Docking of alpha-cobratoxin suggests a basal conformation of the nicotinic receptor

We investigate the interactions between the long chain alpha-cobratoxin (Cbtx) and the nicotinic acetylcholine receptor using a rigid body docking procedure. The method, (i) reproduces the binding of Cbtx to Lymnea acetylcholine-binding protein (AChBP); (ii) shows that most of the structures of AChBP obtained in the presence of antagonists are compatible with Cbtx binding; and (iii) reveals a complex between Cbtx and muscle nAChR that corresponds to the basal "resting" state conformation. The structures are made available for further understanding of the allosteric transitions of the nAChR as well as for drug design.     

Cyclam cored luminescent dendrimers as ligands for Co(II), Ni(II) and Cu(II) ions

We have investigated the photophysical properties of two dendrimers containing a cyclam core decorated with 4 naphthyl units (G0), 12 dimethoxybenzene and 16 naphthyl units (G2). These dendrimers show fluorescence bands that can be assigned to naphthyl localized excited states (λ_max = 337 nm), naphthyl-amine exciplexes (λ_max = 470 nm) and, for G2, naphthyl excimers (λ_max ca. 400 nm). Cyclam is a very good ligand for transition metal ions and we have investigated complex formation between these dendritic ligands and Ni(II), Co(II) and Cu(II), added as nitrate salts. This process can be monitored by the strong changes, both in shape and intensity, observed in the emission spectra of these dendrimers. Complexation with Cu(II) causes not only changes in the relative intensities of the fluorescence bands, but also the appearance of a new absorption band in the near UV spectral region. An analysis of the titration curves has allowed us to obtain clear evidence for the formation of not only 1:1 species, but also 1:2 metal to ligand species. G2 shows a clear preference, compared to G0, in forming complexes with a 1:2 metal-to-ligand stoichiometry, although it possesses very bulky dendrons appended to the cyclam central unit.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q_P) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q_P site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc₁) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe₂S₂ cluster, cyt c₁ and cyt b, forming two catalytic domains, the Q_o (hydroquinone (QH₂) oxidation) and Q_i (quinone (Q) reduction) sites. At the Q_o site, the electron transfer pathways originating from QH₂ oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc₁ and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q_o site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc₁ activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc₁. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH₂ oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe₂S₂ cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH₂ oxidation at the Q_o site of cyt bc₁.     

Exploring the multi-drug resistance in Escherichia coli O157:H7 by gene interaction network: A systems biology approach

In the present study, we have constructed an interaction network of 29 antibiotic resistant genes along with 777 interactions in E. coli O157:H7. Gene ontology analysis reveals that 94, 89 and 67 genes have roles in the cellular process, biological process and molecular function respectively. Gene complexes related to tripartite efflux pumps mdtEF-tolC and ABC family efflux pump macAB-tolC play key roles in multidrug efflux systems. It is noteworthy to mention that, 19 genes are involved in multi-efflux pumps and they play a significant role in multidrug resistance (MDR); while 18 genes are vital for fatty acid synthesis. Interestingly, we found that the four genes arnABCD are involved in both MDR and in fatty acid synthesis. Hence these genes could be targeted for new drug discovery. On the whole, our results provide a detailed understanding of the mode of MDR mechanisms in E.coli O157:H7.     

Metabolomics of Ndufs4−/− skeletal muscle: Adaptive mechanisms converge at the ubiquinone-cycle

Leigh syndrome is one of the most common childhood-onset neurometabolic disorders resulting from a primary oxidative phosphorylation dysfunction and affecting mostly brain tissues. Ndufs4^?/? mice have been widely used to study the neurological responses in this syndrome, however the reason why these animals do not display strong muscle involvement remains elusive. We combined biochemical strategies and multi-platform metabolomics to gain insight into the metabolism of both glycolytic (white quadriceps) and oxidative (soleus) skeletal muscles from Ndufs4^?/? mice. Enzyme assays confirmed severely reduced (80%) CI activity in both Ndufs4^?/? muscle types, compared to WTs. No significant alterations were evident in other respiratory chain enzyme activities; however, Ndufs4^?/? solei displayed moderate decreases in citrate synthase (12%) and CIII (18%) activities. Through hypothesis-generating metabolic profiling, we provide the first evidence of adaptive responses to CI dysfunction involving non-classical pathways fueling the ubiquinone (Q) cycle. We report a respective 48 and 34 discriminatory metabolites between Ndufs4^?/? and WT white quadriceps and soleus muscles, among which the most prominent alterations indicate the involvement of the glycerol-3-phosphate shuttle, electron transfer flavoprotein system, CII, and proline cycle in fueling the Q cycle. By restoring the electron flux to CIII via the Q cycle, these adaptive mechanisms could maintain adequate oxidative ATP production, despite CI deficiency. Taken together, our results shed light on the underlying pathogenic mechanisms of CI dysfunction in skeletal muscle. Upon further investigation, these pathways could provide novel targets for therapeutic intervention in CI deficiency and potentially lead to the development of new treatment strategies.     

Biomanufacturing by in vitro biosystems containing complex enzyme mixtures

Microbial fermentation is the current predominant biomanufacturing platform. However, it suffers from low production yields, slow reaction rates, and scaling-up challenges. In vitro enzymatic biosystems are emerging to expand the traditional biotechnological mode by utilizing more than three enzymes for manufacturing the desired product from cheap substrate. In the past few years, numerous proofs of the concept of in vitro biosystems containing complex enzyme mixtures from different groups worldwide have inspired the development of these platforms for biomanufacturing, these biosystems show advantages such as near-theoretical product yields, faster reaction rates, reduced interference from toxic compounds, and unprecedented level of engineering. In this review, several examples of in vitro systems are presented to illustrate these advantages and possible solutions to overcome the remaining challenges are discussed. The continuing decrease in enzyme cost and improvements in enzyme engineering techniques will make in vitro biosystems a comparable biomanufacturing platform for microbial fermentation in the near future.     

Mobility of lipid in complexes of amylose–fatty acids by deuterium and C solid state NMR

Palmitic and lauric acid complexes with amylose were studied by solid state methods: ¹³C CP/MAS NMR, deuterium NMR, X-ray powder diffraction and differential scanning calorimetry (DSC). The crystalline amylose complexes were found to be in a V-type sixfold single chain helix. The melting points of the complexes were over 100°C, at least 40–50°C higher than the melting points of the free fatty acids. CP/MAS ¹³C NMR spectra revealed two resonance peaks at 33.6 and 32.4 ppm for the palmitic acid, which were assigned as free and complexed fatty acid, respectively. A single resonance peak at 32.4 ppm was found for the lauric acid and assigned to the complex. The chemical shift of 32.4 ppm for the complexed fatty acids suggests a combined trans and gauche conformation for the fatty acid chain in the complex. T₁ relaxation measurements on the two palmitic acid resonances show different behavior: a very slow relaxation for the 33.6 ppm and a much faster relaxation (1.2 s) for the 32.4 ppm resonances. The latter was similar to the relaxation of the single resonance of the lauric acid (1.1 s). Temperature dependent deuterium spectra of the amylose–lauric acid and amylose–palmitic acid complexes suggest a complete complexation for the amylose–lauric acid, whereas the amylose–palmitic acid complex is partially disassociated by the thermal treatment. Based on the overall data, a partially disordered model is proposed: an imperfect helix with the fatty acid partly inside and partly out, depending on crystallization conditions and the necessity of placing the carboxyl head outside the V-helix.     

Models of metamorphic timing: an experimental evaluation with the pond-dwelling salamander Hemidactylium scutatum (Caudata: Plethodontidae)

Amphibian larvae vary tremendously in size at metamorphosis and length of larval period. We raised pond-dwelling four-toed salamander (Hemidactylium scutatum) larvae to test two models that predict a larva’s age and size at metamorphosis. The Wilbur-Collins model proposes that the developmental rate of a larva responds to changes in growth rate in an adaptive manner throughout the larval period, and that metamorphosis can be initiated after a minimum size has been reached. The Leips-Travis or fixed-rate model states that developmental rate is set early in the larval period, perhaps by early growth rate or food availability and their positive correlation with developmental rate, and that changes in growth rate during the larval period affect size at metamorphosis, but have no effect on the age of an individual at metamorphosis. A modified version of the Wilbur-Collins model suggests that a larva’s developmental rate becomes fixed about two-thirds of the way through the larval period, with changes in growth rate after that point only affecting size at metamorphosis. Larvae were raised on eight different feeding regimes which created two constant and six variable growth histories. Growth history did significantly affect size at metamorphosis. However, an a posteriori statistical test revealed a group of seven and an overlapping group of six treatments with indistinguishable lengths of larval period, indicating a general picture of a fixed developmental rate regardless of growth history. This result is unique among similar studies on invertebrates, fish, and frogs. There was no association between early growth or food level and development rates. Neither the Wilbur-Collins nor the Leips-Travis fixed-rate models were supported. The invariable developmental rate of Hemidactylium and recent osteological evidence from the literature suggest that larvae begin the process of metamorphosis as soon as they hatch, probably a trait selected for by strong predation pressure in the aquatic environment. A variety of different approaches (ecological, developmental, phylogenetic) are necessary to fully evaluate the adaptive nature of the timing of transitions between life cycle stages. Received: 3 June 1999 / Accepted: 18 March 2000     

海水养殖含氮废水处理的复合微生态制剂研制

目前应用于海水养殖废水修复的微生态制剂不仅存在菌株针对性、适应性差的缺点,而且很少考虑功能微生物之间的相互作用,因此应用效果难以令人满意。本实验针对海水养殖过程中的氮素循环,选择分离自海水养殖废水环境的亚硝化细菌ZW38、反硝化细菌ZL5和蛋白酶产生菌ZS7进行配伍实验,以混合培养体系中各种类型细菌的数量及氨态氮、亚硝态氮和蛋白质的含量变化为指标,发现3种功能细菌以3:2:1的比例混合,且使用量为109CFU/L时,彼此之间可相互促进生长,经过5d处理,海水养殖废水中的主要污染物质可溶性蛋白和氨态氮的去除率分别达到94.0%和77.5%。