Fluorescence quenching in riboflavin-binding protein and its complex with riboflavin

Fluorescence quenching of tryptophan residues in egg-white riboflavin-binding protein by two typical quenchers (charged iodide and uncharged acrylamide) reveals acid-induced changes of protein conformation. At neutralpH, acrylamide flow in macromolecule, (i.e., the quenching effect) is decisive; tryptophan residue accessibility for iodide is small. At lowpH, some tryptophan residues are exposed to the protein surface and become more accessible to iodide. In contrast, acrylamide is less able to permeate this conformational state of RBP. Fluorescence of tryptophan residues in riboflavin-RBP complex and chemically N-bromosucinimide-modified RBP was quenched by iodide and acrylamide.     

Phenotypic plasticity of the coarse root system of Prosopis flexuosa, a phreatophyte tree, in the Monte Desert (Argentina)

Prosopis flexuosa trees in the Monte Desert grow in dune and inter-dune valleys, where the water table is located at 6–14 m depth. We asked whether trees in the dunes, which are less likely to access the water table, present a coarse surface root architecture that might favor the exploration / exploitation of dune resources, compensating for water table inaccessibility. We characterized the architecture of surface roots of valley and dune trees, together with the soil environment. The dune held 50 % less and deeper gravimetric soil water (along a 4 m profile), 3-times less organic matter, 2-times less available phosphorous, and a sharper contrast of ammonium and nitrate concentration between plant canopies and uncovered soil than the valley. Coarse surface roots of dune trees were highly branched and grew tortuously at 0.56?±?0.16 m depth before sinking downward near the tree crown, suggesting an intensive exploitation of the ephemeral, deep, and canopy-linked resources. In contrast, trees from the valley spread their profuse and less branched surface roots mainly horizontally at 0.26?±?0.08 m depth, several meters outside the crown probably exploring this resource-rich site. A model for the environmental control of root architecture together with potential ecological effects is discussed.     

Bis(dialkylaminophosphino)ferrocenes: Reactivity and electrochemistry

New compounds containing the 1,1′-bis(dialkylaminophosphino)ferrocene ligands 1,1′-bis(dimethylaminophosphino)ferrocene (dmapf) and 1,1′-bis(diethylaminophosphino)ferrocene (deapf) were prepared and characterized by NMR. The oxidative electrochemistries of these compounds were examined in methylene chloride. The transition metal compounds M_nCl₂(P-P) (M = Pd or Pt, n = 1, P-P = dmapf or deapf; M = Au, n = 2, P-P = dmapf) displayed one-electron, Nernstian waves. The corresponding selenides were prepared and the oxidative electrochemistries, along with those of the previously prepared 1,1′-bis(alkylaminophosphine sulfide)ferrocene, were examined. The sulfides displayed one-electron, Nernstian oxidations. The selenides displayed electrochemically irreversible two-electron waves. The X-ray structures of the sulfides and selenides as well as that of the transition metal compounds [(AuCl)₂(dmapf)] and [PtCl₂(dmapf)] were determined.     

Characterization of Spontaneous,Transient Adenosine Release in the Caudate-Putamen and Prefrontal Cortex

Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.17±0.01 µM in the caudate and 0.19±0.01 µM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 µM. The average duration of spontaneous adenosine release was 2.9±0.1 seconds and 2.8±0.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A₁ receptors. The A₁ antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A₁ agonist CPA (N⁶-cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation.     

UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces

UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters revealed structurally dependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium to function efficiently and preserve regulated activity in the cell.     

Localisation of aphidicolin-induced break points in Holstein-Friesian cattle (Bos taurus) using RBG-banding

Fragile sites (FS) seem to play a role in genome instability and may be involved in karyotype evolution and chromosome aberrations. The majority of common fragile sites are induced by aphidicolin. Aphidicolin was used at two different concentrations (0.15 and 0.30 μM) to study the occurrence of FS in the cattle karyotype. In this paper, a map of aphidicolin induced break points and fragile sites in cattle chromosomes was constructed. The statistical analysis indicated that any band with three or more breaks was significantly damaged (P < 0.05). According to this result, 30 of the 72 different break points observed were scored as fragile sites. The Pearson correlation test showed a positive association between chromosome length and the number of fragile sites (r = 0.54). On the contrary, 21 FS were identified on negative R bands while 9 FS were located on positive R bands.     

Dracula orchids exploit guilds of fungus visiting flies: new perspectives on a mushroom mimic

1. Dracula orchids mimic mushrooms in appearance and scent, and are pollinated by mushroom flies. This study examined the guilds of insect visitors to Dracula species and to co‐occurring mushrooms in an Ecuadorean cloud forest. Significant visitor overlap was documented (mostly in the genera Zygothrica and Hirtodrosophila; Drosophilidae). To test the brood‐site mimicry hypothesis, behavioural observations were also performed and rearing success was examined. 2. Many fly species that visit Dracula are also found on mushrooms. Furthermore, the presence of pollinia enabled us to document the fact that particular individuals visiting mushrooms also visit Dracula. Roughly two‐thirds of the visitors to these unusual flowers are fungal‐associated, and the rest appear to be Dracula specialists. 3. A variety of behaviours common on both host groups were observed, including courtship semaphoring, feeding, sheltering, defending territory, and mating. Given this suite of shared reproductive behaviour, it was hypothesised that flies may also oviposit in both substrates. Flies were reared from mushrooms, Dracula spp., and other flowers (controls) to determine which substrate led to the highest fitness for the flies. Very few flies emerged from the Dracula flowers and there was no species overlap between the emergent insects and the known pollinators. 4. Despite the fact that there is evidence for brood‐site mimicry, the Dracula situation is complex. All the flies could derive many of the same fitness benefits from the flowers as they do from mushrooms (shelter, food, mating), but they are clearly not gaining the same fitness benefit, in terms of progeny, on flowers compared with mushrooms.