Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.     

Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.     

A second gene at the tomato Cf-4 locus confers resistance to cladosporium fulvum through recognition of a novel avirulence determinant

The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

The self peptide annexin II (208–223) presented by dendritic cells sensitizes autologous CD4+ T lymphocytes to recognize melanoma cells

Annexin II is known to be over-expressed in different types of tumours. We show here that annexin II protein is expressed by melanoma cell lines in various amounts, consistent with previous findings that an annexin II (208-223) peptide could be eluted from isolated HLA-DR molecules of a constitutively MHC class II-positive melanoma line. T cells sensitized to annexin II (208-223) in vitro using peptide-pulsed autologous dendritic cells responded only to the lines which overexpressed annexin II, in a peptide-specific, HLA-DR-restricted fashion. These CD4+ T cells proliferated strongly and secreted large amounts of type 1 cytokines in response to annexin II (208-223) peptide or annexin II protein-positive melanoma cell lines. These results demonstrate that the annexin II (208-223) peptide, corresponding to a non-mutated sequence of a normal protein, induces antigen-specific T cells which can respond to melanoma cells over-expressing the annexin II molecule. This peptide may therefore be useful in immunotherapy for recruiting CD4+ type 1 helper cells active locally in the tumour environment.