BCR-signaling-induced cell death demonstrates dependency on multiple BH3-only proteins in a murine model of B-cell lymphoma.

Genetic recombination during B-cell development regularly results in the generation of autoreactive, potentially pathogenic B-cell receptors (BCRs). Consequently, multiple mechanisms link inappropriate BCR specificity to clonal deletion. Similar pathways remain in malignant B cells, offering the potential for targeting BCR signaling. Recently, small molecule inhibitors have realized this potential and, therefore, a deeper understanding of BCR-induced signaling networks in malignant cells is vital. The BH3-only protein Bim has a key role in BCR-induced apoptosis, but it has long been proposed that additional BH3-only proteins also contribute, although conclusive proof has been lacking. Here, we comprehensively characterized the mechanism of BCR-induced apoptosis in Eµ-Myc murine lymphoma cells. We demonstrate the upregulation of Bim, Bik, and Noxa during BCR signaling in vitro and that intrinsic apoptosis has a prominent role in anti-BCR antibody therapy in vivo. Furthermore, lymphomas deficient in these individual BH3-only proteins display significant protection from BCR-induced cell death, whereas combined loss of Noxa and Bim offers enhanced protection in comparison with loss of Bim alone. Some but not all of these effects were reversed upon inhibition of Syk or MEK. These observations indicate that BCR signaling elicits maximal cell death through upregulation of multiple BH3-only proteins; namely Bim, Bik, and Noxa.

Molecular cloning and characterization of rat P2Y4 nucleotide receptor.

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y4 (hP2Y4) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y4 receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP = ATP = ADP(pure) = UTP = ATPgammaS = 2-MeSATP = Ap4A > UDP(pure). ADP, ATPgammaS, 2-MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y4 is more like the P2U receptor subtype. The rP2Y4 receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y2 receptor (a known P2U receptor). Thus, rP2Y4 and the P2Y2 subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi-active) but can be distinguished as suramin-insensitive and suramin-sensitive P2U receptors, respectively.

Putative neuropeptides and an EF-hand motif region are encoded by a novel gene expressed in the four giant interneurons of the terrestrial snail.

Nine giant interneurons located in the pleural and parietal ganglia of the terrestrial snail Helix lucorum L. were reported to be a key element in the network controlling withdrawal behaviour of the animal. Using a combination of complementary DNA subtraction cloning and differential screening approaches we have isolated a novel gene named HCS2 which is expressed predominantly in a subset of these interneurons. The predicted amino acid sequence of the HCS2 protein contains at the N-terminus a hydrophobic leader sequence and four putative neuropeptides, and at the C-terminus a perfect match to the consensus motif of the EF-hand family of the Ca2+-binding proteins. All four predicted neuropeptides bear a C-terminal signature sequence Tyr-Pro-Arg-X (where X is Ile, Leu, Val or Pro), and three of them are likely to be amidated. Physiological action of three synthetic peptides corresponding to the predicted mature HCS2 peptides mimics fairly well the described action of parietal interneurons on follower motoneurons controlling pneumostome closure. In situ hybridization experiments demonstrated that the HCS2 gene is selectively expressed in the four parietal giant interneurons, as well as in several small unidentified neurons. The onset of the HCS2 transcription during embryogenesis coincides temporally with the time-point when the first withdrawal responses of the embryo to tactile stimulation appear. We propose that the HCS2 gene encodes a hybrid precursor protein whose processed products act as neuromodulators or neurotransmitters mediating the withdrawal reactions of the snail, and in addition may participate in the calcium regulatory pathways or calcium homeostasis in command neurons.

Early expression of a novel nucleotide receptor in the neural plate of Xenopus embryos.

Extracellular ATP functions as a neurotransmitter and neuromodulator in the adult nervous system, and a signaling molecule in non-neural tissue, acting either via ligand-gated ion channels (P2X) or G-protein-coupled receptors (P2Y). ATP can cause an increase in intracellular Ca2+ (Ca2+i) in embryonic cells and so regulate cell proliferation, migration, and differentiation. We have isolated a Xenopus cDNA encoding a novel P2Y receptor, XlP2Y, which is expressed abundantly in developing embryos. Recombinant XlP2Y responds equally to all five naturally occurring nucleoside triphosphates (ATP, UTP, CTP, GTP, and ITP), which elicit a biphasic Ca2+-dependent Cl- current (ICl,Ca) where the second phase persists for up to 60 min. XlP2Y also causes a continuous release of Ca2+i and a low level persistent activation of ICl,Ca in Xenopus oocytes through the spontaneous efflux of ATP. mRNAs for XlP2Y are expressed transiently in the neural plate and tailbud during Xenopus development, coincident with neurogenesis. This restricted pattern of expression and novel pharmacological features confer unique properties to XlP2Y, which may play a key role in the early development of neural tissue.

Novel gene HCS1 is specifically expressed in the giant interneurones of the terrestrial snail.

The terrestrial snail Helix lucorum is a promising model for molecular neurobiology since its central nervous system (CNS) is simple and contains many morphologically and functionally identified large neurones. Among these, the giant interneurones located in pleural and parietal ganglia are especially interesting because they trigger the withdrawal behaviour of the snail and participate in aversive conditioning. Here we describe the identification and characterization of a gene named HCS1 which is preferentially expressed in these interneurones. It encodes a putative protein 100 amino acids long containing an N-terminal hydrophobic leader peptide. No sequences with significant homology to HCS1 were found in the protein (Swiss-Prot) and nucleotide (EMBLbank) data libraries. We suppose that the product of this gene is a secreted protein, presumably a neuropeptide or a growth factor.

Differential splicing creates a diversity of transcripts from a neurospecific developmentally regulated gene encoding a protein with new zinc-finger motifs.

We have cloned a novel neurospecific gene, named neuro-d4, by differential screening a rat cerebral cortex cDNA library. Northern blot hybridization showed that neuro-d4 expression is restricted to neuronal tissues both in newborn and adult animals. The level of neuro-d4 mRNA in the rat central nervous system is high during the later stages of embryonic development and gradually decreases during the postnatal period. In situ hybridization suggests that the gene transcripts are localized in neuronal cell bodies. Nucleotide sequences of overlapped cDNA clones and all 12 exons in genomic clone were determined. The deduced protein has consensus sequences for a nuclear localization signal, a Krüppel-type zinc-finger and a new type of cysteine/histidine-rich motif resembling zinc-fingers. Several differential splicing variants were found, each of which influences the structure of the encoded protein.