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CD137 (4-1BB)-mediated costimulation plays an important role in directing the fate of Ag-stimulated T cells and NK cells, yet the role of CD137 in mediating B cell function is unknown. We found that CD137 is expressed in vitro on anti-Ig–stimulated peripheral blood B cells and in vivo on tonsillar B cells with an activated phenotype. In vitro CD137 expression is enhanced by CD40 stimulation and IFN-γ and is inhibited by IL-4, -10, and -21. The expression of CD137 on activated human B cells is functionally relevant because engagement with its ligand at the time of activation stimulates B cell proliferation, enhances B cell survival, and induces secretion of TNF-α and -β. Our study suggests that CD137 costimulation may play a role in defining the fate of Ag-stimulated human B cells.

CD137 (4-1BB) is a costimulatory mol-ecule that can be manipulated for the treatment of cancer and autoimmune disease. Although it is known that agonistic antibodies (mAbs) against CD137 enhance the rejection of murine tumors in a natural killer (NK) cell– and T cell–dependent fashion, the mechanism for NK dependence is poorly understood. In this study, we evaluated the ability of 2 different glycoforms of a chimerized antihuman CD137 mAb, an aglycosylated (GA) and a low fucose form (GG), to react with human NK cells. Both mAbs bound similarly to CD137 and partially blocked the interaction between CD137 and CD137 ligand. However, unlike GA mAb, immobilized GG mAb activated NK cells and enhanced CD137 expression. These effects were seemingly dependent on Fc interaction with putative Fc receptors on the NK-cell surface, as only the immobilized Fc-fragment of GG was required for CD137 expression. Furthermore, CD137 expression could be enhanced with antibodies directed against non-CD137 epitopes, and the expression levels directly correlated with patterns of Fc-glycosylation recognized to improve Fc interaction with Fcγ receptors. Our data suggest that CD137 can be enhanced on NK cells in an Fc-dependent fashion and that expression correlates with phenotypic and functional parameters of activation.

The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings.

Unique ATP-inhibitable K+ channels (KATP) in the kidney determine the rate of urinary K+ excretion and play an essential role in extracellular K+ balance. Here, we demonstrate that functionally similar low sulfonylurea affinity KATP channels are formed by two heterologous molecules, products of Kir1.1a and cystic fibrosis transmembrane conductance regulator (CFTR) genes. Co-injection of CFTR and Kir1.1a cRNA into Xenopus oocytes lead to the expression of K+ selective channels that retained the high open probability behavior of Kir1.1a but acquired sulfonylurea sensitivity and ATP-dependent gating properties. Similar to the KATP channels in the kidney but different from KATP channels in excitable tissues, the Kir1.1a/CFTR channel was inhibited by glibenclamide with micromolar affinity. Since the expression of Kir1.1a and CFTR overlap at sites in the kidney where the low sulfonylurea affinity KATP are expressed, our study offers evidence that these native KATP channels are comprised of Kir1.1a and CFTR. The implication that Kir subunits can interact with ABC proteins beyond the subfamily of sulfonylurea receptors provides an intriguing explanation for functional diversity in KATP channels.

We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.

We have analyzed the gene structure that gives rise to tissue-specific isoforms of the Na/Ca exchanger. Five distinct isoforms of the Na/Ca exchanger from rabbit brain, kidney, and heart have been identified previously to which we now add a new brain isoform. Reverse-transcribed polymerase chain reaction, library screening, and sequence analysis of cDNA coding regions indicate that the only significant alteration of the Na/Ca exchanger cDNA in rabbit brain, kidney, and heart isoforms is located in the carboxyl end of the putative intracellular loop of the protein, a region recently linked to ionic and metabolic regulation of the Na/Ca exchanger. Additionally, we find that the Na/Ca exchanger isoforms found in lung and skeletal muscle may arise from among these same six isoforms. Examination of the gene structure of the Na/Ca exchanger in rabbit indicates how the single gene that encodes for the Na/Ca exchanger is alternatively spliced to give rise to the five rabbit isoforms. Specifically, sequence analysis of the intron-exon boundaries reveals the presence of two "mutually exclusive" exons in conjunction with four "cassette" exons in the region of the Na/Ca exchanger gene that codes for the carboxyl end of the predicted intracellular loop region. This unusual arrangement of exons in the Na/Ca exchanger gene could allow for the generation of up to 32 different Na/Ca exchanger mRNAs and accounts for the isoforms identified to date.

Sodium/calcium (Na+/Ca2+) exchangers are membrane transport systems that regulate Ca2+-homeostasis in many eukaryotic cells. In olfactory and vomeronasal sensory neurons ligand-induced olfactory signal transduction is associated with influx and elevation of intracellular Ca2+, [Ca2+]i. While much effort has been devoted to the characterization of Ca2+-related excitation and adaptation events of olfactory chemosensory neurons (OSNs), much less is known about mechanisms that return [Ca2+]i to the resting state. To identify proteins participating in the poststimulus Ca2+-clearance of mouse OSNs, we analyzed the expression of three potassium (K+)-independent (NCX1, 2, 3) and three K+-dependent (NCKX1, 2, 3) Na+/Ca2+ exchangers. In situ hybridization showed that mRNAs of all six Na+/Ca2+ exchangers coexist in neurons of the olfactory and vomeronasal systems, and that some are already detectable in the embryo. Of these, NCX1 and NCKX1 represent the most and least abundant mRNAs, respectively. Moreover, immunohistochemistry revealed that the NCX1, 2, and 3 proteins are expressed in nearly all neurons of the olfactory epithelium, the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. These three exchanger proteins display different expression profiles in dendrites, knobs, and plasma membranes of OSNs and in sustentacular cells. Furthermore, we show that NCX1 mRNA in rat olfactory mucosa is expressed as 8 alternative splice variants. This is the first comprehensive analysis of Na+/Ca2+ exchanger expression in the mammalian olfactory system. Our results suggest that Ca2+-extrusion by OSNs utilizes multiple different Na+/Ca2+ exchangers and that different subtypes are targeted to different subcellular compartments. J. Comp. Neurol. 501:944–958, 2007. © 2007 Wiley-Liss, Inc.

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.

cDNAs for the Na+/Ca2+ exchanger from Drosophila melanogaster (Dmel/Nck) have been cloned by homology screening using the human heart Na+/Ca2+ exchanger cDNA. The overall deduced protein structure for Dmel/Nck is similar to that of mammalian Na+/Ca2+ exchanger genes NCX1 and NCX2, having six hydrophobic regions in the amino terminus separated from six at the carboxy-terminal end by a large intracellular loop. Sequence comparison of the Drosophila exchanger cDNAs with NCX1 and NCX2 Na+/Ca2+ exchangers are approximately 46% identical at the deduced amino acid level. Consensus phosphorylation sites for both protein kinase C and protein kinase A are present on the intracellular loop region of the Dmel/Nck. Alternative splicing for the Dmel/Nck gene is suggested in the same intracellular loop region as demonstrated for NCX1. Functionally, the Drosophila Na+/ Ca2+ exchanger expressed in oocytes differs from expressed mammalian NCX1 with regard to Ca2+ transport in Ca2+/ Ca2+ exchange and the effect of monovalent-dependent Ca2+/ Ca2+ exchange. The Dmel/Nck gene maps to chromosome 3 (93A-B) using in situ hybridization to polytene chromosomes, the same position as the Na(+)-K(+)-ATPase, a related transporter. We conclude that, although extracellular Na+ concentration-dependent Ca2+ transport is subserved by both human and Drosophila Na+/Ca2+ exchangers, there are clear and important differences in the transporters, which should be useful in deducing how the Na+/Ca2+ exchanger protein function depends on its structure.

Short DNA sequence motifs have been demonstrated to interact with DNA binding proteins and regulate flanking genes. The short nature and the lack of continuity of many of these DNA binding sites make it difficult to develop an approach to characterize genes that have the same flanking sequences. We tested various oligonucleotide combinations using an immunoglobulin variable region gene family as a model amplification system. One successful amplification strategy used an oligonucleotide containing two known noncontiguous short sequences connected by random insertion of all four bases to maintain the appropriate spacing. A second approach used an oligonucleotide having a single short homologous sequence with the addition of all four bases randomly placed at the 5′ end to increase the extent of homology. Both strategies will permit the priming of members of a specific gene family, with the two short sequences bridged by all four bases randomly added being more efficient in the amplification process.

To the Editor : We read the report of Lin et al. ([1][1]) with great interest. The authors suggest that observed increases in the Ni2+-sensitive bidirectional current after exposure of the mouse, rat, and guinea pig myocytes to isoproterenol were in fact not attributable to changes in the Na+/Ca2+

The H-2Kbgene is a member of the large major histocompatibility complex class I gene family. Since many members of this family cross-hybridize with class I cDNA probes, the cloned H-2Kbgene was identified by hybridization with specific oligonucleotide probes. This clone was definitively shown to encode the H-2Kbpolypeptide by partial DNA sequencing and by serological and tryptic peptide analyses of the expressed product.

The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.

Genetic interaction as a mechanism for the generation of mutations is suggested by recurrent, multiple nucleotide substitutions that are identical to nucleotide sequences elsewhere in the genome. We have sequenced the mutant K gene from the bm6 mouse, which is one of a series of eight closely related, yet independently occurring mutants known collectively as the "bg series." Two changes from the Kb gene are found, positioned 15 nucleotides apart: an A-to-T change and a T-to-C change in the codons corresponding to amino acids 116 and 121, resulting in Tyr-to-Phe and Cys-to-Arg substitutions, respectively. Hybridization analysis with an oligonucleotide specific for the altered Kbm6 sequence identifies one donor gene, Q4, located in the Qa region of the H-2 complex. The two altered nucleotides that differentiate Kbm6 and Kb are present in Q4 in a region where Kb and Q4 are otherwise identical for 95 nucleotides, delineating the maximum genetic transfer between the two genes. Because the Kbm6 mutation arose in an homozygous mouse these data indicate that the Q4 gene contains the only donor sequence and demonstrates that Q-region gene sequences can interact with the Kb gene to generate variant K molecules.

In many cells including cardiac myocytes, cytoplasmic Ca is importantly controlled by the plasmalemmal Na-Ca exchanger (3, 8). The tissue diversity and differences in cellular environment raise the question whether the same exchanger is found in all tissues. Recent experiments using rod cells have demonstrated that at least two forms of Na-dependent Ca transport exist. We have examined this issue in various rat and human tissues using the cloned human cardiac Na-Ca exchanger cDNA. Northern blot analysis in these two species show that the major transcript of the Na-Ca exchanger is 7.2 kilobases in heart, brain, kidney, liver, pancreas, skeletal muscle, placenta, and lung. Furthermore, ribonuclease protection analysis in rats shows conservation of the 348-base pair segment tested in heart, brain, kidney, skeletal muscle, and liver. Additionally, Southern blot analysis suggests that a single gene encodes this Na-Ca exchanger. Finally, we show that the clone used to generate our probes encodes a completely functional Na-Ca exchanger. With the use of COS cells and 293 cells transfected with the cloned human cardiac Na-Ca exchanger, we tested the Ca transport properties of the Na-Ca exchanger, the voltage dependence of the Na-Ca exchanger, as well as the Na dependence of the transport function of the Na-Ca exchanger. We conclude that the cardiac form of the Na-Ca exchanger is completely functional when the cDNA is expressed in mammalian cell lines, and, furthermore, this "cardiac" form of the Na-Ca exchanger is naturally expressed in all human and rat tissues tested (but at varying levels).

The mechanism of Ni2+block of the Na+/Ca2+exchanger was examined in Sf 9 cells expressing the human heart Na+/Ca2+exchanger (NCX1-NACA1). As predicted from the reported actions of Ni2+, its application reduced extracellular Na+-dependent changes in intracellular Ca2+concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured63Ni2+entry. The63Ni2+entry was observed in Sf 9 cells expressing the Na+/Ca2+exchanger but not in control cells. The established sequential transport mechanism of the Na+/Ca2+exchanger could be compatible with these results if one of the two ion translocation steps is blocked by Ni2+and the other permits Ni2+translocation. We conclude that, because Ni2+entry was inhibited by extracellular Ca2+and enhanced by extracellular Na+, the Ca2+translocation step moved Ni2+, whereas the Na+translocation step was inhibited by Ni2+. A model is presented to discuss these findings.