Differential expression of specific sulphate transporters underlies seasonal and spatial patterns of sulphate allocation in trees

Sulphate uptake and its distribution within plants depend on the activity of different sulphate transporters (SULTR). In long‐living deciduous plants such as trees, seasonal changes of spatial patterns add another layer of complexity to the question of how the interplay of different transporters adjusts S distribution within the plant to environmental changes. Poplar is an excellent model to address this question because its S metabolism is already well characterized. In the present study, the importance of SULTRs for seasonal sulphate storage and mobilization was examined in the wood of poplar (Populus tremula × P. alba) by analysing their gene expression in relation to sulphate contents in wood and xylem sap. According to these results, possible functions of the respective SULTRs for seasonal sulphate storage and mobilization in the wood are suggested. Together, the present results complement the previously published model for seasonal sulphate circulation between leaves and bark and provide information for future mechanistic modelling of whole tree sulphate fluxes.     

Mapping fungal ion channel locations

Ion channel mapping techniques are described and the results for two fungal organisms, Saprolegnia ferax and Neurospora crassa, are presented. In these species, two channel types have been characterized, stretch-activated channels exhibiting significant calcium permeability and spontaneous channels having significant potassium permeability. Two distinct analyses of patch clamp data, analysis of channel self-clustering and association between different channel types, and localization along the hyphae, reveal significant differences between the two organisms. S. ferax maintains a tip-high gradient of both channel types which is lost after disruption of the actin cytoskeleton. There is significant self-clustering of the channels, as well as interactions between channel types. N. crassa on the other hand does not maintain tip-high gradients, and clustered distributions are observed only for the stretch-activated channels. In terms of physiological roles, evidence is quite strong that the stretch-activated channels function as a growth sensor in S. ferax, but have an unknown function in N. crassa. In both organisms, the potassium permeable channels presumably function in potassium uptake. The differences between these two organisms may be due, in part, to differences in their normal environment: aquatic versus terrestrial. Copyright 1998 Academic Press.     

Two new cytotypes reinforce that <Emphasis Type="Italic">Micronycteris hirsuta</Emphasis> Peters, 1869 does not represent a monotypic taxon

Background

The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n?=?26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data.

Results

We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n?=?26 and 25, NF?=?32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA.

Conclusions

The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

     

Occupational exposure of workers to pesticides: Toxicogenetics and susceptibility gene polymorphisms

Farm workers are often exposed to pesticides, which are products belonging to a specific chemical group that affects the health of agricultural workers and is mostly recognized as genotoxic and carcinogenic. The exposure of workers from Piauí, Brazil, to these hazardous chemicals was assessed and cytogenetic alterations were evaluated using the buccal micronucleus assay, hematological and lipid parameters, butyrylcholinesterase (BChE) activity and genetic polymorphisms of enzymes involved in the metabolism of pesticides, such as PON1, as well as of the DNA repair system (OGG1, XRCC1 and XRCC4). Two groups of farm workers exposed to different types of pesticides were evaluated and compared to matched non-exposed control groups. A significant increase was observed in the frequencies of micronuclei, kariorrhexis, karyolysis and binucleated cells in the exposed groups (n = 100) compared to controls (n = 100). No differences were detected regarding the hematological parameters, lipid profile and BChE activity. No significant difference was observed either regarding DNA damage or nuclear fragmentation when specific metabolizing and DNA repair genotypes were investigated in the exposed groups.     

Molecular evolution of olfactomedin

Olfactomedin is a secreted polymeric glycoprotein of unknown function, originally discovered at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently found throughout the mammalian brain. As a first step toward elucidating the function of olfactomedin, its phylogenetic history was examined to identify conserved structural motifs. Such conserved motifs may have functional significance and provide targets for future mutagenesis studies aimed at establishing the function of this protein. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedins in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homopolymer formation such as the CXC motif near the amino terminus are conserved, as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, evolutionary analysis of a region of 53 amino acids of fish, frog, rat, mouse, and human olfactomedins indicates that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian, and mammalian lineages. Indeed, a distant olfactomedin homolog was identified in Caenorhabditis elegans. Although the amino acid sequence of this invertebrate protein is longer and highly divergent compared with its vertebrate homologs, the protein from C. elegans shows remarkable similarities in terms of conserved motifs and posttranslational modification sites. Six universally conserved motifs were identified, and five of these are clustered in the carboxyl terminal half of the protein. Sequence comparisons indicate that evolution of the N-terminal half of the molecule involved extensive insertions and deletions; the C-terminal segment evolved mostly through point mutations, at least during vertebrate evolution. The widespread occurrence of olfactomedin among vertebrates and invertebrates underscores the notion that this protein has a function of universal importance. Furthermore, extensive modification of its N-terminal half and the acquisition of a C-terminal SDEL endoplasmic-reticulum- targeting sequence may have enabled olfactomedin to adopt new functions in the mammalian central nervous system.      

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

ANALYSIS OF SPATIAL DISTRIBUTIONS OF CELLULAR MOLECULES DURING MECHANICAL STRESSING OF CELL SURFACE RECEPTORS USING CONFOCAL MICROSCOPY

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.