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.

Meiotic mutations in rye Secale cereale L.

Spontaneous meiotic mutations of winter rye Secale cereale L. (2n = 14) were revealed in inbred F2 progenies, which were obtained by self-pollination of F1 hybrids resulting from crosses of individual plants of cultivar Vyatka or weedy rye with plants of self-fertile inbred lines. The mutations cause partial or complete sterility, and are maintained in heterozygote condition. Six types of mutations were distinguished as the result of cytological analysis of meiosis and genetic analysis. (1) Plants with nonallelic asynaptic mutations sy1 and sy9 lacked bivalents in 96.8 and 67.0% metaphase I cells, respectively, formed only axial elements but not the mature synaptonemal complex (SC), and had defects in telomere clustering in early prophase I. (2) Weak asynaptic mutant sy3 showed incomplete synapsis at the start of SC degradation at diplotene and lower chiasma number; yet only 2% meiocytes lacked bivalents in MI. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19 caused nonhomologous synapsis; i.e., a varying number of univalents and occasional multivalents were observed in MI, which was preceded by switches of pairing partners and fold-back synapsis at mid-prophase I. (4) Mutation mei6 led to the formation of protrusions and minor branched structures of the SC lateral elements. (5) Allelic mutations mei8 and mei8-10 caused irregular chromatin condensation along the chromosome length in prophase I, which was accompanied by chromosome sticking and fragmentation in MI. (6) Allelic mutations mei5 and mei10 determined chromosome supercondensation, caused the disturbance of meiotic spindle assembly, arrested meiosis at various stages but did not affect formation of the pollen wall, thus arrested meiocytes got covered with the pollen wall. Analysis of double mutants revealed recessive epistatic interactions for some mutations; the epistatic group was sy9 > sy1 > sy3 > sy19. This reflects the sequence of meiotic events controlled by the corresponding genes. The expression of sy2 and sy19 proved to be modified by additional genes. Most meiotic mutations found in rye have analogs in other plants.

Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs.

Voltage-gated calcium channel alpha1 subunits consist of four domains (I-IV), each with six transmembrane segments. A number of truncated isoforms have been identified to occur as a result of alternative splicing or mutation. We have examined the functional consequences for expression of full-length Ca(v)2.2 (alpha1B) of its coexpression with truncated constructs of Ca(v)2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits beta1b and alpha2delta-1, did not form functional channels. When they were coexpressed, low-density whole-cell currents and functional channels with properties similar to wild-type channels were observed. However, when domain I-II, domain III-IV, or domain I alone were coexpressed with full-length Ca(v)2.2, they markedly suppressed its functional expression, although at the single channel level, when channels were recorded, there were no differences in their biophysical properties. Furthermore, when it was coexpressed with either domain I-II or domain I, the fluorescence of green fluorescent protein (GFP)-Ca(v)2.2 and expression of Ca(v)2.2 protein was almost abolished. Suppression does not involve sequestration of the Ca(v)beta subunit, because loss of GFP-Ca(v)2.2 expression also occurred in the absence of beta subunit, and the effect of domain I-II or domain I could not be mimicked by the cytoplasmic I-II loop of Ca(v)2.2. It requires transmembrane segments, because the isolated Ca(v)2.2 N terminus did not have any effect. Our results indicate that the mechanism of suppression of Ca(v)2.2 by truncated constructs containing domain I involves inhibition of channel synthesis, which may represent a role of endogenously expressed truncated Ca(v) isoforms.

Interaction between G proteins and accessory subunits in the regulation of 1B calcium channels in Xenopus oocytes.

The accessory beta subunits of voltage-dependent Ca2+ channels (VDCCs) have been shown to regulate their biophysical properties and have also been suggested to antagonise the G protein inhibition of N-type (alpha1B), P/Q-type (alpha1A) and alpha1E channels. Here we have examined the voltage-dependent involvement of the four neuronal isoforms (beta1b, beta2a, beta3 and beta4) in the process of G protein modulation of alpha1B Ca2+ channels. All beta subunits hyperpolarized alpha1B current activation, and all antagonised the G protein-mediated depolarisation of current activation. However, except in the case of beta2a, there was no generalised reduction by beta subunits in the maximal extent of receptor-mediated inhibition of alpha1B current. In addition, all VDCC beta subunits enhanced the rate of current facilitation at +100 mV, for both receptor-mediated and tonic modulation. The rank order for enhancement of facilitation rate was beta3 > beta4 > beta1b > beta2a. In contrast, the amount of voltage-dependent facilitation during tonic modulation was reduced by beta subunit co-expression, despite the fact that the apparent Gbetagamma dissociation rate at +100 mV was enhanced by beta subunits to a similar level as for agonist-induced modulation. Our data provide evidence that G protein activation antagonises Ca2+-channel beta subunit-induced hyperpolarisation of current activation. Conversely, co-expression of all beta subunits increases the apparent Gbetagamma dimer dissociation rate during a depolarising prepulse. This latter feature suggests the co-existence of bound Ca2+-channel beta subunits and Gbetagamma dimers on the alpha1B subunits. Future work will determine how the interaction between Gbetagamma dimers and Ca2+-channel beta subunits with alpha1B results in a functional antagonism at the molecular level.

The alpha1B Ca2+ channel amino terminus contributes determinants for beta subunit-mediated voltage-dependent inactivation properties.

Co-expression of auxiliary beta subunits with the alpha1B Ca2+ channel subunit in COS-7 cells resulted in an increase in current density and a hyperpolarising shift in the mid-point of activation. Amongst the beta subunits, beta2a in particular, but also beta4 and beta1b caused a significant retardation of the voltage-dependent inactivation compared to currents with alpha1B alone, whilst no significant changes in inactivation properties were seen for the beta3 subunit in this system. Prevention of beta2a palmitoylation, by introducing cysteine to serine mutations (beta2a(C3,4S)), greatly reduced the ability of beta2a to retard voltage-dependent inactivation. Deletion of the proximal half of the alpha1B cytoplasmic amino terminus (alpha1BDelta1-55) differentially affected beta subunit-mediated voltage-dependent inactivation properties. These effects were prominent with the beta2a subunit and, to a lesser extent, with beta1b. For beta2a, the major effects of this deletion were a partial reversal of beta2a-mediated retardation of inactivation and the introduction of a fast component of inactivation, not seen with full-length alpha1B. Deletion of the amino terminus had no other major effects on the measured biophysical properties of alpha1B when co-expressed with beta subunits. Transfer of the whole alpha1B amino terminus into alpha1C (alpha1bCCCC) conferred a similar retardation of inactivation on alpha1C when co-expressed with beta2a to that seen in parental alpha1B. Individual (alpha1B(Q47A) and alpha1B(R52A)) and double (alpha1B(R52,54A)) point mutations within the amino terminus of alpha1B also opposed the beta2a-mediated retardation of alpha1B inactivation kinetics. These results indicate that the alpha1B amino terminus contains determinants for beta subunit-mediated voltage-dependent inactivation properties. Furthermore, effects were beta subunit selective. As deletion of the alpha1B amino terminus only partially opposed beta subunit-mediated changes in inactivation properties, the amino terminus is likely to contribute to a complex site necessary for complete beta subunit function.

Acidic motif responsible for plasma membrane association of the voltage-dependent calcium channel beta1b subunit.

Voltage-dependent calcium channels consist of a pore-forming transmembrane alpha1-subunit, which is known to associate with a number of accessory subunits, including alpha2-delta- and beta-subunits. The beta-subunits, of which four have been identified (beta1-4), are intracellular proteins that have marked effects on calcium channel trafficking and function. In a previous study, we observed that the beta1b-subunit showed selective plasma membrane association when expressed alone in COS7 cells, whereas beta3 and beta4 did not. In this study, we have examined the basis for this, and have identified, by making chimeric beta-subunits, that the C-terminal region, which shows most diversity between beta-subunits, of beta1b is responsible for its plasma membrane association. Furthermore we have identified, by deletion mutations, an 11-amino acid motif present in the C terminus of beta1b but not in beta3 (amino acids 547-556 of beta1b, WEEEEDYEEE), which when deleted, reduces membrane association of beta1b. Future research aims to identify what is binding to this sequence in beta1b to promote membrane association of this calcium channel subunit. It is possible that such membrane association is important for the selective localization or clustering of particular calcium channels with which beta1b is associated.

Coexpression of mRNAs for P2X1, P2X2 and P2X4 receptors in rat vascular smooth muscle: an in situ hybridization and RT-PCR study.

The expression of mRNAs for three P2X receptor subtypes (PX21, P2X2, P2X4) in the rat vascular system was studied by in situ hybridization and RT-PCR. In heart sections mRNAs transcripts for all three receptors were colocalized in smooth muscle cells of coronary vessels, while no specific positivity was apparent in myocardium. Coexpression of P2X receptor mRNA transcripts were also observed in other peripheral vessels, including aorta, pulmonary artery, internal and external iliac arteries, renal artery and femoral artery. By contrast, no mRNA transcripts of the above receptors were found in the superior mesenteric artery. RT-PCR performed on microdissected tissues (coronary arteries, aorta and myocardium from various heart areas) confirmed the presence of P2X1, P2X2 and P2X4 receptor mRNAs. Furthermore, in the same tissues two splice variants of the P2X2 receptor were identified. These results reveal an important molecular heterogeneity of P2X receptors, thus substantiating the possibility of a heteropolymeric assembly of ATP-gated ion channels in the cardiovascular system.

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.

Characterisation of subtypes of the P2X and P2Y families of ATP receptors in the foetal human heart.

ATP exerts a variety of actions within the myocardium, including the regulation of coronary vascular tone and modulation of the autonomic control of the heart. In order to characterise the ATP receptor subtypes involved in these effects, degenerate oligonucleotides were used to clone receptors of both P2X and P2Y families from the human foetal heart. About 1 ng of "Quick-Clone cDNA" from foetal human heart was subjected to amplification with two pairs of degenerate oligonucleotides designed to amplify subtypes of the P2X and P2Y receptor families by means of PCR reactions. The sequence analysis of 34 and 29 clones of the P2X and P2Y receptor families, respectively, demonstrated that P2X1, P2X3 and P2X4 subtypes are present in the human foetal heart together with P2Y6, P2Y2 and P2Y4 receptors. P2X1 and P2Y4 receptor subtypes were here characterised for the first time in the human foetal heart. The present study provides the first molecular characterisation of ATP receptors in the foetal human heart. The results show that many P2 receptor subtypes are expressed in the foetal human heart, perhaps contributing to developmental processes as well as to the activity of the foetal heart.

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.

Meiotic mutants of rye Secale cereale L. II. The nonhomologous synapsis in desynaptic mutants sy7 and sy10.

We studied the expression and inheritance of two spontaneous mutations found in different populations of rye Secale cereale L. that cause high univalent frequency in meiosis and low fertility. Both mutations were inherited as monogenic recessives. For each of the mutations the corresponding gene symbols (sy7 and sy10) were suggested although their allelism has not been studied. These mutants differ in chiasma frequency and in the number of univalents per meiocyte. Electron microscopy of the wholemount surface-spread synaptonemal complexes (SCs) from microsporocytes of both mutants revealed that during meiotic prophase I random synapsis began and progressed that involved not only homologous but also nonhomologous chromosomes. SCs were formed with frequent changes of pairing partners (switches) and intrachromosomal foldbacks of unpaired axial elements. As a result, incompletely synapsed, non-homologous and multivalent SCs were formed in mutants by the stage analogous to pachytene in normal plants. In sy7 a maximum in the number of switches and foldbacks were observed at zygotene, whereas in sy10 this occurred at pachytene. We suggest that it is the process of recognition of homology that is impaired in both mutants. This leads to indiscriminate synapsis and prevents chiasma formation. Both mutants may be classified as desynaptic.

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.

Meiotic mutants of rye Secale cereale L. : I. Synaptic mutant sy-1.

A mutant form of weedy rye characterized by male and female sterility and having a hereditary block in the chromosome synapsis has been found and described. Genetic analysis has shown the synapsis block to be determined by the recessive allele of a gene designated as sy-1. Electron microscopy of surface-spread microsporocyte nuclei revealed the complete absence of the synaptonemal complex over the whole meiotic prophase I, although the axial cores were perfectly formed by each chromosome. Only univalents were observed at metaphase I, their average number ranging from 13.1 to 14.0 per cell. A precocious distribution of univalents at the poles is observed at metaphase I. All of the later stages of meiosis were irregular and resulted in the formation of abnormal microspores. Thus, the mutant proves to be asynaptic because of the blocked initiation of synapses at prophase I.

Dependence on genic balance for synaptonemal complex formation in Drosophila melanogaster.

Electron microscopic examination of gonads of Drosophila melanogaster with different genotypes, including a metafemale 3X;2A and an intersex XXY;3A have revealed that the formation of synaptonemal complexes is controlled by the genic balance, i.e., the ratio of X chromosomes to autosomes. The Y chromosome is not involved in the genetic control of the formation of precursors of the central element of synaptonemal complexes in males, nor does it disturb their formation in XXY females. Hyperploidy for sections 1-3A and 18A-20 of the X chromosome does not lead to the appearance of synaptonemal complexes in males and does not interfere with their formation in females. Females hyperploid for extensive regions of the X chromosome (sections 1-11A, 11A-20, and 8C-20) are fertile and show apparently normal formation of synaptonemal complexes. Hyperploidy for sections 8C-11A of the X results in a sharp decrease in the viability of females, in abnormal differentiation of ovary cells, and in the lack of synaptonemal complexes. These data suggest a possible important role for the sections 8C-11A in the genic balance controlling the formation of synaptonemal complexes in D. melanogaster. The lack of synaptonemal complexes in hypoploid females may be the result of abnormal cell differentiation in gonads.

Synaptonemal complex analysis of B-chromosome behavior in meiotic prophase I in the East-Asiatic mouse Apodemus peninsulae (Muridae, Rodentia).

The mitotic and meiotic chromosomes of four male East-Asiatic mice, Apodemus peninsulae, having three to seven chromosomes in addition to the standard karyotype (2n = 48), were investigated. B-chromosomes were represented by medium-sized metacentric and dotlike chromosomes. Mosaicism of bone marrow cells due to a numerical variation of accessory chromosomes was established for the males examined. Capacity of B-chromosomes to form axial elements and synaptonemal complexes in meiotic prophase I was revealed by electron microscopy. The occurrence of univalents of different morphology, bivalents, and multivalents, corresponding to B-chromosomes, was demonstrated. An increase in the number of B-chromosomes was found in spermatocytes at zygotene-pachytene relative to the number in bone marrow cells, which may be evidence of B-chromosome accumulation in the germ cell line of the East-Asiatic mouse.

Chromosome aberrations in F1 from irradiated male mice studied by their synaptonemal complexes.

Possible implications of surface-spread synaptonemal complex (SC) karyotyping in analysing the causes of sterility of F1 from irradiated male mice are demonstrated in this work. After irradiation by 137Cs gamma-rays at a dose of 5 Gy the males were mated to unirradiated females and genetic analysis of fertility in the F1 progeny was carried out. Males with abnormal fertility were examined for the presence of chromosome aberrations in diakinesis-metaphase I and in pachytene by the method of surface-spread SC karyotyping. In most cases, SC karyotyping provides additional information and permits the detection and analysis of aberrations that are not revealed in diakinesis. Two reciprocal translocations, one X autosomal and one nonreciprocal translocation were discovered in five F1 males studied. It is concluded that the method is efficient in detecting translocations in pachytene in partially fertile F1 hybrids of irradiated and normal mice.