Cytochrome C Oxidase from Rhodobacter Sphaeroides: Oligomeric Structure in the Phospholipid Bilayer and the Structural and Functional Effects of a C-Terminal Truncation in Subunit III

Cytochrome c oxidase (COX) of the mitochondrial electron transport chain catalyzes the reduction of oxygen to water while concomitantly translocating protons across the inner mitochondrial membrane. This two part dissertation is a structural and functional investigation of COX using the bacterial model system from Rhodobacter sphaeroides (R.sph.). First, the oligomeric structure of R.sph. COX within the lipid bilayer was investigated using discontinuous sucrose gradient ultracentrifugation. Utilizing this technique, liposomes containing R.sph. COX (pCOV) were separated from liposomes lacking enzyme (COV). The net buffering capacity and degree of light scattering per COX molecule were reduced in pCOVs, making them well suited for low buffer spectroscopic studies. Also, pCOVs maintained high oxygen reduction and proton pumping activities relative to COVs, indicating minimal damage induced by the centrifugation process. Quantitative lipid and protein concentrations were used to estimate the number of COX molecules per vesicle in the pCOVs. There was only one R.sph. COX molecule per vesicle, indicating that within the lipid bilayer, R.sph. COX exists in the monomeric state in contrast to the bovine enzyme which is dimeric. As a monomer, therefore, R.sph. COX is capable of maximal electron transfer and proton pumping efficiency. Second, the structural and functional effects of a c-terminal subunit III truncation were characterized in R.sph. in order to gain insight into the critical role played by this subunit in proper COX functioning. The mutation was modeled after a human mitochondrial disease mutation which genetically truncates subunit III after the third of its seven helices (Delta114 COX). In R.sph. cells, Delta114 COX had lower expression levels and impeded rates of COX assembly. Altered levels of native in vivo processing of subunits II and IV were observed in Delta114 COX and in COX which had subunit III genetically removed (I-II COX). The truncated subunit III was incorporated into the COX complex with at least 70% stoichiometry and was subject to proteolytic processing at a specific cleavage site. Prior to enzymatic turnover induced inactivation, the proton pumping and oxygen reduction activities of Delta114 COX were half that of wildtype and equivalent to I-II COX at physiological pH. Delta114 and I-II COX had similar catalytic lifetimes in detergent micelle, but when supplemented with phospholipids from soybean, the catalytic lifetime of Delta114 COX was increased compared to I-II COX. Taken together, these results indicate that the c-terminal bundle of subunit III plays a role in the assembly of COX in R.sph. and in the native processing of subunits II and IV. They also highlight the role of the structural lipids within the v-shaped cleft of subunit III as being important for providing protection against turnover induced inactivation

Anomerisation von beta-D-Glucopyranosiden unter Glycosylierungsbedingungen

Unlängst wurde nachgewiesen, daß bei der Polykondensation von tritylierten 1,2–0–(1–Cyano)ethyliden-Zuckerderivaten in Anwesenheit von Tritylethern als Terminator unter Einfluß von Triphenylmethaniumperchlorat das sich bildende polymere Produkt 0–Tritylgruppen enthält, was uns die Möglichkeit zur Synthese von Polysaccharid-Blöcken eröffnete. Zur Verbesserung der Syntheseverfahren für Polysaccharide mit Blockstruktur muß man den Erhaltungsgrad der Tritylethern im ursprünglichen Polykondensationsprodukt bewerten. Zur Lösung dieser Frage wurde von uns eine Polykondensation des Tritylether des 1,2–0–(1–Cyano)ethyliden-Derivats der D–Mannose (I) in Anwesenheit von 0,1 äqu. trityliertem Glucosid (II) als Terminator und 0,1 äqu. TrClO4 durchgeführt und danach das Polykondensationsprodukt ohne seine Abscheidung 5 mal hinsichtlich des ursprünglichen Terminators (II) durch den Cyanoethylidenderivat-Überschuß der D–Galaktose (III) behandelt. Dies mußte zu einem Einbau des beta-D-Galaktopyranose-Restes in die Polysaccharid-Kette führen, wobei man nach dem Galaktose-Glucose-Verhältnis im Endprodukt den 0–Tritylgruppen-Gehalt im ursprünglichen Polykondensationsprodukt, mit 6-Phthalimidohexyl–beta-D-glucopyranosid-Rest am Kettenende, bewerten können sollte

Islands beneath islands: phylogeography of a groundwater amphipod crustacean in the Balearic archipelago

<p>Abstract</p> <p>Background</p> <p>Metacrangonyctidae (Amphipoda, Crustacea) is an enigmatic continental subterranean water family of marine origin (thalassoid). One of the species in the genus, <it>Metacrangonyx longipes</it>, is endemic to the Balearic islands of Mallorca and Menorca (W Mediterranean). It has been suggested that the origin and distribution of thalassoid crustaceans could be explained by one of two alternative hypotheses: (1) active colonization of inland freshwater aquifers by a marine ancestor, followed by an adaptative shift; or (2) passive colonization by stranding of ancestral marine populations in coastal aquifers during marine regressions. A comparison of phylogenies, phylogeographic patterns and age estimations of clades should discriminate in favour of one of these two proposals.</p> <p>Results</p> <p>Phylogenetic relationships within <it>M. longipes </it>based on three mitochondrial DNA (mtDNA) and one nuclear marker revealed five genetically divergent and geographically structured clades. Analyses of cytochrome oxidase subunit 1 (<it>cox1</it>) mtDNA data showed the occurrence of a high geographic population subdivision in both islands, with current gene flow occurring exclusively between sites located in close proximity. Molecular-clock estimations dated the origin of <it>M. longipes </it>previous to about 6 Ma, whereas major cladogenetic events within the species took place between 4.2 and 2.0 Ma.</p> <p>Conclusions</p> <p><it>M. longipes </it>displayed a surprisingly old and highly fragmented population structure, with major episodes of cladogenesis within the species roughly correlating with some of the major marine transgression-regression episodes that affected the region during the last 6 Ma. Eustatic changes (vicariant events) -not active range expansion of marine littoral ancestors colonizing desalinated habitats-explain the phylogeographic pattern observed in <it>M. longipes</it>.</p

Expanding the taxonomic conundrum: three new species of groundwater crustaceans (Syncarida, Bathynellacea, Parabathynellidae) endemic to the Iberian Peninsula

20 pages, 7 figures.Two new species of the genus Iberobathynella and one new species of the cosmopolitan genus Hexabathynella are described from the Iberian Peninsula. The three new species were found in the groundwater of caves and the gravelly bank of a river, two species in the north of Spain (Cantabria and Burgos) and one in the south (Sevilla). The two new species of the genus Iberobathynella present a unique combination of characters. These character combinations for the species from Cantabria are: thoracopod 8 female with wrinkled cuticle and two teeth; seven spines on the furca; nine spines on the sympod, three barbed and one plumose seta on the exopod and two barbed setae on the endopod of the uropod; maxilla with 0, 0 and 15 setae on the three segments, respectively; five teeth on the pars incisiva and six teeth on the pars molaris of the mandible and seven segments on the antennule. The combinations of characters for the species from Burgos are: antennule with seven segments; segment 5 of the antennule with one aesthetasc; mandible with four and five teeth on the pars incisiva and pars molaris respectively; maxillule with one seta on segment 1 and 14 setae on segment 3; segment 1 of the endopod of the thoracopod 1 with two setae; uropod with six spines on the sympod, four barbed setae and one plumose seta on the exopod and no setae on the endopod; furca with five spines and the thoracopod 8 female with wrinkled cuticle and two teeth. This last species has an exclusive character for the genus: the absence of setae on the endopod of the uropod. The new species of the genus Hexabathynella has six spines of similar length on the sympod of the uropod (in all other species within the genus the distal spine is longer than the others); the form of the antennal organ in the males is shown to be a new unique character for the genus; the maxilla has three and 13 setae on segments 2 and 3, respectively; the uropod has six spines on the sympod, three barbed setae on the exopod and two smooth setae on the endopod, the endopod is twice as long as the exopod and the shape of the female thoracopod 8 is unique.This work was supported by projects REN2000- 2040 GLO, EVK2-CT-2001-00121 (European Projects PASCALIS) and Convenio Junta de Castilla y León and CSIC.Peer reviewe

The Pliocene Paludina Lake of Pannonian Basin : new evidence from northern Serbia

This study from the Sremski Karlovci clay pit in northern Serbia sheds new light on the physicochemical conditions, ecology and evolution of the Paludina Lake – the Pliocene successor of the late Miocene giant Lake Pannon hosted by the Pannonian Basin. The multidisciplinary study combines sedimentology, sequence stratigraphy, biostratigraphy, palaeontology, palaeobotany, coal petrology, organic geochemistry and magnetic mineralogy. The sedimentary succession studied represents the lake margin at the foot of the Fruška Gora ridge. Sedimentary facies reveal minor and major lake-level changes, including a forced regression with fluvial valley incision in the succession middle part and the ultimate emergence and covering of the lake floor by Pleistocene loess. Mollusc and ostracod fauna indicates an oligohaline shallow cool-water environment, no deeper than 5–6 m, with an active inflow of spring water. The lake local depth during transgression maxima did not exceed 20 m. Palynological and geochemical analyses indicate a rich and diversified assemblage of gymnosperm plants with a contribution of angiosperms, weeds and microbial biomass in the peat-forming suboxic to oxic coastal swamp environment. Maceral analysis of organic matter shows a prevalence of huminite, accompanied richly by inertinite in lignite and by liptinite in clay. The Pleistocene shift to terrestrial semiarid environment resulted in oxidizing groundwater conditions, with the reddening of sediments around a fluctuating groundwater table and the diagenetic transformation of bacteria-derived greigite into magnetite. In regional stratigraphy, the occurrence of Viviparus neumayri Brusina in the lower half of the succession indicates the Lower Paludina Beds of Dacian Stage (early Zanclean age). Other gastropods and certain ostracodes indicate transition to the Middle Paludina Beds of lower Romanian Stage (late Zanclean–early Piacenzian). The upper half of the succession lacks age-diagnostic fossils and is considered to represent Middle Paludina Beds with a possible relic of Upper Paludina Beds at the top

Development of reciprocating modular electrical machine

The article offers the most advanced design of electrical machines reciprocating action as well as a multi-concept of their design. An algorithm and a computer program based on Microsoft Excel, allowing to determine the number of modules to provide the required power of the electric machine reciprocating. Confirmed performance of the developed software