A thermal unfolding study of plastocyanin from the thermophilic cyanobacterium Phormidium laminosum

The thermal unfolding of the plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium, is herein described. The main objective of this work is to identify structural factors responsible for the higher stability observed in proteins from thermophilic organisms. With the aid of fluorescence spectroscopy, EPR, and NMR, the factors influencing the unfolding process of the protein were investigated, and procedures for its study have been standardized. The different spectroscopic techniques used provided consistent results showing that the thermal unfolding of plastocyanin is irreversible under all the conditions investigated and that this irreversibility does not appear to be related to the presence of oxygen. The oxidized plastocyanin species has proven to be more stable than the reduced one, with respect to both the required temperature for protein unfolding (up to a 9 degrees C difference between the two forms) and the kinetics of the process. The behavior of this plastocyanin contrasts with that of other cupredoxins whose unfolding had previously been studied. The unfolding pH dependence and kinetic studies indicate a process with a tight control around the physiological pH in which plastocyanin plays its redox role and the protein's isoelectric point (5.2), suggesting a close compromise between function and stability.     

Toward a comprehensive model for induced endoreduplication

Both the biological significance and the molecular mechanism of endoreduplication (END) have been debated for a long time by cytogeneticists and researchers into cell cycle enzymology and dynamics alike. Mainly due to the fact that a wide variety of agents have been reported as able to induce endoreduplication and the diversity of cell types where it has been described, until now no clear or unique mechanism of induction of this phenomenon, rare in animals but otherwise quite common in plants, has been proposed. DNA topoisomerase II (topo II), plays a major role in mitotic chromosome segregation after DNA replication. The classical topo II poisons act by stabilizing the enzyme in the so-called cleavable complex and result in DNA damage as well as END, while the true catalytic inhibitors, which are not cleavable-complex-stabilizers, do induce END without concomitant DNA and chromosome damage. Taking into account these observations on the induction of END by drugs that interfere with topo II, together with our recently obtained evidence that the nature of DNA plays an important role for chromosome segregation [Cortes, F., Pastor, N., Mateos, S., Dominguez, I., 2003. The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes. DNA Repair 2, 719-726.], a straightforward model is proposed in which the different mechanisms leading to induced END are considered.     

A unified statistical model for functional mapping of environment-dependent genetic expression and genotype x environment interactions for ontogenetic development

The effects of quantitative trait loci (QTL) on phenotypic development may depend on the environment (QTL x environment interaction), other QTL (genetic epistasis), or both. In this article, we present a new statistical model for characterizing specific QTL that display environment-dependent genetic expressions and genotype x environment interactions for developmental trajectories. Our model was derived within the maximum-likelihood-based mixture model framework, incorporated by biologically meaningful growth equations and environment-dependent genetic effects of QTL, and implemented with the EM algorithm. With this model, we can characterize the dynamic patterns of genetic effects of QTL governing growth curves and estimate the global effect of the underlying QTL during the course of growth and development. In a real example with rice, our model has successfully detected several QTL that produce differences in their genetic expression between two contrasting environments. These detected QTL cause significant genotype x environment interactions for some fundamental aspects of growth trajectories. The model provides the basis for deciphering the genetic architecture of trait expression adjusted to different biotic and abiotic environments and genetic relationships for growth rates and the timing of life-history events for any organism.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil

Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2) in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7%) and transmission (83.3%) rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60%, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1% of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.     

The offshore-ring: A new system design for the open ocean aquaculture of macroalgae

Mass culture of benthic macroalgae under rough offshore conditions in the North Sea requires rigid culture support systems that cannot only withstand rough weather conditions but can also be effectively handled while at the same time retain the cultured species. Various carrier constructions and different mooring systems were tested. Laminaria saccharina grew on all of these carriers with initially high (up to 14.5% per day) and later decreasing length increments. Longlines, ladder and grid systems had certain disadvantages and these are discussed. The study results led to a new ring carrier (patent pending), first used in 1994/1995, which was gradually improved until 2002. This system now emerges as being superior, since it resists not only rough weather conditions (2 m s^–1 current velocity, 6 m wave height) but also permits ease of handling when compared to other constructions. The ring allows various operational modes and can be equipped with culture lines that can be collected offshore or transported to shore facilities for harvesting. The modular nature of the tested ring system lends itself for future use in integrated aquaculture systems located in or attached to offshore wind farms.     

Two sequential phosphates 3' adjacent to the 8-oxoguanosine are crucial for lesion excision by E. coli Fpg protein and human 8-oxoguanine-DNA glycosylase

Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are base excision repair enzymes involved in the 8-oxoguanine (oxoG) repair pathway. Specific contacts between these enzymes and DNA phosphate groups play a significant role in DNA-protein interactions. To reveal the phosphates crucial for lesion excision by Fpg and hOGG1, modified DNA duplexes containing pyrophosphate and OEt-substituted pyrophosphate internucleotide (SPI) groups near the oxoG were tested as substrate analogues for both proteins. We have shown that Fpg and hOGG1 recognize and specifically bind the DNA duplexes tested. We have found that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the 8-oxoguanosine (oxodG) and one nucleotide 3' away from it. In contrast, they efficiently incised DNA duplexes bearing the same phosphate modifications 5' to the oxodG and two nucleotides 3' away from the lesion. The effect of these phosphate modifications on the substrate properties of oxoG-containing DNA duplexes is discussed. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or SPI groups immediately 3' to the oxodG or one nucleotide 3' away from it are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a wild-type enzymes bound to oxoG-containing DNA.     

Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.