Imaging intracellular pH in a reef coral and symbiotic anemone.

The challenges corals and symbiotic cnidarians face from global environmental change brings new urgency to understanding fundamental elements of their physiology. Intracellular pH (pHi) influences almost all aspects of cellular physiology but has never been described in anthozoans or symbiotic cnidarians, despite its pivotal role in carbon concentration for photosynthesis and calcification. Using confocal microscopy and the pH sensitive probe carboxy SNARF-1, we mapped pHi in short-term light and dark-incubated cells of the reef coral Stylophora pistillata and the symbiotic anemone Anemonia viridis. In all cells isolated from both species, pHi was markedly lower than the surrounding seawater pH of 8.1. In cells that contained symbiotic algae, mean values of pHi were significantly higher in light treated cells than dark treated cells (7.41 +/- 0.22 versus 7.13 +/- 0.24 for S. pistillata; and 7.29 +/- 0.15 versus 7.01 +/- 0.27 for A. viridis). In contrast, there was no significant difference in pHi in light and dark treated cells without algal symbionts. Close inspection of the interface between host cytoplasm and algal symbionts revealed a distinct area of lower pH adjacent to the symbionts in both light and dark treated cells, possibly associated with the symbiosome membrane complex. These findings are significant developments for the elucidation of models of inorganic carbon transport for photosynthesis and calcification and also provide a cell imaging procedure for future investigations into how pHi and other fundamental intracellular parameters in corals respond to changes in the external environment such as reductions in seawater pH.

Cytogenetic abnormalities associated with the t(12;21): a collaborative study of 169 children with t(12;21)-positive acute lymphoblastic leukemia.

The t(12;21)(p13;q22) is a cryptic abnormality observed in 25% of children with B-lineage acute lymphoblastic leukemia (ALL), associated with a favorable prognosis. To determine whether specific cytogenetic abnormalities accompany the t(12;21), we analyzed the cytogenetic profiles of blast cells from 169 ALL cases positive for the t(12;21), previously identified by molecular methods. Only 13.6% of samples had normal karyotypes. Structural changes were detected in 89.7% of abnormal karyotypes, and numerical abnormalities in 47%. Rearrangements of 12p were the most frequent structural aberration (57 out of 146 patients with chromosomal abnormalities). Nonspecific deletions of chromosomes 6 and 9 were also found. The most frequent numerical abnormalities was trisomy for chromosomes 21. Blast cells were pseudodiploid (45.6%), hyperdiploid with 47 to 51 chromosomes (24.3%), hypodiploid with 44 to 45 chromosomes (10%), near-triploid (0.6%), or near-tetraploid (5.9%). Our results show that the t(12;21) is not associated with hyperdiploidy of 52 to 68 chromosomes or with the prognostic t(1;19), t(4;11) or t(9;22). Only children with B-lineage ALL who lack these abnormalities detected by conventional cytogenetics will probably benefit from additional testing by molecular methods to detect the t(12;21).

Soluble organic matrix of two Scleractinian corals: partial and comparative analysis.

This study is a biochemical and molecular analysis of the soluble organic matrix (SOM) of two Scleractinian corals differing in their morphological characteristics: Stylophora pistillata, a branched robust coral and Pavona cactus, a leafy complex coral. Soluble organic matrix of both coral species were shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analyses of soluble organic matrix proteins were very different. Three proteins of S. pistillata and at least five proteins of P. cactus were detected by silver staining, some of them being able to bind calcium. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species. This domain suggests an essential role for this protein in the control of mineralization.

Morphological plasticity of the coral skeleton under CO2-driven seawater acidification.

Ocean acidification causes corals to calcify at reduced rates, but current understanding of the underlying processes is limited. Here, we conduct a mechanistic study into how seawater acidification alters skeletal growth of the coral Stylophora pistillata. Reductions in colony calcification rates are manifested as increases in skeletal porosity at lower pH, while linear extension of skeletons remains unchanged. Inspection of the microstructure of skeletons and measurements of pH at the site of calcification indicate that dissolution is not responsible for changes in skeletal porosity. Instead, changes occur by enlargement of corallite-calyxes and thinning of associated skeletal elements, constituting a modification in skeleton architecture. We also detect increases in the organic matrix protein content of skeletons formed under lower pH. Overall, our study reveals that seawater acidification not only causes decreases in calcification, but can also cause morphological change of the coral skeleton to a more porous and potentially fragile phenotype.

Cloning of a calcium channel alpha1 subunit from the reef-building coral, Stylophora pistillata.

While the mechanisms of cellular Ca2+ entry associated with cell activation are well characterized, the pathway of continuous uptake of the large amount of Ca2+ needed in the biomineralization process remains largely unknown. Scleractinian corals are one of the major calcifying groups of organisms. Recent studies have suggested that a voltage-dependent Ca2+ channel is involved in the transepithelial transport of Ca2+ used for coral calcification. We report here the cloning and sequencing of a cDNA coding a coral alpha1 subunit Ca2+ channel. This channel is closely related to the L-type family found in vertebrates and invertebrates. Immunohistochemical analysis shows that this channel is present within the calicoblastic ectoderm, the site involved in calcium carbonate precipitation. These data and previous results provide molecular evidence that voltage-dependent Ca2+ channels are involved in calcification. Cnidarians are the most primitive organisms in which a Ca2+ channel has been cloned up to now; evolutionary perspectives on Ca2+ channel diversity are discussed.

The distribution of the CDW52 molecule on blood cells and characterization of its involvement in T cell activation.

The O97 mouse mAb was used to define, together with the CAMPATH-1 mAb series, the CDw52 in the course of the Fourth International Workshop on Human Leucocyte Differentiation Antigens. O97 and CAMPATH-1M produce full competitive inhibition and react with an epitope dependent on O-linked sugar residues. The two mAb, however, display significant differences in reactivity with leukocyte populations--in fact the reactivity of O97, which also exerts a powerful cytotoxic effect with human C', is similar to mAb from the CAM-PATH-1 series having the broadest one. Noteworthy, O97 spares PBL, NK, and LAK precursors, permitting an easy purification of these cells; O97 is able to kill long-term colony-forming cells in bone marrow culture and characteristically reacts, in contrast to CAMPATH-1M, with erythrocytes. Western blotting has revealed a strikingly different molecular size on red cells, since CDw52 mAb revealed a broad band ranging from 6-16 kDa, instead of the 21-28 kDa revealed from leukocyte extracts. In agreement with immunofluorescence data, O97 revealed abundant material from red cells in Western blotting, while reactivity of CAMPATH-1M was very faint. Overall, these results show that distinct molecular forms of the CDw52 molecules are present on different blood cells. We have also characterized an activation signal that can be delivered to T cells via the CDw52 molecule by CAMPATH-1M but not by O97. This is an accessory signal that can complement a primary activation signal delivered via the CD2 pathway but not via the CD3-TcR pathway. This is similar to the effects obtained with the CD45R (2H4) mAb, 2H4 and CAMPATH-1M mAb having additive effects on T cell activation via CD2.

[Lymphocytic molecules implicated in rosette phenomenon. Differential effect of antilymphocyte serum on T (CD2+E2+) and B (CD2-E2+) lymphocytes]. / Molécules lymphocytaires impliquées dans le phénomène des rosettes. Effet différentiel du sérum anti-lymphocytaire sur les lymphocytes T (CD2+E2+) et B (CD2-E2+).

The molecule E2 is present on T lymphocytes and thymocytes and is implicated in rosette phenomenon most probably via interaction with CD2. The discrepancy observed between rosette levels and lymphocyte phenotypes in the follow-up of kidney-transplanted patients treated with antilymphocyte globulins (ALG), methylprednisolone and azathioprine leaded us to study the effect of ALG on E2 molecule.

Comparative analysis of the soluble organic matrix of axial skeleton and sclerites of Corallium rubrum: insights for biomineralization.

We analysed the soluble organic matrix (SOM) of two biominerals formed by the same organism but differing by their morphological characteristics: the axial skeleton and the sclerites of Corallium rubrum. The results of 1D SDS-PAGE electrophoresis show for the two biominerals that SOM proteins bands have similar apparent molecular weight but differ in quantity. Further analysis by 2D electrophoresis reveals each protein band as a line of spots with different isoelectric points. Our results suggest that each SOM protein band consists of a mix of proteins and/or one unique protein with post-translational modifications. By immunohistochemistry, we show that antibodies raised against the SOM of axial skeleton and sclerites label the SOM of the two biominerals but also label the insoluble organic matrix suggesting the presence of common epitopes between the two biominerals and the two organic fractions.

Evidence of low molecular weight components in the organic matrix of the reef building coral, Stylophora pistillata.

Biominerals contain both inorganic and organic components. Organic components are collectively termed the organic matrix, and this matrix has been reported to play a crucial role in mineralization. Several matrix proteins have been characterized in vertebrates, but only a few in invertebrates, primarily in Molluscs and Echinoderms. Methods classically used to extract organic matrix proteins eliminate potential low molecular weight matrix components, since cut-offs ranging from 3.5 to 10 kDa are used to desalt matrix extracts. Consequently, the presence of such components remains unknown and these are never subjected to further analyses. In the present study, we have used microcolonies from the Scleractinian coral Stylophora pistillata to study newly synthesized matrix components by labelling them with 14C-labelled amino acids. Radioactive matrix components were investigated by a method in which both total organic matrix and fractions of matrix below and above 5 kDa were analyzed. Using this method and SDS-PAGE analyses, we were able to detect the presence of low molecular mass matrix components (<3.5 kDa), but no free amino acids in the skeletal organic matrix. Since more than 98% of the 14C-labelled amino acids were incorporated into low molecular weight molecules, these probably form the bulk of newly synthesized organic matrix components. Our results suggest that these low molecular weight components may be peptides, which can be involved in the regulation of coral skeleton mineralization.

Monoclonal antibodies directed against the E2 protein (MIC2 gene product) induce exposure of phosphatidylserine at the thymocyte cell surface.

Monoclonal antibodies (mAbs) directed against E2, a 32-kDa transmembrane protein encoded by the MIC2 gene located in the pseudoautosomal region, induce a transbilayer movement of phosphatidylserine and, to a lesser extent, phosphatidylethanolamine in human thymocytes and a Jurkat T lymphocytes. The translocation of phosphatidylserine has been evidenced by using either derivatization of anionic phospholipids with trinitrobenzenesulfonate (TNBS) or cytofluorimetry after labeling of cells with antiphosphatidylserine antibodies. The perturbation of membrane phospholipids induced by anti-E2 mAbs was further evidenced by labeling the cells with merocyanine 540. The specificity of anti-E2-induced perturbations of membrane asymmetry was tested by using a number of mAbs able to activate T cells, including CD3 and CD2. The results strongly suggest that anti-E2-induced changes in PtdSer are related to cell aggregation since the same mAbs specifically induce the aggregation of both thymocytes and Jurkat cells and since the E2 molecule has been previously implicated in the adhesive properties of human T cells with erythrocytes.

Engagement of the CD45 molecule induces homotypic adhesion of human thymocytes through a LFA-1/ICAM-3-dependent pathway.

Cell-cell interactions play a central role during differentiation and development of the immune system. T or B lymphocyte homotypic adhesions can be induced via several surface molecules which, in some cases, are known to trigger the LFA-1/ICAM-1 adhesion pathway. We show here that mAbs reacting with the CD45 common epitopes or restricted RO epitope lead to a strong and rapid aggregation of all human thymocytes, and of T cell lines with an immature phenotype, but not of peripheral T lymphocytes. Aggregation requires energy, a physiologic temperature, Mg2+ divalent cations, and an intact cytoskeleton. It is LFA-1 dependent because CD11a and CD18 mAbs inhibit homotypic aggregation, whereas CD11b and CD11c mAbs do not. Homotypic thymocyte adhesion, however, is not decreased by CD54 mAb (anti-ICAM-1) but is inhibited by CDw50 mAb (anti-ICAM-3). Soluble CD22 fails to induce thymocyte adhesion, suggesting that CD45-induced aggregation is triggered by another ligand. Finally, because inhibitions observed with mAbs against LFA-1 and ICAM-3 are only partial, it can be assumed that another adhesion pathway is involved in thymocyte adhesion. The adhesion event specific for thymocytes we describe here is likely to play an important role in T cell differentiation.

The E2 molecule (CD99) specifically triggers homotypic aggregation of CD4+ CD8+ thymocytes.

We have previously described E2 as a 32-kDa transmembrane glycoprotein displaying an isomorphism, as two epitopes (defined by mAbs O662 and L129) are widely distributed on T cells whereas two epitopes are restricted to T cell subsets (defined by mAbs D44 and 12E7). E2, the MIC-2 gene product, is involved in T cell adhesion because anti-E2 mAbs against pan T epitopes block spontaneous T cell rosettes. Pan T E2 mAbs are also able to induce exposure of the phosphatidylserine at the thymocyte surface but not at the surface of mature T lymphocytes, an event most likely linked to adhesion phenomena. We now show here that the anti-E2 mAbs (0662 and L129) that block rosettes and induce phosphatidylserine exposure at the thymocyte surface, and not those reacting with epitopes not involved in adhesion, also trigger aggregation of certain immature T cell lines and no other cell lines tested. Among the normal cells tested, anti-E2 mAbs exclusively induce homotypic aggregation of CD4+ CD8+ human thymocytes. This phenomenon is temperature, energy, and Mg++ dependent, and requires an intact cytoskeleton. These adhesion properties are rather characteristic of integrins. Nevertheless, mAb against beta 1, beta 2, and beta 3 integrin chains, as well as those against alpha-chains known to be present on thymocytes, are unable to block corticothymocyte aggregation. We conclude that E2 triggers on corticothymocytes and no other T cells a homotypic adhesion pathway most likely mediated by an uncharacterized integrin.

Isoforms of the E2 molecule: D44 monoclonal antibody defines an epitope on E2 and reacts differentially with T cell subsets.

Human T cell rosetting with erythrocytes is clearly dependent on the CD2-CD58 interaction. We have previously demonstrated that other T cell molecules are involved in the rosette phenomenon, including the E2 molecule, a 32-kDa transmembrane glycoprotein. In this report we show that the D44 monoclonal antibody (mAb), previously shown to subdivide T cells into subpopulations with distinct functional repertoires and to identify 70% of lymphocytes from bronchoalveolar lavage from HIV+ patients, defined a new epitope on the E2 molecule. This was illustrated by the reactivity of the D44 mAb in Western blot experiments performed with the immunoaffinity purified E2 molecule. Moreover, double-labeling experiments showed that the E2 molecule exhibited varying epitopes when expressed in different cell types. The D44 and 12E7 epitopes were restricted to distinct subpopulations of T cells and, more importantly, the D44 expression was limited to the CD29+ population including the memory subset. The O662 and L129 epitopes are present on all T cells. Thus, the E2 molecule has both common and variable epitopes in its extracellular domain, and only the common epitopes seem to be involved in T cell adhesion processes.

CD59 molecule: a second ligand for CD2 in T cell adhesion.

The T cell surface molecule CD2 forms, with its counter-receptor CD58 (LFA-3), a powerful adhesion/activation pathway. There is some evidence that CD2 might bind more than a single ligand. Chinese hamster ovary cells (CHO) expressing human CD59 after cDNA transfection (CD59+CHO) form rosettes with human T cells; these rosettes are inhibited in a dose-dependent fashion by the CD59 monoclonal antibody (mAb) H19 and by the CD2 mAb O275 known to block natural rosettes, but not by the CD2R mAb D66, a poor rosette blocker. CD2+CHO transfectants form rosettes with human erythrocytes; these rosettes are inhibited by the CD59 mAb H19 in addition to CD2 mAb O275 and CD58 mAb; murine thymoma cells expressing human CD2 form rosettes with CD59+CHO cells that again are blocked by CD59 H19 and by CD2 O275 mAb. In a marked contrast with H19, two others CD59 mAb, YTH 53.1 and MEM 43, which react with a different epitope on CD59, led to a 50%-70% increase of the number of cells forming rosettes. In addition to rosette experiments, the binding of 125I-labeled CD59 molecules to CD2+CHO cells was specifically inhibited by unlabeled CD59 molecule and CD2 mAb. Furthermore, the binding of CD59 molecules to resting T cells induced expression of CD2R epitopes. These results directly show that CD2 binds CD59 and that subtle molecular changes occur upon binding.