service grant NS-03777 and grant GB-16330 X from the National Science Foundation     

Transcriptional regulation of vascular bone morphogenetic protein by endothelin receptors in early autoimmune diabetes mellitus

Endothelin (ET) and bone morphogenic proteins (BMP) have been implicated in the development of micro- and macrovascular complications of type 2 diabetes mellitus due to atherosclerosis. This study investigated vascular BMP-expression during early development of experimental autoimmune diabetes mellitus and whether ET(A) receptors are involved in its regulation, using the selective ET(A) receptor antagonist BSF461314. Specificity of BSF461314 was confirmed through ET-mediated p44/42 mitogen-activated protein kinase (ERK1/2) phosphorylation experiments. For animal studies, non-obese diabetic (NOD) and control mice at 16 weeks of age were treated with BSF461314 for 6 weeks. Plasma glucose levels were measured before and after treatment and vascular gene expression of BMP-2, BMP-7, and BMP-type II receptor was determined in the aorta by quantitative real-time polymerase chain reaction analysis. At the beginning of the study in all animals, plasma glucose levels were within the normal range. After 6 weeks gene expression of vascular BMP-2, BMP-7 and BMP-type II receptor was almost doubled in NOD mice compared with non-diabetic controls (p < 0.05). Concomitant treatment with BSF461314 significantly reduced expression of all BMPs and lowered plasma glucose levels in NOD mice close to controls (all p < 0.05 versus untreated). In conclusion, vascular BMP-2, BMP-7, and BMP-type II receptor expression is upregulated in early stages of autoimmune diabetes mellitus. The data further indicate that ET(A) receptors inhibit diabetes-associated activation of vascular BMPs and regulate plasma glucose levels suggesting that ET(A) receptors might provide a new therapeutic target to interfere with the early development of atherosclerosis in patients with type 1 diabetes mellitus.     

Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis

Light‐harvesting complex II (LHCII) contains three highly homologous chlorophyll‐a/b‐binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo‐ and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so‐called state‐transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10 sec of ‘state 2 light’ results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non‐phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super‐ and mega‐complexes that comprised both photosystem (PS)I and PSII, and the state 2‐specific PSI–LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting.     

Isolation and partial characterization of a protein kinase NII from wheat germ chromatin

A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (-³²P)ATP and (-³²P)GTP.