Relevance of CD6-mediated interactions in T cell activation and proliferation

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.     

Antiapoptotic effects of roscovitine in cerebellar granule cells deprived of serum and potassium: a cell cycle-related mechanism

Neuronal apoptosis may be partly due to inappropriate control of the cell cycle. We used serum deprivation as stimulus and reduced potassium from 25 to 5mM (S/K deprivation), which induces apoptosis in cerebellar granule neurons (CGNs), to evaluate the direct correlation between re-entry in the cell cycle and apoptosis. Roscovitine (10 microM), an antitumoral drug that inhibits cyclin-dependent kinase 1 (cdk1), cdk2 and cdk5, showed a significant neuroprotective effect on CGNs deprived of S/K. S/K deprivation induced the expression of cell cycle proteins such as cyclin E, cyclin A, cdk2, cdk4 and E2F-1. It also caused CGNs to enter the S phase of the cell cycle, measured by a significant incorporation of BrdU (30% increase over control cells), which was reduced in the presence of roscovitine (10 microM). On the other hand, roscovitine modified the expression of cytochrome c (Cyt c), Bcl-2 and Bax, which are involved in the apoptotic intrinsic pathway induced by S/K deprivation. We suggest that the antiapoptotic effects of roscovitine on CGNs are due to its anti-proliferative efficacy and to an action on the mitochondrial apoptotic mechanism.     

Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase

Thiosulphate is one of the products of the initial step of the elemental sulphur oxidation pathway in the thermoacidophilic archaeon Acidianus ambivalens. A novel thiosulphate:quinone oxidoreductase (TQO) activity was found in the membrane extracts of aerobically grown cells of this organism. The enzyme was purified 21-fold from the solubilized membrane fraction. The TQO oxidized thiosulphate with tetrathionate as product and ferricyanide or decyl ubiquinone (DQ) as electron acceptors. The maximum specific activity with ferricyanide was 73.4 U (mg protein)(-1) at 92 degrees C and pH 6, with DQ it was 397 mU (mg protein)(-1) at 80 degrees C. The Km values were 2.6 mM for thiosulphate (k(cat) = 167 s(-1)), 3.4 mM for ferricyanide and 5.87 micro M for DQ. The enzymic activity was inhibited by sulphite (Ki = 5 micro M), metabisulphite, dithionite and TritonX-100, but not by sulphate or tetrathionate. A mixture of caldariella quinone, sulfolobus quinone and menaquinone was non-covalently bound to the protein. No other cofactors were detected. Oxygen consumption was measured in membrane fractions upon thiosulphate addition, thus linking thiosulphate oxidation to dioxygen reduction, in what constitutes a novel activity among Archaea. The holoenzyme was composed of two subunits of apparent molecular masses of 28 and 16 kDa. The larger subunit appeared to be glycosylated and was identical to DoxA, and the smaller was identical to DoxD. Both subunits had been described previously as a part of the terminal quinol:oxygen oxidoreductase complex (cytochrome aa3).     

Widespread distribution of a lexA-regulated DNA damage-inducible multiple gene cassette in the Proteobacteria phylum

The SOS response comprises a set of cellular functions aimed at preserving bacterial cell viability in front of DNA injuries. The SOS network, negatively regulated by the LexA protein, is found in many bacterial species that have not suffered major reductions in their gene contents, but presents distinctly divergent LexA-binding sites across the Bacteria domain. In this article, we report the identification and characterization of an imported multiple gene cassette in the Gamma Proteobacterium Pseudomonas putida that encodes a LexA protein, an inhibitor of cell division (SulA), an error-prone polymerase (DinP) and the alpha subunit of DNA polymerase III (DnaE). We also demonstrate that these genes constitute a DNA damage-inducible operon that is regulated by its own encoded LexA protein, and we establish that the latter is a direct derivative of the Gram-positive LexA protein. In addition, in silico analyses reveal that this multiple gene cassette is also present in many Proteobacteria families, and that both its gene content and LexA-binding sequence have evolved over time, ultimately giving rise to the lexA lineage of extant Gamma Proteobacteria.     

Genetic regulation of mouse glycosylphosphatidylinositol-phospholipase D

Glycosylphosphatidylinositol phospholipase D (GPI-PLD) has been proposed to be responsible for cleaving membrane-associated glycosylphosphatidyl inositol (GPI) molecules to generate inositol phosphoglycan (IPGs), which have growth factor-mimetic properties. We have cloned the mouse liver GPI-PLD cDNA, which has a sequence that differs from that previously isolated from a mouse glucagonoma cell library. Using a highly specific and very sensitive RNase protection assay, we found that the GPI-PLD expressed in adult/post-natal brain, antrum and insulin-producing cells is identical to that isolated from liver. The expression of mouse GPI-PLD in liver shows a complex genetic regulation with a mouse strain-specific variation. In addition, GPI-PLD mRNA levels were higher in 4-week old animals compared to older animals, and the GPI-PLD mRNA levels increased in mice that developed insulin dependent type 1 diabetes spontaneously. This suggests that the expression of liver GPI-PLD in mice is highly regulated.     

Thermotolerant poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characterization of the PHB depolymerase of Schlegelella sp. KB1a

Eighteen gram-negative thermotolerant poly(3-hydroxybutyrate) (PHB)-degrading bacterial isolates (T _max60°C) were obtained from compost. Isolates produced clearing zones on opaque PHB agar, indicating the presence of extracellular PHB depolymerases. Comparison of physiological characteristics and determination of 16S rRNA gene sequences of four selected isolates revealed a close relatedness of three isolates (SA8, SA1, and KA1) to each other and to Schlegelella thermodepolymerans and Caenibacterium thermophilum. The fourth strain, isolate KB1a, showed reduced similarities to the above-mentioned isolates and species and might represent a new species of Schlegelella. Evidence is provided that S. thermodepolymerans and C. thermophilum are only one species. The PHB depolymerase gene, phaZ, of isolate KB1a was cloned and functionally expressed in Escherichia coli. Purified PHB depolymerase was most active around pH 10 and 76°C. The DNA-deduced amino acid sequence of the mature protein (49.4 kDa) shared significant homologies to other extracellular PHB depolymerases with a domain substructure: catalytic domain type 2—linker domain fibronectin type 3—substrate-binding domain type 1. A catalytic triad consisting of S₂₀, D₁₀₄, and H₁₃₈ and a pentapeptide sequence (GLS₂₀AG) characteristic for PHB depolymerases (PHB depolymerase box, GLSXG) and for other serine hydrolases (lipase box, GXSXG) were identified.This contribution is dedicated to Hans G. Schlegel in honor of his 80th birthday.Fabian Romen and Simone Reinhardt share first authorship.     

Comparative Analysis of <Emphasis Type="Italic">Alu</Emphasis> Insertion Sequences in the APP 5′ FlankingRegion in Humans and Other Primates

Overexpression of the amyloid precursor protein gene (APP) may play a role in the neuropathology of Alzheimers disease. Therefore, elucidating the mechanisms involved in APP gene regulation is of primary importance, and various cis-acting regulatory elements located in 5 distal regions are known to play a main role. Some of them lie within Alu elements, one of which (Alu1) is only found in humans and apes while the other (Alu2) has a much older history and is also found in rhesus. These Alu insertions harbor sequence motifs that may act as cis-regulatory elements, which may cause differences in APP regulation among primate species and whose functionality may be ascertained through their conservation in a comparative analysis. We have performed a comparative analysis of the region comprising the two Alu elements of the APP promoter in several primates, including humans. We have found a significant decrease in nucleotide diversity in the Alu2 element (inserted in all the species analyzed) compared to the Alu1 (inserted only in apes). This finding can be interpreted as a constriction in the Alu2 sequence variation as a consequence of a functional role of this element in the APP expression. The present results suggest a wider extension of the regulatory elements than the known short consensus regulatory sequences. Moreover, the different conservation of two highly similar and neighboring sequences suggests that, besides the importance of the sequence motifs, their position in relation to the gene suggests that they have played a role in being recruited as regulatory elements.     

High variety of different simian T-cell leukemia virus type 1 strains in chimpanzees (Pan troglodytes verus) of the Taï National Park, Côte d'Ivoire

We found human T-cell leukemia virus type 1- and simian T-cell leukemia virus type 1 (STLV-1)-related infections in 5 of 10 chimpanzees originating from three groups of wild chimpanzees. The new virus isolates showed a surprising heterogeneity not only in comparison to STLV-1 described previously in other primate species but also between the different chimpanzee groups, within a group, or even between strains isolated from an individual animal. The interdisciplinary combination of virology, molecular epidemiology, and long-term behavioral studies suggests that the primary route of infection might be interspecies transmission from other primates, such as red colobus monkeys, that are hunted and consumed by chimpanzees.     

Genetic structure of Flores island (Azores, Portugal) in the 19th century and in the present day: evidence from surname analysis

The island of Flores is the most westerly of the Azores archipelago (Portugal). Despite its marked geographic isolation and reduced population size, biodemographic and genetic studies conducted so far do not support the idea that its population constitutes a genetic isolate. In this study we conducted a surname analysis of the Flores population for two time periods: the second half of the 19th century and the present day. Our main purposes were (1) to biodemographically and genetically characterize the island, taking into account the strong reduction in population observed from the middle of the 19th century to the present day; and (2) to analyze the influence that the effective population size and geographic distance have on the genetic structure of populations. For both periods analyzed, all indicators of diversity revealed a high level of surname diversity. Our results are in accordance with the diversity estimates obtained from both monoparental genetic markers located in the Y chromosome and frequencies of mtDNA haplogroups. Contrary to what could be expected, considering the strong reduction of population in the last 150 years, we observed that diversity was maintained and that microdifferentiation decreased. Both observations support a higher openness of parishes as a consequence of the increase in communication routes. From the first to the second period analyzed, a change in surname composition is evident, although the more frequent surnames in Flores are almost the same for both periods and some of them are reported to be surnames present in the first settlers of Flores. This result testifies to the impact of founders on the present-day gene pool of Flores island and allows us to infer that the genetic characterization of the present-day population of Flores could provide reliable information about the history of the peopling of the Azores.     

Endozoochorous dispersal of aquatic plants: does seed gut passage affect plant performance?

The ingestion of seeds by vertebrates can affect the germinability and/or germination rate of seeds. It is, however, unclear if an earlier germination as a result of ingestion affects later plant performance. For sago pondweed, Potamogeton pectinatus, the effects of seed ingestion by ducks on both germinability and germination rate have been previously reported from laboratory experiments. We performed an experiment to determine the effects of seed ingestion by ducks on germination, seedling survival, plant growth and asexual multiplication. Both at the start and end of the winter, seeds were fed to three captive shovelers (Anas clypeata) and planted outdoors in water-filled containers. Plant biomass and its allocation to vegetative parts (shoot and roots), tubers, and seeds were determined in autumn. More duck-ingested seeds than control (uningested) seeds germinated in early winter, but this difference disappeared for seeds planted in late winter, when the treatments were first stratified for 3 mo. None of the variables for measuring seedling survival and plant performance varied between treatments. Under our experimental conditions (no herbivory or competition), ingestion by ducks in early winter resulted in increased performance for seeds surviving gut passage due to enhanced seed germinability, without other costs or benefits for the seedlings.