Structural characterization of novel cobalt corrinoids synthesized by enzymes of the vitamin B12 anaerobic pathway

Investigation on the use of the oxidized form (factor 3 (3a)) of the trimethylated intermediate (precorrin 3 (2)) as a substrate for the enzymes of the anaerobic pathway to vitamin B12 led to the synthesis of three pairs of novel cobalt corrinoids. The products were made with the aid of the Salmonella typhimurium enzymes CbiH, CbiF, CbiG, and CbiT, were synthesized in several 13C labeled versions, and were isolated as methylesters after esterification. Structures were determined by detailed NMR and MS analyses. Each set of products was obtained in the decarboxylated (RMe) and non-decarboxylated (R=CH2COOCH3) forms (at the C-12 position of the porphyrinoid).     

Expression of the crucifer-infecting TMV-Cg movement protein in tobacco plants complements in trans a TMV-U1 trafficking-deficient mutant

Tobamovirus movement proteins play a determinant role in the establishment of infections in plants, allowing the local movement of viral RNA genome through plasmodesmatas. We expressed the movement protein (MP) of the crucifer- and garlic-infecting Tobacco Mosaic Virus strain Cg (TMV-Cg) in both resistant Xanthi NN and sensitive Xanthi nn Nicotiana tabacum plants. MP-Cg function was assayed by inoculating transgenic plants with a trafficking-deficient mutant of TMV strain U1. Following infection, local necrotic lesions were developed in resistant transgenic plants, and a systemic infection was produced in sensitive tobaccos. Thus, movement function of the mutant virus was complemented in trans by MP-Cg expressed in transgenic plants, causing the same symptoms as wild-type strain. We demonstrated that the function of MP-U1 could be replaced efficiently by MP-Cg, even though these proteins share only 36% of identity. Similar hydrophobic patterns of MP-Cg and MP-U1 suggests structure and function conservations of both proteins. This work is an example of how two tobamoviruses differing in their host range help to understand viral movement mechanism during the infection.     

Five Entry Points of the Mitochondrially Encoded Subunits in Mammalian Complex I Assembly

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.Complex I (CI) (NADH-ubiquinone oxidoreductase; EC 1.6.5.3) is one of the main electron entry points in the mitochondrial respiratory electron transport chain catalyzing the oxidation of NADH to reduce ubiquinone to ubiquinol (31, 39, 40), contributing to the proton motive force to synthesize ATP by the process called oxidative phosphorylation (OXPHOS).CI assembly is a difficult problem to address due to the large size of the complex and its dual genomic nature, as 7 out of its 45 subunits are encoded by the mitochondrial DNA (mtDNA) (10, 11). Until very recently, mammalian CI assembly was explained using two different and apparently contradictory models. One model was proposed by following the time course of formation of CI intermediates in human cells in culture once mitochondrial protein synthesis had recovered after its inhibition by doxycycline (36). Based on these observations, human CI was proposed to be assembled through two different modules corresponding to the membrane and peripheral arms. The other model was proposed after analysis of a cohort of four CI-deficient patients in which seven putative assembly intermediates containing a combination of both peripheral- and membrane arm subunits were identified. Thus, an assembly pathway in which the peripheral- and membrane arm subassemblies came together before the completion of each of the arms was proposed (4). However, the most recent studies have refined the previous models and propose an overlapping view of the process. One study, by green fluorescent protein (GFP) tagging of the NDUFS3 subunit, identified six peripheral-arm intermediates. The second and third smaller NDUFS3-containing subassemblies were accumulated and could not advance into higher-molecular-mass species when mitochondrial protein synthesis was inhibited, thus determining the entry point of the mitochondrially encoded subunits in the CI assembly pathway (37). The most recent study analyzed the incorporation of the mitochondrial subunits in a time course to the fully assembled CI and, on the other hand, the incorporation of the nuclear subunits by importing them into isolated mitochondria (24). Although these two models differ in the order in which some subunits are incorporated, they agree on the general human CI assembly pathway, which takes place via evolutionarily conserved modular subassemblies (14, 25, 28, 37).However, the specific entry points of all the mtDNA-encoded CI subunits (ND subunits) in the CI assembly pathway and their roles in the stability of the complex remained to be clarified. Structural studies related to mutations in the ND subunits in pathological cases have given some hints as to the importance of each of them for CI assembly/stability. In this case, defects in specific ND subunits do not have the same effect: ND1, ND4, and ND6 seem to be fundamental to CI assembly, while ND3 and ND5 are important for its activity but not for assembly. On the other hand, mutations in ND2 alter CI assembly, with abnormal intermediate accumulation (19).In this article, we present new insights into the roles of the ND subunits by using mouse cells deficient for ND4, ND6, and a combination of ND6 and ND5. This study has allowed us to propose the five different entry points by which the mtDNA-encoded subunits are sequentially incorporated into the CI assembly pathway, completing the current view of the process. We conclude that ND4 and ND6 are required for the proper function and assembly of CI, although at different degrees due to their different entry points and roles in the CI assembly pathway.     

Thermoregulatory behavior, heat gain and thermal tolerance in the periwinkle Echinolittorina peruviana in central Chile

The amount of solar radiation absorbed by an organism is a function of the intensity of the radiation and the area of the organism exposed to the source of the radiation. Since the prosobranch gastropod Echinolittorina peruviana is longer than it is wide, its areas of the lateral sides are approximately twice as large as the areas of the frontal and dorsal faces. We quantified the orientation of the intertidal prosobranch E. peruviana with respect to the position of the sun and solar heat gain in the different orientations. In the field, 80.9% of the E. peruviana monitored on sunny summer days tended to face the sun frontally or dorsally while only 19.1% faced the sun with the larger lateral sides. On overcast summer or on winter days, this trend was not observed. We then show that the body temperature of individuals increases more rapidly and reaches higher equilibriums when the lateral sides are facing the sun than when they face the sun with either of the smaller frontal or dorsal sides. These results therefore show that the orientation behavior of E. peruviana is thermoregulatory and that it permits the organisms to maintain lower temperatures on hot summer days.     

Demography and fishery of the sea urchin Loxechinus albus (Echinodermata: Echinidae) in south-austral Chile region

In the Magellan Region of southern Chile (52 degrees 20'S - 55 degrees 30'S), the edible urchin Loxechinus albus is collected by 1200 artisanal fishermen, of whom 450 are divers. About 360 small fishing boats and 54 transport vessels carry the fresh product to 16 processing plants. Landings of about 27 000 tons were recorded between January and December 1995. Test diameters of urchins harvested monthly were measured for a total of 119 239 specimens, and 36 406 specimens were individually weighed; sex determination was carried out on 2 314 specimens. Field data indicate that the harvest was about 6.6 x 106 dozen urchins (this is a measuring method employed by fishermen in the region), with an extractive effort of 14 753 diver/days. The fisheries yield ranged from an annual minimum of 235 DUDD (dozen urchins per diver/day) to a maximum of 660 DUDD. In overall terms, the lowest average yields were between January and April (415-427 DUDD), and the highest yields between May and December (456-510 DUDD). Mean sizes increased from June to November and decreased from December to June. Size frequency of males and females were polymodal, with the most relevant modes at 72-84 mm in males, and at 79-88 mm in the females. The percentage of individuals below the minimum legal size (70 mm) did not exceed 4.9% for males and 3.6% for females. The size-weight records fit a power model which suggested that this species has a negative allometric growth (b = 2.007). Regarding weight, urchins in the size range from 80.0 to 84.9 mm were those with the maximum contribution to the regional landings. The highest values recorded for the utilized condition factor were: Average Condition Factor (ACF) = May to July, and November; Isometric (or Cubic) Condition Factor (ICF) = July; and Allometric Condition Factor (ACF) = June. Spawning occurred mainly between August and September, and ended by the end of October. Exploitation of this species represents one of the main sources of employment for the artisanal fisheries sector in the Magellan Region. The main difficulty observed in this fishery was obtaining a sufficient supply of urchins with a yellow-gold colored gonadic material, which forms the basis for demand of this urchin by the international market.     

Local distribution and thermal ecology of two intertidal fishes

Geographic variability in the physiological attributes of widely distributed species can be a result of phenotypic plasticity or can reflect evolutionary responses to a particular habitat. In the field, we assessed thermal variability in low and high intertidal pools and the distribution of resident fish species Scartichthys viridis and transitory Girella laevifrons along this vertical intertidal gradient at three localities along the Chilean coast: Antofagasta (the northernmost and warmest habitat), Carrizal Bajo (central coast) and Las Cruces (the southernmost and coldest habitat). In the laboratory, we evaluated the thermal sensitivity of fish captured from each locality. The response to temperature was estimated as the frequency of opercular movements and as thermal selectivity in a gradient; the former being a indirect indicator of energy costs in a particular environment and the latter revealing differential occupation of habitat. Seawater temperature in intertidal pools was greatest at Antofagasta, and within each site was greatest in high intertidal pools. The two intertidal fish species showed opposite patterns of local distribution, with S. viridis primarily inhabiting the lower sectors of the intertidal zone, and G. laevifrons occupying the higher sectors of the intertidal zone. This pattern was consistent for all three localities. Locality was found to be a very important factor determining the frequency of opercular movement and thermal selectivity of both S. viridis and G. laevifrons. Our results suggest that S. viridis and G. laevifrons respond according to: (1) the thermal history of the habitat from which they came, and (2) the immediate physical conditions of their habitat. These results suggest local adaptation rather than plasticity in thermoregulatory and energetic mechanisms.