Effects of copper sulfate on growth and physiological responses of Limoniastrum monopetalum.

A glasshouse study of the coastal shrub Limoniastrum monopetalum was carried out to evaluate its tolerance and capacity to accumulate copper. We investigate the effects of Cu from 0 to 60 mmol l(-1) on the growth, photosynthetic apparatus, and nutrient uptake of L. monopetalum, by measuring gas exchange, chlorophyll fluorescence parameters, photosynthetic pigments, and total copper, nitrogen, phosphorus, sulfur, calcium, and magnesium content in the plant tissues. Although L. monopetalum did not survive at 60 mmol l(-1) Cu, the species demonstrated a high tolerance to Cu-induced stress, since all plants survived external Cu concentrations of up to 35 mmol l(-1) and displayed similar growth in the Cu-enriched medium as in the control treatment of up to the external level of 15 mmol Cu l(-1) (1,000 mg Cu l(-1)). The reduced growth registered in plants exposed to 35 mmol Cu l(-1) can be attributed to reduced photosynthetic carbon assimilation associated with the adverse effect of the metal on the photochemical apparatus and a reduction in the absorption of essential nutrients. Copper tolerance was associated with the capacity of the plant to accumulate the metal in its roots and effectively prevent its translocation to photosynthetic tissues. L. monopetalum has the characteristics of a Cu-excluder plant and could be used in the revegetation of Cu-contaminated soils.

Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy.

Bioartificial lungs re-engineered from decellularized organ scaffolds are a promising alternative to lung transplantation. Critical features for improving scaffold repopulation depend on the mechanical properties of the cell microenvironment. However, the mechanics of the lung extracellular matrix (ECM) is poorly defined. The local mechanical properties of the ECM were measured in different regions of decellularized rat lung scaffolds with atomic force microscopy. Lungs excised from rats (n=11) were decellularized with sodium dodecyl sulfate (SDS) and cut into ~7µm thick slices. The complex elastic modulus (G(∗)) of lung ECM was measured over a frequency band ranging from 0.1 to 11.45Hz. Measurements were taken in alveolar wall segments, alveolar wall junctions and pleural regions. The storage modulus (G', real part of G(∗)) of alveolar ECM was ~6kPa, showing small changes between wall segments and junctions. Pleural regions were threefold stiffer than alveolar walls. G' of alveolar walls and pleura increased with frequency as a weak power law with exponent 0.05. The loss modulus (G″, imaginary part of G(∗)) was 10-fold lower and showed a frequency dependence similar to that of G' at low frequencies (0.1-1Hz), but increased more markedly at higher frequencies. Local differences in mechanical properties and topology of the parenchymal site could be relevant mechanical cues for regulating the spatial distribution, differentiation and function of lung cells.

Tolerance and accumulation of copper in the salt-marsh shrub Halimione portulacoides.

The present study evaluated the tolerance and accumulation potential in the salt-marsh shrub Halimione portulacoides under moderate and high external Cu levels. A greenhouse experiment was conducted in order to investigate the effects of a range of external Cu concentrations (0 to 60 mmol l(-1)) on growth and photosynthetic performance by measuring gas exchange, chlorophyll fluorescence parameters and photosynthetic pigments. We also determined total copper, nitrogen, phosphorus and sulfur concentrations in the plant tissues. H. portulacoides survived with external Cu concentrations of up to 35 mmol Cu l(-1), although the excess of metal resulted in a biomass reduction of 48%. The effects of Cu on growth were linked to a drastic reduction in net photosynthesis. However, H. portulacoides tolerated Cu levels of up to 15 mmol Cu l(-1) without suffering adverse physiological effects. Our results indicate that this species could play an important role in the restoration of Cu-contaminated soils.

Zinc tolerance and accumulation in the salt-marsh shrub Halimione portulacoides.

The halophytic shrub Halimione portulacoides is known to be capable of growth in soils containing extremely high concentrations of Zn. This study evaluated in detail the tolerance and accumulation potential of H. portulacoides under moderate and high external Zn levels. A greenhouse experiment was conducted in order to investigate the effects of a range of Zn concentrations (0-130 mmol L(-1)) on growth and photosynthetic performance by measuring relative growth rate, total leaf area, specific leaf area, gas exchange, chlorophyll fluorescence parameters and photosynthetic pigment concentrations. We also determined the total zinc, nitrogen, phosphorus, calcium, magnesium, sodium, potassium, iron and copper concentrations in the plant tissues. H. portulacoides demonstrated hypertolerance to Zn stress, since it survived with leaf concentrations of up to 2300 mg Zn kg(-1)dry mass, when treated with 130 mmol Zn L(-1). Zinc concentrations greater than 70 mmol L(-1) in the nutrient solution negatively affected plant growth, in all probability due to the recorded decline in net photosynthesis rate. Our results indicate that the Zn-induced decline in the photosynthetic function of H. portulacoides may be attributed to the adverse effect of the high concentration of the metal on photosynthetic electron transport. Growth parameters were virtually unaffected by leaf tissue concentrations as high as 1500 mg Zn kg(-1)dry mass, demonstrating the strong capability of H. portulacoides to protect itself against toxic Zn concentrations. The results of our study indicate that this salt-marsh shrub may represent a valuable tool in the restoration of Zn-polluted areas.

Growth and photosynthetic responses to zinc stress of an invasive cordgrass, Spartina densiflora.

Spartina densiflora Brongn. is found in coastal marshes of southwest Spain, growing over sediments containing 100-4800 ppm Zn. A glasshouse experiment was designed to investigate the effect of Zn from 0 to 100 mmol.l(-1) on the growth and photosynthetic apparatus of S. densiflora, by measuring relative growth rate, leaf elongation rate, number of tillers, height of tillers, chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. We also determined total ash, Zn, calcium, magnesium and phosphorus concentrations, and the C/N ratio. At 100 mmol.l(-1) Zn, S. densiflora showed a 48% biomass reduction after 1 month of treatment. Long-term effects of Zn on growth of S. densiflora consisted mainly of variations in net photosynthesis. Modification of the Zn/Mg ratio was linked to a strong decrease in RuBP carboxylase (Zn was favoured in local competition with Mg, so that the affinity of RuBisCO for CO(2) decreased), oxygenase activity of RuBisCO acting as a substitute for the photosynthetic function. Also, Zn had a marked overall effect on the photochemical (PSII) apparatus and the synthesis of photosynthetic pigments. However, the results indicate that S. densiflora is capable of tolerating very high and continued exposure to Zn, as this species lowers the translocation of Zn from the nutrient solution to roots and controls Zn ion transport into leaves. Therefore, S. densiflora could be useful in the phytostabilization of soils.

Expression of the Cydia pomonella granulovirus iap3 gene.

The IAP3 protein of Cydia pomonella granulovirus (CpGV) was the first identified member of the baculovirus IAP family of proteins, which have been shown to block apoptosis in diverse systems. However, little is known of the expression and subcellular localisation of CpGV IAP3 during a viral infection. This study examined IAP3 in cells infected by CpGV and in cells infected by an Autographa californica nucleopolyhedrovirus (AcMNPV) recombinant that carried the CpGV iap3 gene. The levels of iap3 specific transcripts were monitored and production of the protein was assessed using an IAP3-specific antiserum. The data showed that iap3 is expressed during both early and late phases of infection, with a switch occurring from distal early transcription start sites to proximal late start sites. Protein levels are highest after DNA replication. IAP3 is localised exclusively in the cytoplasm. Subcellular fractionation experiments demonstrated that the protein is present in both soluble and membrane-bound cytosolic fractions. The membrane-bound fraction includes IAP3 that is associated with the mitochondria. However, the data do not support the hypothesis that release of cytochrome C from the mitochondria is involved in baculovirus-induced apoptosis.

Use of whole genome sequence data to infer baculovirus phylogeny.

Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) and Granulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.

Insect-virus relationships: sifting by informatics.

Several groups of large DNA viruses successfully utilise the rich resource provided by insect hosts. Defining the mechanisms that enable these pathogens to optimise their relationships with their hosts is of considerable scientific and practical importance, but our understanding of the processes involved is, as yet, rudimentary. Here we describe an informatics-based approach that uses comparison of viral genomic sequences to identify candidate genes likely to be specifically involved in this process. We hypothesise that such genes should satisfy two essential criteria, namely, that they should be (i) present in those members of a virus family that infect insects, but absent from those that infect other hosts, and (ii) found in at least two unrelated taxa of insect viruses. These criteria currently identify six groups of viral genes, including one that encodes the fusolin/gp37 proteins. Demonstration that the fusolin/gp37 proteins can enhance oral infectivity of insect viruses provides a primary validation of this approach to the examination of insect-virus relationships.

Effect of cisapride on intestinal bacterial overgrowth and bacterial translocation in cirrhosis.

Deranged intestinal motility, which occurs in cirrhosis, may facilitate the development of intestinal bacterial overgrowth (IBO), which can lead to bacterial translocation (BT). To assess the effect of cisapride on IBO and BT in cirrhosis, cirrhotic rats received cisapride or a placebo for 7 days, and measurements of jejunal bacterial content and BT studies were performed. In addition, jejunal fluid from 46 cirrhotic patients was obtained for quantitative bacterial culture. Those patients in whom gram-negative IBO was detected were randomized to receive or not to receive cisapride (20 mg twice per day) for 1 week. Cisapride significantly reduced IBO in cirrhotic rats. In addition, no BT was documented in treated animals, whereas it occurred in 40% in nontreated cirrhotic rats. Total IBO was documented in 23 of 46 cirrhotic patients, which was caused by gram-negative organisms in 10 cases. Orocecal transit time (OCT) significantly decreased after cisapride therapy, and was associated with the abolishment of bacterial overgrowth caused by gram-negative organisms in 4 out of 5 treated patients, whereas it persisted in nontreated cases. Cisapride administration to cirrhotic rats resulted in a reduction of the IBO, which is associated with a marked decrease in BT. On the other hand, cisapride facilitates the abolition of IBO caused by gram-negative organisms in cirrhotic patients.

Generation of baculovirus expression vectors.

The baculovirus expression system has become an important tool for the expression of heterologous genes because it has several positives attributes. First, high quantities of protein are produced because the target genes are driven by strong viral promoters. Second, most eukaryotic posttranslational modifications are carried out in insect cells in an authentic manner. Thus, proteins expressed with the baculovirus expression system usually have the same activities as the authentic protein. Several approaches have been developed to obtain recombinant baculoviruses easily and nowadays many modified baculoviral DNAs and a huge variety of transfer plasmids are available. Here, we described the rapid generation of recombinant baculoviruses using parental viral DNA that incorporates a lethal deletion and can be selected against. This basic approach should be suitable for the majority of applications.

Sorbitol dehydrogenase of Drosophila. Gene, protein, and expression data show a two-gene system.

The Drosophila melanogaster sorbitol dehydrogenase (SDH) is characterized as a two-enzyme system of the medium chain dehydrogenase/reductase family (MDR). The SDH-1 enzyme has an enzymology with Km and kcat values an order of magnitude higher than those for the human enzyme but with a similar kcat/Km ratio. It is a tetramer with identical subunits of approximately 38 kDa. At the genomic level, two genes, Sdh-1 and Sdh-2, have a single transcriptional start site and no functional TATA box. Expression is greater in larvae and adults than in pupae, where it is very low. At all three stages, Sdh-1 constitutes the major transcript. Sdh-1 and Sdh-2 genes were located at positions 84E-F and 86D in polytene chromosomes. The deduced amino acid sequences of the two genes show 90% residue identity. Evaluation of the sequence and modeling of the structure toward that of class I alcohol dehydrogenase (ADH) show altered loop and gap arrangements as in mammalian SDH and establishes that SDH, despite gene multiplicity and larger variability than the "constant" ADH of class III, is an enzyme conserved over wide ranges.

Characterization and molecular analysis of Adh retrosequences in species of the Drosophila obscura group.

Retrosequences, genes, and pseudogenes originated by retrotranscription are frequent components of vertebrate genomes, but they have only occasionally been described in invertebrates. In Drosophila, very few retrosequences have been reported, among them those of alcohol dehydrogenase (Adh) and phosphoglyceromutase (Pglym). Although 52 Adh gene sequences are available for comparison, Adh retrosequences have been described only in the sibling species D. teissieri and D. yakuba (melanogaster subgroup) and in D. subobscura (obscura subgroup). Here, we report the presence of Adh retrosequences in two closely related species of D. subobscura: D. madeirensis and D. guanche. Extensive sequence comparisons with their functional paralogs suggest separate retrotranscriptional events: one in the melanogaster subgroup in the ancestor of D. teissieri and D. yakuba, and the other in the obscura subgroup before the radiation of the lineages leading to D. subobscura, D. madeirensis, and D. guanche. In the former, the Adh retrotranscript originated a new expressed gene, named jingwei. However, in the obscura Adh retrosequences, retention of codon bias and higher Ks than Ka values, both distinctive evolutionary features supporting functionality, have to be considered together with a frameshift, premature stop codons, and other nucleotide substitutions, which, added to the lack of the original promoter elements, suggest that they are pseudogenes. At least two different Adh retrosequences have been characterized in each of the obscura species, and their phylogenetic analysis indicates that paralogs and their flanking genomic regions share a higher degree of similarity than orthologous sequences. Two alternative hypotheses could explain this current organization and structure: either a multiplication event occurred independently in each species, or gene conversion events should be invoked after a single duplication in the species ancestor. The significance of retrotranscriptional events in the evolution of invertebrate genomes is discussed.

Structure of the Drosophila melanogaster glutathione-dependent formaldehyde dehydrogenase/octanol dehydrogenase gene (class III alcohol dehydrogenase). Evolutionary pathway of the alcohol dehydrogenase genes.

The glutathione-dependent formaldehyde dehydrogenase gene (gfd) of Drosophila melanogaster encodes an enzyme that is active toward S-hydroxymethylglutathione, an adduct of formaldehyde with glutathione, and also with long-chain primary alcohols, both properties typical of class III alcohol dehydrogenases, gfd hybridizes at the 86D division of the third chromosome, in agreement with the known location of the Drosophila octanol dehydrogenase gene (odh), gfd/odh was isolated from a lambda EMBL-4 genomic library and consists of three exons (with coding segments of 21, 90 and 1029 bp) and two introns (69 bp and 70 bp, respectively). The introns are small in size like the Drosophila interrupting sequences and are located at the 5' end of the coding region. Comparisons with the homologous genes of Saccharomyces, Candida and humans provide information on the evolution of the class III alcohol dehydrogenases. Moreover, results from analysis of exon/intron distributions in eleven dehydrogenases are compatible with the hypothesis of intron loss accounting for aspects of the present structure of these genes.

Fundamental molecular differences between alcohol dehydrogenase classes.

Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism.