Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.      

EpCAM: Structure and function in health and disease

Injection of tumor cells in mice more than 30 years ago resulted in the discovery of an epithelial antigen, later defined as a cell adhesion molecule (EpCAM). Although EpCAM has since evoked significant interest as a target in cancer therapy, mechanistic insights on the functions of this glycoprotein have been emerging only very recently. This may have been caused by the multitude of functions attributed to the glycoprotein, its localization at different subcellular sites and complex posttranslational modifications. Here, we review how EpCAM modifies cell–cell contact adhesion strength and tissue plasticity, and how it regulates cell proliferation and differentiation. Major knowledge derived from human diseases will be highlighted: Mutant EpCAM that is absent from the cell surface leads to fatal intestinal abnormalities (congenital tufting enteropathy). EpCAM-mediated cell proliferation in cancer may result from signaling (i) via regulated intramembrane proteolysis and/or (ii) the localization and association with binding partners in specialized membrane microdomains. New insight in EpCAM signaling will help to develop optimized cancer therapies and open new avenues in the field of regenerative medicine.     

Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence

The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet-isolation might contribute to longevity-variations of immunoisolated islet-grafts.     

Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell-cell communication

Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK(+)) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK(+) and, to a lesser extent, with wild-type c-Src, but not with kinase-dead c-Src. Mutation of residue Cx43 Tyr(265) (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK(+) in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43Delta263) that lacks residue Tyr(265). Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr(265), resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.     

Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas RhoA is activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors, it is unknown how LPA receptors may signal to Rac. Here we report that the prototypic LPA(1) receptor (previously named Edg2), when expressed in B103 neuroblastoma cells, mediates transient activation of RhoA and robust, prolonged activation of Rac leading to cell spreading, lamellipodia formation, and stimulation of cell migration. LPA-induced Rac activation is inhibited by pertussis toxin and requires phosphoinositide 3-kinase activity. Strikingly, LPA fails to activate Rac in cell types that lack the Rac-specific exchange factor Tiam1; however, enforced expression of Tiam1 restores LPA-induced Rac activation in those cells. Tiam1-deficient cells show enhanced RhoA activation, stress fiber formation, and cell rounding in response to LPA, consistent with Tiam1/Rac counteracting RhoA. We conclude that LPA(1) receptors couple to a G(i)-phosphoinositide 3-kinase-Tiam1 pathway to activate Rac, with consequent suppression of RhoA activity, and thereby stimulate cell spreading and motility.     

Connexin-43 interactions with ZO-1 and alpha- and beta-tubulin

Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication. (ii) the ZO-1 'scaffold' protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of alpha/beta-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds alpha-tubulin equally well as beta-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.     

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Mitochondrial Dysfunction in Human Leukemic Stem/Progenitor Cells upon Loss of RAC2

Leukemic stem cells (LSCs) reside within bone marrow niches that maintain their relatively quiescent state and convey resistance to conventional treatment. Many of the microenvironmental signals converge on RAC GTPases. Although it has become clear that RAC proteins fulfill important roles in the hematopoietic compartment, little has been revealed about the downstream effectors and molecular mechanisms. We observed that in BCR-ABL-transduced human hematopoietic stem/progenitor cells (HSPCs) depletion of RAC2 but not RAC1 induced a marked and immediate decrease in proliferation, progenitor frequency, cobblestone formation and replating capacity, indicative for reduced self-renewal. Cell cycle analyses showed reduced cell cycle activity in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Moreover, a decrease in mitochondrial membrane potential was observed upon RAC2 downregulation, paralleled by severe mitochondrial ultrastructural malformations as determined by automated electron microscopy. Proteome analysis revealed that RAC2 specifically interacted with a set of mitochondrial proteins including mitochondrial transport proteins SAM50 and Metaxin 1, and interactions were confirmed in independent co-immunoprecipitation studies. Downregulation of SAM50 also impaired the proliferation and replating capacity of BCR-ABL-expressing cells, again associated with a decreased mitochondrial membrane potential. Taken together, these data suggest an important role for RAC2 in maintaining mitochondrial integrity.     

Gap junction protein connexin-43 interacts directly with microtubules.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.